TEX101, a germ cell-marker glycoprotein, is associated with lymphocyte antigen 6 complex locus k within the mouse testis

In adult male mice, the glycosylphosphatidyl inositol-anchored glycoprotein TEX101 is expressed only in germ cells and is thought to be involved in spermatogenesis. However, the details regarding the function of TEX101 remain to be clarified. We previously identified Ly6k as a candidate TEX101-associated protein, but as molecular probes are not currently available to detect Ly6k, we do not have conclusive evidence of the association between TEX101 and Ly6k. In this study, we confirmed the biological interaction between TEX101 and Ly6k using an established anti-mouse Ly6k polyclonal antibody (pAb). A combination of immunoprecipitation, Western blot, and immunohistochemical analyses using the pAb revealed that TEX101 is physically associated with Ly6k within the testis. In addition, these proteins simultaneously co-migrate into the detergent-resistant membrane fractions, suggesting that TEX101 collaborates with Ly6k on the cell membrane and may play a role in spermatogenesis.     

Modification of protein with BGL06, a novel branched oligoglycerol derivative

A branched oligoglycerol derivative, BGL06, with a cascade-like structure of glycerol units was used as a novel reagent for protein modification. Modification reaction with a recombinant human granulocyte-colony stimulating factor derivative, ND28, was carried out successfully in aqueous conditions to obtain a coupled form, BGL06-ND28. Characterization of the modified ND28 suggests that two types of products were obtained by controlling the reaction; one was H(BGL06)-ND28, a highly modified version coupled with 4.34 molecules of BGL06 units on average, and the other was L(BGL06)-ND28, a moderately modified version coupled with 2.58 molecules of BGL06 units on average, respectively. The properties of these products were compared to the known polyethylene glycol (PEG)-modified ND28. In the cell proliferation assays, unlike PEGylation, modification with BGL06 did not produce a significant loss of biological activity even when the modification extent was elevated. Under such conditions, 76.0% of the activity was in fact maintained for H(BGL06)-ND28, while PEGylated ND28 retained only 24.6% of biological activity in vitro even though the extent of modification was smaller. In addition, H(BGL06)-ND28 showed comparable thermostability to a 20 kDa PEG-modified counterpart. Therefore, the BGL06 derivative will be a useful alternative as a protein modification reagent where PEGylation is not effective.     

Local anesthetics, antipsychotic phenothiazines, and cationic surfactants shut down intracellular reactions through membrane perturbation in yeast

High osmolarity and glucose deprivation cause rapid shutdowns of both actin polarization and translation initiation in yeast. Like these stresses, administration of local anesthetics and of antipsychotic phenothiazines caused similar responses. All these drugs have amphiphilic structures and formed emulsions and permeabilized the cell membrane, indicating that they have the same features as a surfactant. Consistently with this, surfactants induced responses similar to those of local anesthetics and phenothiazines. Benzethonium chloride, a cationic surfactant, showed a more potent shutdown activity than phenothiazines, whereas SDS, an anionic surfactant, transiently depolarized actin without inhibiting translation initiation, suggesting that a cationic charge in the amphiphile is important to the shutdown of both reactions. The clinical drugs and the cationic surfactants at low concentrations caused shutdown without membrane permeabilization, suggesting that these compounds and stresses activate shutdown, via perturbation rather than disruption of the cell membrane.     

Petiolins A-C, phloroglucinol derivatives from Hypericum pseudopetiolatum var. kiusianum

Two new phloroglucinol derivatives possessing chromane skeleton, petiolins A (1) and B (2), and a new phloroglucinol derivative containing a dihydrofuran ring, petiolin C (3), were isolated from aerial parts of Hypericum pseudopetiolatum var. kiusianum. The gross structures of 1-3 were elucidated by spectroscopic data, and the relative stereochemistry of 3 was elucidated by NOESY data. Petiolins A-C (1-3) showed modest cytotoxicity, while petiolin C (3) exhibited antifungal activity.     

