Essential role of Pro 74 in stefin B amyloid-fibril formation: Dual action of cyclophilin A on the process

We report that Pro74 in human stefin B is critical for fibril formation and that proline isomerization plays an important role. The stefin B P74S mutant did not fibrillate over the time of observation at 25 °C, and it exhibited a prolonged lag phase at 30 °C and 37 °C. The peptidyl prolyl cis/trans isomerase cyclophilin A, when added to the wild-type protein, exerted two effects: it prolonged the lag phase and increased the yield and length of the fibrils. Addition of the inactive cyclophilin A R55A variant still resulted in a prolonged lag phase but did not mediate the increase of the final fibril yield. These results demonstrate that peptidyl prolyl cis/trans isomerism is rate-limiting in stefin B fibril formation.     

Proton-linked subunit heterogeneity in ferrous nitrosylated human adult hemoglobin: an EPR study

The effect of pH on the X-band electron paramagnetic resonance (EPR) spectrum of ferrous nitrosylated human adult tetrameric hemoglobin (HbNO) as well as of ferrous nitrosylated monomeric alpha- and beta-chains has been investigated, at -163 degrees C. At pH 7.3, the X-band EPR spectrum of tetrameric HbNO and ferrous nitrosylated monomeric alpha- and beta-chains displays a rhombic shape. Lowering the pH from 7.3 to 3.0, tetrameric HbNO and ferrous nitrosylated monomeric alpha- and beta-chains undergo a transition towards a species characterized by a X-band EPR spectrum with a three-line splitting centered at 334mT. These pH-dependent spectroscopic changes may be taken as indicative of the cleavage, or the severe weakening, of the proximal HisF8-Fe bond. In tetrameric HbNO, the pH-dependent spectroscopic changes depend on the acid-base equilibrium of two apparent ionizing groups with pK(a) values of 5.8 and 3.8. By contrast, the pH-dependent spectroscopic changes occurring in ferrous nitrosylated monomeric alpha- and beta-chains depend on the acid-base equilibrium of one apparent ionizing group with pK(a) values of 4.8 and 4.7, respectively. The different pK(a) values for the proton-linked spectroscopic transition(s) of tetrameric HbNO and ferrous nitrosylated monomeric alpha- and beta-chains suggest that the quaternary assembly drastically affects the strength of the proximal HisF8-Fe bond in both subunits. This probably reflects a 'quaternary effect', i.e., structural changes in both subunits upon tetrameric assembly, which is associated to a relevant variation of functional properties (i.e., proton affinity).     

Binding of beta-D-glucopyranosyl bismethoxyphosphoramidate to glycogen phosphorylase b: kinetic and crystallographic studies

In an attempt to identify a new lead molecule that would enable the design of inhibitors with enhanced affinity for glycogen phosphorylase (GP), beta-D-glucopyranosyl bismethoxyphosphoramidate (phosphoramidate), a glucosyl phosphate analogue, was tested for inhibition of the enzyme. Kinetic experiments showed that the compound was a weak competitive inhibitor of rabbit muscle GPb (with respect to alpha-D-glucose-1-phosphate (Glc-1-P)) with a Ki value of 5.9 (+/-0.1) mM. In order to elucidate the structural basis of inhibition, we determined the structure of GPb complexed with the phosphoramidate at 1.83 A resolution. The complex structure reveals that the inhibitor binds at the catalytic site and induces significant conformational changes in the vicinity of this site. In particular, the 280s loop (residues 282-287) shifts 0.4-4.3 A (main-chain atoms) to accommodate the phosphoramidate, but these conformational changes do not lead to increased contacts between the inhibitor and the protein that would improve ligand binding.     

Comparative analysis of circulating hemocytes of the freshwater snail Pomacea canaliculata

