Update August 2007

Mal's Musings

Malcolm A Traill

IN  THE  PUBLIC  INTEREST
Updated 6th August 2007

                                                 CENTRIOLES AND THE RETINA

 

 

. . . . one is not able to establish the pathway of the degradation of the glucose without having recourse or access to an enzymatic glycolytic extract from the tissues, permitting the study of the reaction intermediates in solution.”  (Translated from the French)

Meyerhof & Perdigon[1] (1940)

 

. . . What was formerly only probable has now become certain . . .  The era in which the fermentation of cancer cells or its importance could be disputed is over, and no one today can doubt that we understand the origin of cancer cells if we know how their large fermentation originates, or, to express it more fully, if we know how the damaged respiration and excessive fermentation of the cancer cells originate. . . We now understand the chemical mechanism of respirations and fermentation almost completely . . . . ” !

Otto Warburg[2] (1956)

 

Preamble: In the Submission to the NH&MRC Review into the use of UHF in cancer treatment (elsewhere, this site), there was presented an hypothesis to explain the resonance phenomenon described by Dr John Holt. To do this, the conclusion was that :-

·        The centriole seemed to be the only organelle within cells that would have the structural features capable of supporting a resonance at 434 MHz when under an intense UHF field of that frequency and,

·        The energy source, as suggested by Holt, would most likely be the glucose to lactate breakdown as described by Needham and his group in 1938,

·        This energy pathway would produce radiant energy such as heat and/or electro-magnetic waves,

·        When tissues are irradiated with 434 MHz UHF, there can be set up a resonance along the lines as described by Herbert Fröhlich (for an unnatural stress situation). The longitudinal waves would be envisaged as involving the “membrane” joining the triad microtubule groups in the circumference of the distal centriole, described by Anderson & Brenner (1971)[3], as an “A-C linking sheet” of “dense material,” [which could be a problem for Albrecht-Buehler’s hypothesis (1990)[4].] These workers seem to have used Osmium tetroxide alone for most photomicrographs. (The method quoted[5] made no mention of stains, but an example photomicrograph of cilia, with good clarity of microtubules, was with Osmium tetroxide alone) This sheet would seem likely to have an unsaturated lipid component and constitute an unit membrane. Only a few other workers seem to have applied mono-staining using Osmium tetroxide. [Such mono-staining examples are rare; that by De Robertis (1956)[6] is slightly blurred and that by Calarco-Gillam et al. (1983)[7] is weak and unclear. However, the overall centriolar staining by the latter exceeds that of the nearby nuclear membrane.] Strangely, the nature of this barrier does not seem to have been studied specifically. By examination of the various EM photomicrographs, the impression is that the membrane is more complete and better represented in the middle-to-distal parts of the centriole, where there is usually stainable material in the core, now known to contain Centrin, γ-tubulin and Sfi1 which, presumably, needs to be separated from the material between the centriolar blades. The staining reaction with Osmium tetroxide implies that unsaturated lipids are an appreciable component of the membrane, and able to act as a dielectric insulator.

·        In normal physiology, the centrosome/centriole may, under certain conditions, be associated with the same D-Glucose to L-Lactate reaction, but produce shorter bursts of radiation, possibly in the near infrared (NIR) region. That is because the frequency would be set by smaller structural intervals within the centrosome/centriole, rather than the full centriole circumference.

·        The infra-red produced may have some physiological function.

·        The centrosome/centriole may act as a physical-chemical transducer. In the previous hypothesis (located elsewhere on the website), there was suggested that the centriole may respond to calcium waves by contracting, thereby modifying other centriole-related activities. Some readers may be aware that the centriole/basal body has been implicated in sensory detection for some time[8] and that one response to tweaking a primary cilium is to produce a calcium wave. However, this is relatively slow, in 10-20 second[9] for a prod with a pipette, 20-40 second for a flow in the medium. These responses are well outside the expected responses that can be linked to stimulated contractile proteins like Centrin where, in organisms such as Naegleria flagellates, Paramecium and particularly Vorticella, contractions by Centrin or Centrin-related proteins are very rapid in response to calcium[10]. Localization of the detector components for chemical stimuli may, in part, be by the Polycystins (Teilmann et al. 2005[11]). Sadly these workers did not examine in detail the portions of the basal body-to-cilium distribution of the Polycystins. Examination of their photomicrographs would suggest that the distal basal body may be associated (eg Fig. 2C). In the vertebrate centriole there could be a two stage response – an initial, short calcium wave that stimulates the Centrin in the centriolar core to contract rapidly, which then tweaks the centriole, as can be done physically with the primary centrioles or chemically[12]. This tweak then induces (increased) writhing or spasm in the centriole, inducing a slower, more prolonged and intense calcium wave by the actions of components that are possibly in association with the centriolar blades, perhaps along the lines of the autophosphorylation of Calmodulin-dependent protein kinase II[13]. (You may recall that UHF+GBA to patients with large cell lymphoma precipitated malignant hypercalcaemia. The involvement of the centrioles with calcium may be relevant.) The writhing or spasms of the centrin both within the centrioles and around the γ-TuRC-PCM-1 (Dammermann & Merdes 2002[14]) in the outer part of the centrosomal matrix may “push-pull” or “squeeze” the microtubules in and around their “follicles.” These pass through the centrosomal matrix located over the centriolar subdistal appendages (Fuller et al. 1995[15]; Kenney 1997[16]), and may provide mechanical leverage. Intermittently, this would bring together the components carried by the microtubules and those bound to the centrosome. This intermittent physical apposition could, in the low frequency of normal physiology, activate further messenger types in an organized way. Under the extreme conditions of UHF at 434 MHz, the components would be effectively fused together. The ramifications of this possibility will be discussed later. The earlier hypothesis that there could be an energy source within the wall or core of the centrioles seems implausible, and is not considered further. 

 

Given that Nature uses electo-magnetic radiation already in numerous ways, such as fire-flies, glow worms and deep sea organisms, the possibility that NIR could be used in some way would seem likely, possible and plausible. The concept that infrared may be involved in neural networks is not new, and has been examined by my brother Dr Robert Traill[17]. The most likely wavelength (in air, λa) would seem to be ~1.6 μm, being the wavelength closest to visual light but still having reasonably low attenuation in water. The lesson from the Holt experience is that certain unsolved enigmas may now be explained, if only in an hypothesis form, particularly in relation to the big enigma of retinal (and cochlear hair cell) physiology : – how does the retina (or hair cell receptor) manage to process the huge range of light (or sound) strengths from barely detectable, to photo-bleaching (or deafening) intensity ? Both retinal and hair cell functions may, in this regard, be quite similar (Zenisek et al 2003[18], Fuchs 2005[19]) but, since the former seem to have been far more extensively studied, most consideration shall be confined to the retina, in particular, the rods, cones and bipolar cells. The enigma of the control has been mentioned by a number of authors, some being Smith et al. (2001)[20] and Dunn et al. (2006)[21].

 

To summarize what is to be addressed, namely, the signalling by the centrioles of the rods and cones by means of NIR, by which the latter provides negative feedback via modulating the functions of the ribbon synapses and pre-synaptic function :

 

               Feature                                            Role/function__________________

·        Origin of infra-red within cells             Centrosome

·        Likely cells                                        Rods, Cones and Bipolar Cells

·        Likely roles                                        Chemo-radiation transducer

Provides negative feedback, (modify plasma membrane characteristics)

·        Likely emission type                           Short bursts and spike runs of photons

(waveguide role) &/or voltage (subdistal appendage/microtubules)

·        Energy source                                    Glucoseè L-lactate (Needham pathway)

·        Transmission                          Along “axons,” acting as waveguides

·        Detectors                                          Ribbon synapses; (plasma membrane)

·        General action                                   Slow the discharge of presynaptic

secretory vesicles; (change characteristics of the plasma membrane and its receptor)

·        Biochemistry/Biophysics                    IR (la ~1.6 mm) activates lactate into

vibration mode with an higher energy level. D-lactate (a by-product of glycolysis) is at low concentrations and is probably rate limiting. It has affinity for the D-LDH enzymes of the GAPDH/CtBP class and, by being energized, attaches to the enzyme binding site(s), modifying the molecular shape, characteristics and responsiveness, thereby modifying Ribbon (and plasma membrane) characteristics.  

