Temperature and pH dependence of the metarhodopsin I-metarhodopsin II equilibrium and the binding of metarhodopsin II to G protein in rod disk membranes.

The equilibria between metarhodopsins I and II (MI and MII) and the binding of MII to retinal G protein (G) were investigated, using the dual wavelength absorbance response of rod disk membrane (RDM) suspensions to a series of small bleaches, together with a nonlinear least-squares fitting procedure that decouples the two reactions. This method has been subjected to a variety of theoretical and experimental tests that establish its validity. The two equilibrium constants, the amount of active G protein (that can bind to and stabilize MII) and the fraction bleached by the flash, have been determined without a priori assumptions about these values, at temperatures between 0 and 15 degrees C and pHs from 6.2 to 8.2. Binding of G to MII in normal RDM exhibits 1:1 stoichiometry (not cooperative), relatively weak, 2-4 x 10(4) M-1 affinity on the membrane, with a pH dependence maximal at pH 7.6, and a low thermal coefficient. The reported amount of active G remained constant even when its binding constant was reduced more than 10-fold at low pH. The method can readily be applied to the binding of MII to other proteins or polypeptides that stabilize its conformation as MII. It appears capable of determining many of the essential physical constants of G protein coupled receptor interaction with immediate signaling partners and the effect of perturbation of environmental parameters on these constants.     

A new form of crystalline rubisco and the conversion to its common dodecahedral form

In this paper, we present a new purification procedure that yields a new crystalline form of rubisco and has enabled us to completely remove this most abundant protein from tobacco leaf extract. The crystals formed within 48 h after refrigeration at 4 degrees C at pH 5.6. However, these crystals were not well-ordered crystals and lacked well-defined facets or edges. The remaining leaf extract (fraction 2 protein) was void of rubisco. Conversion of this new crystalline form of rubisco to its common dodecahedral form was achieved by dialysing the protein solution in Tris buffer at pH 8.0 or purified water. Since the molecular size of its large subunit of rubisco (55 kD) is similar to that of the papillomavirus capsid protein, L1 (57 kD), its complete removal from fraction 2-protein may facilitate the detection, purification, and recovery of the Li protein.     

Physical and chemical studies of a low-molecular-weight form of urease

1. A new form of enzymically active jack-bean [Canavalia ensiformis (L.) DC] urease corresponding to an S_20,w value of 11·8s and a molecular weight of 260000 was investigated. 2. Conversion of 18s urease (EC 3.5.1.5) into the 12s form depends on both low protein concentration and pH. Above pH5·3 urease exists in the 18s form and below pH4·8 in the 12s form; between these two pH values a 12s–18s rapid-equilibrium process is observed. 3. Comparison of the properties of 18s and 12s urease indicated no major differences. 4. A survey of other good urease sources revealed that the 12s form can also be obtained from soya bean (Glycine soja Sieb. and Zucc. cultivar Biloxi) and the bacterium Bacillus pasteurii (Miguel) Migula, but not from watermelon (Citrullus vulgaris Schrad. cultivar Congo). 5. The 12s forms from jack bean and Bacillus pasteurii did not hybridize.     

Phosphorylation alters the pH-dependent active state equilibrium of rhodopsin by modulating the membrane surface potential.

Phosphorylation reduces the lifetime and activity of activated G protein-coupled receptors, yet paradoxically shifts the metarhodopsin I-II (MI-MII) equilibrium (K(eq)) of light-activated rhodopsin toward MII, the conformation that activates G protein. In this report, we show that phosphorylation increases the apparent pK for MII formation in proportion to phosphorylation stoichiometry. Decreasing ionic strength enhances this effect. Gouy-Chapman theory shows that the change in pK is quantitatively explained by the membrane surface potential, which becomes more negative with increasing phosphorylation stoichiometry and decreasing ionic strength. This lowers the membrane surface pH compared to the bulk pH, increasing K(eq) and the rate of MII formation (k(1)) while decreasing the back rate constant (k(-)(1)) of the MI-MII relaxation. MII formation has been observed to depend on bulk pH with a fractional stoichiometry of 0.6-0.7 H(+)/MII. We find that the apparent fractional H(+) dependence is an artifact of altering the membrane surface charge during a titration, resulting in a fractional change in membrane surface pH compared to bulk pH. Gouy-Chapman calculations of membrane pH at various phosphorylation levels and ionic strengths suggest MII formation behavior consistent with titration of a single H(+) binding site with 1:1 stoichiometry and an intrinsic pK of 6.3 at 0.5 degrees C. We show evidence that suggests this same site has an intrinsic pK of 5.0 prior to light activation and its protonation before activation greatly enhances the rate of MII formation.     