Molluscs are invertebrates of great relevance for economy, environment and public health. The numerous studies on molluscan immunity and physiology registered an impressive variability of circulating hemocytes. This study is focused on the first characterization of the circulating hemocytes of the freshwater gastropod Pomacea canaliculata, a model for several eco-toxicological and parasitological researches.Flow cytometry analysis identified two populations of hemocytes on the basis of differences in size and internal organization. The first population contains small and agranular cells. The second one displays major size and a more articulated internal organization. Light microscopy evidenced two principal morphologies, categorized as Group I (small) and II (large) hemocytes. Group I hemocytes present the characteristics of blast-like cells, with an agranular and basophilic cytoplasm. Group I hemocytes can adhere onto a glass surface but seem unable to phagocytize heat-inactivated Escherichia coli. The majority of Group II hemocytes displays an agranular cytoplasm, while a minority presents numerous granules. Agranular cytoplasm may be basophilic or acidophilic. Granules are positive to neutral red staining and therefore acidic. Independently from their morphology, Group II hemocytes are able to adhere and to engulf heat-inactivated E. coli. Transmission electron microscopy analysis clearly distinguished between agranular and granular hemocytes and highlighted the electron dense content of the granules. After hemolymph collection, time-course analysis indicated that the Group II hemocytes are subjected to an evident dynamism with changes in the percentage of agranular and granular hemocytes. The ability of circulating hemocytes to quickly modify their morphology and stainability suggests that P. canaliculata is endowed with highly dynamic hemocyte populations able to cope with rapid environmental changes as well as fast growing pathogens.     

Programmed cell death and stem cell differentiation are responsible for midgut replacement in <Emphasis Type="Italic">Heliothis virescens</Emphasis> during prepupal instar

We have analyzed midgut development during the fifth larval instar in the tobacco budworm Heliothis virescens. In prepupae, the midgut formed during larval instars undergoes a complete renewal process. This drastic remodeling of the alimentary canal involves the destruction of the old cells by programmed cell-death mechanisms (autophagy and apoptosis). Massive proliferation and differentiation of regenerative stem cells take place at the end of the fifth instar and give rise to a new fully functioning epithelium that is capable of digesting and absorbing nutrients and that is maintained throughout the subsequent pupal stage. Midgut replacement in H. virescens is achieved by a balance between this active proliferation process and cell-death mechanisms and is different from similar processes characterized in other insects. This work was supported by FAR 2006 (University of Insubria) to G.T., by a MIUR-FIRB-COFIN grant (no. RBNE01YXA8/2004077251), and by the Centro Grandi Attrezzature (University of Insubria).     

Taxonomic Status of Four Rare Alien Fish Species of the Kapchagay Reservoir (Balkhash Basin,Central Asia)

Journal of Ichthyology - Based on the morphological features, four rare alien species in the Kapchagai reservoir were identified: Coregonus peled, Parasalmo mykiss, Megalobrama mantschuricus, and...     

Acute exposure to long-chain fatty acids impairs {alpha}2-adrenergic receptor-mediated antilipolysis in human adipose tissue

The acute in vitro and in vivo effects of long-chain fatty acids (LCFAs) on the regulation of adrenergic lipolysis were investigated in human adipose tissue. The effect of a 2 h incubation, without or with LCFA (200 mumol/l), on basal and hormonally induced lipolysis was tested in vitro on isolated fat cells. The lipolytic response to epinephrine was enhanced by suppression of the antilipolytic alpha(2)-adrenergic effect. Then, healthy lean and obese male subjects performed a 45 min exercise bout at 50% of their heart rate reserve either after an overnight fast or 3 h after a high-fat meal (HFM: 95% fat, 5% carbohydrates). Subcutaneous adipose tissue lipolysis was measured by microdialysis in the presence or absence of an alpha-antagonist (phentolamine). In vivo, a HFM increased plasma levels of nonesterified fatty acids in lean and obese subjects. In both groups, the HFM did not alter hormonal responses to exercise. Under fasting conditions, the alpha(2)-adrenergic antilipolytic effect was more pronounced in obese than in lean subjects. The HFM totally suppressed the alpha(2)-adrenergic antilipolytic effect in lean and obese subjects during exercise. LCFAs per se, in vitro as well as in vivo, suppress alpha(2)-adrenergic-mediated antilipolysis in adipose tissue. LCFA-mediated suppression of antilipolytic pathways represents another mechanism whereby a high fat content in the diet might increase adipose tissue lipolysis.     

Altered transarcolemmal Ca transport modifies the myofibrillar ultrastructure and protein metabolism in cultured adult ventricular cardiomyocytes

