Binding potency of peptide fragments of type 1 collagen cross-linked N-telopeptide measured by an enzyme-linked immunosorbant assay

Osteoporosis represents a major healthcare problem affecting elderly person. Urinary level of the crosslinked N-telopeptide of type I collagen is a sensitive marker of bone resorption. Ten overlapping peptides covering the N-telopeptide of alpha-2 type I collagen were synthesized, purified, and assayed for their relative binding response to anti-type I collagen cross-linked N-telopeptide (NTX) antibody by using a competitive-inhibition enzyme-linked immunosorbent assay (ELISA). Peptides 1, 2, and 3, containing the N-terminal sequence of N-telopeptide, showed higher binding potency than peptides 4-10, suggesting that these peptides may contain binding sites for anti-NTX antibodies, and can serve as the lead for further preparation of their antibodies in order to develop novel bioassays for monitoring the bone loss in humans.     

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.     

An approach for cloning biosynthetic genes of 2-deoxystreptamine-containing aminocyclitol antibiotics: isolation of a biosynthetic gene cluster of tobramycin from Streptomyces tenebrarius

Genes homologous to 2-deoxystreptamine (DOS) biosynthetic genes were isolated from aminoglycoside producers, Micromonospora and Streptomyces spp., using PCR primers based on the core sequences of 2-deoxy-scyllo-inosose (DOI) synthase and L-glutamine: scyllo-inosose aminotransferase (GIA). Identities of 40-45% were observed for DOI synthases, and 65-75% were observed for GIAs. The gene cluster of tobramycin biosynthesis was isolated from the genomic library of Streptomyces tenebrarius using DOI synthase as a probe. Sequencing of 33.9 kb revealed 24 putative open reading frames including the tobramycin biosynthetic gene cluster (13.8 kb) and a transport protein. This cluster encodes proteins homologous to 2-deoxystreptamine biosynthetic enzymes, glycosyltransferase and other aminocyclitols biosynthetic enzymes. Sequence analysis revealed the evolution of DOI synthases from 3-dehydroquinate (DHQ) synthases in actinomycetes. DOI synthases and GIA are therefore useful for cloning biosynthetic genes of DOS-containing aminocyclitol antibiotics or for screening such metabolites producers.     

Phosphatidylinositol 4,5-bisphosphate and intracellular pH regulate the ROMK1 potassium channel via separate but interrelated mechanisms

ROMK channels are responsible for K(+) secretion in kidney. The activity of ROMK is regulated by intracellular pH (pH(i)) with acidification causing channel closure (effective pK(a) approximately 6.9). Recently, we and others reported that a direct interaction of the channels with phosphatidyl-4,5-bisphosphate (PIP(2)) is critical for opening of the inwardly rectifying K(+) channels. Here, we investigate the relationship between the mechanisms for regulation of ROMK by PIP(2) and by pH(i). We find that disruption of PIP(2)-ROMK1 interaction not only decreases single-channel open probability (P(o)) but gives rise to a ROMK1 subconductance state. This state has an increased sensitivity to intracellular protons (effective pK(a) shifted to pH approximately 7.8), such that the subconductance channels are relatively quiescent at physiological pH(i). Open probability for the subconductance channels can then be increased by intracellular alkalinization to supra-physiological pH. This increase in P(o) for the subconductance channels by alkalinization is not associated with an increase in PIP(2)-channel interaction. Thus, direct interaction with PIP(2) is critical for ROMK1 to open at full conductance. Disruption of this interaction increases pH(i) sensitivity for the channels via emergence of the subconductance state. The control of open probability of ROMK1 by pH(i) occurs via a mechanism distinct from the regulation by PIP(2).     

Structural and functional properties of a Bacillus subtilis temperature-sensitive sigma(A) factor

Bacillus subtilis DB1005 is a temperature-sensitive (Ts) sigA mutant containing double-amino-acid substitutions (I198A and I202A) on the hydrophobic face of the promoter -10 binding helix of sigma(A) factor. We have analyzed the structural and functional properties of this mutant sigma(A) factor both in vivo and in vitro. Our data revealed that the Ts sigma(A) factor possessed predominantly a multimeric structure which was prone to aggregation at restrictive temperature. The extensive aggregation of the Ts sigma(A) resulted in a very low core-binding activity of the Ts sigma(A) factor and a markedly reduced sigma(A)-RNA polymerase activity in B. subtilis DB1005, suggesting that extensive aggregation of the Ts sigma(A) is the main trigger for the temperature sensitivity of B. subtilis DB1005. Partial proteolysis, tryptophan fluorescence and 1-anilinonaphthalene-8-sulfonate-binding analyses revealed that the hydrophobic face of the promoter -10 binding helix and also the hydrophobic core region of the Ts sigma(A) factor were readily exposed on the protein surface. This hydrophobic exposure provides an important cue for mutual interaction between molecules of the Ts sigma(A) and allows the formation of multimeric Ts sigma(A). Our results also indicate that Ile-198 and Ile-202 on the hydrophobic face of the promoter -10 binding helix are essential to ensure the correct folding and stabilization of the functional structure of sigma(A) factor.     

