p90 RSK-1 associates with and inhibits neuronal nitric oxide synthase

Evidence is presented that RSK1 (ribosomal S6 kinase 1), a downstream target of MAPK (mitogen-activated protein kinase), directly phosphorylates nNOS (neuronal nitric oxide synthase) on Ser847 in response to mitogens. The phosphorylation thus increases greatly following EGF (epidermal growth factor) treatment of rat pituitary tumour GH3 cells and is reduced by exposure to the MEK (MAPK/extracellular-signal-regulated kinase kinase) inhibitor PD98059. Furthermore, it is significantly enhanced by expression of wild-type RSK1 and antagonized by kinase-inactive RSK1 or specific reduction of endogenous RSK1. EGF treatment of HEK-293 (human embryonic kidney) cells, expressing RSK1 and nNOS, led to inhibition of NOS enzyme activity, associated with an increase in phosphorylation of nNOS at Ser847, as is also the case in an in vitro assay. In addition, these phenomena were significantly blocked by treatment with the RSK inhibitor Ro31-8220. Cells expressing mutant nNOS (S847A) proved resistant to phosphorylation and decrease of NOS activity. Within minutes of adding EGF to transfected cells, RSK1 associated with nNOS and subsequently dissociated following more prolonged agonist stimulation. EGF-induced formation of the nNOS-RSK1 complex was significantly decreased by PD98059 treatment. Treatment with EGF further revealed phosphorylation of nNOS on Ser847 in rat hippocampal neurons and cerebellar granule cells. This EGF-induced phosphorylation was partially blocked by PD98059 and Ro31-8220. Together, these data provide substantial evidence that RSK1 associates with and phosphorylates nNOS on Ser847 following mitogen stimulation and suggest a novel role for RSK1 in the regulation of nitric oxide function in brain.     

Low-temperature dynamic light scattering. I. Structural reorganization and physical gel formation in cellulose triacetate/methyl acetate dilute solution at -99 - 45 degrees C

Curious low-temperature solubility of cellulose triacetates (CTA; here we use nominally "CTA," but the sample still contains 7% of C-6 position hydroxyls) in an organic solvent, methyl acetate (MA), was studied by a newly designed low-temperature type of DLS apparatus, which enabled for the first time to investigate the structural change of CTA in solution from 45 degrees C down to -100 degrees C. A molecularly dissolved CTA was found to coexist with three types of self-assemblies over all the temperature ranges except for the three specific temperatures T* of 30, -10, and -75 degrees C. However, these multiple self-assemblies are not in real thermodynamic equilibrium but in a metastable state, which could be stabilized effectively by the intermolecular hydrogen bonding (HB) with the help of the dipole interaction at low temperatures. In more detail, with decreasing temperature, these assemblies performed the structural reorganization drastically at three T*'s and would finally be frozen in a physical gel structure at -99 degrees C; around the freezing temperature of MA, CTA molecules could be trapped homogeneously in the frozen MA. The crucial role in such structural reorganizations is played by the balance between the intermolecular HB and the dipole interaction worked in the highly electronegative solvent. Because these interactions, which are mediated by the solvent electronegativity, change drastically with temperature, they result in the control of not only the single CTA chain conformation (= the intramolecular HB) but also the binding ways of the intermolecular HBs between CTA molecules and they induce multitudinous metastable structures in solution. Here it is noted that HB could work mainly between the C-6 position hydroxyls in the anhydroglucose units of CTA and are essentially effective at low temperatures.     

Process optimization for increased yield of surface-expressed protein in Escherichia coli

The autotransporter family of Gram-negative protein exporters has been exploited for surface expression of recombinant passenger proteins. While the passenger in some cases was successfully translocated, a major problem has been low levels of full-length protein on the surface due to proteolysis following export over the cytoplasmic membrane. The aim of the present study was to increase the surface expression yield of the model protein SefA, a Salmonella enterica fimbrial subunit with potential for use in vaccine applications, by reducing this proteolysis through process design using Design of Experiments methodology. Cultivation temperature and pH, hypothesized to influence periplasmic protease activity, as well as inducer concentration were the parameters selected for optimization. Through modification of these parameters, the total surface expression yield of SefA was increased by 200 %. At the same time, the yield of full-length protein was increased by 300 %, indicating a 33 % reduction in proteolysis.     