 

These concepts are all presented at the start, with a plan to discuss the points in greater detail now and over time.

The centrioles of the retina are chosen for examination because the centrioles within the connecting cilium of the rods and cones clearly present a pivotal location in micro- structure at least, and seem likely to fulfil a more active role than merely as a conduit for transducin between the outer and inner segments (Geiss et al. 2004[22]) in response to light.

 

1.                  Origin of infra-red within cells. The origin of NIR within cells would be, as stated earlier, from the centrosomes modulated by centrioles responding, most likely, to Calcium waves, mechanical tweaking and other chemical stimuli and acting as transducers. This requires an hitherto unexplained energy source – which must now be addressed and, for this, an answer MUST be found ! Holt suggested the Glucolysis of Needham (1937[23]), supporting this contention by his experimental quenching of the UHF resonance spectra by intravenous L-glyceraldehyde (see elsewhere on the website) in patients with cancer. This seemed a reasonable conclusion, but historically, the pathway led nowhere: Needham and his group seemed to be establishing for England a metabolic pathway to rival the German dominance; having seen the titans Warburg, Embden and Meyerhof. As presented, the Germans had elucidated the Embden-Meyerhof-Parnas pathway of phosphorylating glycolysis, to which we now belatedly add the name of the Ukraine/Polish biochemist Jakub Parnas (1884-1949). Since Needham’s pathway did not appear to be coupled to any intracellular energy-transfer mechanism, there was no apparent application to cellular processes. If real, it has been an enigma :-

There are three main possibilities :

·        That the work by Needham and his group (simply referred-to as Needham) had major flaws and should be discarded. This is a possibility that will be examined, but noting that the contemporaneous comments about his group’s work were respectful. The first doubts that appeared to be printed were in 1939. An Englishwoman Marjorie MacFarlane[24] compared the work with the metabolism of yeast which, today, can be questioned (see Appendix – in preparation). Then, the German school (moved to France) Meyerhof and Perdigon1 1940, raised doubts, after studying brei that had been frozen (an issue also to be examined in detail later in the Appendix – in preparation). The concerns were, in particular, that NAD+/NADH and many other glycolytic enzymes were present in the chick embryo at day three (E3) but that NAD+/NADH was labile, and reactions could proceed in the brei (ie broyage, prepared in phosphate buffer) if NAD+ were added. A review by Dorfman[25] followed in 1943, referring to the glycolytic pathway as the Embden-Meyerhof-Parnas-Cori-Warburg cycle.” In effect, his view was that all noted discrepancies, including the Needham work, as appreciated then, and found in the future, may be explained by the known Embden-Meyerhof-Parnas pathway using (the then) current knowledge or, if there were any inconsistencies, with future knowledge yet to be gleaned. This enshrined the Embden-Meyerhof-Parnas pathway as Holy Writ and any deviation from it to be an heresy. Such a “keep it simple stupid” approach may well be appropriate for undergraduates preparing for a multiple-choice written examination but, as is now known with GAPDH (see later), Mother Nature has failed to follow this policy ! Stumpf (1947)[26] and Novikoff & Potter (1948)[27] then provided data which, they believed, finished the Needham heresy for ever (see Appendix – in preparation).

·        There is an enzyme (or are enzymes) involved that has(have) not yet been discovered. Such would have to be organelle-bound and presumably in an appreciable amount, particularly in the early (chick) embryo. An occult and appreciable cellular component would seem unlikely to have been missed at this stage, and may be considered improbable.

·        There is a pre-existing enzyme (or are enzymes) which can be used in a way (or ways) that has(have) not yet been considered and/or studied. The most likely contender for this is Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH; EC 1.2.1.12). This is an ubiquitous, well represented, highly conserved and ancient enzyme[28],[29], best known for a key role in the Embden-Meyerhof-Parnas glycolytic pathway, but now also recognized as having an increasingly large number of functions (see later). It is related to the large family of D-Lactate Dehydrogenases (D-LDH; not to be confused with the L-LDH isoenzymes with different structure, and the enzyme that is measured in clinical practice). GAPDH, its roles and features shall be examined in detail later. There are many reasons for suspecting the key roles for GAPDH, which should become apparent as this dissertation proceeds. The likelihood of such an association has some support from the clinical observations on patients that, (as detailed earlier) :

a)      UHF caused a fall or plateau in the blood level of tumour markers. This was attributed to the disruption of the Golgi during a presumed prolonged mitosis. The mutant GAPDH in the CHO cells (Robbins 1995[30]) caused a phenotypic change, with an inability to process exocytosis from the Golgi. Heat or ion-damaged GAPDH at or near the centrosome may reproduce a similar effect, and

b)      UHF precipitated a spectacular malignant hypercalcaemia in two patients with non-Hodgkin’s Lymphoma, and lesser calcium changes in other cancer patients. GAPDH has been linked to the release of Calcium from the Endoplasmic reticulum, the major intracellular store for Calcium (Patterson et al. 2005[31]).

 

The Needham Pathway.

As proposed by Needham and his associates, D-Glucose could be converted ~directly to L-Lactate without the involvement of phosphate. Such a claimed reaction was (seemingly) demonstrated under anaerobic conditions, during the first ~3 days of chick embryo life, considered to require glutathione as a co-factor and was inhibited by fluoride and Iodoacetate.

An enzyme activity could not be identified in solution; the activity being restricted to the sediment (grana). Without any isolated enzyme, conclusions had to be based on collateral information and deduction; in particular that, in the chick embryo (E0-5), the Embden-Meyerhof-Parnas pathway had minimal flux and that there was very little effective “cozymase” (=NAD+/NADH). Later workers and critics claimed that enzymes of the Embden-Meyerhof-Parnas were present and active and that the NAD+/NADH had been present, but was unstable and destroyed in vitro; the reactions proceeding if the supply of the cofactor was supplemented to the artificial witches’ brew that was used (see later and Appendix [in preparation] for further comment).

So, for the moment, let us assume that there is some truth to the existence of such a pathway (as described) and look at observations in relation to the chick embryo and a mammalian example; the well-studied mouse embryo. In Figure 1 the Needham pathway product, L-Lactate, is shown in red, showing some similarities with the heat output, being a more absolute measure of energy production (provided the determinations are valid). No determination of mammalian zygote or embryo heat output for comparison has been identified to date.

There is a need to show caution is assessing many of the experiments with embryos. That is because phosphate-containing media, particularly when combined with glucose, were found to be toxic (Schini & Bavister 1988[32], see review by Bavister 1995[33]) and resulted in death or various delays to different degrees in different species and strains, a susceptibility seemingly passed largely maternally. Since almost all physiological buffers prior to 1988 included phosphate, the degree to which this may have influenced experimental results is probably unclear. There has been no work found on this aspect with regard to the Chick embryo. Since, in mammals, the ultimate test is successful implantation and live birth, and this test is not possible with a Chick embryo that has floated on a phosphate buffer; any subtle effects of phosphate and glucose on Chick metabolism and viability would seem untested to date. 

                                                                        Figure 1

Figure 1: Thick lines are from Chick Embryo [C]: Needham’s anaerobic measurements; Heat[34],[35]; Enzymes[36]. The Needham et al. glucolytic activity is as Lactate produced anaerobically from glucose by chick brei per mg/dry weight, the heat as cal/mg dry weight/24h. Needham’s Glucolysis activities before E2½ are probably higher, but figures are unreliable/absent. (The vertical scales cannot be related, merely the relative trends with time.) The thin lines are from Mouse embryo [M]: Aerobic metabolism, by μL/mg dry weight, Brinster (1974)[37]; Enzyme activities[38], are for comparison, and indicate that the aerobic +/- glycolytic metabolism is not appreciably activated until ~E4. The time for late morula in the mouse embryo is indicated by the vertical, dashed lines, which also corresponds roughly with the first detection of Centrin associated with the centrioles in the mouse (Hiraoka & Golden 1989[39]) and the α-subunit mRNA of the Na+/K+-ATPase pump to form the blastocoel (Gardiner et al. 1990[40]). The need for glucose (minimum 5.5 mM, a level toxic at earlier E times) to stimulate the mouse transition from morula to blastocyst, with maximum effect at 42h (Chatot et al. 1994[41]) is shown by the faint pink vertical line.