The Root Extract of Gentiana macrophylla Pall. Alleviates Cardiac Apoptosis in Lupus Prone Mice

The roots of the perennial herb Gentiana macrophylla Pall. (GM) are known as Qinjiao, which has been used for centuries to treat systemic lupus erythematosus (SLE). However, little is known about the effects of GM on cholesterol-aggravated cardiac abnormalities in SLE, and the mechanisms thereof. This study investigates whether GM exhibits anti-apoptotic effects, focusing on the left ventricle (LV) of NZB/W F1 mice fed with high-cholesterol diet. The morphology and apoptotic status of ventricular tissues were determined by microscopy and Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Levels of apoptotic biomarkers were determined by immunoblotting. The results thus obtained revealed that GM significantly reduced the cholesterol-aggravated apoptosis of LV in NZB/W F1 mice by suppressing both intrinsic and extrinsic apoptotic pathways. Additionally, GM significantly increased the cardiac insulin-like growth factors (IGF)-1 survival signaling and anti-apoptotic proteins in LV tissues. Accordingly, GM is considered to be beneficial in alleviating cholesterol-aggravated cardiac damage in SLE, and therefore constitute an alternative treatment for SLE patients with cardiac abnormalities.     

MT-4 Suppresses Resistant Ovarian Cancer Growth through Targeting Tubulin and HSP27

Objective

In this study, the anticancer mechanisms of MT-4 were examined in A2780 and multidrug-resistant NCI-ADR/res human ovarian cancer cell lines.

Methods

To evaluate the activity of MT-4, we performed in vitro cell viability and cell cycle assays and in vivo xenograft assays. Immunoblotting analysis was carried out to evaluate the effect of MT-4 on ovarian cancer. Tubulin polymerization was determined using a tubulin binding assay.

Results

MT-4 (2-Methoxy-5-[2-(3,4,5-trimethoxy-phenyl)-ethyl]-phenol), a derivative of moscatilin, can inhibit both sensitive A2780 and multidrug-resistant NCI-ADR/res cell growth and viability. MT-4 inhibited tubulin polymerization to induce G2/M arrest followed by caspase-mediated apoptosis. Further studies indicated that MT-4 is not a substrate of P-glycoprotein (p-gp). MT-4 also caused G2/M cell cycle arrest, accompanied by the upregulation of cyclin B, p-Thr161 Cdc2/p34, polo-like kinase 1 (PLK1), Aurora kinase B, and phospho-Ser10-histone H3 protein levels. In addition, we found that p38 MAPK pathway activation was involved in MT-4-induced apoptosis. Most importantly, MT-4 also decreased heat shock protein 27 expression and reduced its interaction with caspase-3, which inured cancer cells to chemotherapy resistance. Treatment of cells with SB203580 or overexpression of dominant negative (DN)-p38 or wild-type HSP27 reduced PARP cleavage caused by MT-4. MT-4 induced apoptosis through regulation of p38 and HSP27. Our xenograft models also show the in vivo efficacy of MT-4. MT-4 inhibited both A2780 and NCI-ADR/res cell growth in vitro and in vivo.

Conclusion

These findings indicate that MT-4 could be a potential lead compound for the treatment of multidrug-resistant ovarian cancer.     

Major Adverse Cardiovascular Events in Treated Periodontitis: A Population-Based Follow-Up Study from Taiwan

Background

The aim of the present study was to identify the long-term major adverse cardiovascular events (MACE) in treated periodontitis patients in Taiwan.

Methods

From the National Health Insurance Research Database (2001-2010), adult patients (≥ 18 years) with treated periodontitis were identified. Comparison was made between patients with mild form and severe form of treated periodontitis after propensity score matching. The primary end point was the incidence of MACE.

Results

A total of 32,504 adult patients with treated periodontitis were identified between 2001 and 2010. After propensity score matching, 27,146 patients were preserved for comparison, including 13,573 patients with mild form and 13,573 patients with severe form of treated periodontitis. During follow-up, 728 individuals in mild treated periodontitis group and 1,206 individuals in severe treated periodontitis group had at least 1 MACE event. After adjustment for gender, hyperlipidemia, hypertension and diabetes mellitus, severe treated periodontitis was associated with a mildly but significantly increased risk of MACE among older patients > 60 years of age (incidence rate ratio, 1.26; 95% confidence interval, 1.08–1.46). No association was found among younger patients ≤ 60 years of age.

Conclusions

Severe form of treated periodontitis was associated with an increased risk of MACE among older Taiwanese patients, but not among younger Taiwanese patients. We should put more efforts on the improvement of periodontal health to prevent further MACE.