Synergistic effect of basic residues at positions 14-15 of nociceptin on binding affinity and receptor activation

Nociceptin is an endogenous ligand that activates a G protein-coupled receptor ORL1 and contains two indispensable Arg-Lys (RK) dipeptide units at positions 8-9 and 12-13. By replacing an additional RK unit at positions 6-7, 10-11, 14-15, or 16-17, of the peptide we have identified the analog, [RK(14-15)]nociceptin as a superagonist. In fact, this peptide exhibits 3-fold higher binding affinity and 17-fold greater potency in a functional GTPgammaS-binding assay compared to wild-type nociceptin. Here, we have further investigated the role of basic residues in position 14-15. The replacement of three other possible basic dipeptides, KR, RR, and KK, into nociceptin at positions 14-15 resulted in similar enhancements of binding affinity (3-5-fold) and biological potency (10-12-fold in the GTPgammaS assay). However, when only a single basic residue (Arg or Lys) was replaced in either position 14 or 15, all the resulting analogs showed moderate enhancements of binding and biological activity (2-4-fold in both). These results indicate that the addition of basic charges in positions 14 and 15 enhance in a synergistic fashion the interaction of nociceptin with the receptor and only the simultaneous presence of two adjacent basic residues yields an optimal effect. This suggests that specific electrostatic interactions between both amino acids present in 14-15 and corresponding residues in the receptor are responsible for the enhancement of nociceptin activity.     

Delayed Treatment with Arundic Acid Reduces the MPTP-induced Neurotoxicity in Mice

The authors investigated the protective effects of a novel astrocyte-modulating agent, arundic acid, in a 1-methyl-4-phenyl-1,2,3,6-tetrahyropyridine (MPTP) mouse model of Parkinson’s disease. Male mice received four intraperitoneal (i.p.) injections of MPTP (20 mg/kg) at 2 h intervals. The content of dopamine and its metabolites in the striatum was reduced markedly 7 days after MPTP treatment. The delayed treatment with arundic acid (30 mg/kg, i.p.) administered 3, 4, 5 and 6 days after MPTP treatment did not affect the depletion of dopamine and its metabolites in the striatum. Our immunohistochemical study with anti-tyrosine hydroxylase antibody, anti-neuronal nuclei antibody, anti-glial fibrillary acidic protein antibody, anti-S100β antibody and anti-nestin antibody showed that the delayed treatment with arundic acid had a protective effect against MPTP-induced neuronal damage in the striatum and the substantia nigra of mice. Furthermore, this agent ameliorated the severe reductions in number of isolectin reactive microglia in the striatum and the substantia nigra 7 days after MPTP treatment. These results demonstrate that the inhibition of S100β synthesis in astrocytes may be the major component of the beneficial effect of arundic acid. Thus, our present findings provide that the therapeutic strategies targeted to astrocytic modulation with arundic acid offers a great potential for restoring the functional capacity of the surviving dopaminergic neurons in individuals affected with Parkinson’s disease.     

Hydroxymethylglutaryl--CoA reductase inhibitor inhibits induction of nitric oxide synthase in 3T3--L1 preadipocytes

Preadipocytes are considered to play a role in adipose tissue inflammation in obesity. The purpose of this study was to determine whether hydroxymethylglutaryl-CoA reductase inhibitor (statin) modulates the nitric oxide (NO) production via inducible NO synthase (iNOS) in preadipocytes. Undifferentiated 3T3-L1 cells, a model of preadipocytes, significantly produced NO by the treatment with the combination of lipopolysaccharide (L), tumor necrosis factor-alpha (T) and interferon-gamma (I). Pre-incubation with simvastatin, a lipophilic statin, or pravastatin, a hydrophilic one, dose-dependently inhibited the NO production in the LTI-treated cells. The effect of simvastatin was offset by mevalonate or geranylgeranyl pyrophosphate (GGPP) but not by squalene. The mRNA level for iNOS paralleled the NO production. The nuclear factor-kappaB (NF-kappaB) was activated by the LTI-treatment, and was inhibited by addition of simvastatin or pravastatin. Mevalonate or GGPP completely offset the effect of simvastatin. Simvastatin or pravastatin also decreased the LTI-stimulated interleukin-6 (IL-6) secretion. These effects of pravastatin were relatively weak compared with those of simvastatin. Y27632, an inhibitor of Rho kinase, also inhibited the LTI-induced NF-kappaB activation and iNOS expression, and decreased the production of NO and IL-6 in 3T3-L1 preadipocytes. These results suggest that statins, especially lipophilic types, inhibit induction of iNOS by inhibiting the small GTP-binding protein signal in preadipocytes.