Acute interleukin-6 infusion increases IGFBP-1 but has no short-term effect on IGFBP-3 proteolysis in healthy men

Human conditions of elevated interleukin-6 (IL-6) and transgenic mice overexpressing IL-6 have increased proteolytic degradation of insulin-like growth factor binding protein (IGFBP)-3. In addition, IL-6 alters the hepatic expression of insulin-like growth factor-I (IGF-I) and the IGFBPs in vitro. The aim of the present study was to investigate whether moderately elevated IL-6 levels have short-term effects on circulating IGF-I, IGFBP-1 and IGFBP-3 proteolysis in vivo. Healthy men received a 3-h IL-6 (n = 6) or saline (n = 6) infusion and blood samples were collected prior to and up to 8 h after the start of infusion. Free IGF-I, total IGF-I, IGFBP-1, insulin and cortisol were measured using immunoassays. Serum IGFBP-3 proteolysis was analyzed by Western immunoblot and by in vitro degradation of (125)I-IGFBP-3. We found that IL-6 concentrations reaching approximately 100 pg/ml significantly increased IGFBP-1 after the end of infusion in the absence of changes in insulin. In addition, plasma levels of cortisol were increased in response to IL-6 during and after infusion compared to saline. There was no effect of IL-6 on IGFBP-3 proteolysis, total IGF-I or free dissociable IGF-I. These data suggest that moderately elevated levels of IL-6 such as in the post-operative state or after exercise may contribute to increased levels of IGFBP-1. Although this study does not exclude that high levels and/or prolonged exposure to IL-6 may induce IGFBP-3 proteolysis in sepsis or chronic inflammatory disease, it suggests that IL-6 released from exercising skeletal muscle is not directly involved in proteolysis of circulating IGFBP-3.     

Increased expression of the dnaA gene has no effect on DNA replication in a dnaA+ strain of Escherichia coli

We have constructed a pBR322 plasmid derivative which expresses dnaA protein under the control of the E. coli lac UV5 promotor. Expression of the dnaA protein from the plasmid is inducible by isopropyl-beta-D-thiogalactoside. In a dnaA+ strain induction has no effect on the accumulation of DNA. In contrast, in a thermosensitive dnaA46 strain, induction, at either the permissive or the nonpermissive temperature, results in an immediate stimulation of DNA accumulation. We conclude that, while in a dnaA46 strain dnaA protein limits DNA replication, in a dnaA+ strain dnaA protein activity does not control the timing of replication initiation.     

The apolipoprotein B Q3405E polymorphism has no effect on its low-density-lipoprotein receptor binding affinity

A better understanding of the apolipoprotein B100 (apoB100) sequences involved in binding to the low-density lipoprotein (LDL) receptor will be achieved by studying the effects of polymorphisms and rare mutations of apoB100. Upon re-examination of apoB100 DNA sequencing discrepancies, a charge-change polymorphism, Q3405E, was found in the putative LDL receptor binding domain of the protein. Positively charged lysine and arginine side chains of the protein have been demonstrated to participate in the ligand. This led us to propose that the presence of an additional negative charge in close proximity could have an impact on the binding affinity. The polymorphism is the result of a C-to-G transition at nucleotide 10422. Population screening revealed 20 of the less common glutamate alleles at an allele frequency of 0.9%. The effect of the presence of one glutamate allele on the binding affinity of LDL for the LDL receptor was investigated in seven heterozygous individuals by a competitive dual-label fibroblast binding assay. One individual who was homozygous for the glutamate allele was discovered and her LDL examined in a competitive displacement binding assay. The additional negative charge at residue 3405 had no detectable affect on the binding affinity. Received: 8 May 1996 / Revised: 9 July 1996     

Peripheral obestatin has no effect on feeding behavior and brain Fos expression in rodents

Obestatin is produced in the stomach from proghrelin by post-translational cleavage. The initial report claimed anorexigenic effects of obestatin in mice. Contrasting studies indicated no effect of obestatin on food intake (FI). We investigated influences of metabolic state (fed/fasted), environmental factors (dark/light phase) and brain Fos response to intraperitoneal (ip) obestatin in rats, and used the protocol from the original study assessing obestatin effects in mice. FI was determined in male rats injected ip before onset of dark or light phase, with obestatin (1 or 5 μmol/kg), CCK8S (3.5 nmol/kg) or 0.15 M NaCl, after fasting (16 h, n = 8/group) or ad libitum (n = 10–14/group) food intake. Fos expression in hypothalamic and brainstem nuclei was examined in freely fed rats 90 min after obestatin (5 μmol/kg), CCK8S (1.75 nmol/kg) or 0.15 M NaCl (n = 4/group). Additionally, fasted mice were injected ip with obestatin (1 μmol/kg) or urocortin 1 (2 nmol/kg) 15 min before food presentation. No effect on FI was observed after obestatin administration during the light and dark phase under both metabolic conditions while CCK8S reduced FI irrespectively of the conditions. The number of Fos positive neurons was not modified by obestatin while CCK8S increased Fos expression in selective brain nuclei. Obestatin did not influence the refeeding response to a fast in mice, while urocortin was effective. Therefore, peripheral obestatin has no effect on FI under various experimental conditions and did not induce Fos in relevant central neuronal circuitries modulating feeding in rodents.     