Proteomic profiling of murine oocyte maturation

In an effort to better understand oocyte function, we utilized two-dimensional (2D) electrophoresis and mass spectrometry to identify proteins that are differentially expressed during murine oocyte maturation. Proteins from 500 germinal vesicle (GV) and metaphase II-(MII) arrested oocytes were extracted, resolved on 2D electrophoretic gels, and stained with silver. Analysis of the gels indicated that 12 proteins appeared to be differentially expressed between the GV and MII stage. These proteins were then cored from the 2D gels and identified by mass spectrometry as: transforming acidic coiled-coil protein 3 (TACC3), heat shock protein 105 (HSP105), programmed cell death six-interacting protein (PDCD6IP), stress-inducible phosphoprotein (STI1), importin alpha2, adenylsuccinate synthase (ADDS), nudix, spindlin, lipocalin, lysozyme, translationally controlled tumor protein (TCTP), and nucleoplasmin 2 (NPM2). Interestingly, PDCD6IP, importin alpha2, spindlin, and NPM2 appear slightly larger in mass and more acidic on the MII oocyte gel compared to the GV oocyte gel, suggesting that they may be post-translationally modified during oocyte maturation. Given NPM2 is an oocyte-restricted protein, we chose to further investigate its properties during oocyte maturation and preimplantation development. Real-Time RT-PCR showed that NPM2 mRNA levels rapidly decline at fertilization. Indirect immunofluorescence analysis showed that, with the exception of cortical localization in MII-arrested oocytes, NPM2 is localized to the nucleus of both GV stage oocytes and all stages of preimplantation embryos. We then performed one-dimensional (1D) western blot analysis of mouse oocytes and preimplantation embryos and found that, as implicated by the 2D gel comparison, NPM2 undergoes a phosphatase-sensitive electrophoretic mobility shift during the GV to MII transition. The slower migrating NPM2 form is also present in pronuclear embryos but by the two-cell stage, the majority of NPM2 exists as the faster migrating form, which persists to the blastocyst stage.     

Evidence from electron paramagnetic resonance for function-related conformation changes in the anion-transport protein of human erythrocytes

The erythrocyte membrane protein involved in anion transport (band 3) was isolated in its native lipid milieu in the form of leaky vesicles and then was spin-labelled with N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)-meleimide (MalMe4PipO). The resulting electron paramagnetic resonance spectrum of band-3-bound MalMe4PipO was resolved into a rapid tumbling component and another, relatively immobile component. The percentage of the signal contributed by the mobile component (Q), was sensitive to various characteristic factors known to affect erythrocyte anion transport: Q was a hyperbolic function of chloride concentration displaying a half-saturation constant K1/2 similar to that of chloride transport. On the other hand Q showed a biphasic response to sulfate concentration, in line with the relatively high affinity of sulfate for the anion modifier site. Q was a saturable function of pH, either in presence of Cl- or SO4(-2), showing a pKa between pH 6.0 and 6.5, in analogy with the pH titration curve of Cl- and SO4(-2), transport. Spin-labelled vesicles treated with a covalent inhibitor of anion transport, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid, were markedly less susceptible to changes in Cl- concentration. It is suggested that the electron paramagnetic resonance spectrum of MalMe4PipO covalently bound to the band-3 protein, reports conformational changes which are related to the anion-transport function of this protein.     

Chromatography of oxidized and reduced cytochrome c on carboxymethylcellulose

1. Cytochrome c was isolated from horse heart by a chromatographic method. 2. Oxidized and reduced cytochrome c were chromatographed on CM-cellulose that was in equilibrium with several buffer systems of constant composition at pH values of 8.4, 6.75 and 4.9. 3. Separation was better at the higher pH values; the oxidized form was retarded more than twice as much as the reduced form, though they differed by only a single charge. 4. Self-competition between cytochrome molecules is suggested to account for the peak distortion observed at high loads (above 20mum protein concentration).     

pH effects on the hyaluronan hydrolysis catalysed by hyaluronidase in the presence of proteins: Part I. Dual aspect of the pH-dependence