 

To dispel the impression that the Chick’s energy source is largely from glucose and/or mannose, the energy supplied during the incubation of the hen’s egg has been set out in the table below, which is taken from Romanoff & Romanoff (1967):

                                                      Energy (in Kilocalories)

                                                In the Egg         In the Chick      Expended

Chemical Component                          (with spare yolk)

Proteins            35.4                 34.9                 3.5

Lipids               50.6                 27.6                 19.9

Carbohydrates  1.9                   1.5                   0.5

            Total    87.9                 64                    23.9

From this we can conclude that the carbohydrate pool stays relatively small, but with an active flux. We must presume that the L-Lactate that is produced through the Needham pathway is, to a considerable degree, to provide 3-carbon intermediates ultimately for purposes such a lipid synthesis and protein production, such as the major production of MPF[42], with some to be used for oxidative respiration in the (Krebs) Tricarboxylic acid cycle (TCA) and mitochondrial cytochromes.

The GAPDH and Hexokinase activities are artificial and in vitro, created by introducing to the suspensions of enzymes NAD+ +/- substrate, with no consideration of compartmentalization, and may simply represent the added NAD+’s competitive and (possibly) unphysiological access to the enzyme structures present in the witches’ brew at that developmental stage. [In suspensions made without a detergent and divalent cation chelator, many gross, organized tissue structures would be disrupted, and some cell plasma membranes, and possibly some nuclear membranes would be expected to be damaged[43],[44] but sub-cellular, smaller organelles and their organization, such as the nuclear-centrosome-centriole associations would probably be largely structurally intact[45],[46],[47],[48] within the cytosol gel – being still “vital,” (having limited function) in the grana (granular sediment). Mitochondria, by the above method would be “not morphologically identifiable” as assessed by electron microscopy of the time.[49] (It was the traditional addition of sand that destroyed the nuclei before the addition of any water. Distilled water obliterated the mitochondria). Freezing, without cryo-preservatives and appropriate care, would be expected to break-up micro-compartments[50], dissociate microtubule-centrosomal association[51] and produces lipid peroxidation, reduction of SOD and membrane stress with embrittlement in human spermatozoa[52],[53]. Grieg et al. 1939[54], in giving some support to the work of Needham but using 6 day old chick embryos, noted that merely cutting the embryos with scissors (“several times”) resulted in increased anaerobic glycolysis, or a better exit of intermediate products, as would be expected if there were breakdown of micro-compartments.]

Considering the greater cell mass and differentiation stage in the chick, (which has reached the 8 somite pair stage at ~28 h, whilst the Mouse is at ~2-cell) the transition in metabolism seems based largely upon embryonic age (“clock”), although Brinster (1974) considered that the cleavage divisions were the major influence in the mammalian comparisons (see Figures 3-4 for further elaboration). The activation of the glycolytic (EMP) pathway seems to coincide with the appearance of Centrin and centrioles within the MTOC/Centrosomes. Perhaps the arrival of these stabilize and bring under control the microtubule/TuRC/MTOC/PCM relationships which, up until then were influenced by the number, potential and dilution of MTOCs (Gard et al.1990[55]).  Notable are :

a)      The dominance of the heat production by the very young chick. Whilst we may wonder about the accuracy of the value on the first day, the trend thereafter seems consistent. No similar measurements upon mammalian embryos have been found to date. This can be regarded as an absolute and direct measure of energy production.[Lokhorst  & Romijn, (1960)  provide the results obtained from one egg ! (Possibly representative.) The Respiratory Quotients (RQs)[56] are 1.63, 0.84, 1.0, 1.0, 0.94, 0.86, 0.80, for day 1 è 7. The RQ of 1.63 would probably indicate that carbohydrate is being converted to fat, as from C2 units, to which may be contributed CO2 from the conversion of Pantothenate to CoA (McKiernan  & Bavister  2000[57])].

b)      Despite the mixtures of vertical scales, the Needham pathway activity shows some similarity with the fall of the heat production after E2½.

c)      GAPDH and HK in vitro activities are possibly present and low at E2 but fall, before rising.

Whilst some enzymes may be present E1-3 and can be driven to activity artificially by adding substrates and cofactors, such findings do not establish that the flux through the entire glycolytic (EMP) pathway is fully active. Bavister (1995) noted the seemingly excessive exit (leakage) of lactate and pyruvate from the early mouse embryos, consistent with an excess production over the ability to utilize the intermediates, and hence an exit leak. Lactate could be produced via the Needham pathway and be converted to Pyruvate by LDH, with the requirement for NAD+. Consistent with this, is the high level of activity of LDH until ~E2, when it falls exponentially (Epstein et al. 1969). Needham maintained that the EMP pathway was effectively inactive up until ~E5, attributing this to either lack of specific enzymes or lack of cofactor NAD+/NADH. He may have been correct about the enzyme deficiencies and/or the absent flux :

·        Rinaudo (1966)[58]; Hexokinase (HK), phosphofructokinase (PFK), Triosephosphate isomerase (TPI), Phosphoglycerate kinase (PGK), phosphopyruvate carboxylase and pyruvate kinase (PK) do not appear in the chicken liver until about E8 as assessed by in vitro individual enzyme activity. [The liver was homogenized in 0.15M KCl, centrifuged and the supernatant tested for TPI, PGK PK and phosphopyruvate kinase. An homogenate in 0.02 M K2HPO4-KH2PO4 buffer, pH=7 centrifuged (x2) for the HK assay. No homogenate or extract is recorded as having been frozen.]

·         Ledig et al. (1982)[59] noted effectively no ENO enzyme activity at E6, being detectable at E8, and not rising appreciably until 4 days before hatching.

·        Maxwell et al. (1982)[60] could not detect lumbar spinal cord neuron-specific enolase until E9-10, by histochemical staining.

·        Kusuhara & Ishida (1974)[61] reported that, at E12 (the earliest stage examined), chicken yolk sac membrane had “weak” levels of GAPDH by histochemical staining.

None of the above studies used the very sensitive techniques that would be necessary to identify small, but concentrated levels of enzyme in a selected compartment, such as at or on the centrosome, and testing in the crucial E1-2 interval is not documented.

Needham’s critics used very destructive cellular processing to make their witches’ brews, quite remote from the modern concept of the compartmentalization of reactions and their reactants (see Appendix – in preparation.). So, the documentation for the early Chick embryo (pre-E5) seems to support one of Needham’s major contentions, namely, that the flux through the EMP pathway is probably very small, if present at all, because there is little, if any, evidence of a full set of functional enzymes in situ.

The graphs for some of the enzymes in the mouse zygote and embryo are presented for comparison, with some similarities to the Chick embryo trends. The mouse model is extended in more detail in Figure 2, for which there is much more detailed information.

                                                           Figure 2

Figure 2. Mouse embryo metabolic changes. The glucose 5.56mM to CO2 data are from Brinster (1967)[62], with results also doubled to equate to 3-carbon intermediates (remember 1 mole of glucose è 2 mole Lactate è 6 mole CO2).  Pyruvate and lactate to CO2 data are from Brinster (1967)[63].  GAPDH mRNA (in vivo and in vitro) data are from Mamo et al. (2007)[64], their early 2-cell data are placed in the late Zygote stage for graphical reasons and because there may be very early transcription at that stage (Bouniol et al. 1995[65], Hamatani et al. 2004[66]), with the onset of transcription some 4 h prior to the detection of mRNA. Glucose (2) to CO2 Total & via PPP data are from O’Fallon & Wright (1986)[67], expressed as per embryo over 4 h.  Conspicuous, are the graphs for GAPDH mRNA, with an early rise ~late zygote-early 2-cell embryo, more marked in the in vivo data.

