Substrate specificity in HIV-1 protease by a biased sequence search method

Drug resistance in HIV-1 protease can also occasionally confer a change in the substrate specificity. Through the use of computational techniques, a relationship can be determined between the substrate sequence and three-dimensional structure of HIV-1 protease, and be utilized to predict substrate specificity. In this study, we introduce a biased sequence search threading (BSST) methodology to analyze the preferences of substrate positions and correlations between them that might also identify which positions within known substrates can likely tolerate sequence variability and which cannot. The potential sequence space was efficiently explored using a low-resolution knowledge-based scoring function. The low-energy substrate sequences generated by the biased search are correlated with the natural substrates. Octameric sequences were predicted using the probabilities of residue positions in the sequences generated by BSST in three ways: considering each position in the substrate independently, considering pairwise interdependency, and considering triple-wise interdependency. The prediction of octameric sequences using the triple-wise conditional probabilities produces the most accurate results, reproducing most of the sequences for five of the nine natural substrates and implying that there is a complex interdependence between the different substrate residue positions. This likely reflects that HIV-1 protease recognizes the overall shape of the substrate more than its specific sequence.     

The relationship between serum levels of total IgE, IL-18, IL-12, IFN-gamma and disease severity in children with atopic dermatitis

Studies about the role of cytokines on the immunopathogenesis of atopic dermatitis (AD) are generally based on in vitro observations and this role has not been completely clarified yet. Serum levels of total IgE, IL-18, IL-12, IFN-gamma and the relationship between these parameters and disease severity, determined using the SCORAD index, in a group of atopic patients were investigated in this study. Serum levels of total IgE were measured by the nephelometric method and serum levels of IL-18, IL-12/p40 and IFN-gamma were measured by ELISA method. Serum levels of total IgE and IL-18 were found significantly higher in study group than in controls (P<.001). There was no statistically significant difference between patients and controls in respect of serum levels of IL-12/p40 (P = .227). A statistically significant relationship between SCORAD values and serum levels of total IgE (P < .001), IL-18 (P < .001), and IL-12/p40 (P < .001) was determined. These results show that serum levels of IL-18 can be a sensitive parameter that importantly correlates with clinical severity of AD, can play a role in the immunopathogenesis of AD, and furthermore may be used in the diagnosis and follow-up of the disease in addition to other parameters.     

Analysis of the Wnt/B-catenin/TCF4 pathway using SAGE,genome-wide microarray and promoter analysis: Identification of BRI3 and HSF2 as novel targets

The Wnt signaling pathway is involved in many differentiation events during embryonic development and can lead to tumor formation after aberrant activation of its components. β-catenin, a cytoplasmic component, plays a major role in the transduction of canonical Wnt signaling. The aim of this study was to identify novel genes that are regulated by active β-catenin/TCF signaling in hepatocellular carcinoma-derived Huh7 cells with high (transfected) and low β-catenin/TCF activities. High TCF activity Huh7 cells led to earlier and larger tumor formation when xenografted into nude mice. SAGE (Serial Analysis of Gene Expression), genome-wide microarray and in silico promoter analysis were performed in parallel, to compare gene expression between low and high β-catenin/TCF activity clones, and also those that had been rescued from the xenograft tumors. SAGE and genome-wide microarray data were compared and contrasted. BRI3 and HSF2 were identified as novel targets of Wnt/β-catenin signaling after combined analysis and confirming experiments including qRT-PCR, ChIP, luciferase assay and lithium treatment.     

Evaluation of the Potential Therapeutic Properties of Liquidambar orientalis Oil

Liquidambar orientalis Mill., commonly called the Anatolian sweetgum or Sigla tree, is endemic to southwestern Turkey. It has been historically significant in traditional medicine. In our research, we delved into the therapeutic attributes of its oil, emphasizing its antioxidant, antimicrobial, and antitumor properties. The primary chemical constituent of the gum is styrene, accounting for 78.5 %. The gum demonstrated antioxidant capabilities in several assays, including in 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), cupric reducing antioxidant capacity (CUPRAC) and ferric reducing antioxidant power (FRAP). It displayed bactericidal actions against various gram-positive bacteria, such as Staphylococcus aureus, and gram-negative strains, including Escherichia coli. Additionally, the oil showcased potent antitumor effects against breast (MDA-MB-231), lung (A549), and prostate (PC3) cancer cell lines. These effects were found to be both time- and dose-dependent. L. orientalis Mill. oil showed the best antitumor activity against breast, lung, and prostate cancer cell lines after the 24 h and 48 h treatment. Its oil might induce autophagy in the PC3 prostate cancer cell line, whereas its cytotoxicity against MDA-MB-231 and A549 cancer cell lines might not be correlated with autophagy or apoptosis pathways. In conclusion, the oil from the Sigla tree offers promising therapeutic potential and warrants further exploration.     

