Calpain 6 is involved in microtubule stabilization and cytoskeletal organization

The calpains are a family of Ca(2+)-dependent cysteine proteases implicated in various biological processes. In this family, calpain 6 (Capn6) is unique in that it lacks the active-site cysteine residues requisite for protease activity. During the search for genes downstream of the endothelin 1 (ET-1) signaling in pharyngeal-arch development, we identified Capn6. After confirming that the expression of Capn6 in pharyngeal arches is downregulated in ET-1-null embryos by in situ hybridization, we investigated its function. In Capn6-transfected cells, cytokinesis was retarded and was often aborted to yield multinucleated cells. Capn6 overexpression also caused the formation of microtubule bundles rich in acetylated alpha-tubulin and resistant to the depolymerizing activity of nocodazole. Green fluorescent protein-Capn6 overexpression, immunostaining for endogenous Capn6, and biochemical analysis demonstrated interaction between Capn6 and microtubules, which appeared to be mainly mediated by domain III. Furthermore, RNA interference-mediated Capn6 inactivation caused microtubule instability with a loss of acetylated alpha-tubulin and induced actin reorganization, resulting in lamellipodium formation with membrane ruffling. Taken together, these results indicate that Capn6 is a microtubule-stabilizing protein expressed in embryonic tissues that may be involved in the regulation of microtubule dynamics and cytoskeletal organization.     

Detection of bikunin mRNA in limited portions of rat brain.

Tissue distribution of bikunin mRNA, which encodes a Kunitz-type serine protease inhibitor of the inter-alpha-inhibitor family (IalphaI), was studied in rats and mice by the reverse-transcripsion polymerase chain reaction (RT-PCR). We found that the liver as well as other tissues, such as the kidney, testis and adrenal gland, expressed bikunin mRNA. Although signals of bikunin mRNA were faint in the whole brain of rats and mice, distinct signals were found in limited portions of rat brain, such as the hippocampus, cerebral cortex and pituitary, but undetectable in cerebellum, medulla oblongata, hypothalamus, striatum, midbrain and choroid plexus. In three distinct types of cells, such as neurons, astrocytes and meningeal cells, in primary cultures isolated from the cerebral cortex and meninges of 1-day-old newborn rats, only neurons positively expressed bikunin mRNA. These results suggest that, in addition to peripheral tissues, neurons in the hippocampus and cerebral cortex produce bikunin, suggesting a potential role of bikunin/IalphaI family in these brain regions.     

Inherited deficiency of the seventh component of complement associated with meningococcal meningitis: lack of serum bactericidal activity against Neisseria meningitidis in a girl with C7 deficiency and HLA studies of a C7-deficient Japanese family

An 8-year-old girl with meningococcal meningitis lacked serum complement activity. The seventh component of complement (C7) could not be detected in her serum by either functional or immunochemical analysis. The levels of the other components were within the normal range. Her serum complement activity was restored by the addition of purified C7. Her fresh serum showed a total absence of bactericidal activity against Neisseria meningitidis, group Y, but her serum bactericidal activity was restored by the addition of purified C7. The restoration of her serum bactericidal activity was completely inhibited in the presence of Mg2+ EGTA. These findings suggest that restoration of the bactericidal activity of her serum against N. meningitidis might be mediated by the specific antibody against N. meningitidis and the reconstituted complement system in her serum. Heterozygous deficiency of C7 was found in 10 of her family members. Genetic studies showed that the mode of inheritance might be an autosomal codominant trait. No genetic linkage between deficiency of C7 and the HLA system was found.     

Telmisartan Activates Endothelial Nitric Oxide Synthase via Ser1177 Phosphorylation in Vascular Endothelial Cells

Because endothelial nitric oxide synthase (eNOS) has anti-inflammatory and anti-arteriosclerotic functions, it has been recognized as one of the key molecules essential for the homeostatic control of blood vessels other than relaxation of vascular tone. Here, we examined whether telmisartan modulates eNOS function through its pleiotropic effect. Administration of telmisartan to mice significantly increased the phosphorylation level of eNOS (Ser1177) in the aortic endothelium, but administration of valsartan had no effect. Similarly, telmisartan treatment of human umbilical vein endothelial cells significantly increased the phosphorylation levels of AMP-activated protein kinase (Thr172) and eNOS and the concentration of intracellular guanosine 3′,5′-cyclic monophosphate (cGMP). Furthermore, pretreatment with a p38 mitogen-activated protein kinase (p38 MAPK) inhibitor suppressed the increased phosphorylation level of eNOS and intracellular cGMP concentration. These data show that telmisartan increases eNOS activity through Ser1177 phosphorylation in vascular endothelial cells mainly via p38 MAPK signaling.     

A novel role of IL-15 in early activation of memory CD8+ CTL after reinfection

A rapid induction of effector functions in memory T cells provides rapid and intensified protection against reinfection. To determine potential roles of IL-15 in early expansion and activation of memory CD8+ T cells in secondary immune response, we examined the cell division and cytotoxicity of memory CD8+ T cells expressing OVA(257-264)/Kb-specific TCR that were transferred into IL-15-transgenic (Tg) mice, IL-15 knockout (KO) mice, or control C57BL/6 mice followed by challenge with recombinant Listeria monocytogenes expressing OVA (rLM-OVA). In vivo CTL activities and expression of granzyme B of the transferred CD8+ T cells were significantly higher in the IL-15 Tg mice but lower in the IL-15 KO mice than those in control mice at the early stage after challenge with rLM-OVA. In contrast, there was no difference in the cell division in IL-15 Tg mice and IL-15 KO mice compared with those in control mice. In vivo administration of rIL-15 conferred robust protection against reinfection via induction of granzyme B in the memory CD8+ T cells. These results suggest that IL-15 plays an important role in early activation of memory CD8+ T cells.     