Hyaluronan (HA) hydrolysis catalysed by hyaluronidase (HAase) is strongly inhibited when performed at a low ratio of HAase to HA concentrations and at low ionic strength. This is because long HA chains can form non-active complexes with HAase. Bovine serum albumin (BSA) is able to compete with HAase to form electrostatic complexes with HA so freeing HAase which then recovers its catalytic activity. This BSA-dependence is characterised by two main domains separated by the optimal BSA concentration: below this concentration the HAase activity increases when the BSA concentration is increased, above this concentration the HAase activity decreases. This occurs provided that HA is negatively charged and BSA is positively charged, i.e. in a pH range from 3 to 5.25. The higher the pH value the higher the optimal BSA concentration. Other proteins can also modulate HAase activity. Lysozyme, which has a pI higher than that of BSA, is also able to compete with HAase to form electrostatic complexes with HA and liberate HAase. This occurs over a wider pH range that extends from 3 to 9. These results mean that HAase can form complexes with HA and recover its enzymatic activity at pH as high as 9, consistent with HAase having either a high pI value or positively charged patches on its surface at high pH. Finally, the pH-dependence of HAase activity, which results from the influence of pH on both the intrinsic HAase activity and the formation of complexes between HAase and HA, shows a maximum at pH 4 and a significant activity up to pH 9.     

Effects of different cryoprotectants and carbohydrates on freezing of matured and unmatured bovine oocytes

Cumulus cell-enclosed bovine oocytes in germinal vesicle (GV) and in metaphase II (MII) stages were cryopreserved. Different concentrations (1 M; 1.5 M) of various cryoprotectants (glycerol, PROH, DMSO) were tested. After thawing, the oocytes were exposed to various carbohydrates (sucrose, lactose, trehalose) at a concentration of 0.1 M and 0.25 M for cryoprotectant removal. Developmental capacity of the frozen-thawed oocytes was studied by in vitro maturation, fertilization and culture. We found no difference in subsequent development using glycerol or PROH for GV and MII oocytes. The DMSO treatment led to significantly better cleavage and development up to 4-cell stage in MII oocytes. Development beyond the 8-cell stage was obtained only when unmatured oocytes were frozen. No difference in the efficiency of the 3 cryoprotectants was detected in MII oocytes. However, in GV oocytes, glycerol and PROH yielded significantly better cleavage and 4-cell rate compared to DMSO (P<0.001). Influence of the concentration of a cryoprotectant on development was not observed in GV or MII oocytes. Among the 3 cryoprotectants, DMSO was less suitable, at both concentrations, than PROH and glycerol for the development of 6- to 8-cell stage embryos in the GV group. In the MII group, 1.5 M DMSO was as efficient as PROH and as glycerol at a 1.5-M concentration, and it was more efficient than 1 M glycerol. The use of carbohydrates during rehydration did not render a beneficial effect at either of the 2 concentrations, and when no carbohydrates were used in the MII group the oocytes cleaved better than GV oocytes.     

Complex-formation between triphosphoinositide and experimental allergic encephalitogenic protein

1. Reactions between triphosphoinositide and the basic experimental allergic encephalitogenic (EAE) protein were examined in aqueous solution and in a biphasic solvent system (chloroform-methanol-water, 8:4:3, by vol.). 2. In the absence of salt an insoluble complex (I) is formed containing triphosphoinositide and EAE protein in proportions that represent complete neutralization of lipid and protein at the pH concerned. 3. In the presence of a low concentration (0.05m) of sodium chloride an insoluble positively charged complex (II) forms. It contains triphosphoinositide and EAE protein in a lower concentration ratio than complex I. This complex, which has a constant composition between pH7.5 and pH10, can take up additional micellar triphosphoinositide producing complex I, which can then be solubilized by excess of triphosphoinositide. 4. The complexes are dissociated by more concentrated sodium chloride solutions and low concentrations of calcium chloride, suggesting that they are largely stabilized by electrostatic bonds. The protein recovered after dissociation is immunologically active and has the same electrophoretic mobility as the original. 5. Water-insoluble ternary complexes containing triphosphoinositide, EAE protein and large amounts of phosphatidylcholine can be prepared. From these, chloroform-methanol (2:1, v/v) extracts only phosphatidylcholine. 6. An insoluble ternary complex of Ca(2+) ion, EAE protein and triphosphoinositide can be prepared by adding calcium chloride to a complex I preparation solubilized by excess of triphosphoinositide. 7. EAE protein will also form complexes with other acidic phospholipids, e.g. phosphatidic acid, phosphatidylserine and phosphatidylinositol, but not with phosphatidylcholine or phosphatidylethanolamine. The phosphatidylinositol and phosphatidylserine complexes are chloroform soluble, i.e. proteolipids. 8. The possibility that complexes between EAE protein and acidic phospholipids occur in vivo is discussed. Triphosphoinositide and EAE protein occur in ox brain myelin in approximately the same concentration ratios as they do in complex II, formed at physiological salt concentration and pH.     