Ischemia-reperfusion leads to depletion of glutathione content and augmentation of malondialdehyde production in the rat heart from overproduction of oxidants: Can caffeic acid phenethyl ester (CAPE) protect the heart?

During restoration of blood flow of the ischemic heart induced by coronary occlusion, free radicals cause lipid peroxidation with myocardial injury. Lipid peroxidation end-products, such as malondialdehyde (MDA), have been used to assess oxygen free radical-mediated injury of the ischemic-reperfused (I/R) myocardium in rats. This experimental study assessed the preventive effect of caffeic acid phenthyl ester (CAPE), antioxidant, on I/R-induced lipid peroxidation in the rat heart. We are also interested in the role of CAPE on glutathione (GSH) levels, an antioxidant whose levels are influenced by oxidative stress. I/R leads to the depletion of GSH which is the major intracellular nonprotein sulphydryl and plays an important role in the maintenance of cellular proteins and lipid in their functional state and acts primarily to protect these important structures against the threat of oxidation. In addition, we also examined morphologic changes in the heart by using light microscopy. The left coronary artery was occluded for 30 min and then reperfused for 120 min more before the experiment was terminated. CAPE (50 M kg^–1) was administered 10 min prior to ischemia and during occlusion by infusion. At the end of the reperfusion period, rats were sacrificed, and the heart was quickly removed for biochemical determination and histopathological analysis. I/R was accompanied by a significant increase in MDA production and decrease in GSH content in the rat heart. Administration of CAPE reduced MDA production and prevented depletion of GSH content. These beneficial changes in these biochemical parameters were also associated with parallel changes in histopathological appearance. These findings imply that I/R plays a causal role in heart injury due to overproduction of oxygen radicals or insufficient antioxidant and CAPE exert cardioprotective effects probably by the radical scavenging and antioxidant activities.     

Octreotide improves burn-induced intestinal injury in the rat

The local thermal trauma activates a number of systemic mediator cascades, e.g. a complement activation, cytokine production, resulting in a generalized sequestration and a priming of local and systemic neutrophils and macrophages. We aimed to determine the possible protective effect of octreotide (OCT), a synthetic somatostatin analogue, against burn-induced intestinal tissue damage possibly by inhibiting neutrophil infiltration. Under brief ether anaesthesia, shaved dorsum of the rats was exposed to 90 degrees C bath for 10s to induce burn injury. Rats were decapitated either 3, 24 or 72 h after burn injury. Octreotide (10 microg/kg) or saline was administered subcutaneously (s.c.) immediately after the burn injury. In the 24- and 72-h burn groups, OCT injections were repeated three times daily. In the sham group the same protocol was applied except that the dorsum was dipped in a 25 degrees C water bath for 10 s Malondialdehyde (MDA) and glutathione (GSH) levels and myeloperoxidase (MPO) activity were determined in the intestinal tissue. The results demonstrate that burn injury results in significant neutrophil accumulation, as evidenced by increases in MPO activity. The increase in MDA and the concomitant decrease in GSH levels demonstrate the role of oxidative mechanisms in burn injury. OCT may have some beneficial therapeutic effects by reducing neutrophil-dependent injury and related lipid peroxidation following burn trauma.     

Temperature-sensitive mutants of Balb/3T3 cells: description of a mutant affected in cellular and polyoma virus DNA synthesis.

A temperature-sensitive Dna- mutant (ts-2) of the mouse cell Balb/3T3 is characterized. Studies with synchronized cells indicate that the defect is in DNA synthesis itself, rather than in progress toward its initiation. ts-2 supports polyoma DNA synthesis after infection at 33degreesC but not at 38degreesC. Viral DNA synthesis begun at 33degreesC is inhibited upon shift to 38degreesC. A procedure is proposed by which viral DNA synthesis can be used to distinguish different classes of cell Dna- mutants.     