Interference with murein turnover has no effect on growth but reduces beta-lactamase induction in Escherichia coli

Physiological studies of a mutant of Escherichia coli lacking the three lytic transglycosylases Slt70, MltA, and MltB revealed that interference with murein turnover can prevent AmpC beta-lactamase induction. The triple mutant, although growing normally, shows a dramatically reduced rate of murein turnover. Despite the reduction in the formation of low-molecular-weight murein turnover products, neither the rate of murein synthesis nor the amount of murein per cell was increased. This might be explained by assuming that during growth in the absence of the major lytic transglycosylases native murein strands are excised by the action of endopeptidases and directly reused without further breakdown to muropeptides. The reduced rate of murein turnover could be correlated with lowered cefoxitin-induced expression of beta-lactamase, present on a plasmid carrying the ampC and ampR genes from Enterobacter cloacae. Overproduction of MltB stimulated beta-lactamase induction, whereas specific inhibition of Slt70 by bulgecin repressed ampC expression. Thus, specific inhibitors of lytic transglycosylases can increase the potency of penicillins and cephalosporins against bacteria inducing AmpC-like beta-lactamases.     

Removal of serine phosphates from simian virus 40 large T antigen increases its ability to stimulate DNA replication in vitro but has no effect on ATPase and DNA binding.

The effect of phosphorylation on the ability of simian virus 40 large T antigen to stimulate DNA synthesis in vitro was tested. Treatment of affinity-purified large T antigen with calf intestinal alkaline phosphatase resulted in the removal of 70 to 80% of the phosphate residues. Only serine-bound phosphate residues were affected. Phosphatase-treated large T antigen stimulated in vitro DNA synthesis fourfold over the untreated control. The stimulation was strongest at early times of DNA replication. At later times, DNA replication proceeded at equal rates with dephosphorylated and untreated large T antigen. The ATPase activity of large T antigen was not affected by phosphatase treatment. The origin-binding activity of large T antigen was tested over a wide range of large T antigen to DNA ratios, including DNA excess, and in the presence and absence of carrier DNA. Under no condition was an effect of dephosphorylation of large T antigen on its DNA-binding activity observed. These findings might indicate that phosphorylation at serine residues modulates the interaction of large T antigen with cellular factors. During DNA synthesis large T antigen was substantially rephosphorylated by kinases in the HeLa cell extract. As shown by two-dimensional peptide mapping, this phosphorylation occurred at all known in vivo sites. No phosphatase and protease activities were detectable in the HeLa cell extract.     

Estrogen directly attenuates human osteoclastogenesis, but has no effect on resorption by mature osteoclasts

Estrogen deficiency arising with the menopause promotes marked acceleration of bone resorption, which can be restored by hormone replacement therapy. The inhibitory effects of estrogen seem to involve indirect cytokine- mediated effects via supporting bone marrow cells, but direct estrogen-receptor mediated effects on the bone-resorbing osteoclasts have also been proposed. Little information is available on whether estrogens modulate human osteoclastogenesis or merely inhibit the functional activity of osteoclasts. To clarify whether estrogens directly modulate osteoclastic activities human CD14+ monocytes were cultured in the presence of M-CSF and RANKL to induce osteoclast differentiation. Addition of 0.1-10 nM 17beta-estradiol to differentiating osteoclasts resulted in a dose-dependent reduction in tartrate resistant acid phosphatase (TRACP) activity reaching 60% at 0.1 nM. In addition, 17beta-estradiol inhibited bone resorption, as measured by the release of the C-terminal crosslinked telopeptide (CTX), by 60% at 0.1 nM, but had no effect on the overall cell viability. In contrast to the results obtained with differentiating osteoclasts, addition of 17beta-estradiol (0.001-10 nM) to mature osteoclasts did not affect bone resorption or TRACP activity. We investigated expression of the estrogen receptors, using immunocytochemistry and Western blotting. We found that ER-alpha is expressed in osteoclast precursors, whereas ER- beta is expressed at all stages, indicating that the inhibitory effect of estrogen on osteoclastogenesis is mediated by ER-alpha for the major part. In conclusion, these results suggest that the in vivo effects of estrogen are mediated by reduction of osteoclastogenesis rather than direct inhibition of the resorptive activity of mature osteoclasts.     