In Figure 2, the uptake and conversion of pyruvate and lactate to CO2 are shown, with what appear to be relatively stable and consistent states. On the other hand, the uptake and conversion of glucose shows a jump (boost) at sperm penetration/fertilization. This seems associated with the gene activation for GAPDH, at a time when emphasis is on the TCA metabolism, prior to the “programmed waves” of gene activation associated with anaerobic glycolysis (Hamatani et al. 2004). The Pentose Phosphate Pathway (PPP), though only tested from the 2-cell stage onwards, indicates a moderate activity, then falling at the 8-cell stage before rising. The GAPDH mRNA shows an early burst, then a fall, with a later steep rise. Based upon the G-6-PD activity changes (Epstein 1969)[68], the likely PPP activity in the Zygote may be slightly higher than that in the 2-cell stage. Using the data from Lane & Gardner (2001), in which the inhibition of the 3-Phosphoglycerate kinase step of the glycolytic pathway by EDTA and Cibacron blue (with no reference, but Billington et al. 2004[69] may assist) produce remarkably similar results, when we consider that these agents could inhibit other steps by different mechanisms. There was also a discrepancy in the inhibition of 3-PGK activity as opposed to the inhibition of the EMP flux : -

 

2-Cell

  3-PGK activity                                     PPP component of total glucose use

Post EDTA 10 μM   ~ 81.9%

15.8%

The inhibition of glycolysis (EMP, see next table below) is disproportionate to the inhibition of 3-PGK

 (Extracted from Lane & Gardner 2001[70] & O’Fallon et al. 1986)

 

Accordingly, we may estimate the contribution that the EMP and PPP pathways give to the zygote, and find that there is some 19.9 – 24.6 % activity in the utilization of glucose that is unaccounted. This would be consistent with a pathway direct to L-Lactate, as by the proposed Needham pathway : -

 

Zygote       

Non-Glycolysis; (EMP)”        è        Less PPP component (guestimated)

(Left after inhibitor)                              Glucolysis* (unexplained)

Post EDTA       10 μM ~ 42%

Post EDTA     10 μM ≈ 24.6% ?

Post Cibacron# 10 μM ~ 37.3%

Post Cibacron 10 μM ≈ 19.9% ?

*PPP in Zygote (~15%), extrapolated from 2-cell; presumed maternal contribution. Based upon the enzyme level of G-6-PD (Epstein et al 1969), the PPP might be ~+11% over the 2-cell level; 17.4%.  Epstein et al. (1969) noted that LDH activity was high è 60 h post ovulation, then fell over 2 days (exponentially) [as though switched-off]. #Cibacron blue.

 

Assessments of Glucose uptake by authors differ; Brinster (1967), using a radioactive method obtained a much higher reading for the 1-cell zygote than Gardner & Leese (1988)[71], who used colorimetric methods. Whilst the micro-colorimetric techniques are technical triumphs, they may be at the lower limits of sensitivity, and have not been used for graphical presentation.

 Measure                  1-cell                            2-cell                        8-cell                        Blastocyst

Glucose uptake (in 1 mM);

Chemical test#

0.05 +/- ? pmole/E/h

0.73 +/- ~0.09 pmole/E/h

0.82 pmole/E/h

4.38 pmole/E/h

U14C Glucose (in 0.556 mM) to Carbon dioxide*

0.30 +/- 0.01 pmole/E/h

0.68 +/- 0.04 pmole/E/h

1.25 +/- 0.12 pmole/E/h

7.2 +/- 0.23 pmole/E/h

U14C Glucose (in 5.56 mM) to Carbon dioxide*

0.68 +/- 0.02 pmole/E/h

1.19 +/- 0.09 pmole/E/h

2.16 +/- 0.08 pmole/E/h

10.9 +/- 1.11

pmole/E/h

#Garner & Leese (1988); * Brinster RL Exp. Cell Res. 1967; 47: 271-77

The Gardner & Leese (1988) work, however, presents a graph of glucose uptake, showing an uptake peak in 3 mM Glucose, with the maximum Standard Error derived in 2.0 mM Glucose medium. The latter could represent a critical point, at which pathways may diverge; that enzyme systems switch from the preferred zygote-embryo type, to the later embryo type. Consistent with this, Martin & Leese (1995)[72] found that lower concentrations of Glucose (0.5 – 2.0 mM) were stimulatory to the zygote-embryo but, when >~2.0 mM, Glucose became toxic. The hypothesis that GAPDH in a particular metabolic compartment, can change from one type of function to anther, as part of the Zygote-Embryo maturation, may be illustrated : -

GAPDH PHASES

 Product                 Glucose              NAD+/NADH     Pinorganic#           GSH                    Magnesium[73]

L-Lactate

0.5-2 mM

Nil

Nil

Co-factor

? Nil

GAP*

>3.0 mM

Involved

Required

? not needed

Needed

*GAP= Glyceraldehyde-3-phosphate. #Inorganic Phosphate

To apply higher concentrations of glucose, NAD+/NADH, inorganic Phosphate, and less (reduced) Glutathione (GSH) (to list a sample of factors relevant here) could change the phase of GAPDH within the compartment and comply with the EMP pathway inappropriately. This could have flow-on, disastrous effects upon the subsequent embryo development.

Agents that block this switch such as EDTA added to the culture medium (Lane & Gardner 2001), &/or (hypo) taurine (Barnett & Bavister 1992[74] [hamster]), &/or reducing oxygen tension etc. (Bavister 1995) may be seen to be factors that directly or indirectly set the phase of GAPDH to be more appropriate for that particular stage in development in the compartment concerned. Sensitivity to different amino acids may likewise reflect the ability for the zygotes and embryos to use those that are beneficial without disturbing the appropriate GAPDH phase.

Otherwise inexplicable observations may be explained by this phase shift, such as :

a)      The ability that 2-cell embryos of sensitive, blocking strains, being able to proceed after a small transfer of cytoplasm from a non-blocking strain Muggleton-Harris & Whittington (1982)[75]. (At the 1-cell stage, 22pL cytoplasm had only minor benefit when assessed at the 8-cell/morula. However, at the 2-cell stage, 8pL (~4%) of egg volume transferred produced much better results. Since GAPDH can autophosphorylate (Kawamoto & Caswell 1986[76], Laschet et al. 2004[77]), the injected cytoplasm could act as an infectious-like nidus causing an expanding halo and wave of phosphorylation, appropriate for the 2-cells stage to move on along its life-line.

b)      The ability of a one minute dip of an embryo at about 42 h in a glucose-containing medium was adequate to stimulate the embryo to develop further (Chatot et al. 1994). The authors noted that this seemed to imply a switching process. The explanation may be that, at that stage in development, an high level of Glucose exposure triggers a full conversion to phosphorylating glycolysis through GAPDH in the compartment concerned, appropriate for the following stages.

c)      Other compounds, such as Nicotinamide, may be detrimental in some species, such as mice. (Bavister 1995). Nicotinamide may occupy the Rossmann fold and bind to the NAD+/NADH binding site thereby inhibiting the phosphorylating pathway’s GAPDH function.

d)      An elevated oxygen tension in the Zygote stage may allow consumption of reducing compounds (Johnson & Nasr-Esfahani 1994[78], particularly Glutathione, and deprive the GAPDH of its cofactor needed at that stage in the compartment concerned.

                                                            Figure 3

Figure 3. Metabolism in the mouse. The pyruvate and lactate that exit the cells into the media are figures from Wales (1969)[79]. The pyruvate and lactate to CO2 data are from Brinster (1967, as in Figure 2).  The horizontal time scale is very approximate. The data for the oocytes were taken from oocytes at about the time that other oocytes were being inseminated. Because the metabolism (energy production) of the oocyte seems to be steady until sperm penetration, the value at this time was used retrospectively for the ovulation point (contentious). With these points, there would seem to be reasonable lines of interpolation between the oocyte activity and 8-cell & blastocyst stage – “life lines.” The deviations from and above the lines at the zygote and 2-cell stage will be presented again in Figure 4.

