Kinin generation from exogenous kininogens at the surface of retinoic acid-differentiated human neuroblastoma IMR-32 cells after stimulation with interferon-γ

Bradykinin-related peptides, kinins, ubiquitously occur in the nervous system and together with other pro-inflammatory mediators contribute to pathological states of that tissue such as edema and chronic pain. In the current work we characterized the kinin-forming system of neuronal cells obtained by differentiation of human neuroblastoma cell line IMR-32 with retinoic acid. These cells were shown to concentrate exogenous kinin precursors, kininogens, on the surface, release kinins from kininogens and subsequently convert kinins to their des-Arg metabolites. Significantly higher amounts of kinins and des-Arg-kinins were produced after cell stimulation with interferon-γ, a potent pro-inflammatory mediator involved in many neurological disorders. The expression of the major tissue kininogenase (the human kallikrein 1) and the major cell membrane-bound kininase (the carboxypeptidase M) also increased after cell stimulation with interferon-γ, suggesting the involvement of these enzymes in the kinin production and degradation, respectively. Interferon-γ was also able to up-regulate the expression of two known subtypes of kinin receptors. On the protein level, the changes were only observed in the expression of the des-Arg-kinin-specific type 1 receptor which functions in the propagation of the inflammatory state. Taken together, these results suggest a novel way for local kinin and des-Arg-kinin generation in the nervous tissue during pathological states accompanied by interferon-γ release.     

Altered insulin receptor signalling and β-cell cycle dynamics in type 2 diabetes mellitus

Insulin resistance, reduced β-cell mass, and hyperglucagonemia are consistent features in type 2 diabetes mellitus (T2DM). We used pancreas and islets from humans with T2DM to examine the regulation of insulin signaling and cell-cycle control of islet cells. We observed reduced β-cell mass and increased α-cell mass in the Type 2 diabetic pancreas. Confocal microscopy, real-time PCR and western blotting analyses revealed increased expression of PCNA and down-regulation of p27-Kip1 and altered expression of insulin receptors, insulin receptor substrate-2 and phosphorylated BAD. To investigate the mechanisms underlying these findings, we examined a mouse model of insulin resistance in β-cells--which also exhibits reduced β-cell mass, the β-cell-specific insulin receptor knockout (βIRKO). Freshly isolated islets and β-cell lines derived from βIRKO mice exhibited poor cell-cycle progression, nuclear restriction of FoxO1 and reduced expression of cell-cycle proteins favoring growth arrest. Re-expression of insulin receptors in βIRKO β-cells reversed the defects and promoted cell cycle progression and proliferation implying a role for insulin-signaling in β-cell growth. These data provide evidence that human β- and α-cells can enter the cell-cycle, but proliferation of β-cells in T2DM fails due to G1-to-S phase arrest secondary to defective insulin signaling. Activation of insulin signaling, FoxO1 and proteins in β-cell-cycle progression are attractive therapeutic targets to enhance β-cell regeneration in the treatment of T2DM.     

Design, synthesis, and evaluation of small molecule Hsp90 probes

A number of compounds from different chemical classes are known to bind competitively to the ATP-pocket of Hsp90 and inhibit its chaperone function. The natural product geldanamycin was the first reported inhibitor of Hsp90 and since then synthetic inhibitors from purine, isoxazole and indazol-4-one chemical classes have been discovered and are currently or soon to be in clinical trials for the treatment of cancer. In spite of a similar binding mode to Hsp90, distinct biological profiles were demonstrated among these molecules, both in vitro and in vivo. To better understand the molecular basis for these dissimilarities, we report here the synthesis of chemical tools for three Hsp90 inhibitor classes. These agents will be useful for probing tumor-by-tumor the Hsp90 complexes isolated by specific inhibitors. Such information will lead to better understanding of tumor specific molecular markers to aid in their clinical development. It will also help to elucidate the molecular basis for the biological differences observed among Hsp90 inhibitors.     

Imbalanced circulating matrix metalloproteinases in polycystic ovary syndrome

Altered levels of matrix metalloproteinases (MMPs) may reflect relevant pathogenetic mechanisms of disease conditions. The objective of this study was to compare the plasma levels of MMPs and tissue inhibitors of MMPs (TIMPs) in polycystic ovary syndrome (PCOS) patients with those found in healthy ovulatory controls and to examine whether the levels of these biomarkers are associated with clinical and biochemical features of this syndrome. Sixty-five healthy ovulatory subjects (controls) and 80 patients with PCOS were include in this study. MMP-2, MMP-8, MMP-9, TIMP-1, TIMP-2 concentrations were measured in plasma samples by gelatin zymography or enzyme-linked immunoassays. MMP-2, MMP-8, MMP-9, and TIMP-1 levels were similar in PCOS patients and in healthy controls (P > 0.05). PCOS patients had lower plasma TIMP-2 levels than healthy controls (P < 0.05). We found higher MMP-2/TIMP-2 and MMP-9/TIMP-1 ratios in PCOS patients than in healthy controls (all P < 0.05). Testosterone levels correlated positively with the MMP-9/TIMP-1 ratio and negatively with TIMP-2 levels (r = 0.26, P < 0.01 and r = -0.21, P = 0.02, respectively). In addition, only testosterone was an independent predictor of TIMP-2 levels (estimate = -0.35, P = 0.04) and the MMP-9/TIMP-1 ratio (estimate = 0.01, P = 0.04). We found evidence indicating that the balance between MMPs and TIMPs in women with PCOS is altered, probably due to androgen excess found in these women.     