Interleukin 1 protects against the lethal effects of irradiation of mice but has no effect on tumors in the same animals

Interleukin 1 (IL-1) is a radioprotector of bone marrow and is cytotoxic to some tumor cells. This investigation examines these two properties in the same host animals and gives evidence of radioprotection against localized x-irradiation of the head and neck region. By LD50 analyses, recombinant human IL-1 (100 ng/mouse, approximately 3 micrograms/kg) was found to be radioprotective against whole-body irradiation for both C3H/Km and C57BL/Ka mice. The combined potency ratio for the two strains was 1.07 (95% confidence limit: 1.02-1.12). It was also radioprotective against the injury leading to acute lethality resulting from localized head and neck irradiation of C3H/Km mice; 100 ng of IL-1/mouse produced a potency ratio of 1.05 (95 confidence limit: 1.03-1.07). However, two tumors that originated in C3H/Km mice, RIF-1 and SCCVII, showed neither in vitro nor in vivo response to IL-1. Also, there was no IL-1-induced reduction in in vivo growth of the RL 12NP lymphoma in C57BL/Ka mice.     

Transgenic expression of CD36 in the spontaneously hypertensive rat is associated with amelioration of metabolic disturbances but has no effect on hypertension

Spontaneously hypertensive rats (SHR/NIH strain) harbor a deletion variant in the Cd36 fatty acid transporter and display defective fatty acid metabolism, insulin resistance and hypertension. Transgenic rescue of Cd36 in SHR ameliorates insulin resistance and improves dyslipidemia. However, the role of Cd36 in blood pressure regulation remains controversial due to inconsistent blood pressure effects that were observed with transgenic expression of Cd36 on the SHR background. In the current studies, we developed two new SHR transgenic lines, which express wild type Cd36 under the control of the universal Ef-1 alpha promoter, and examined the effects of transgenic expression of wild type Cd36 on selected metabolic and cardiovascular phenotypes. Transgenic expression of Cd36 in the new lines was associated with significantly decreased serum fatty acids, amelioration of insulin resistance and glucose intolerance but failed to induce any consistent changes in blood pressure as measured by radiotelemetry. The current findings confirm the genetic association of defective Cd36 with disordered insulin action and fatty acid metabolism in the SHR/NIH strain and suggest that Cd36 is linked to other gene(s) on rat chromosome 4 that regulate blood pressure.     

猪β干扰素在毕赤酵母中的分泌表达及其对伪狂犬病毒的抑制作用

为了研制高活性的重组猪β干扰素,对PoIFN-β成熟蛋白第3、7和164位的3个氨基酸密码子进行毕赤酵母偏嗜性改造并构建了酵母表达载体pPICZαA-PIB。pPICZαA-PIB经SacⅠ酶切线性化后电转化导入毕赤酵母菌株X-33。多株PCR鉴定为阳性的酵母转化子经甲醇诱导发酵分泌表达了PoIFN-β,其中B1株酵母的PoIFN-β产量最高,约为2.5×105U/mL,其表达量约为60μg/mL,比活为4.17×106U/mg。将发酵上清液用PEG20000浓缩后进行SDS-PAGE和Western blot检测,结果表明表达产物是分子量约为28kDa和25kDa蛋白的混合物,两者均可与PoIFN-β阳性抗血清发生特异反应。表达产物比PoIFN-β理论推导分子量(约20.8kDa)大,推测可能是表达产物发生了不同程度的糖基化。重组PoIFN-β对伪狂犬病毒在细胞中增殖可呈现抑制作用,并且rPoIFN-β对伪狂犬病毒在MDBK细胞上早期增殖的抑制效果最为明显。     

VR-1 receptor blockade attenuates the pressor response to capsaicin but has no effect on the pressor response to contraction in cats