Aging changes the chromatin configuration and histone methylation of mouse oocytes at germinal vesicle stage

Aging decreases the fertility of mammalian females. In old oocytes at metaphase II stage (MII) there are alterations of the chromatin configuration and chromatin modifications such as histone acetylation. Recent data indicate that alterations of histone acetylation at MII initially arise at germinal vesicle stage (GV). Therefore, we hypothesized that the chromatin configuration and histone methylation could also change in old GV oocytes. In agreement with our hypothesis, young GV oocytes had non-surrounded nucleolus (NSN) and surrounded nucleolus (SN) chromatin configurations, while old GV oocytes also had chromatin configurations that could not be classified as NSN or SN. Regarding histone methylation, young GV and MII oocytes showed dimethylation of lysines 4, 9, 36 and 79 in histone 3 (H3K4me2, H3K9me2, H3K36me2, H3K79me2), lysine 20 in histone H4 (H4K20me2) and trimethylation of lysine 9 in histone 3 (H3K9me3) while a significant percentage of old GV and MII oocytes lacked H3K9me3, H3K36me2, H3K79me2 and H4K20me2. The percentage of old oocytes lacking histone methylation was similar at GV and MII suggesting that alterations of histone methylation in old MII oocytes initially arise at GV. Besides, the expression of the histone methylation-related factors Cbx1 and Sirt1 was also found to change in old GV oocytes. In conclusion, our study reports changes of chromatin configuration and histone methylation in old GV oocytes, which could be very useful for further understanding of human infertility caused by aging.     

Aging changes the chromatin configuration and histone methylation of mouse oocytes at germinal vesicle stage

Aging decreases the fertility of mammalian females. In old oocytes at metaphase II stage (MII) there are alterations of the chromatin configuration and chromatin modifications such as histone acetylation. Recent data indicate that alterations of histone acetylation at MII initially arise at germinal vesicle stage (GV). Therefore, we hypothesized that the chromatin configuration and histone methylation could also change in old GV oocytes. In agreement with our hypothesis, young GV oocytes had non-surrounded nucleolus (NSN) and surrounded nucleolus (SN) chromatin configurations, while old GV oocytes also had chromatin configurations that could not be classified as NSN or SN. Regarding histone methylation, young GV and MII oocytes showed dimethylation of lysines 4, 9, 36 and 79 in histone 3 (H3K4me2, H3K9me2, H3K36me2, H3K79me2), lysine 20 in histone H4 (H4K20me2) and trimethylation of lysine 9 in histone 3 (H3K9me3) while a significant percentage of old GV and MII oocytes lacked H3K9me3, H3K36me2, H3K79me2 and H4K20me2. The percentage of old oocytes lacking histone methylation was similar at GV and MII suggesting that alterations of histone methylation in old MII oocytes initially arise at GV. Besides, the expression of the histone methylation-related factors Cbx1 and Sirt1 was also found to change in old GV oocytes. In conclusion, our study reports changes of chromatin configuration and histone methylation in old GV oocytes, which could be very useful for further understanding of human infertility caused by aging.     

Folding of pyrimidine-enriched RNA fragments from the vicinity of the internal ribosomal entry site of hepatitis A virus.

Two RNA fragments from the region just upstream of the internal ribosome entry site of Hepatitis A virus (HAV) were studied, a 35mer (HAV-35), 5'U4C3U3C3U4C3U3C2UAU2C3U33(4), and a 23mer (HAV-23), 5(4)U4C3U3C3U4C3U33(4). Secondary structural predictions and nuclease digestion patterns obtained with genomic RNAs suggested that they link two stable Watson-Crick (WC) hairpins in the genomic RNA and do not form conventional WC secondary structure, but do fold to form a condensed, stacked 'domain'. To obtain more information, folding of HAV-23 and -35 RNA fragments was characterized using 1H nuclear magnetic resonance, in H2O as a function of pH and temperature, circular dichroism as a function of NaCl concentration, pH and temperature, and square-wave voltammetry as a function of pH. The results indicate that these oligo-nucleotides form intramolecular structures that contain transient U*U base pairs at pH 7 and moderate ionic strength (100 mM NaCl). This folded structure becomes destabilized and loses the U*U base pairs above and below neutral pH, especially at ionic strengths above 0.1. All of the cytidine protons exchange relatively rapidly with solvent protons (exchange lifetimes shorter than 1 ms), so the structure contains few if any C*CH+base pairs at neutral pH, but can apparently form them at pH values below 6. We present a series of possible models in which chain folding draws the strand termini closer together, possibly serving to pull the attached WC hairpin domains together and providing a functional advantage by nucleating reversible formation of a more viable RNA substrate.     

