Role of vitelline envelope during fertilization in the black tiger shrimp, Penaeus monodon

Animal eggs possess investments through which sperm must penetrate. The aim of the present study was to investigate the role of the egg coating, the vitelline envelope, during sperm-egg interactions in the black tiger shrimp, Penaeus monodon. The site(s) of primary binding between sperm and egg and the possible binding molecule(s) for sperm were identified. In vitro adsorption of the vitelline envelope protein onto the sperm surface showed that primary binding occurred between the sperm anterior spike of acrosome intact sperm and the vitelline envelope. Results from streptavidin blotting revealed that the component of the vitelline envelope that interacts with the sperm integral membrane protein is a 370 kDa protein. In addition, it was shown that the vitelline envelope protein had no ability to induce acrosome reaction. These results suggest that the function of the vitelline envelope is as a primary binding site for sperm in shrimp, but not a sole trigger for the acrosome reaction.     

Acetyl-L-carnitine cytoprotection against 1-methyl-4-phenylpyridinium toxicity in neuroblastoma cells

Acetyl-L-carnitine (ALCAR) plays an integral role in the transport of long chain fatty acids across the inner mitochondrial membrane for oxidative phosphorylation. In non-human primates, administration of ALCAR was reported to prevent 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurological injury to the substantia nigra. The present study investigates the effects of ALCAR against the toxicity of 1-methyl-4-phenylpyridinium (MPP(+)), the neurotoxic metabolite of MPTP, in murine brain neuroblastoma cells. MPP(+), a potent mitochondrial toxin, induced a dose-dependent reduction in mitochondrial oxygen consumption and cell viability, corresponding to an accelerated rate of cellular glucose utilization. Treatment with ALCAR, but not L-carnitine, prevented MPP(+) toxicity and partially restored intracellular ATP concentrations, but did not reverse the MPP(+)-induced loss of mitochondrial oxygen consumption. These data indicate that protective effects are independent of oxidative phosphorylation. ALCAR had a substantial glucose sparing effect in both controls and MPP(+)-treated groups, demonstrating a potential role in enhancing glucose utilization through glycolysis. Antagonizing the entry of fatty acids into the mitochondria, with either insulin or malonyl CoA, did not interfere with ALCAR protection against MPP(+). On the contrary, insulin potentiated the protective effects of ALCAR. In conclusion, these data indicate that ALCAR protects against MPP(+) toxicity, independent of mitochondrial oxidative capacity or beta-oxidation of fatty acids. In contrast, the protective effects of ALCAR appear to involve potentiation of energy derived from glucose through anaerobic glycolysis.     

Antioxidant and pro-oxidant properties of pyrroloquinoline quinone (PQQ): implications for its function in biological systems

Pyrroloquinoline quinone (PQQ) is a novel redox cofactor recently found in human milk. It has been reported to function as an essential nutrient, antioxidant and redox modulator in cell culture experiments and in animal models of human diseases. As mitochondria are particularly susceptible to oxidative damage we studied the antioxidant properties of PQQ in isolated rat liver mitochondria. PQQ was an effective antioxidant protecting mitochondria against oxidative stress-induced lipid peroxidation, protein carbonyl formation and inactivation of the mitochondrial respiratory chain. In contrast, PQQ caused extensive cell death to cells in culture. This surprising effect was inhibited by catalase, and was shown to be due to the generation of hydrogen peroxide during the autoxidation of PQQ in culture medium. We conclude that the reactivities of PQQ are dependent on its environment and that it can act as an antioxidant or a pro-oxidant in different biological systems.     