Vanilloid type 1 (VR-1) receptors are stimulated by capsaicin and hydrogen ions, the latter being a by-product of muscular contraction. We tested the hypothesis that activation of VR-1 receptors during static contraction contributes to the exercise pressor reflex. We established a dose of iodoresinaferatoxin (IRTX), a VR-1 receptor antagonist, that blocked the pressor response to capsaicin injected into the arterial supply of muscle. Specifically, in eight decerebrated cats, we compared pressor responses to capsaicin (10 mug) injected into the right popliteal artery, which was subsequently injected with IRTX (100 mug), with those to capsaicin injected into the left popliteal artery, which was not injected with IRTX. The pressor response to capsaicin injected into the right popliteal artery averaged 49 +/- 9 mmHg before IRTX and 9 +/- 2 mmHg after IRTX (P < 0.05). In contrast, the pressor response to capsaicin injected into the left popliteal artery averaged 46 +/- 10 mmHg before and 43 +/- 6 mmHg after (P > 0.05). We next determined whether VR-1 receptors mediated the pressor response to contraction of the triceps surae. During contraction without circulatory occlusion, the pressor response before IRTX (100 mug) averaged 26 +/- 3 mmHg, whereas it averaged 22 +/- 3 mmHg (P > 0.05) after IRTX (n = 8). In addition, during contraction with occlusion, the pressor responses averaged 35 +/- 3 mmHg before IRTX injection and 49 +/- 7 mmHg after IRTX injection (n = 7). We conclude that VR-1 receptors play little role in evoking the exercise pressor reflex.     

Temperature acclimation has no effect on ryanodine receptor expression or subcellular localization in rainbow trout heart

In cardiomyocytes, ryanodine receptors (RYRs) mediate Ca²⁺-induced Ca²⁺-release (CICR) from the sarcoplasmic reticulum (SR) during excitation–contraction (e–c) coupling. In rainbow trout heart, the relative importance of CICR increases with cold-acclimation. Thus, the aim of this study was to investigate the effect of temperature acclimation (4, 11 and 18°C) on RYR intracellular localization and expression density. We used immunocytochemistry to assess intracellular localization in ventricular myocytes and Western blotting to assess RYR expression in both atrial and ventricular tissue. In ventricular myocytes, RYRs were localized peripherally in transverse bands aligning with sarcomeric m-lines and centrally around mitochondria and the nucleus. Localization did not change with temperature acclimation. RYR expression was also unaffected by temperature acclimation. The localization of RYRs at the m-line is similar to neonatal mammalian cardiomyocytes. We suggest this positioning is indicative of myocytes which rely predominantly on transsarcolemmal Ca²⁺-influx, rather than CICR, during e–c coupling.     

PDGF-AA effectively stimulates early events but has no mitogenic activity in AKR-2B mouse fibroblasts.

The response of AKR-2B mouse fibroblasts, which express approximately equal numbers of platelet-derived growth factor (PDGF)-alpha and -beta receptors on their surface (V. Hoppe et al. Eur. J. Biochem. 187, 207-214, 1990) to all three isoforms of PDGF, was studied. All isoforms stimulated early events, i.e., receptor autophosphorylation on tyrosine, total cellular phosphorylation, increase in 32P-labeled phospholipid content, but there was no correlation between the extents measured for the different effects. Although rPDGF-AA effectively stimulated these early events, it was unable to induce [3H]thymidine incorporation and cell growth whereas rPDGF-BB and -AB stimulated the division of more than 90% of the cells. This activity was restored by addition of insulin-like growth factor I (IGF-I), which itself exhibited only a low mitogenic activity. rPDGF-AB or -BB did not require the presence of IGF-I to fully stimulate cells for [3H]thymidine incorporation and cell division. Apparently, rPDGF-AA induced only a "competence" state of the cells whereas rPDGF-AB or -BB was also able to initiate "progression". It is speculated that some early events occurring during the competence phase might be part of a "maintenance" program elicited by growth factors.     

Perinatal BPA exposure demasculinizes males in measures of affect but has no effect on water maze learning in adulthood

Bisphenol A (BPA) is an endocrine disrupting agent that can alter the normal gonadal steroid-sensitive sexual differentiation of the brain and behavior. While reproductive behavior and physiology are known to be altered by perinatal exposure to this compound, less is known about BPA's effects on sex differences in learning and measures of affect. In order to evaluate the effects of perinatal BPA treatment on learning and affect in adulthood, we exposed rats to one of five doses of BPA through gestation and lactation then examined adult behavior in the Morris Water Maze (MWM), the Elevated Plus Maze (EPM) and the Forced Swim Test (FST). No effect of BPA was observed in the MWM, but on both the EPM and FST, low doses (5 μg/kg) of BPA eliminated sex differences found between controls; furthermore, a non-monotonic dose-response observed in previous studies was confirmed for these tasks. Overall, our study adds to the growing data suggesting that BPA interferes with the normal development of affective behaviors in a non-linear, dose-dependent manner.