Specificity of elongation factor EF-TU for hydrophobic peptides

The elongation factor EF-Tu carries aminoacyl-tRNAs to the A-site of the ribosome during the elongation process of protein biosynthesis. We, and others, have recently reported that the Escherichia coli EF-Tu interacts with unfolded and denatured proteins and behaves like a chaperone in protein folding and protection against protein thermal denaturation. In this study, we have identified EF-Tu binding sites in protein substrates by screening cellulose-bound peptides scanning the sequences of several proteins. The binding motifs recognized by EF-Tu in protein substrates are also recognized by the chaperone DnaK and mainly consist of hydrophobic clusters. EF-Tu interacts as efficiently as DnaK with the membrane spanning sequence of the membrane protein phospholemman and with the signal sequence of alkaline phosphatase. It interacts less efficiently with several other hydrophobic clusters of lysozyme and alkaline phosphatase, which are also DnaK substrates and fails to bind to several DnaK binding sites. Our results suggest that EF-Tu, like DnaK, interacts albeit more weakly with the hydrophobic regions of substrate protein and are consistent with the hypothesis that it possesses chaperone properties.     

Estrogen activates cyclin-dependent kinases 4 and 6 through induction of cyclin D in rat primary osteoblasts

Estrogen plays important roles in maintaining bone density and protecting against osteoporosis, but the underlying mechanisms of estrogen action via estrogen receptors (ERs) in bone remain to be clarified. In the present study, we isolated primary osteoblasts derived from transgenic rats harboring a dominant negative ER mutant, rat ERalpha (1-535) cDNA, and from their wild-type littermates. We observed that the rate of cell growth of osteoblasts from the transgenic rats was reduced compared to that of wild-type osteoblasts. Utilizing cDNA microarray analysis, we found that mRNA level of cyclin D2 was lower in the osteoblasts from the transgenic rats. D-type cyclins including cyclin D1, cyclin D2, and cyclin D3 are cell cycle regulators that promote progression through the early-to-mid G1 phase of the cell cycle. The protein levels of D-type cyclins including cyclin D2 and cyclin D3 but not cyclin D1 were elevated in wild-type osteoblasts with 17beta-estradiol treatment, resulting in the activation of cyclin-dependent kinases 4 and 6 (Cdk4/6) activities and the promotion of cell growth. Moreover, an anti-estrogen ICI 182,780 abolished the induction of the expression of D-type cyclins by 17beta-estradiol. Our findings indicate that estrogen and its receptors enhance Cdk4/6 activities through the induction of D-type cyclins, leading to the growth promotion of osteoblasts.     

Expression and functional analysis of rat P23, a gut hormone-inducible isoform of trypsin,reveals its resistance to proteinaceous trypsin inhibitors

Rat P23 is an isoform of trypsin (ogens) synthesized by rat acinar cells. Expression of P23 is stimulated strongly by caerulein, an analogue of cholecystokinin (CCK). However, the physiological relevance of rat P23 in healthy and pathological conditions such as caerulein-induced pancreatitis is largely unknown. Using recombinant P23 trypsinogen and reconstitution analysis of zymogen autoactivation, unique inhibitor-resistance characteristics of P23 were elucidated. P23 cDNA was expressed in Escherichia coli periplasm, yielding recombinant P23 trypsinogen. Autoactivation of zymogen granule contents from caerulein-induced rat pancreas was also studied. Activation kinetics of P23 by enterokinase was similar to those of rat anionic trypsinogen, which is a major isoform of trypsinogen. Interestingly, rat pancreatic secretory trypsin inhibitor (PSTI), which protects against deleterious activation of trypsinogens in zymogen granules, failed to inhibit P23 trypsin even with four-fold molar excess, at which concentration it effectively inhibited rat anionic trypsin to almost 100%. P23 trypsin also showed marked resistance to proteinaceous trypsin inhibitors such as soybean trypsin inhibitor and aprotinin. P23 trypsin activated by enterokinase dramatically accelerated the cascade of autoactivation of anionic trypsinogen even in the presence of PSTI. Taken together with a previous observation that P23 is specifically upregulated 14-fold by 24-h caerulein infusion, these results suggest that elevated levels of P23 should be taken into consideration in the mechanism of trypsinogens within the pancreas in pathological conditions.     