Figure 3 is drawn to highlight the “boost” to glucose uptake following spermatozoan penetration. Despite some contentious estimations, there seem to be “life-lines” for both glucose input and lactate output (exit), which represents a leakage probably associated with disproportionate lactate production to ability to utilize lactate. These “life-lines” raise the possibility that the concept of a “zygotic clock” (Van Bleckom 1981[80], Nothias et al. 1995[81], Day et al. 2001[82] & Zuccotti et al. 2002[83]) may extend back to ovulation and forward to the early blastocyst.  The former may be supported by Wang et al. (2004)[84], although Hamatani et al. (2004) do not favour the latter process. Given that some mammalian embryos proceed onwards by a parthenogenic pathway, the formality of fertilization for others may be seen to be an introduced hurdle in an otherwise continuous process of development along the “life line.”

                                                                    Figure 4

Figure 4. The Glucose to CO2 data, 5.56 & 0.556 mM, are from Brinster 1967 as in the earlier Figures. The O2 uptake is quoted from Mills & Brinster (1967)[85].  The boosts to metabolism brought about by spermatozoan penetration &/or fertilization are shown, with the lactate leak (exit) peaking after that for pyruvate.

The graphs in Figure 4 are to highlight the boosts to Glucose uptake and L-Lactate exit (loss), the former seeming to peak first. This is at a time when the “programmed wave” of gene expression favours oxidative (TCA) metabolism (Hamatani et al. 2004). The boost (rise) in Glucose utilization is consistent with the observations of Martin & Leese (1995)[86], who found that Glucose on E2 +/- 1 day was needed, with deficiency resulting in a subsequent failure to move from compaction to blastocyst (see also Du & Wales 1993[87],[88]; Watson 1992[89] for a review).  With a level of Glucose of 1.5 mM in the rabbit oviduct fluid available for the cumulus cells to utilize to produce Pyruvate,  the rabbit unfertilized ova would have all three nutrients, Lactate, Pyruvate and Glucose, but the latter at a level lower than that frequently used in culture media (Leese & Barton 1985[90]).

 

Summary to the present.

There have been presented analyses of data on Chick embryo and mouse embryo to show that glycolytic flux in the early Zygote/Embryo is likely to be minimal or absent. Instead, there are reasons to believe that there is a pathway from Glucose to L-Lactate that cannot be linked to the EMP phosphorylating pathway. There is circumstantial evidence that GAPDH may be present and possibly active at these stages. Such a pathway and GAPDH involvement would appear to have largely disappeared in the overall metabolism by the 8-cell stage in the mouse (about E2+), which may correspond to a similar age in the Chick embryo (with some flexibility). With GAPDH very much under the spotlight, we may now have a closer look at more recent studies on this remarkable “master” enzyme : -

 

RÔLES FOR GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE

(GAPDH: EC 1.2.1.12: Functional gene, chromosome 12)

 

SOME ROLES FOR GAPDH[91],[92],[93] (some are probably overlapping, eg site and action)

Possibly incomplete:

Red = intranuclear, Green = glycolysis/enzymatic, Blue = membrane function, Pink = conditions

 

1)            Glycolytic pathway enzyme (Embden-Myerhoff-Parnas; EMP)[94]

2)            Conversion D-lactate « pyruvate (weak)

3)            Reduction of Arsenate to Arsenite, needing NAD+ & Glutathione[95]

4)            Membrane fusion/fission functions

5)            Mobilization of Calcium from Endoplasmic Reticulum stores

6)            GABAA receptor inhibition by kinase action

7)            Membrane trafficking; endoplastic reticulum to Golgi[96],[97]

8)            ATP-dependant charging of neuronal synaptic vesicles with glutamate[98]

9)            Nuclear RNA export

10)        DNA fault recognition and repair

11)        Transcriptional control of histone gene expression[99]

12)        Telomere structure maintenance

13)        Apoptosis (nuclear translocation)[100],[101]

14)        Microtubule bundling

15)        Microtubule binding with GAPDH[102],[103] (C-terminal α-tubulin) and function 

16)        Actin-binding

17)        Bacterial, yeast, Mycoplasma, and parasitic cell wall component and virulence factor[104]

18)        Viral pathogenesis

19)        RNA regulation

20)        Phosphotransferase/kinase[105]

21)        Nitric oxide physiology/biochemistry

22)        Immunoglobulin production stimulating factor

23)        Hyperglycaemic stress reaction

24)        The metabolic syndrome[106]

25)        Promyelocytic leukaemia

26)        Amyotrophic lateral sclerosis[107], Alzheimer’s disease, Parkinson’s disease and Huntington’s disease

With such numbers of diverse rôles arising from the presumed progenitor of GAPDH, in number probably eclipsing all other enzymes, we may conclude that it may have been the master enzyme of the cell.

Not only do the photoreceptors of the retina contain relatively large quantities of GAPDH, but they also have considerable quantities of the related enzymes CtBP and Ribeye, located in the ribbon synaptic terminals[108]. And they have diverse rôles :

 

RÔLES FOR THE C-TERMINAL BINDING PROTEIN (CtBP FAMILY)[109],[110],[111]

1)            Transcription co-repressors for some ~20 transcription factors (may need dehydrogenase domain.)

2)            Context-dependent transcription stimulation

3)            Involved in DNA repair (?)

4)            Interaction with nitric oxide synthase

5)            Nuclear sensor for NADH/NAD+ redox state

6)            Acetyl transferase activity in the Golgi

7)            Anti-viral action – combines with PXDLS motif

8)            Related to Ribeye & component of retinal ribbon synapse protein

9)            Microtubule cytoskeleton (in plants)[112] (?)

10)        Catalysis of pyruvate « D-lactate

Whilst Chinnadurai (2002) stated that acid dehydrogenase activity had not been detected in CtBP, Kumar et al. (2002)[113] and Achouri et al. (2007)[114] established that it had D-lactate enzyme action, with the dehydrogenase domain the gene co-repressor.

Given that GAPDH, CtBP and Ribeye are all derived from the highly conserved D-LDH family, there should be surprise that LDH enzyme activity (lactate « pyruvate), if occurring at all, is apparently, very minor: this function seems to be vestigial and not the means of eradicating, what generally is presented as the waste product, D-Lactate[115], since there is probably a more proficient mitochondrial enzyme system close to the point of D-Lactate creation from the Methylglyoxal/Glyoxalase II pathway[116]; – or the D-LDH equipment and capability are used in another way – see later.

 

To clarify the reasons for believing that GAPDH may lie at the heart of the Needham glucolysis, below are some observations by Needham :

 

 Feature                            Needham’s glucolysis                Embden-Meyerhof         Affected ?

Sensitivity to IAA*

(Embryo) Very sensitive:

             2x10-4 M

(Muscle) Sensitive:

            2x10-3 M

GAPDH

Sensitivity to inhibition

      by Fluoride#

45% at 5x10-3 M

100% at 5x10-3 M

Enolase or

phosphatase@

Co-factor

Glutathione

NAD+/NADH

 

*Iodoacetate; an indicator of –SH groups in critical position(s). It blocks reactions

#An indicator of Mg2+ (&/or other divalent ions) – the traditional explanation being that it formed fluorophosphate, which has a low dissociation. This has been studied more recently[117]

@An example of a protein phosphatase with several functions involving variations in substrates, cofactors and inhibitors is given by Nakai & Thomas (1974)[118]

 

Earlier researchers were concerned with the “straight” and “housekeeping” roles for the enzymes of the main metabolic pathways They were little concerned with the “discrepancies” that were found, inconsistencies which seemed irrelevant to the enzyme’s perceived main role. [Refer to the Appendix (in preparation) for elaboration.]