Reductive cleavage and regeneration of the disulfide bonds inStreptomyces subtilisin inhibitor (SSI) as studied by the carbonyl13C NMR resonances of cysteinyl residues

Summary Four enhanced carbonyl carbon resonances were observed whenStreptomyces subtilisin inhibitor (SSI) was labeled by incorporating specifically labeled [1-¹³C]Cys. The¹³C signals were assigned by the¹⁵N,¹³C double-labeling method along with site-specific mutagenesis. Changes in the spectrum of the labeled protein ([C]SSI) were induced by reducing the disulfide bonds with various amounts of dithiothreitol (DTT). The results indicate that, in the absence of denaturant, the Cys⁷¹-Cys¹⁰¹ disulfide bond of each SSI subunit can be reduced selectively. This disulfide bond, which is in the vicinity of the reactive site scissile bond Met⁷³-Val⁷⁴, is more accessible to solvent than the other disulfide bond. Cys³⁵-Cys⁵⁰, which is embedded in the interior of SSI. This half-reduced SSI had 65% of the inhibitory activity of native SSI and maintained a conformation similar to that of the fully oxidized SSI. Reoxidation of the half reduced-folded SSI by air regenerates fully active SSI which is indistinguishable with intact SSI by NMR. In the presence of 3 M guanidine hydrochloride (GuHCl), however, both disulfide bonds of each SSI subunit were readily reduced by DTT. The fully reduced-unfolded SSI spontaneously refolded into a native-like structure (fully reduced-folded state), as evidenced by the Cys carbonyl carbon chemical shifts, upon removing GuHCl and DTT from the reaction mixture. The time course of disulfide bond regeneration from this state by air oxidation was monitored by following the NMR spectral changes and the results indicated that the disulfide bond between Cys⁷¹ and Cys¹⁰¹ regenerates at a much faster rate than that between Cys³⁵ and Cys⁵⁰.Nomenclature of the various states of SSI that are observed in the present study Fully oxidized-folded native or intact (without GuHCl or DTT) - half reduced-folded (Cys⁷¹-Cys¹⁰¹ reduced; DTT without GuHCl) - inversely half reduced-folded (Cys³⁵-Cys⁵⁰ reduced; a reoxidation intermediate from fully reduced-folded state) - fully reduced-unfolded (reduced by DTT in the presence of GuHCl) - fully reduced-folded (an intermediate state obtained by removing DTT and GuHCl from the fully reduced-unfolded SSI reaction mixture)     

Pysicochemical studies of taste reception. V. Suppressive effect of salts on sugar response of the frog.

The tast responses of frog to various kinds of sugars were measured quantitatively by use of the glossopharyngeal nerve activity under an appropriate condition where the water response was completely suppressed. The concentration dependences of response of frog tongue to D-fructose, D-glucose, and sucrose were almost the same, D-galactose, however, elicited a much larger response in comparison with the other sugars in the whole range of concentrations examined. The sugar response was suppressed extensively by the presence of small amount of salts in the stimulating sugar solution. The suppressive effects of NaCl, KCl, MgCl2, MgSO4, and K4Fe(CN)6 were examined with a fixed concentration of sugar. The results obtained with these salts, added in various concentrations, fell on a single curve when the data were plotted against the ionic strength in the stimulating solution. The present results were consistent with the notion that the taste receptor potential for salts or acids is attributable to a change in the phase boundary potential at the membrane-solution interface as proposed in the previous papers of this series.     

Area-based analyzing technique at cell array experiment using neuronal cell line

We propose a simple procedure for the identification and quantitative analysis of neurite outgrowth in neuronal cell lines that were uniformly differentiated. Upon stimulation most neuronal cell lines extend neurites in the differentiation process, resulting, according to our observation, in the increase of cell surface area. This increase is dependent on the length and the number of extended neurites. Furthermore, we use this method for the phenotype analysis of cell array experiments to perform large-scale functional evaluation of genes involved in the neurite outgrowth during neuronal differentiation.     

Structural requirement of leucine for activation of p70 S6 kinase

The addition of leucine induced activation of p70S6k in amino acid-depleted H4IIE cells. Whereas the activation of p70S6k by leucine was transient, the complete amino acid stimulated p70S6k more persistently. The effect of leucine on p70S6k was sensitive to rapamycin, but less sensitive to wortmannin. Using various amino acids and derivatives of leucine, we found that the chirality, the structure of the four branched hydrocarbons, and the primary amine are required for the ability of leucine to stimulate p70S6k, indicating that the structural requirement of leucine to induce p70S6k activation is very strict and precise. In addition, some leucine derivatives exhibited the ability to stimulate p70S6k and the other derivatives acted as inhibitors against the leucine-induced activation of p70S6k.