Characterization of cytoplasmic arginine-rich basic protein of Guerin epithelioma

Summary Arginine-rich basic protein from cytoplasma of Guerin epitheliomas has been isolated and characterized. It contains five amino acids: arginine, lysine, glycine, alanine and glutamic acid which make together 74 per cent of all amino acid residues. The protein has a cationic character with an isoelectric point of 8.2. No carbohydrate component was found in this protein. The significance of arginine-rich basic protein in the cytoplasma of Guerin epithelioma is discussed briefly.     

Radiobiology of the acute radiation syndrome

Acute radiation syndrome or acute radiation sickness is classically subdivided into three subsyndromes: the hematopoietic, gastrointestinal and neurovascular syndrome but many other tissues can be damaged. The time course and severity of clinical signs and symptoms are a function of the overall body volume irradiated, the inhomogeneity of dose exposure, the particle type, the absorbed dose and the dose rate. Classical pathophysiology explain the failure of each of these organs and the timing of appearance of their signs and symptoms due to radiation-induced cytocidal effects of a great number of parenchymal cells of hierarchically organized tissues. Contemporaneously, many other radiation-induced effects has been described and all of them may lead to tissue injury with their corresponding signs and symptoms that can be expressed after short or long period of time. Radiation-induced multi-organ involvement is thought to be due to radiation-induced systemic inflammatory response mediated by released pro-inflammatory cytokines.     

Chemometric analysis of in-line multi-wavelength fluorescence measurements obtained during cultivations with a lipase producing Aspergillus oryzae strain

The filamentous fungus, Aspergillus oryzae, was cultivated in batch and fed-batch cultivations in order to investigate the use of multi-wavelength fluorescence for monitoring course of events during filamentous fungi cultivations. The A. oryzae strain applied expressed a fungal lipase from Thermomyces lanuginosus. Spectra of multi-wavelength fluorescence were collected every 5 min with the BioView system (DELTA, Denmark) and both explorative and predictive models, correlating the fluorescence data with cell mass and lipase activity, were built. During the cultivations, A. oryzae displayed dispersed hyphal growth and under these conditions no fouling of the multi-wavelength fluorescence sensor was observed. The scores of a parallel factor analysis (PARAFAC) model, based on the fluorescence spectra, gave clear evidence of, for example, the on-set of the feeding phase. The predictive models, estimating the cell mass, showed correlations between 0.73 and 0.97 with root mean square error of cross validation (RMSECV) values between 1.48 and 0.77 g . kg(-1). A model estimating the lipase activity was also constructed for the fed-batch cultivations with a correlation of 0.93. The results presented here clearly show that multi-wavelength fluorescence is a useful tool for monitoring fed-batch cultivations of filamentous fungi.     

Allele-specific inhibition of divergent protein tyrosine phosphatases with a single small molecule

A central challenge of chemical biology is the development of small-molecule tools for controlling protein activity in a target-specific manner. Such tools are particularly useful if they can be systematically applied to the members of large protein families. Here we report that protein tyrosine phosphatases can be systematically 'sensitized' to target-specific inhibition by a cell-permeable small molecule, Fluorescein Arsenical Hairpin Binder (FlAsH), which does not inhibit any wild-type PTP investigated to date. We show that insertion of a FlAsH-binding peptide at a conserved position in the PTP catalytic-domain's WPD loop confers novel FlAsH sensitivity upon divergent PTPs. The position of the sensitizing insertion is readily identifiable from primary-sequence alignments, and we have generated FlAsH-sensitive mutants for seven different classical PTPs from six distinct subfamilies of receptor and non-receptor PTPs, including one phosphatase (PTP-PEST) whose three-dimensional catalytic-domain structure is not known. In all cases, FlAsH-mediated PTP inhibition was target specific and potent, with inhibition constants for the seven sensitized PTPs ranging from 17 to 370 nM. Our results suggest that a substantial fraction of the PTP superfamily will be likewise sensitizable to allele-specific inhibition; FlAsH-based PTP targeting thus potentially provides a rapid, general means for selectively targeting PTP activity in cell-culture- or model-organism-based signaling studies.     

The gap between the concept and definitions in the Evolutionarily Significant Unit: the need to integrate neutral genetic variation and adaptive variation

The Evolutionarily Significant Unit (ESU) was conceptualized in 1986 as a conservation unit below the species level, theoretically applicable to a wide range of taxa. The concept has gained support, and various definitions or criteria, some of which are inconsistent with each other, have since been proposed. Recent critiques of the ESU have pointed out the dominance of definitions biased to the identification of long-term isolation or neutral genetic variation, which has largely ignored the adaptive components. We present here the validity of such claims and show how the ESU definitions have actually been applied in research. We surveyed scientific journals for original papers supporting ESU designations and determined who among the proponents of ESU definitions have gained wider support. Our results indicate that indeed there are inconsistencies with the original concept and with the existing definitions. Although the original concept recommended both ecological and genetic data as the basis for identification of ESUs, which reflect true evolutionary variation, recent definitions have become biased to either neutral genetic variation or adaptive variation. The definition which uses genetic data to assess neutral genetic variation (long-term isolation) has gained major support, and therefore validates the earlier claims. To bridge the gap between the original concept and the practical application, we propose the use of partial ESU and full ESU designations. The application of full ESU should be limited solely to when both information about neutral genetic variation and adaptive variation are available. In other cases, in which only a part of the variation is examined, we should use the term partial ESU (e.g., molecular-based ESU) and continue to investigate focal populations from other aspects of variations to designate full ESU.