 

Dehydration

The involvement of an Enolase (ENO, or similar) function is necessary to explain these findings. This enzyme removes water, and belongs to a superfamily, all sharing the basic mechanistic step whereby a proton is removed from the α-carbon of a carboxyl group[119],[120],[121], with the hydroxyl group following it. Two Magnesium atoms are required for each catalytic site – one is positioned first as “conformational” and the “catalytic” other follows the substrate to the site, with this Magnesium atom’s removal inhibited if the solution ligand substrate concentration is high – a trapdoor-like effect. They provide the sensitivity to Fluoride inhibition when inorganic Phosphate is present. The mobile catalytic Magnesium atom may function as an enable « disable switch, sensitive to electrostatic forces. It could act like the computer processor “clock,” locking the enzyme steps into an applied frequency, possibly such as 434 MHz, or by amplitude modulation on a carrier frequency[122]. The Enolase molecule is barrel-shaped, with the reactive groups that line the interior attached to amino acid loops. ENO is not generally found bound to GAPDH, and is generally not found attached to microtubules (Walsh et al. 1989[123]) except in myocytes (Keller et al. 2007[124]) that are deficient in centrosomes (Tassin et al. 1985[125],[126]). High speed, controlled and cooperative function would require special physical requirements. However, the ENO location and concentration (in part) at the centrosome[127] and its involvement as an autoantigen in certain autoimmune diseases[128],[129] is consistent with such a rôle, if the GAPDH association can be postulated.

A step leading to dehydration also occurs in the scheme proposed by Needham, however, the first carbon atom of glucose has a carbonyl group, so that there is a clear difference, unless the GAPDH can modify this into an appropriate intermediate or accept it as is. Needham’s suggested intermediate is that which may be obtained following the extraction of water  :

 

            CH=O                         CH2

            │                                 || 

         HCOH                            COH

            │                                 │                                 COOH  

      HOCH                         HOC                                  │

            │           è                  ||               è        HOCH

         HCOH    – H2O              COH                            │

            │                                 │                                 CH3 

         HCOH                         HCOH                2 x L-Lactic acid

            │                                 │ 

            CH2OH                        CH2OH

          D-Glucose                 Intermediate ?                  

           

For the postulated reactions of these two enzymes to proceed rapidly (as under the overall clocking influence of 434 MHz) they would need to be in very close proximity. In the primitive dinoflagellates, both enzymes are incorporated within the same protein chain[130] – to the puzzlement of the authors. This arrangement would be fine for a very simple organism with a need to produce L-Lactate quickly by the Needham glucolysis, but would reduce flexibility and lock the organism into a metabolism which may not be easily varied over time. That organisms with flagella and their basal bodies (~centrioles) should develop such complex combined enzymes would seem consistent with the development of some special function(s). In Streptococci, GAPDH and ENO are bonded on the outer surface of the organism, and may play a role in pathogenesis (Pancholi & Chhatwal 2003[131]). How they reach that location, and the nature of their bonding are mysteries to date.  In higher organisms, the enzymes PGK, GAPDH, ENO & PK, dealing with the three-carbon intermediates, are loosely bound by Annexin A2[132] (p36) (producing a complex later referred-to by the latter authors as the “metabolome[133]), which may include RNA and S100 isoforms. There would be scope for the closer association of particular enzymes at appropriate times. The linker protein p22 could bring the GAPDH to the microtubules of the centrioles in a loose association (Andrade et al 2004x2 – see earlier, and Walsh et al. 1989[134], and others[135],[136],[137],[138]) as may be demonstrated with red cell membranes (Kliman & Steck 1980[139], Ryazanof et al. 1988[140]), difficult to show by standard chemical tests, and needing tests such as immune fluorescent decoration. However, perinuclear cytoplasmic fluorescence for GAPDH swamps the centrosomal region[141],[142],[143]. (Enlightenment on this was sought by e-mail from the Andrade et al.’s corresponding author Margarida Barroso but, sadly, there was no reply. Perhaps there is a bright yellow spot (p22+tubulin) at right and above the nucleus in their Figure 2c that could be the centrosome; and, again, the slight possibility of a red spot (indicating p22) between the nucleus and the Golgi in their Figure 2f, is in the right position to be a centrosome. The interpretation of their Figure 20 is not possible because of the intensity of staining.) There is documented binding of GAPDH to microtubules extending from the plasma membrane[144] via the microtubules to the perinuclear region where the centrosome is found. In addition to the GAPDH-microtubular association already listed, MAP1B also associates with neuronal microtubules (Cueille et al. 2007). Whilst the MAP1B heavy and light chains show no clear gross co-localization and dispersion similarities, the light chain does seem to co-localize with GAPDH in the probable centrosomal region, with some streaming into the cytoplasm, consistent with the possibility that the association is by the microtubular binding. The MAP (210 kDa) of vertebrate non-neural cells also binds with microtubules, but does not bind to the PCM or centrioles[145]. Since MAP competes with GAPDH for binding, exclusion of MAP (210 kDa) from the centrosome creates a protected region for potential GAPDH binding to the centriolar microtubules. With all this microtubular binding or association, there is the distinct possibility of some form of binding to, or around, the most concentrated microtubular structure in the cell, the centriole. But has anyone looked ? [Riederer reported that the diffuse GAPDH concentration makes difficult the immune co-localization decoration testing for centrosomal antigens. (Personal communication 2007[146])]. This group also noted that H2O2 or Colcemid treatment caused the GAPDH decoration to disappear from the cytoplasm. Given the quantity and applications of GAPDH, one may wonder if the immune epitopes were hidden by non-catalytic –S–S– site bonding caused by the H2O2 treatment and dislodgement from the microtubules resulted in the transfer of GAPDH to the endoplasmic reticulum by covalent bonding at non-catalytic sites. Binding to the –SH groups of the endoplasmic reticulum as a result of intravenous oxidized glutathione administration could set the stage for subsequent release of calcium (Jafri & Keiser 1995[147]; Patterson et al. 2003) and activation of apoptosis following UHF treatment. Such an effect by H2O2 may also have some relevance to the “2-cell block” in mammalian embryology[148],[149],[150]. Simply because the GAPDH dimer can be bound to the microtubules, a congregation of enzyme molecules at or near the nucleating sites (Chrétien et al. 1997)[151] can be presumed. Their Figure 8c, created in vitro from purified centrosomes and purified tubulin, shows a “halo” or space measuring ~ 6.2 - 6.7 nm (~ ¼-⅓ the diameter of the microtubules) around the negative ends of the microtubules, between the microtubule and the pericentriolar material (PCM) and uniformly through the PCM shown. There was some patchy accentuation of the staining of the PCM abutting the space, but no “root bulb” and no clear transverse space or structure at the proximal end. Unknown, is whether the GAPDH could be in the spaces surrounding the microtubules in vivo (assuming it not an artefact) or at least at/in the microtubule/PCM interface and mass, and whether MAP molecules are there also. With the nucleating activity (Dammermann 2003[152]), free minus ends are stable or depolarize – they do not grow. Centrosomal minus ends do not depolymerize – can only grow or be stable.  

Tensions, growth and movements of the microtubules could modulate the effective concentration of GAPDH at the sites and the apposition to ENO. The general structure of γ-tubulin is very similar to that of the α- & b-tubulins (Alprez 2005[153], Downing & Nogales 1999[154]), so that γ-tubulin attached to the PCM as part of the microtubule-associated ring complexes (TuRCs) could be cross-linked to the GAPDH dimer bound to the microtubules and form the GAPDH tetramer link (a variant of microtubular clumping), helping to tether the negative end of the microtubules to the centrosome. ENO, known to be bound to the centrosome (Johnstone et al. 1992), but reported to be not associated with GAPDH in red blood cell membranes (Chu & Low 2006)[155] at least, would be in close proximity (if not loosely bound) as components in the fragile γ-TuBC/pericentrin lattice [Dictenberg et al. 1998[156], Manandhar et al. 2000[157](Fig 1D)], and molecules of substrate could “ping-pong” between the enzymes. This arrangement would comprise a special compartment of an organoid type, where ATP, which dissociates the GAPDH-microtubular links, would not be required, and may be excluded (in part) by the location in the peripheral PCM. Consistent with this is the observation that ATPase activity at the centrosome is not a conspicuous feature[158] (although the photomicrograph detail is small), and where it occurs, it seems to involve the region between the centriolar blades, where it could associate with CaM protein kinase II[159] (or similar), rather than the PCM further removed, where the microtubular nucleation occurs (Chrétein et al. 1997; Moritz et al. 1995[160]). (The tubulins bind and use GTP for polymerization, Downing & Nogales 1999.) Also, the cognate HSP70, which is found at the centriole and has been shown to associate with α-,b- and γ-tubulin[161], does not need co-factors to process γ-tubulin (Melki et al. 1986)[162], consistent with a safe-haven from nucleotides. The Lactate that may be formed by this organoid arrangement can be processed by the L-LDH (type B) attached to the centrosome[163] in human transformed and untransformed cell lines. Importantly, there is a high level of L-LDH (type B) in the mouse embryo for the first 2 days[164] (which may reflect a primitive arrangement that may reappear in cancers), until there is a fall and change to type M[165].  An immunochemical decoration for GAPDH (if technically possible) would be expected to show a speckled pattern at the negative ends of the microtubules in the PCM, around the subdistal appendages and between the mother and daughter centrioles, at least, and not too close to the core in the distal centriole. Whether or not there is any loose GAPDH attachment to the microtubules of the centriole awaits further research. The inability of any documentation of an appreciable quantity of ENO (and where it can be found) in the zygote and early embryo poses a problem. There remains the possibility that GAPDH under the appropriate conditions (with the exclusion of phosphate) and in the right compartment, may be able to perform this dehydration step, presumably still with Magnesium as a co-factor. Such a rôle would be likely to be relatively primitive and inefficient, to be replaced by the mature ENO at an appropriate stage.