Dietary Zn deficiency does not influence systemic blood pressure and vascular nitric oxide signaling in normotensive rats

Because zinc (Zn) is an important component for cell protection against certain oxygen species, it has been suggested that Zn deficiency impairs the potent oxidant defense capacity, which is constitutively provided in the vascular system. However, the influence of dietary Zn deficiency on systemic blood pressure and vascular system is controversial and unclear. We therefore examine the effect of dietary Zn deficiency on systemic blood pressure, a potent superoxide scavenger, aortic Cu/Zn superoxide dismutase (SOD) activity, a most representative synthase of the endothelium-derived relaxing factor, and aortic endothelial nitric oxide synthase (eNOS) expression. Furthermore, the direct effects of intravenous administration of NOS inhibitor, N ^ω-nitro-l-arginine methyl ester (l-NAME), and a SOD mimetic compound, tempol, in normotensives were tested in Wistar-Kyoto (WKY) rats. A Zn-deficient diet (4 wk) contributed to growth retardation, the decrease in thymus weight, and the lower levels of serum Zn compared with the standard diet group. However, no significant difference in conscious systolic and diastolic blood pressure was found in the Zn-deficiency group. The administration of l-NAME caused an increase in the mean arterial pressure (MAP) levels in the two groups of rats and the involvement of the vasodilator nitric oxide (NO) in the regulation of systemic BP in the normotensive state. On the other hand, administration of the superoxide scavenger, tempol, led to a decrease in MAP levels in the two groups of rats, indicating the participation of the oxygen free radical, superoxide, in the maintenance of the systemic BP in a normotensive state. There were no significant differences between the Zn-deficient diet group and the standard diet group in the normotensive state. eNOS expression and Cu/Zn SOD activity in the aorta were also intact in Zn-deficient normotensive rats. These findings suggest that the 4 wk of Zn deficiency was inadequate to alter systemic blood pressure and focal NO signaling in the normotensive state. Long-term Zn deficiency affects the neuronal, immune, and hematopoietic systems, which contribute to systemic and/or local circulation. However, Zn deficiency alone does not cause hypertension and local vascular dysfunction in the normotensive state.     

Connexin-30 deletion analysis in connexin-26 heterozygotes

Mutations in the Connexin-26 gene (Cx 26, GJB2) are the most common cause of hereditary nonsyndromic sensorineural hearing loss (SNHL). DNA analysis of the Cx 26 gene in deaf or hard-of-hearing individuals frequently demonstrates heterozygosity despite the fact that most mutations are known to be recessive. A 342-kb deletion in a gene adjacent to Cx 26, the Connexin-30 gene (Cx 30, GJB6), has been reported to cause deafness in the homozygous state or in combination with heterozygous mutations in Cx 26 (digenic inheritance). We have analyzed deaf or hard-of-hearing Cx 26 heterozygotes and individuals with no mutations in Cx 26 for this Cx 30 deletion. We found that 4/20 (20%) of the Cx 26 heterozygotes are heterozygous for this deletion and that no individuals were homozygous for the Cx 30 deletion. Cx 30 deletion analysis is recommended for all individuals with nonsyndromic SNHL following Cx 26 sequencing that does not demonstrate two recessive mutations.     