Endonucleases and simian virus 40 virions: origin of a virion-associated endonuclease.

The origin and role of the endonuclease activity associated with purified virions of simian virus 40, previously described by this and other laboratories, have been further investigated. We found that the enzymatic activity from virions of temperature-sensitive (ts) mutants is not more heat labile than that from wild-type virions. This result was obtained for a variety of ts mutants, including three of the tsA class, and in experiments in which the enzyme was tested in both the presence and absence of viral particles. Comparison of the viron enzyme with endonucleases prepared from either serum or nuclei of uninfected cells reveals a similarity between the viron and serum enzymes based on chromatographic behavior and relative activity with different cations. Virus particles prepared free of this endonuclease were still infectious. We were unsuccessful in uncovering endonuclease in such preparations upon disruption. These data emphasize the necessity for caution in interpreting the role of particle-associated enzymes.     

Subunit level crosslinking of rabbit muscle pyruvate kinase by o-phthaldialdehyde

o-Phthaldialdehyde caused irreversible inhibition of rabbit muscle pyruvate kinase following preliminary formation of an enzyme-reagent complex. At pH 7.5, 35 degrees C, the dissociation constant for the complex and the maximal pseudo-first-order rate constant for covalent modification were 0.32 +/- 0.08 mM and 2.54 +/- 0.23 min-1, respectively. The inactivation was accompanied by uv-spectral changes pointing to isoindole formation, with a limiting stoichiometry of 1 isoindole linkage per enzyme subunit. Phosphoenolpyruvate, ADP, and ATP effectively protected the enzyme against inactivation, suggesting that the active site is the target of o-phthaldialdehyde action. As native and modified enzymes were indistinguishable with respect to mobility of the major band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it was concluded that the crosslinkage was intrasubunit in character, and that the amino acid residues involved must be closely positioned in the polypeptide backbone. Lysine 366, previously shown to be selectively reactive toward 2',3'-dialdehyde ADP (Bezares et al., 1987, Arch, Biochem. Biophys. 253, 133-137), and cysteine 325 or 357 are implicated.     

Differential gene expression in SV40-mediated immortalization of human fibroblasts

Normal human diploid fibroblasts (HF) have a limited life span, undergo senescence, and rarely, if ever, spontaneously immortalize in culture. Introduction of the gene for T antigen encoded by the DNA virus SV40 extends the life span of HF and increases the frequency of immortalization; however, immortalization requires both T-dependent and T-independent functions. We previously generated independent SV40-transformed non-immortal (pre-immortal) HF cell lines from which we then obtained immortal sublines as part of a multifaceted approach to identify functions responsible for immortalization. In this study we undertook a search for cellular mRNAs which are differentially expressed upon immortalization. A λcDNA library was prepared from a pre-immortal SV40-transformed HF (HF-C). We screened the library with a subtracted probe enriched for sequences present in HF-C and reduced in immortal AR5 cells. A more limited screen was also employed for sequences overexpressed in AR5 using a different strategy. Alterations in the level of mRNAs in AR5 encoding functions relevant to signal transduction pathways were identified; however, most cDNAs encoded novel sequences. In an effort to clarify which of the altered mRNAs are most relevant to immortalization, we performed Northern analysis with RNA prepared from three paired sets of independent pre-immortal and immortal (4 cell lines) SV40-transformants using eight cloned cDNAs which show reduced expression in AR5. Three of these were reduced in additional immortal cell lines as well; one, J4-4 (unknown function) is reduced in all the immortal cell lines tested; a second, J4-3 (possible PP2C type phosphatase) is reduced in 2 of the 3 matched sets; and a third, J2-2 (unknown function) is redu ced in 2 unrelated immortal cell lines. Although the roles of these genes are as yet unclear, their further analysis should extend our understanding of the molecular bases for immortalization. In particular, the patterns of expression of J4-4 and J4-3 strongly suggest that they are involved in the process of immortalization and/or can serve as target genes for assessing regulators of gene expression in this process. J. Cell. Physiol. 171:325–335, 1997. © 1997 Wiley-Liss, Inc.