A thermodynamic analysis of the interaction between the mitochondrial coupling adenosine triphosphatase and its naturally occurring inhibitor protein

1. The naturally occurring ATPase (adenosine triphosphatase)-inhibitor protein, from bovine heart mitochondria, was obtained as a single pure protein. It was not identical with any of the five subunits (alpha-epsilon) of the isolated ATPase, and appeared to be a single polypeptide chain. 2. The inhibitor combined with the ATPase in a 1:1 molar ratio, producing a completely inhibited ATPase molecule. The affinity of the ATPase for its inhibitor is high; the K(d) is of the order of 10(-8)m. 3. The enthalpy of the ATPase-inhibitor complex-formation is positive, the value of K(d) decreasing as the temperature is raised. This suggests that the forces involved are largely hydrophobic in nature. 4. Hydrolysis of a nucleoside triphosphate promoted formation of the ATPase-inhibitor complex, although the equilibrium position was almost unaffected by the rate of hydrolysis. At low salt concentration, less than 200 turnovers of the ATPase suffice for the ATPase to combine with the inhibitor protein. At higher salt concentrations, a larger number of turnovers is required. It is suggested that the inhibitor binds to a form of the ATPase that is produced transiently during hydrolysis. 5. In the presence of 75mm-K(2)SO(4), the rates of association and dissociation are slow enough to allow their kinetics to be studied. Association is first-order in inhibitor concentration, but fractional order in ATPase concentration. Dissociation is first-order in ATPase-inhibitor complex concentration. The temperature coefficients of the ;on' and ;off' processes were also measured. 6. A simple kinetic model for the ATPase-inhibitor interaction is proposed that can be extended to take into account release of inhibitor protein under energized conditions on the membrane. 7. The isolated ATPase is inhibited by preincubation with Mg(2+), reversible by subsequent addition of EDTA, and by ADP, reversible by subsequent addition of ATP. These effects are not found on the membrane-bound ATPase. The mechanism of these effects is discussed.     

A protease-like permeability factor in the guinea pig skin. 2. In vitro activation of the latent form permeability factor by weakly acidic phosphate buffer.

Conditions for the in vitro activation of the latent form of a protease-like permeability factor in the pseudoglobulin fraction from guinea pig skin were examined. (1) The factor was activated by dialysis against 67 mM phosphate buffer at pH 5.8--6.4, not at pH 7.0--8.0. (2) High salt concentration (200 mM or greater phosphate buffer or 67 mM phosphate buffer containing 200 mM or greater KCl or NaCl) prevented the activation at pH 6.2. (3) High osmotic pressure (sucrose at 1 M) did not affect activation at pH 6.2. (4) Reconversion of the activated permeability factor into an inactive form was not observed under high salt conditions, under which the latent permeability factor was stable in its own form. (5) The molecular size of the latent permeability factor was estimated as approx. 80 000 by Sephadex G-100 gel filtration at high salt concentration.     

The kinetics of the reduction of cytochrome c by the superoxide anion radical.