Inhibition of recombinant dipeptidyl peptidase III by synthetic hemorphin-like peptides

In order to find the most effective antagonist for dipeptidyl peptidase III degrading enkephalin, we synthesized hemorphin-like pentapeptides with aliphatic or aromatic amino acids at the N-termini, such as VVYPW, LVYPW, IVYPW, YVYPW, FVYPW and WVYPW. Among those pentapeptides, IVYPW and WVYPW showed the strongest inhibitory activity toward rDPP III. The K(i) values of IVYPW and WVYPW were 0.100+/-0.011 and 0.126+/-0.015 microM (mean+/-S.E.), respectively. The order of K(i) values was Ile> or =Trp>Phe> or =Tyr>Leu>Ala>Val>Ser>Gly. rDPP III activity is inhibited in a non-competitive manner by these peptides. The peptide VYPW did not inhibit rDPP III activity, but the sequence is essential for the expression of inhibitory activity.     

Atlas of the embryonic brain in the pygmy squid,Idiosepius paradoxus

Gross structural changes and neuropil formation in the brain during development were described in Idiosepius paradoxus, a sepioid that we chose as a model cephalopod. The brain originates in 4 pairs of ectodermal placodes, which occur separately in the embryonic surface undergoing epiboly. In the final period of epiboly, neuroblasts internalize from the placodes and gather into 4 pairs of ganglionic masses. The ganglionic masses assemble into a ring-like cluster encircling the inner yolk and the foregut anlage, gradually integrated into the 4 domains of a massive brain, a subesophageal mass (SBM), a supraesophageal mass (SPM), and a pair of optic lobes. In the early brain, neuropil forms a framework composed of a longitudinal ladder lying in the SBM, and a transverse arch standing on the lateral sides of the SBM and crossing the SPM. Differentiation of brain lobes proceeds from ventral to dorsal along this framework; first the magnocellular lobes and the posterior pedal lobe appear first in the SBM, the other lobes in the SBM and the basal lobes follow in the proximal region of the SPM, and the accessory lobes develop last in the most dorsal zone of the SPM. In the hatchlings, the brain lobes show almost the same arrangement as in the adults, but the accessory lobes, particularly the vertical lobe, are much smaller than those in the adults. Comparison of the present results with those in the teuthoid and the octopod indicates that developmental sequences of the brain are highly conserved in the coleoid cephalopods.     

Molecular cloning and sequences of cDNAs encoding alpha (large) and beta (small) isoforms of human pancreatic zymogen granule membrane-associated protein GP2

cDNA clones encoding two different (alpha and beta) forms of human pancreatic zymogen granule membrane-associated protein glycoprotein 2 (GP2) (also referred to as ZAP75), a critical component in regulated membrane trafficking along the apical secretory pathway in pancreatic acinar cells, have been isolated. Structural analysis of the clones revealed that the alpha and beta forms of GP2 consist of 527 and 380 amino acid (aa) residues, respectively. The beta form lacks a 147 aa domain that corresponds to the 25-171 region of the alpha form, suggesting that it is a product of an alternative splicing event. Expression of both forms of GP2 in the human pancreas was confirmed. A unique isoform of GP2 is reported for the first time in humans.     

Phylogenetic identification of n-alkane assimilating Candida yeasts based on nucleotide divergence in the 59 end of LSU rDNA gene

Phylogenetic relationships of several species within the n-alkane assimilating Candida yeasts were investigated by using characters from the nucleotide sequence of the variable D1/D2 region at the 5' end of a large-subunit (26S) ribosomal DNA (rDNA) gene. First the nucleotide sequences of D1/D2 domain of Candida sp. 1098 (formerly identified as C. tropicalis 1098) and its dicarboxylic acid-producing-mutant strain M1210 were investigated. These two nucleotide sequences were identical and lacked only one base pair compared with that of C. maltosa CBS 5611 (type strain), and they were identified as C. maltosa. We then showed that C. maltosa IFO 1978 (formerly identified as C. cloacae) and C. maltosa IFO 1975 (formerly identified as C. subtropicalis) had the same nucleotide sequence and had only one base pair substitution compared with C. maltosa CBS 5611 (type strain), which is consistent with conventional classification. We also found that another widely studied n-alkane assimilating Candida yeast, C. tropicalis pk233, to be C. viswanathii.