Therapeutic effect of risedronate on cancellous and cortical bone in ovariectomized osteopenic rats: a comparison with the effects of alfacalcidol.

The purpose of the present study was to compare the therapeutic effects of risedronate (RIS) and alfacalcidol (ALF) on cancellous and cortical bone in ovariectomized osteopenic rats. Forty-two female Sprague-Dawley rats, 7 months of age, were randomized by the stratified weight method into six groups: the sham-operated control (Sham) group, and five ovariectomized groups: treated with vehicle, RIS (0.1, 1.0, or 2.5 mg/kg, p.o., daily), and ALF (0.5 microg/kg, p.o., daily). Treatment was started 6 weeks after surgery and continued for 6 weeks. Evaluation at 12 weeks after surgery revealed that ovariectomy (OVX) decreased the cancellous bone volume/total tissue volume (BV/TV) of the proximal tibial metaphysis as a result of an increase of the bone formation rate/bone surface (BFR/BS), BFR/BV, and eroded surface (ES/BS), while having no effect on the cortical area (Ct Ar) of the tibial diaphysis. OVX also decreased the maximum load of the femoral distal metaphysis, while having no effect on any mechanical property parameters of the femoral diaphysis. RIS (at all the doses) increased the BV/TV relative to the value in the OVX-Vehicle group, but the value was not restored to that observed in the Sham group. The effects of RIS (1.0 mg/kg and 2.5 mg/kg) were similar, and greater than those of RIS (0.1 mg/kg). ALF also increased the BV/TV relative to the OVX-Vehicle group, but the value was not restored to that observed in the Sham group, similar to the results of RIS (1.0 mg/kg and 2.5 mg/kg) treatment. The alterations of the structural parameters induced by RIS (at the doses) were attributable to suppression of the increase of ES/BS, BFR/BS, and BFR/BV. The alterations of the structural parameters induced by ALF were attributable to suppression of the increase of ES/BS and attenuation of the increase of BFR/BV, while the BFR/BS was maintained. ALF also increased the Ct Ar to beyond the value observed in the Sham group. RIS (at all the doses) had no effect on the mechanical properties of the femoral distal metaphysis, whereas ALF prevented the loss of the maximum load of the femoral distal metaphysis. Thus, the results of the present study show differential effects of RIS and ALF on cancellous and cortical bone in ovariectomized osteopenic rats.     

The new generation dihydropyridine type calcium blockers, bearing 4-phenyl oxypropanolamine, display alpha-/beta-adrenoceptor antagonist and long-acting antihypertensive activities

A new series of dihydropyridine derivatives, bearing oxypropanolamine moiety on phenyl ring at the 4-position of the dihydropyridine base, were prepared. Oxypropanolamine was synthesized by replacing the phenolic OH of vanillin or other compounds, having a phenyl aldehyde group, with epichlorohydrin, followed by cleavaging the obtained epoxide compounds with tert-butylamine, n-butylamine or 2-methoxy-1-oxyethylamino benzene (guaiacoxyethylamine), respectively. Obtained various oxypropanolamine compounds, still remaining a phenyl aldehyde moiety, were then performed by Hantzsch condensation reaction with methylacetoacetate or ethylacetoacetate, respectively, to give our new series of dihydropyridine linked with the 4-phenyl ring. These compounds were evaluated for inotropic, chronotropic, and aorta contractility that associated with calcium channel and adrenoceptor antagonist activities. Dihydropyridine derivatives that with oxypropanolamine side chain on their 4-phenyl ring associated alpha-/beta-adrenoceptor blocking activities created a new family of calcium entry and the third generation beta-adrenoceptor blockers. Optimizing this research to obtain more potent alpha-/beta-adrenoceptor blocking and long-acting antihypertensive oxypropanolamine on the 4-phenyl ring of dihydropyridine series compounds was thus accomplished and classified as third generation dihydropyridine type calcium channel blockers, in comparison with previous short-acting type nifedipine and long-acting type amlodipine. We concluded that compounds 1a, 1b and 1g showed not only markedly high calcium-antagonistic activity but also the highest antihypertensive effect; compounds 1b, 1c, 1f, 1g, 1i and 1j induced sustained antihypertensive effects are major and attributed to their calcium entry and alpha-adrenoceptor blocking activities in the blood vessel due to their introduction of 2-methoxy, 1-oxyethylamino benzene moiety in the side chain on the 4-phenyl ring of dihydropyridine. Bradycardiac effects of all the compounds 1a-1j resulted from calcium entry and beta-adrenoceptor blocking, which attenuate the sympathetic activation-associated reflex tachycardia in the heart. We selected compound 1b as candidate compound for further pharmacological and pre-clinical evaluation studies.     