Cytotoxicity studies of 2‐hydroxymethyl‐1‐naphthol diacetate on K+ currents in neoplastic plasma cells

The present study investigated the effects of 2‐hydroxymethyl‐1‐naphthol diacetate (TAC) on cell proliferation and K⁺ currents in RPMI‐8226 human myeloma cells. In cells with intracellular Ca²⁺ concentration ([Ca²⁺]_i) = 10 nM, depolarizing square pulses from a holding potential of –80 mV elicited instantaneous outward current with slow inactivation, corresponding to voltage‐activated K⁺ current. TAC (1–100 μM) inhibited I_K(V) in a concentration‐dependent manner. A23187 (1 μM), a Ca²⁺ ionophore, can potentiate Ca²⁺‐activated K⁺ current (I_K(Ca)). Tetraethylammonium chloride (10 mM) caused a small decrease in the amplitude of I_K(Ca) elicited by A23187, whereas TAC (30 μM) and quinidine (10 μM) decreased I_K(Ca) more effectively. The present results show that TAC directly blocks voltage‐ and Ca²⁺‐activated K⁺ currents in human myeloma cells. TAC inhibited both cell proliferation and voltage‐activated K⁺ current with an effective dose inducing half‐maximum effects at 3.8 ± 0.8 μM and 10 ± 1.5 μM, respectively. The present study suggests that the cytotoxic effect of TAC in cancer cells may be partially explained by blockade of K⁺ channels. The delocalization energy of TAC and other analogs was employed to compare their ability to block the voltage‐activated K⁺ channel in myeloma cells. It was found that naphthol derivatives‐mediated blockade of voltage‐activated K⁺ channel might relate to the level of delocalization energy and molecular volume. Drug Dev. Res. 47:1–8, 1999. © 1999 Wiley‐Liss, Inc.     

Determination of creatine kinase isoenzyme MB (CK-MB): Comparison of methods and clinical evaluation

An ion-exchange chromatography method based on the method of Mercer is advocated for the routine determination of serum CK-MB. This method has some prominent advantages over other methods with which it is compared, and which include electrophoresis and an immunological technique. This method proves to be reliable and highly reproducible, while it allows a rather large number of samples to be analyzed within a relatively short period of time.Some parameters of the release pattern of CK-MB after acute myocardial infarction are characterized: normal values, time of first rise, time of peak value and rate-constant of inactivation.The clinical significance of serum CK-MB determination is evaluated.     

Properties of urophysial proteins (urophysins) from the white sucker, Catostomus commersoni.

Polyacrylamide gel electrophoresis, polyacrylamide gel electrofocusing and equilibrium dialysis studies on extracts of the urophysis of the white sucker, Catostomus commersoni, have shown the existence of a group of proteins which are specific to the urophysis and which bind two biologically active urophysial peptides, urotensin I (rat hypctensive principle) and urotensin II (fish smooth muscle stimulating principle).     

3'-axial CH2 OH substitution on glucopyranose does not increase glycogen phosphorylase inhibitory potency. QM/MM-PBSA calculations suggest why

Glycogen phosphorylase is a molecular target for the design of potential hypoglycemic agents. Structure‐based design pinpointed that the 3′‐position of glucopyranose equipped with a suitable group has the potential to form interactions with enzyme’s cofactor, pyridoxal 5′‐phosphate (PLP), thus enhancing the inhibitory potency. Hence, we have investigated the binding of two ligands, 1‐(β‐d ‐glucopyranosyl)5‐fluorouracil (GlcFU) and its 3′‐CH₂OH glucopyranose derivative. Both ligands were found to be low micromolar inhibitors with K_i values of 7.9 and 27.1 μm , respectively. X‐ray crystallography revealed that the 3′‐CH₂OH glucopyranose substituent is indeed involved in additional molecular interactions with the PLP γ‐phosphate compared with GlcFU. However, it is 3.4 times less potent. To elucidate this discovery, docking followed by postdocking Quantum Mechanics/Molecular Mechanics – Poisson–Boltzmann Surface Area (QM/MM‐PBSA) binding affinity calculations were performed. While the docking predictions failed to reflect the kinetic results, the QM/MM‐PBSA revealed that the desolvation energy cost for binding of the 3′‐CH₂OH‐substituted glucopyranose derivative out‐weigh the enthalpy gains from the extra contacts formed. The benefits of performing postdocking calculations employing a more accurate solvation model and the QM/MM‐PBSA methodology in lead optimization are therefore highlighted, specifically when the role of a highly polar/charged binding interface is significant.