Variations in the C3, C3a receptor, and C5 genes affect susceptibility to bronchial asthma

Bronchial asthma (BA) is a common chronic inflammatory disease characterized by hyperresponsive airways, excess mucus production, eosinophil activation, and the production of IgE. The complement system plays an immunoregulatory role at the interface of innate and acquired immunities. Recent studies have provided evidence that C3, C3a receptor, and C5 are linked to airway hyperresponsiveness. To determine whether genetic variations in the genes of the complement system affect susceptibility to BA, we screened single nucleotide polymorphisms (SNPs) in C3, C5, the C3a receptor gene (C3AR1), and the C5a receptor gene (C5R1) and performed association studies in the Japanese population. The results of this SNP case-control study suggested an association between 4896C/T in the C3 gene and atopic childhood BA (P=0.0078) as well as adult BA (P=0.010). When patient data were stratified according to elevated total IgE levels, 4896C/T was more closely associated with adult BA (P=0.0016). A patient-only association study suggested that severity of childhood BA was associated with 1526G/A of the C3AR1 gene (P=0.0057). We identified a high-risk haplotype of the C3 gene for childhood (P=0.0021) and adult BA (P=0.0058) and a low-risk haplotype for adult BA (P=0.00011). We also identified a haplotype of the C5 gene that was protective against childhood BA (P=1.4×10^–6) and adult BA (P=0.00063). These results suggest that the C3 and C5 pathways of the complement system play important roles in the pathogenesis of BA and that polymorphisms of these genes affect susceptibility to BA.     

Geographic variation in the growth of white croaker,Pennahia argentata,off the coast of northwest Kyushu,Japan

We examined geographic variation in the growth of white croaker,Pennahia argentata, off the coast of northwest Kyushu, Japan, Ariake Sound, Tachibana Bay, Omura Bay and the Goto Sea by examination of otoliths. The outer margins of the otoliths showed that a opaque zone was formed once a year, with its peak in June, and could be used as an annulus. White croaker caught during this study reached a maximum age of 10years in the Goto Sea. The growth curves for both sexes in all localities were expressed by the von Bertalanffy growth equations from back-calculated total length of fish. We found significant sexual differences in growth curves in Ariake Sound, Tachibana Bay and the Goto Sea. For both sexes, white croaker in the Goto Sea reached the largest length at each estimated age of the four localities. The growth curves for both sexes showed significant differences among four localities, suggesting that several stocks may exist in the study area although the greatest distance between each locality was at most 30 km.     

Tissue-specific pyruvate dehydrogenase complex deficiency causes cardiac hypertrophy and sudden death of weaned male mice

Pyruvate dehydrogenase complex (PDC) plays an important role in energy homeostasis in the heart by catalyzing the oxidative decarboxylation of pyruvate derived primarily from glucose and lactate. Because various pathophysiological states can markedly alter cardiac glucose metabolism and PDC has been shown to be altered in response to chronic ischemia, cardiac physiology of a mouse model with knockout of the alpha-subunit of the pyruvate dehydrogenase component of PDC in heart/skeletal muscle (H/SM-PDCKO) was investigated. H/SM-PDCKO mice did not show embryonic lethality and grew normally during the preweaning period. Heart and skeletal muscle of homozygous male mice had very low PDC activity (approximately 5% of wild-type), and PDC activity in these tissues from heterozygous females was approximately 50%. Male mice did not survive for >7 days after weaning on a rodent chow diet. However, they survived on a high-fat diet and developed left ventricular hypertrophy and reduced left ventricular systolic function compared with wild-type male mice. The changes in the heterozygote female mice were of lesser severity. The deficiency of PDC in H/SM-PDCKO male mice greatly compromises the ability of the heart to oxidize glucose for the generation of energy (and hence cardiac function) and results in cardiac pathological changes. This mouse model demonstrates the importance of glucose oxidation in cardiac energetics and function under basal conditions.     

Uptake of L-cystine via an ABC transporter contributes defense of oxidative stress in the L-cystine export-dependent manner in Escherichia coli

Intracellular thiols like L-cystine and L-cystine play a critical role in the regulation of cellular processes. Here we show that Escherichia coli has two L-cystine transporters, the symporter YdjN and the ATP-binding cassette importer FliY-YecSC. These proteins import L-cystine, an oxidized product of L-cystine from the periplasm to the cytoplasm. The symporter YdjN, which is expected to be a new member of the L-cystine regulon, is a low affinity L-cystine transporter (K _m = 1.1 μM) that is mainly involved in L-cystine uptake from outside as a nutrient. E. coli has only two L-cystine importers because ΔydjNΔyecS mutant cells are not capable of growing in the minimal medium containing L-cystine as a sole sulfur source. Another protein YecSC is the FliY-dependent L-cystine transporter that functions cooperatively with the L-cystine transporter YdeD, which exports L-cystine as reducing equivalents from the cytoplasm to the periplasm, to prevent E. coli cells from oxidative stress. The exported L-cystine can reduce the periplasmic hydrogen peroxide to water, and then generated L-cystine is imported back into the cytoplasm via the ATP-binding cassette transporter YecSC with a high affinity to L-cystine (K _m = 110 nM) in a manner dependent on FliY, the periplasmic L-cystine-binding protein. The double disruption of ydeD and fliY increased cellular levels of lipid peroxides. From these findings, we propose that the hydrogen peroxide-inducible L-cystine/L-cystine shuttle system plays a role of detoxification of hydrogen peroxide before lipid peroxidation occurs, and then might specific prevent damage to membrane lipids.