Knockdown of the aryl hydrocarbon receptor attenuates excitotoxicity and enhances NMDA-induced BDNF expression in cortical neurons

NMDA receptors play dual and opposing roles in neuronal survival by mediating the activity-dependent neurotrophic signaling and excitotoxic cell death via synaptic and extrasynaptic receptors, respectively. In this study, we demonstrate that the aryl hydrocarbon receptor (AhR), also known as the dioxin receptor, is involved in the expression and the opposing activities of NMDA receptors. In primary cultured cortical neurons, we found that NMDA excitotoxicity is significantly enhanced by an AhR agonist 2,3,7,8-tetrachlorodibenzo- p -dioxin, and AhR knockdown with small interfering RNA significantly reduces NMDA excitotoxicity. AhR knockdown also significantly reduces NMDA-increases intracellular calcium concentration, NMDA receptor expression and surface presentation, and moderately decreases the NMDA receptor-mediated spontaneous as well as miniature excitatory post-synaptic currents. However, AhR knockdown significantly enhances the bath NMDA application– but not synaptic NMDA receptor-induced brain-derived neurotrophic factor (BDNF) gene expression, and activating AhR reduces the bath NMDA-induced BDNF expression. Furthermore, AhR knockdown reveals the calcium dependency of NMDA-induced BDNF expression and the binding activity of cAMP-responsive element binding protein (CREB) and its calcium-dependent coactivator CREB binding protein (CBP) to the BDNF promoter upon NMDA treatment. Together, our results suggest that AhR opposingly regulates NMDA receptor-mediated excitotoxicity and neurotrophism possibly by differentially regulating the expression of synaptic and extrasynaptic NMDA receptors.     

Parasympathetic nervous activity mirrors recovery status in weightlifting performance after training

Heart rate variability (HRV) and parasympathetic power are closely related to the well-being and health status in humans. The main goal of the study was to determine whether these measures can reflect recovery status after weight training. After a 10-day detraining period, 7 weightlifters were challenged with a 2-hour weight training which elicited approximately fourfold increases in circulating muscle creatine kinase level and protracted pain feeling (p < 0.05). Weightlifting performance was then evaluated 3, 24, 48, and 72 hours after training to determine the degree of recovery from fatigue. Heart rate variability, circulating dehydroepiandrostendione sulfate (DHEA-S), and muscle damage markers were measured before each performance test. An electrocardiogram was recorded for 5 minutes continuously at rest in seated positions. After training, weightlifting performance of the subjects decreased below baseline in paralleled with suppressed parasympathetic power (high-frequency [HF] HRV), whereas sympathetic power (normalized low-frequency HRV) was slightly elevated at 3 hours of recovery (p < 0.05). Both weightlifting performances and parasympathetic power returned to baseline values in 24 hours and further increased above baseline during 48-72 hours of recovery in a similar fashion (p < 0.05). Circulating DHEA-S level dropped at 24 hours (p < 0.05) and returned to normal values by 48 hours. Muscle pain increased at 3 hours after training and remained higher than baseline values for the 72-hour recovery period (p < 0.05). Our data suggest that parasympathetic power, indicated by HF HRV, is able to reflect the recovery status of weightlifters after training.     

Optimizing Human Synovial Fluid Preparation for Two-Dimensional Gel Electrophoresis

Background

Prenatal screening for Down Syndrome (DS) would benefit from an increased number of biomarkers to improve sensitivity and specificity. Improving sensitivity and specificity would decrease the need for potentially risky invasive diagnostic procedures.

