Serotriflin, a CRISP family protein with binding affinity for small serum protein-2 in snake serum

Habu (Trimeresurus flavoviridis) serum contains 3 small serum proteins (SSP-1, SSP-2, and SSP-3) with molecular masses of 6.5 to 10 kDa. Gel filtration analysis showed that all the SSPs exist in high molecular mass forms of approximately 60 kDa in the serum. Ultrafiltration of Habu serum showed that SSPs dissociated from the complex below a pH of 4. An SSP-binding protein was purified from Habu serum by gel filtration, ion exchange, and reverse-phase HPLC. N-terminal sequencing yielded a 39-amino acid sequence, similar to the N-terminal region of triflin, which is a snake venom-derived Ca2+ channel blocker that suppresses smooth muscle contraction. The amino acid sequence of this protein, termed serotriflin, was established by peptide analysis and cDNA cloning. Serotriflin is a glycosylated protein and consists of 221 amino acids. Among the 3 SSPs, only SSP-2 formed a noncovalent complex with serotriflin. It was bound to triflin and serotriflin with high affinity, as evidenced by surface plasmon resonance. SSP-2 is considered to be a protein that prevents self injury by accidental leaking of venom into the blood.     

Short-term preservation of mouse oocytes at 5 degrees C.

The temporary preservation of oocytes without freezing would be useful for some experiments. ICR mouse oocytes were kept in a preservation medium under mineral oil for 1, 2, 3, 4 or 7 days at 5 degrees C, and 1 or 2 days at 37 degrees C. In vitro fertilization was attempted on oocytes rinsed with TYH medium after preservation. More than 70% of morphologically normal oocytes were recovered from each preservation group. Fertilization rates of oocytes preserved for 1, 2, 3, 4 or 7 days at 5 degrees C were 69.9, 66.5, 45.3, 26.7 and 8.8% respectively. Fertilization rates of oocytes preserved for 1 or 2 days at 37 degrees C were 9.6 and 1.6%, respectively. Preservation of oocytes at 5 degrees C has some capability as a method of short-term storage without freezing.     

Flexibility of small molecular CD4 mimics as HIV entry inhibitors

CD4 mimics such as YIR-821 and its derivatives are small molecules which inhibit the interaction between the Phe43 cavity of HIV-1 gp120 with host CD4, an interaction that is involved in the entry of HIV to cells. Known CD4 mimics generally possess three structural features, an aromatic ring, an oxalamide linker and a piperidine moiety. We have shown previously that introduction of a cyclohexyl group and a guanidine group into the piperidine moiety and a fluorine atom at the meta-position of the aromatic ring leads to a significant increase in the anti-HIV activity. In the current study, the effects of conformational flexibility were investigated by introduction of an indole-type group in the junction between the oxalamide linker and the aromatic moiety or by replacement of the oxalamide linker with a glycine linker. This led to the development of compounds with high anti-HIV activity, showing the importance of the junction region for the expression of high anti-HIV activity. The present data are expected to be useful in the future design of novel CD4 mimic molecules.     

Electrophysiology and kinesiology for health and disease.

This paper summarizes my Basmajian keynote presentation at the 2004 International Society of Electrophysiology and Kinesiology Conference. I dedicate this paper to Dr. Herbert A. deVries, the mentor of my research career. The following topics will be covered from the standpoint of Electrophysiology and Kinesiology for health and disease: (1) electromechanical manifestations of neuromuscular fatigue and muscle soreness, (2) cardiac depolarization-repolarization characteristics of normal and patients, (3) etiology of obesity and diabetes and autonomic nervous system, and (4) functional electrical stimulation for health and disease, respectively.     

Production of functional spermatids from mouse germline stem cells in ectopically reconstituted seminiferous tubules

Testicular germ cell transplantation into the seminiferous tubules is at present the only way to induce spermatogenesis from a given source of spermatogonial stem cells. Here we show an alternative method that harnesses the self-organizing ability of testicular somatic cells. The testicular cells of embryonic or neonatal mice or rats and of newborn pigs were dissociated into single cells. Each of them reorganized into a tubular structure following implantation into the subcutis of immunodeficient mice. When mouse germline stem (GS) cells derived from spermatogonial stem cells and expanded in culture were intermingled with testicular cells of rodents, they were integrated in the reconstituted tubules and differentiated beyond meiosis into spermatids. Normal offspring were produced by the microinjection of those spermatids into oocytes. This method could be applicable to various mammalian species and useful for producing functional gametes from GS cells in a xenoectopic environment.     

Anti- and pro-oxidative effects of flavonoids on metal-induced lipid hydroperoxide-dependent lipid peroxidation in cultured hepatocytes loaded with α-linolenic acid

Lipid hydroperoxide (LOOH)–dependent lipid peroxidation was induced in α-linolenic acid (LNA)-loaded hepatocytes by adding Fe, Cu, V, or Cd ions at concentrations from 20 to 500 μM. The effects of structurally related flavonoids at concentrations from 10 to 500 μM on the lipid peroxidation were examined. The results with regard to each flavonoid subclass are as follows: (i) Flavonols such as myricetin, quercetin, fisetin, and kaempferol, but not morin, showed dose-dependent antioxidative activity against metal-induced lipid peroxidation at all metal concentrations. Myricetin, quercetin, and fisetin were the most effective antioxidants, although their efficacies depended on the metal ion. Kaempferol and morin had antioxidative activity equal to the other flavonols in the presence of Cu ions, but were much less effective for the other three metal ions. (ii) Flavones, luteolin, apigenin, and chrysin were antioxidative at low Fe concentrations, but were pro-oxidative at high Fe concentrations. Luteolin exhibited antioxidative activity similar to that of catechol-containing flavonols in the presence of the other three metal ions. Apigenin and chrysin also acted as pro-oxidants with V or with all metal ions, respectively. (iii) Taxifolin, a flavanone, also showed both anti- and prooxidative activity, depending on Fe concentrations, but with other metal showed only antioxidative activity ions. (iv) Epigallocatechin, a flavanol, was antioxidative with all metal ions, and its activity was similar to that of catechol-containing flavonols. The various effects of flavonoids on metal-induced lipid peroxidation in LNA-loaded hepatocytes is discussed with regard to the change in redox potential of flavonoid–metal complexes.     

Paradoxical gain‐of‐function mutant of the G‐protein‐coupled receptor PROKR2 promotes early puberty

The human genome encodes ~750 G‐protein‐coupled receptors (GPCRs), including prokineticin receptor 2 (PROKR2) involved in the regulation of sexual maturation. Previously reported pathogenic gain‐of‐function mutations of GPCR genes invariably encoded aberrant receptors with excessive signal transduction activity. Although in vitro assays demonstrated that an artificially created inactive mutant of PROKR2 exerted paradoxical gain‐of‐function effects when co‐transfected with wild‐type proteins, such a phenomenon has not been observed in vivo. Here, we report a heterozygous frameshift mutation of PROKR2 identified in a 3.5‐year‐old girl with central precocious puberty. The mutant mRNA escaped nonsense‐mediated decay and generated a GPCR lacking two transmembrane domains and the carboxyl‐terminal tail. The mutant protein had no in vitro signal transduction activity; however, cells co‐expressing the mutant and wild‐type PROKR2 exhibited markedly exaggerated ligand‐induced Ca²⁺ responses. The results indicate that certain inactive PROKR2 mutants can cause early puberty by enhancing the functional property of coexisting wild‐type proteins. Considering the structural similarity among GPCRs, this paradoxical gain‐of‐function mechanism may underlie various human disorders.