The present study was designed to investigate how prolonged (24-72 h) exposure to modifiers of Ca transarcolemmal transport affects the myofibrillar structure, protein turnover and content of myofibrillar proteins in adult guinea pig cardiomyocytes maintained beating synchronously in long-term cultures. First we established the functional responses (the contractile activity and [Ca]_i transients) of the cultured myocytes to acute exposures to several drugs used in this study. The ultrastructural characteristics of these cultures under the various treatments were determined using immunohistochemistry and confocal scanning laser microscopy, and their biochemical properties were evaluated using analysis of total cellular protein content, myofibrillar protein content and SDS-PAGE electrophoretic examination. We compared the effects of 24, 36 and 72 h-long exposures to the various specific Ca-flux modifiers. Increased Ca influx via Ca_L-channel agonist (Bay K 8644) or via the reversed- mode of the Na/Ca exchanger (veratrine) did not alter the myofibrillar structure or the specific protein profiles or proteosynthesis. However, when cytosolic Ca was increased by three different types of inhibitors of Ca extrusion from the cells via Na/Ca exchange, (Na-free solution, 5 mM NiCl₂ and 10^-6 M ouabain), very significant changes in all investigated parameters occurred almost immediately. Twenty-four h-long exposure to Na-free did not affect significantly the total cellular protein (TCP), but the protein synthesis was decreased by 87% and the total myofibrillar protein (TMP) content was decreased by 38%. The myofibrils were heavily fragmented. Similarly, 24 h-long exposure to 5 mM NiCl₂ did not affect the TCP, but it reduced protein synthesis by about 90% and decreased the total myofibrillar protein content by 30%. These effects were even more pronounced at 72 h of exposure and they were accompanied with a complete disassembly of myofilaments. Exposure to 10^-6 M ouabain over 72 h resulted in > 80% inhibition of protein synthesis, a 45% decrease in TCP content and a 53% in TMP content. In contrast, 10^-7 M ouabain did not produce any such changes. The changes produced by the Na/Ca-exchange inhibitors were accompanied by only minor changes in DNA content, indicating that the myocytes remained viable. Moreover, these effects were not due to the associated contractile arrest, since exposure to Ca_L-channel antagonists (5-20 M nifedipine or 10 M verapamil) produced only very minor changes in the myofibrillar structure and in protein profiles.Our data demonstrate that short-term (up to 72 h) increased Ca influx or contractile arrest of well-interconnected, spontaneously beating adult cardiomyocytes does not affect their ultrastructural characteristics or their myofibrillar protein turnover greatly, while any situations leading to Ca accumulation (via inhibition of Na/Ca exchange) affect cardiomyocyte function and ultrastructure almost immediately. These data are in sharp contrast to those previously reported from immature, neonatal myocytes.     

Biohydrogen production by dark fermentation of glycerol using Enterobacter and Citrobacter Sp

Glycerol is an attractive substrate for biohydrogen production because, in theory, it can produce 3 mol of hydrogen per mol of glycerol. Moreover, glycerol is produced in substantial amounts as a byproduct of producing biodiesel, the demand for which has increased in recent years. Therefore, hydrogen production from glycerol was studied by dark fermentation using three strains of bacteria: namely, Enterobacter spH1, Enterobacter spH2, and Citrobacter freundii H3 and a mixture thereof (1:1:1). It was found that, when an initial concentration of 20 g/L of glycerol was used, all three strains and their mixture produced substantial amounts of hydrogen ranging from 2400 to 3500 mL/L, being highest for C. freundii H3 (3547 mL/L) and Enterobacter spH1 (3506 mL/L). The main nongaseous fermentation products were ethanol and acetate, albeit in different ratios. For Enterobacter spH1, Enterobacter spH2, C. freundii H3, and the mixture (1:1:1), the ethanol yields (in mol EtOH/mol glycerol consumed) were 0.96, 0.67, 0.31, and 0.66, respectively. Compared to the individual strains, the mixture (1:1:1) did not show a significantly higher hydrogen level, indicating that there was no synergistic effect. Enterobacter spH1 was selected for further investigation because of its higher yield of hydrogen and ethanol. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013     

The surface charge of NKLy ascitic tumor cells differing in their proliferative activity, in their place in the cell cycle and in their ploidy

The NKLy ascitic tumor cells in the stationary phase of growth were fractionated by velocity sedimentation method. Cells from the obtained fractions were characterized by measurements of DNA contents and 3H-thymidine incorporation. The surface properties of the cells from five fractions, differing in proliferative capacity, stage of the cell cycle and ploidy were considered using cell electrophoresis, two polymer aqueous phase system and Alcian blue sorption. A correlation between electrophoretic mobility and cell partition constant for these fractions has been obtained. No correlation was found between these parameters and dye absorption. The surface charge of the cells from G0/G1 and S fractions was higher than that of other cells. The polyploid NKLy cells demonstrated a lower surface charge. The surface properties of the tumor cells differing in proliferative capacity, stage of the cell cycle and ploidy are discussed.