Needham studied the early chick embryo which, fortunately, has only one gene and one mRNA for GAPDH[166]. This means that any changes to function will be post-transcriptional. This is unlike the situation for Homo sapiens and rats/mice, where there is a single functional gene but some ~10 or ~200 presumed non-functional pseudogenes respectively. He claimed that, in the early chick embryo, the phosphorylating processes for the glycolytic pathway are very immature, with effectively no NAD+/NADH. If that is the case, then the Rossmann fold, which typically holds the NAD+/NADH cofactors, will be empty, leaving plenty of space for the glucose (or mannitol) and Glutathione as cofactor. Not only would there be more space, but the bonding and rigidity of the two components of GAPDH will be lessened, permitting greater freedom. Already, there is documentation that DNA and/or RNA (particularly with AUUUA sequences)[167],[168] can occupy and compete for the binding site in the cleft typically used for NAD+/NADH, with specificity created by tertiary structure, rather than set amino-acid sequences; the RNA binding to GAPDH monomers inhibiting homotetrameric formation. Later in development, when the production of NAD+/NADH becomes appreciable (&/or there is better access), the cleft will be occupied by one or other of these, blocking it to variable degrees and, depending upon location, the Needham glucolysis and other processes that use DNA or RNA[169]. Needham concluded that the only co-factor required was Glutathione. If it occupied the cleft near the site to be used later in development by NAD+/NADH with or without other factors such as DNA and/or RNA, we may be able to apply, by analogy, the reactions noted in the reduction of Arsenate to Arsenite. This reaction in mammals has been examined by Németi et al. (2005)[170], Gregus et al. (2005)[171] and Németi et al. (2006)[172]. Their work firmly establishes a place for Glutathione as a co-factor for at least some reactions involving GAPDH. NAD+ was required as the ultimate electron/proton recipient. They postulated that a reaction similar to those seen in bacterial or yeast plasmids may operate; but in these, NAD+ is not directly involved. The plasmid enzymes’ catalytic reaction is centred on three Cysteine residues, two being supplied by the enzyme, one (generally) being provided by Glutathione[173]. Arginine residues probably stabilize the catalytic site and attach to the Carbonyl of the Glutathione Glycine and the γ-Carbonyl of Glutathione, although there are other possibilities. The Glutathione will not attach unless the Arsenic ion is present. Based upon energy considerations, the Glutathione step is irreversible. Water is produced, but reutilized. They note the presence of Sulphate initially at the reaction site, which is replaced by Arsenate. Sulphate reduction is recognized in plants (Weber et al. 2000[174]) but is not considered a mammalian pathway. Consideration may now be given to the possibility that, under certain special circumstances, such as in the Zygote and early Embryo, Sulphate may be reduced to Sulphite, along analogous lines shown in the flow diagram given by DeMel et al. 2004 (their Figure 5), with covalently attached sugar components, giving unstable intermediates.

 

  Plasmid                          Source                   Initial Co-factor                Later reductant

R773

E. coli

Glutathione

Glucoredoxin

pI258

S. aureus

Thioredoxin

 

pI258 homologue

S. aureus

Glutathione

Glucoredoxin

Acr2p

S. cereviciae

Glutathione

Glucoredoxin

 

Presumably, Glutathione could be hydrogen bounded with the GAPDH along the lines outlined by DeMel et al. 2004, but which may cover and protect other –SH group(s). Since Iodoacetate (IAA) blocks sulphydral sites, the greater sensitivity of the reaction to IAA may be explained by any such extra group involved, if not protected.

The Sulphite that may be produced could be eliminated by Glutathione, along the lines suggested by Kågedal et al. (1986)[175]. The administration of oxidized Glutathione in Holt’s “glucose blocking agent” prior to the application of UHF may inhibit this elimination pathway to S-Sulphocysteine, leaving the toxic Sulphite to affect cellular wellbeing. The beneficial effects of (hypo)taurine on embryo development (Barnett & Bavister 1992[176]) may derive from breakdown to inorganic sulphur compounds capable of sustaining the exclusion of phosphate from the enzyme. The toxic effects of Phosphate on the embryo can be explained by it occupying a site inappropriate for that stage of development and blocking the Sulphate/sulphite molecules.

We are to presume that the C1 of D-Glucose and D-Mannose occupy the location as the C1 of D-Lactate or Pyruvate. To draw analogies with the catalytic site in the D-LDH of Lactobacillus pentosus[177], the =O on C1 would presumably line up with Arg235 and Val78, with the –OH/=O of C2 with the second NH2- of Arg235, close to Hist296, which would probably be involved in the proton/electron transfer most likely coming from Glutathione +/-RNA. Since there is no gross loss or gain of electrons/protons (other than as water), these can be stored temporarily by chargeable groups, as in Glutathione; the site is known to be capable of accommodating larger molecular chains, particularly without the presence of the dinucleotide co-factor. If the Enolase is held in apposition to the catalytic cleft by micro-anatomical association, the dehydration and fission the Needham pathway may be able to proceed.

The inhibition by L-glyceraldehyde, as claimed by Needham, may not be by competition at the catalytic site. Grant et al (1996)[178] studied the D-LDH from E. coli and a relatively specific inhibition that L-Serine had upon it. They envisaged a roughly oval-shaped tetrameric structure, in which the ends have some hinge-like action, which can be impeded by L-serine. The L-Serine is presumed to use its three functional hydrogen-binding groups to lock the adjacent enzyme molecules and prevent closure of the catalytic sites.  Two molecules of L-Serine can inhibit the tetramer by 85%. Serine homologues with two functional sites had some effect, but ~ 200 times weaker. Strangely, the authors did not seem to test with D-Serine, and did not test with L-Glyceraldehyde. An examination of the structures of L-Serine and L-Glyceraldehyde show some similarity. This form of inhibition may the type observed by Needham (though not discounting the possibility of competition at the catalytic site). Both Iodoacetate and L-Glyceraldehyde were used as inhibitors of GAPDH in experiments looking into the 2-cell block in Hamster embryos due to phosphate and glucose[179], with results indicating a reduction of the block effect by these inhibitors at low concentrations, consistent with the involvement of GAPDH.

