Molecular docking analysis of flavonoids with aldose reductase

Diabetes mellitus is a group of metabolic disorders that has risen to become the third most common cause in humans in recent years. The development of new bioactive substances from natural sources is a relatively new area. Flavonoids are believed to have a variety of beneficial properties in nature, including anti-inflammatory, antimicrobial, anticancer, antioxidant, neuroprotective, and anti-HIV properties. 15 naturally occurring flavonoids docked with the selected target aldose reductase. We report the optimal binding of Acumitin, Agathisflavone, Agehoustin B, and alpha-Toxicarol with aldose reductase for further consideration in drug discovery for T2DM.     

c-IAP1 is cleaved by caspases to produce a proapoptotic C-terminal fragment

Although human c-IAP1 and c-IAP2 have been reported to possess antiapoptotic activity against a variety of stimuli in several mammalian cell types, we observed that full-length c-IAP1 and c-IAP2 failed to protect cells from apoptosis induced by Bax overexpression, tumor necrosis factor alpha treatment or Sindbis virus infection. However, deletion of the C-terminal RING domains of c-IAP1 and c-IAP2 restored antiapoptotic activity, indicating that this region negatively regulates the antiapoptotic function of the N-terminal BIR domain. This finding is consistent with the observation by others that the spacer region and RING domain of c-IAP1 functions as an E3 ligase, promoting autoubiquitination and degradation of c-IAP1. In addition, we found that c-IAP1 is cleaved during apoptosis to 52- and 35-kDa fragments. Both fragments contain the C-terminal end of c-IAP1 including the RING finger. In vitro cleavage of c-IAP1 with apoptotic cell extracts or with purified recombinant caspase-3 produced similar fragments. Furthermore, transfection of cells with the spacer-RING domain alone suppressed the antiapoptotic function of the N-terminal BIR domain of c-IAP1 and induced apoptosis. Optimal death-inducing activity of the spacer-RING required both the spacer region and the zinc-binding RING domain of c-IAP1 but did not require the caspase recruitment domain located within the spacer region. To the contrary, deletion of the caspase recruitment domain increased proapoptotic activity, apparently by stabilizing the C-terminal fragment.     

The permeability transition pore controls cardiac mitochondrial maturation and myocyte differentiation

Although mature myocytes rely on mitochondria as the primary source of energy, the role of mitochondria in the developing heart is not well known. Here, we find that closure of the mitochondrial permeability transition pore (mPTP) drives maturation of mitochondrial structure and function and myocyte differentiation. Cardiomyocytes at embryonic day (E) 9.5, when compared to E13.5, displayed fragmented mitochondria with few cristae, a less-polarized mitochondrial membrane potential, higher reactive oxygen species (ROS) levels, and an open mPTP. Pharmacologic and genetic closing of the mPTP yielded maturation of mitochondrial structure and function, lowered ROS, and increased myocyte differentiation (measured by counting Z bands). Furthermore, myocyte differentiation was inhibited and enhanced with oxidant and antioxidant treatment, respectively, suggesting that redox-signaling pathways lie downstream of mitochondria to regulate cardiac myocyte differentiation.     

Oxidative stress induces vascular heme oxygenase-1 expression in ovariectomized rats

Heme oxygenase-1 (HO-1), an inducible stress protein, has been implicated in cytoprotection against oxidative stress in vitro and in vivo. Estrogens also have antioxidant effects. This study investigated the time course of HO-1 and inducible nitric oxide synthase (iNOS) expression in the aortas of ovariectomized rats, and the regulatory relationship between the NO/NOS and the carbon monoxide/HO systems. HO-1 and iNOS protein expression was induced by ovariectomy (Ovx) and was extremely high 2-6 weeks after Ovx compared with the sham-operated group. Expression of the constitutive enzymes HO-2 and endothelial NOS did not differ significantly between sham-operated and Ovx rats. 17beta-Estradiol (E(2)) replacement reversed these changes in rats after Ovx. Long-term treatment with the antioxidant tempol significantly inhibited HO-1 and iNOS expression. The iNOS inhibitor aminoguanidine significantly suppressed the induction of HO-1. Oxidized glutathione in the hearts of Ovx rats increased gradually, with significant elevation at 3-6 weeks after Ovx compared with the sham-operated group, whereas plasma levels of NO metabolites were significantly reduced 4-6 weeks after Ovx. Treatment with the HO inhibitor zinc protoporphyrin IX blocked HO-1 induction, but significantly increased the plasma levels of NO metabolites. In conclusion, HO-1 is induced by oxidative stress resulting from E(2) depletion. The NO/iNOS system contributes to the induction of HO-1, which may subsequently suppress iNOS activity to modulate vasculoprotective effects after menopause.     