Results

We have performed an in depth two-dimensional difference gel electrophoresis (2D DIGE) study to identify potential biomarkers. We have used maternal plasma samples obtained from first and second trimesters from mothers carrying DS affected fetuses compared with mothers carrying normal fetuses. Plasma samples were albumin/IgG depleted and expanded pH ranges of pH 4.5 - 5.5, pH 5.3 - 6.5 and pH 6 - 9 were used for two-dimensional gel electrophoresis (2DE). We found no differentially expressed proteins in the first trimester between the two groups. Significant up-regulation of ceruloplasmin, inter-alpha-trypsin inhibitor heavy chain H4, complement proteins C1s subcomponent, C4-A, C5, and C9 and kininogen 1 were detected in the second trimester in maternal plasma samples where a DS affected fetus was being carried. However, ceruloplasmin could not be confirmed as being consistently up-regulated in DS affected pregnancies by Western blotting.

Conclusions

Despite the in depth 2DE approach used in this study the results underline the deficiencies of gel-based proteomics for detection of plasma biomarkers. Gel-free approaches may be more productive to increase the number of plasma biomarkers for DS for non-invasive prenatal screening and diagnosis.     

The F-box protein SKP2 binds to the phosphorylated threonine 380 in cyclin E and regulates ubiquitin-dependent degradation of cyclin E

Cyclin E is required for S phase entry. The subsequent ubiquitin-dependent degradation of cyclin E contributes to an orderly progression of the S phase. It has been shown that phosphorylation of threonine 380 (Thr380) in cyclin E provides a signal for its ubiquitin-dependent proteolysis. We report that SKP2, an F-box protein and a substrate-targeting component of the SCF(SKP2) ubiquitin E3 ligase complex, mediates cyclin E degradation. In vitro, SKP2 specifically interacted with the cyclin E peptide containing the phosphorylated-Thr380 but not with a cognate nonphosphorylated peptide. In vivo, expression of SKP2 induced cyclin E polyubiquitination and degradation. Conversion of Thr380 into nonphosphorylatable amino acids caused significant resistance of cyclin E to SKP2. The presence of the CDK inhibitor p27(Kip1) also prevented the SKP2-dependent degradation of cyclin E. Our findings suggest that SKP2 regulates cyclin E stability, thus contributing to the control of S phase progression.     

Biochemical properties and cDNa cloning of two new lectins from the plasma of Tachypleus tridentatus: Tachypleus plasma lectin 1 and 2+

A Sepharose CL-4B-binding protein, Tachypleus plasma lectin 1 (TPL-1), and a lipopolysaccharide (LPS)-binding protein, Tachypleus plasma lectin-2 (TPL-2), have been isolated from the plasma of Tachypleus tridentatus and biochemically characterized. Each protein is coded by a homologous family of multigenes. TPL-1 binds to Sepharose CL-4B and was eluted with buffer containing 0.4 m GlcNAc. The deduced amino acid sequence of TPL-1 consisted of 232 amino acids with an N-glycosylation site, Asn-Gly-Ser at residues 74-76. It shares a 65% sequence identity and similar internal repeats of about 20 amino acid motifs with tachylectin-1. Tachylectin-1 was identified as a lipopolysaccharide-agarose binding nonglycosylated protein from the amebocytes of T. tridentatus. TPL-2 was eluted from the LPS-Sepharose CL-4B affinity column in buffer containing 0.4 m GlcNAc and 2 m KCl. The deduced amino acid sequence of TPL-2 consisted of 128 amino acids with an N-glycosylation site, Asn-Cys-Thr, at positions 3-5. It shares an 80% sequence identity with tachylectin-3, isolated from the amebocytes of T. tridentatus. TPL-2 purified by LPS-affinity column from the plasma predominantly exists as a dimer of a glycoprotein with an apparent molecular mass of 36 kDa. Tachylectin-3 is an intracellular nonglycosylated protein that also exists as a dimer in solution with an apparent molecular mass of 29 kDa. It recognizes Gram-negative bacteria through the 0-antigen of LPS. Western blot analyses showed that, in the plasma, TPL-1 and TPL-2 exist predominantly as oligomers with molecular masses above 60 kDa. They both bind to Gram-positive and Gram-negative bacteria, and this binding is inhibited by GlcNAc. Possible binding site of TPL-1 and TPL-2 to the bacteria could be at the NAc moiety of GlcNAc-MurNAc of the peptidoglycan. The physiological function of TPL-1 and TPL-2 is most likely related to their ability to form a cluster of interlocking molecules to immobilize and entrap invading organisms.     