      

            CH=O                         CH=O

            │                                 │ 

         HCOH                      HOCH

            │                                 │                                 COOH                         COOH  

      HOCH                         HOCH                               │                                 │

            │                                 │                          H2NCH                         HOCH

         HCOH                         HCOH                            │                                 │

            │                                 │                                 CH2OH                        CH2OH 

         HCOH                         HCOH                       L-Serine                L-Glyceraldehyde    

            │                                 │                           (IC50=0.008) 

            CH2OH                        CH2OH                               

          D-Glucose                 D-Mannose                           

 

COOH                         COOH                         COOH                         COOH

│                                 │                                 │                                 │

    H2NCH2                       H2NCH                        H2NCH                        H2NCH

       D-Glycine                        │                                 │                                 │            

           (IC50=1.8)                           CH2                       HOCH                               CH2

│                                 │                                 │        

            COOH                        O                                  CH3                             S=O

│                                 │                     L-Allothreonine                      │

     H2NCH                               C=O                       (IC50=1.5)                             CH3

            │                                 │                                                          S-Methyl-L-Cysteine

CH3                             CH3                                                         Sulphoxide

     L-Alanine               O-Acetyl-L-Serine

         (IC50=3.0)                              (IC50=1.4)

 

            COOH                            CH2SH

            │                                    │

     NH2CH-CH2-CH2-CO¾NHCH-CO¾NHCH2-COOH

 Glutathione  =  γ-L-Glutamyl-L-cysteinyl-glycine = “GSH”

 

To Summarize this Part:

a)      The relevant literature has been reviewed

b)      The suggestion is that there may be a pathway for D-Glucose metabolism (The Needham pathway) that provides a rapid means to supply L-Lactate, particularly in the zygote or very early embryo

c)      There seem grounds for believing that GAPDH may play a major rôle in this pathway, possibly by means of a modulated physical association with ENO at the centrosome

d)      This glucolysis pathway releases energy, which may be in the NIR region in normal physiology but, if driven by UHF 434 MHz, may emit at or about that activation frequency, based upon resonance around the circumference of the centrioles

e)      The requirement for Glutathione as a cofactor, and the effects of the inhibitors Iodoacetate and Fluoride are discussed, as is the reported in vivo inhibition by L-Glyceraldehyde.   

 

2.         The retinal rods and cones. Activation of the Centrins in and around the centrioles of the retinal rods and cones may, in turn activate the GAPDH-ENO glucolysis energy source that has been described earlier. The normal functioning of the photodetectors (rods and cones) is based fundamentally upon modulations of intracellular calcium (Pugh & Lamb 2000[180]), making a direct effect of calcium a likely contender, and linked directly with the photodetection cascade. Whether the NIR can be directed by the small centriole as an evanescent radiation as can occur with silica “wires” as waveguides (Tong et al. 2003[181]) becomes more speculative. The subdistal appendages (basal feet) and microtubules or foot processes (alar sheets) radiating from the distal end of the distal centriole (basal body) near the junction with the base of the cilial axoneme (Marshall 2001[182], Anderson 1972[183]) may likewise serve as “aerials.” In less specialized cells, the suggestion earlier was that the NIR could be directed into the peri-centriolar matrix at the proximal end of the centriole, which could then act as a diffuser, but front-to-back differences would not seem likely to be great. The radiation would, in the more specialized retinal cells, primarily be pointed directly towards the vitreous, through the cellular processes (“axons”) and to the synaptic terminals and bulbs in the outer ganglion layer. It is there that cones have ribbon synapses with horizontal and cone bipolar cells and the rods have ribbon synapses with rod bipolar cells. These are chemical synapses. There are also interconnections of electrical type with amacrine cells, bipolar and horizontal cells by connexon [gap] junctions and calcium connexin hemichannel involvement which may provide ephaptic inhibition[184],[185]. For the purposes of simplicity, these will not be considered in detail further. The slender cell processes passing through to the outer plexiform layer towards the vitreous are generally 0.6 – 1.0 μm thick. Given that an NIR wavelengthair (la) of 1.6 μm in air, when passing through water with a refractive index (RI) of ~1.336 (as in tears[186]) will have a wavelengthwater of λw/RI ~ 1.2 μm, and that a waveguide needs to have a diameter of ≥ λw/2, (being 0.6 μm), the conditions would seem compatible for transmission of such NIR through to the synaptic terminal, and setting as a limit, la ≤ ~1.6 mm.

3.         Likely cells. Discussion here is stimulated by the pivotal position of the centriole(s) in rods and cones. However, there is no reason to limit the conclusions to these cells – the bipolar cells have similar ribbon synapses and, accordingly similar physiological processes involved in signal attenuation. Since the NIR produced, as outlined above, would reflect calcium responsiveness to the light intensity changes, there is the capability of the NIR to be able to act as a modulating control of activities closer to the vitreous, as in the synaptic terminals and bulbs. In other cells (eg neurons), the NIR may have effects upon nuclear molecules and functions, and on plasma membrane structure and functions (see later).

4                    Likely rôles. The likely heat production at the centrioles would preclude continuous NIR production. Short trains of very short spikes of radiation would seem most likely and could be adequate to produce athermal heat (molecular agitation) at a distance, as has been used to produce brief temperature rises of up to 15oC in experimental studies on L-LDH[187], using temperature jump relaxation spectroscopy. The radiation would arise, as suggested earlier, from the moving microtubular sites in the outer centrosome brought about by local contraction of the contractile, Calcium-binding centrin molecules, thus bringing into apposition the GAPDH and ENO molecular groups that are capable of forming active sites for Needham’s glucolysis.

5.                  Likely emission type. The physical dimensions of the axon would set the wavelengths that could be utilized, with an upper limit wavelength of ~1.6 μm in air.

6.                  The Energy source would be the Needham glucolysis, along the lines elaborated earlier, cytosolic D-Glucose converting to L-Lactate, with the production of free energy – not into chemical bonds.

7.                  Transmission would be along “axons,” acting as waveguides. The distal end of the axon is a relatively large bulb, which is essentially a bag of secretory vesicles[188]. The signal would be diffused through these, to reach the detectors, the Ribbon synapses.

8.                  Detectors would be the Ribbon synapses[189],[190], but possibly also at the plasma membrane; wherever related molecules of the GAPDH/CtBP/Ribeye[191]-type molecules may occur. The NIR, which can activate Lactate at NIR wavelength of ~1556 nm (in air; a lesser absorption peak at 1844 nm) would lift the  D-Lactate and possibly NAD+[192] to higher energy states (Suehara & Yano 2004[193]), favouring binding of D-Lactate to the detector molecules. This would change their physical +/- chemical characteristics, and modify their function[194] – as negative feedback.

9.                  General action of NIR would lower the discharge rate of presynaptic secretory vesicles, by changing, globally and cooperatively within the pre-synaptic terminal, the labile characteristics of all ribbon synapses[195],[196] changes in response to light which, hitherto, are not well explained. There could be changed characteristics of the plasma membrane[197] and its receptors.

10.              Biochemistry/Biophysics. NIR (la ~1.6 mm) activates Lactate into a vibration mode with an higher energy level. D-lactate is at low concentrations, is linked to and considered a by-product of glycolysis, and is probably rate limiting. In the current hypothesis, this breakdown product may have a function. This is not new in nature: in some animals, such as snails, it would seem to have an established major rôle, seemingly a major circulating and tissue metabolite, as shown in the Table: -  

Tissue D-Lactate and haemolymph in snails (Helix lucorum)[198]:

Tissue (n=3)                            D-Lactate aerobic         D-Lactate anaerobic, at 48 h

Foot (mmol g-1 wet mass)          0.25 ±0.03                   17.02 ±2.1

Heart (mmol g-1 wet mass)         0.16 ±0.02                   9.67 ±0.11

Haemolymph (mmol L-1)           0.25 ±0.03                   56.46 ±7.5

With an affinity for the D-LDH enzymes of the GAPDH/CtBP/Ribeye class, D-Lactate, by being energized and attaching to the enzyme binding site(s), could modifying the molecular shape, characteristics and responsiveness of this large group of related molecules, thereby modifying Ribbon and plasma membrane function.

 

Concluding Comment

The observations and studies done by Dr John Holt with the UHF of 434 MHz on cancer patients, have opened up new ideas and concepts relevant to cellular functions and energy sources. The Holt resonance phenomenon introduces completely new concepts and, accordingly, should open up new fields of research and treatment in the future.

 

Malcolm A Traill

Copyright © MA Traill; 7th of August, 2007

matraill@iprimus.com.au

 

 

 

 

 

 

 

 

 

 

 


 

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