1. At neutral pH ferricytochrome c is reduced by the superoxide anion radical (O2-), without loss of enzymatic activity, by a second order process in which no intermediates are observed. The yield of ferrocytochrome c (82-104%), as related to the amount of O2- produced, is slightly dependent on the concentration of sodium formate in the matrix solution. 2. The reaction (k1 equals (1.1+/-0.1) - 10(6) M-1 - s-1 at pH 7.2, I equals 4 mM and 21 degrees C) can be inhibited by superoxide dismutase and trace amounts of copper ions. The inhibition by copper ions is removed by EDTA without interference in the O2- reduction reaction. 3. The second-order rate constant for the reaction of O2- with ferricytochrome c depends on the pH of the matrix solution, decreasing rapidly at pH greater than 8. The dependence of the rate constant on the pH can be explained by assuming that only the neutral form of ferricytochrome c reacts with O2- and that the alkaline form of the hemoprotein is unreactive. From studies at pH 8.9, the rate for the transition from the alkaline to the neutral form of ferricytochrome c can be estimated to be 0.3 s-1 (at 21 degrees C and I equals 4 mM). 4. The second-order rate constant for the reaction of O2- with ferricytochrome c is also dependent on the ionic strength of the medium. From a plot of log k1 versus I1/2-(I + alphaI1/2)-1 we determined the effective charge on the ferricytochrome c molecule as +6.3 and the rate constant at I equals 0 as (3.1+/-0.1) - 10(6) M-1 - s-1 (pH 7.1, 21 degrees C). 5. The possibility that singlet oxygen is formed as a product of the reaction of O2- with ferricytochrome c can be ruled out on thermodynamic grounds.     

Characteristics of the methylammonium/ammonium transport systems of the nitrogen-fixing cyanobacterium Anabaena 7120 (ATCC 27893)

NH4(+)-transport in Anabaena 7120 was studied using the NH4+ analogue, 14CH3NH3+. At pH 7, two energy-dependent NH4(+)-transport systems were detected in both N2- and NO3(-)-grown cells, but none in NH4(+)-grown cells. Both transport systems showed a low and a high affinity mode of operation depending on the substrate concentration. One of the transport systems showed Km values of 8 microM (Vmax = 1 nmole min-1mg-1protein) and 80 microM (Vmax = 7 nmole min-1mg-1protein), and was insensitive to L-methionine-DL-sulphoximine, a glutamate analogue and irreversible inhibitor of glutamine synthetase. The other transport system showed Km values of 2.5 microM (Vmax = 0.1 nmole min-1mg-1protein) and 70 microM (Vmax = 0.7 nmole min-1mg-1protein), and was sensitive to L-methionine-DL-sulphoximine. Intracellular accumulation of free 14CH3NH3+ showed a biphasic pattern in response to variation in external 14CH3NH3+ concentrations. A maximum intracellular concentration of 2.5 mM and 7.5 mM was reached in the external 14CH3NH3+ concentration range of 1-50 microM and 1-500 microM, respectively. At pH 9, an energy-independent diffusion of 14CH3NH2 leading to a higher intracellular accumulation and assimilation rate, than that at pH 7, was observed.     

Meiotic progression of rat spermatocytes requires mitogen-activated protein kinases of Sertoli cells and close contacts between the germ cells and the Sertoli cells

Progression of germ cells through meiosis is regulated by phosphorylation events. We previously showed the key role of cyclin dependent kinases in meiotic divisions of rat spermatocytes co-cultured with Sertoli cells (SC). In the present study, we used the same culture system to address the role of mitogen-activated protein kinases (MAPKs) in meiotic progression. Phosphorylated ERK1/2 were detected in vivo and in freshly isolated SC and in pachytene spermatocytes (PS) as early as 3 h after seeding on SC. The yield of the two meiotic divisions and the percentage of highly MPM-2-labeled pachytene and secondary spermatocytes (SII) were decreased in co-cultures treated with U0126, an inhibitor of the ERK-activating kinases, MEK1/2. Pre-incubation of PS with U0126 resulted in a reduced number of in vitro formed round spermatids without modifying the number of SII or the MPM-2 labeling of PS or SII. Conversely, pre-treatment of SC with U0126 led to a decrease in the percentage of highly MPM-2-labeled PS associated with a decreased number of SII and round spermatids. These results show that meiotic progression of spermatocytes is dependent on SC-activated MAPKs. In addition, high MPM-2 labeling was not acquired by PS cultured alone in Sertoli cell conditioned media, indicating a specific need for cell-cell contact between germ cells and SC.     

Borate esters of the methyl D-glucopyranosides

Methyl alpha- and beta-D-glucopyranoside act as moderate chelators of a boron centre through their O-4/6 binding site whereas the trans/trans arrangement of the O-2/3/4 hydroxy functions is not suited to form a chelate with the small boron central atom. In this work we report the crystal structure of the bisdiolatoborate Na2[B(Me alpha-D-Glcp4,6H(-2))(Me alpha-D-Glcp3,4,6H(-3))].NaOH.8H2O (1) along with a combined 11B and 13C NMR spectroscopic studies of borate-pyranoside solutions at different concentrations and pH. It is shown that crystallisation of a bisdiolatoborate needs both a high pH and a high total concentration.