Molecular genetic manipulation of Pichia pastoris SEC4 governs cell growth and glucoamylase secretion

We have previously engineered a recombinant Pichia pastoris GS115 transformant, MSPGA-7, harboring seven copies of glucoamylase (GA) fused with modified signal peptide. High yield secretion of GA was achieved as an extra copy of SEC4 was integrated to the transformant. To elucidate the physiological role of SEC4, a dominant-negative mutant of SEC4, SEC4(S28N), was overexpressed under the control of alchohol oxidase 1 (AOX1) promoter in P. pastoris strain MSPGA-7 as well as a set of host cells harboring multi-copy of wild type SEC4. We found that SEC4(S28N) mutation in the key guanine nucleotide binding domain reduced guanine nucleotide binding affinity, hence it blocked the transport of vesicles required for targeting and fusion to the plasma membrane. The inhibitory levels of cell growth and GA secretion were correlated with the dosage of SEC4(S28N) gene. In addition, overexpression of SEC4 driven by AOX1 promoter in MSPGA-7 improved the secretory production of GA, but demonstrated the delay of cell growth by increased gene dosage of SEC4. Interestingly, a limited level of Sec4p did not disturb the cell growth. It was because expression of only one copy of SEC4 resulted in delay of cell growth at an early stage while still maintaining high level Sec4p at long-term incubation. Accordingly, as glyceraldehyde-3-phosphate dehydrogenase promoter was used to substitute AOX1 promoter to drive the SEC4 expression, enhanced GA secretion but not inhibition of cell growth was achieved. Taken together, our results demonstrate that SEC4 is essential for P. pastoris in regulating cell growth and heterologous protein secretion in a dosage-dependent manner.     

Enhanced nuclear factor-kappa B-associated Wnt-1 expression in hepatitis B- and C-related hepatocarcinogenesis: identification by functional proteomics

Summary Chronic infections with hepatitis B and C viruses (HBV and HCV) are etiologically linked to hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC). Both viruses may induce activation of nuclear factor-kappa B (NF-κB) in hepatocytes that plays a crucial role in the regulation of cell growth and apoptosis. Functional proteomics analysis of proteins associated with NF-κB signaling complexes in both viruses-related HCC tumor and non-tumor tissues may disclose possible common mechanisms in hepatocarcinogenesis. By functional proteomics, we analyzed proteins associated with NF-κB-signaling complexes in four-paired human HCC tumor and non-tumor tissues from HBV- and HCV-infected patients, respectively, and in one-paired tissue with dual viral infection. The quantity of NF-κB-associated proteins was semi-quantitatively measured by protein spot intensity on the gels of two-dimensional polyacrylamide gel electrophoresis. The results showed that overexpression of NF-κB-associated Wnt-1 protein in tumor part was detected in the␣majority of HBV- and HCV-infected HCC samples. These data suggest that enhanced expression of NF-κB-associated Wnt-1 protein might be a mechanism of hepatocarcinogenesis common to HBV- and HCV-infected patients. NF-κB signaling pathway and Wnt-1 protein could be potential targets for designing highly effective therapeutic agents in treating HCC and for chemoprevention of hepatocarcinogenesis.     

TIMP2 deficient mice develop accelerated osteoarthritis via promotion of angiogenesis upon destabilization of the medial meniscus

Vascular invasion into the normally avascular articular surface is a hallmark of advanced osteoarthritis (OA). In this study, we demonstrated that the expression of tissue inhibitor of metalloproteinases-2 (TIMP2), an anti-angiogenic factor, was present at high levels in normal articular chondrocytes, and was drastically decreased shortly after destabilization of the medial meniscus (DMM). We also investigated the anti-angiogenic properties of TIMP2 via knockout. We hypothesized that the loss of TIMP2 could accelerate osteoarthritis development via promotion of angiogenesis. Loss of TIMP2 led to increased periarticular vascular formation 1 month post DMM, compared to wild-type mice, and did so without altering the expression pattern of matrix metalloproteinases and vascular endothelial growth factors. The increased vascularization eventually resulted in a severe degeneration of the articular surface by 4 months post DMM. Our findings suggest that reduction of TIMP2 levels and increased angiogenesis are possible primary events in OA progression. Inhibiting or delaying angiogenesis by TIMP2 expression or other anti-angiogenic therapies could improve OA prevention and treatment.