FADD null mouse embryonic fibroblasts undergo apoptosis after photosensitization with the silicon phthalocyanine Pc 4

Oxidative stress, such as photodynamic therapy with the silicon phthalocyanine Pc 4 (Pc 4-PDT), can induce apoptosis and tumor necrosis factor alpha (TNF) production. TNF receptors, as well as other death receptors, have been implicated in stress-induced apoptosis. To assess directly the role of FADD, a death receptor-associated protein, in induction of apoptosis post-Pc 4-PDT, embryonic fibroblasts from FADD knock out (k/o) and wild-type (wt) mice were used. Pc 4-PDT induced casp-3 activation and apoptosis in both cell types. In the presence of zVAD, a pancaspase inhibitor, Pc 4-PDT-induced apoptosis was abrogated in both cell lines. Fumonisin B1 (FB), an inhibitor of ceramide synthase, had no effect on apoptosis after Pc 4-PDT in either cell line. Similar to Pc 4-PDT, exogenous C6-ceramide bypassed FADD deficiency and induced zVAD-sensitive apoptosis. In contrast to Pc 4 photosensitization, TNF did not induce either apoptosis or ceramide accumulation in FADD k/o cells. In the absence of FADD deficiency, TNF-induced apoptosis was zVAD-sensitive and FB-insensitive. Induced ceramide levels remained elevated after cotreatment with TNF and zVAD in FADD wt cells. Taken together, these data provide genetic evidence for a lack of FADD requirement in Pc 4-PDT- or C6-ceramide-induced apoptosis. FB-sensitive ceramide production accompanies, but does not suffice, for apoptosis after Pc 4 photosensitization or TNF.     

A second-site mutation at glutamate-257 that restores the function of the mutant yeast ribosomal protein L5 containing lysine-270,271-->arginine

A genetic approach was used to identify interacting regions of yeast ribosomal protein L5 (also known as L1, L1a, or YL3). Previous studies from our laboratory showed that residues K270 and K271 in protein L5 are essential for its function. The mutant L5 protein in which both residues were replaced by arginine residues (K270,271R) exhibited about 80% RNA binding capability compared to the wild-type and the mutant protein was assembled into the 60S ribosomal subunits in vivo. The yeast strain expressing this mutant protein in a homozygous form was lethal (Biochim. Biophys. Acta 1308 (1996) 133-141). In the present study, this non-functional mutant was used to select intragenic suppressors. A spontaneous, intragenic suppressor which contained an E257K substitution (in addition to the primary mutations) was identified. The suppressor protein bound about 60% of yeast 5S rRNA in vitro compared to the wild-type. To gain more insight into the nature of the intragenic suppressor, additional mutant proteins in which E257 was substituted by a variety of amino acids were produced by site-directed mutagenesis. The ability of each mutant protein to bind yeast 5S rRNA in vitro and to suppress the lethal effect of the double K270,271 mutation in vivo were examined. Results suggest communication between two non-contiguous domains on protein L5 and that several factors, such as electrostatic interaction and hydrogen bonding are likely to play a role in this global communication. Mutation studies on E257 alone also reveal that substitutions of this residue in L5 protein could affect cell growth under specified conditions, but a variety of changes could be tolerated without serious deleterious effects. We propose a working model in which E257 is located in a loop and the dynamic as well as the flexibility of this loop is important for L5 function.