Identification of serotonin-sensitive aryl acylamidase activity with cobra venom acetylcholinesterase

Acetylcholinesterase purified from cobra (Naja naja) venom exhibits a serotonin-sensitive aryl acylamidase activity. Both acetylcholinesterase and aryl acylamidase activities co-eluted in column chromatographic procedures (Sephadex G-75 and Zinc-Sepharose), co-migrated on polyacrylamide gel electrophoresis, co-immunoprecipitated by anti-snake venom antibody and showed the same heat denaturation profile at 40 degrees C. Further, several potent acetylcholinesterase inhibitors at different concentrations inhibited the cholinesterase and aryl acylamidase activities to the same extent. It is concluded that in cobra venom, acetylcholinesterase is associated with a serotonin-sensitive aryl acylamidase activity similar to earlier observations made with acetylcholinesterase from different sources.     

Expression and activity of protein kinase C isoenzymes during normal and abnormal murine palate development

Protein kinase C (PKC) plays a critical role in signal transduction, mediating various cellular events critical for normal development, including that of the palate. In vivo and in vitro studies suggest the relevance of the inhibition of PKC by the mycotoxin, secalonic acid D (SAD), to its induction of cleft palate (CP) in mice. In the present study, temporal and spatial expression and the activity of various PKC isoenzymes were studied in the control and SAD-exposed murine embryonic palate during gestational days (GD) 12-14.5 by western blotting, immunohistochemistry, and phosphotransfer assay. The Ca2+-dependent isoenzymes, PKC alpha and PKC betaII, showed significant expression on GD 12.0, which gradually decreased through GD 14.5, whereas PKC betaI and PKC gamma were negligible throughout. All Ca2+-independent isoenzymes (epsilon, delta, and zeta) were expressed more abundantly and, in contrast to the Ca2+-dependent ones, progressively increased with age. SAD failed to alter this pattern of expression but enhanced the phosphorylation of PKC epsilon throughout development. Immunohistochemical analysis revealed an isoenzyme-specific distribution of PKC between the epithelium and mesenchyme. As expected, SAD significantly inhibited the total Ca2+-dependent PKC activity in palatal extracts. Although total Ca2+-independent PKC activity in palatal extracts was unaffected by SAD, individual pure isoenzymes were either selectively inhibited (PKC zeta), stimulated (PKC delta), or unaffected (PKC epsilon) by SAD. These results show that PKC isoenzymes exhibit dynamic temporal and spatial patterns of expression and activity in the developing palate and that the induction of CP by SAD is associated with an alteration in their activation and/or activity.     

Somatic embryogenesis from leaf derived callus of Tylophora indica (Burm. f.) Merrill

Mature leaf explant derived callus of Tylophora indica (Burm. f.) Merrill yielded somatic embryos on MS medium supplied with BA(1-2 mg/L) or kinetin(1-5 mg/L) or kinetin/BA (1-2 mg/L) used along with IAA(0.1-1 mg/L). Maximum somatic embryos (30) could be recovered from 100 mg of embryogenic callus within 60 days at an optimum concentration of 2 mg/L of BA which was also best suited for providing the maximum conversion rate (90%) of embryoids to plantlets. Kinetin (1-5 mg/L), used as the sole growth hormone, induced the development of embryoids showing either shoot or root primordia in 30% of the cultures. However, embryoids with shoot primordia developed roots upon transfer to medium containing IAA(0.1 mg/L) and kinetin(2 mg/L). Embryoids from all cultures germinated in the initiation medium and were transplanted to sterile vermiculite for hardening. After two weeks of hardening, the plantlets were transferred to the green house where they grew and established well showing a high rate of survival (90%).     

Type II myosin regulatory light chain relieves auto-inhibition of myosin-heavy-chain function

The F-actin based motor protein myosin II has a key role in cytokinesis. Here we show that the Schizosaccharomyces pombe regulatory light chain (RLC) protein Rlc1p binds to Myo2p in manner that is dependent on the IQ sequence motif (the RLC-binding site), and that Rlc1p is a component of the actomyosin ring. Rlc1p is important for cytokinesis at all growth temperatures and is essential for this process at lower temperatures. Interestingly, all deleterious phenotypes associated with the loss of Rlc1p function are suppressed by deletion of the RLC binding site on Myo2p. We conclude that the sole essential function of RLCs in fission yeast is to relieve the auto-inhibition of myosin II function, which is mediated by the RLC-binding site, on the myosin heavy chain (MHC).     

Proteomics of Mycoplasma genitalium: identification and characterization of unannotated and atypical proteins in a small model genome

We present the results of a comprehensive analysis of the proteome of Mycoplasma genitalium (MG), the smallest autonomously replicating organism that has been completely sequenced. Our aim was to identify and characterize all soluble proteins in MG that are structurally and functionally uncharacterized. We were particularly interested in identifying proteins that differed significantly from typical globular proteins, for example, proteins which are unstructured in the absence of a ‘partner’ molecule or those that exhibit unusual thermodynamic properties. This work is complementary to other structural genomics projects whose primary aim is to determine the three-dimensional structures of proteins with unknown folds. We have identified all the full-length open reading frames (ORFs) in MG that have no homologs of known structure and are of unknown function. Twenty-five of the total 483 ORFs fall into this category and we have expressed, purified and characterized 11 of them. We have used circular dichroism (CD) to rapidly investigate their biophysical properties. Our studies reveal that these proteins have a wide range of structures varying from highly helical to partially structured to unfolded or random coil. They also display a variety of thermodynamic properties ranging from cooperative unfolding to no detectable unfolding upon thermal denaturation. Several of these proteins are highly conserved from mycoplasma to man. Further information about target selection and CD results is available at http://bioinfo.mbb.yale.edu/genome     

beta(2)-glycoprotein I-dependent alterations in membrane properties

beta(2)-Glycoprotein I (beta(2)GP1), a 50 kDa serum glycoprotein, binds anionic phospholipids and plays a role in phosphatidylserine (PS)-dependent coagulation and apoptotic processes. To characterize the molecular consequences that occur to target membranes upon binding of beta(2)GP1, the interaction between beta(2)GP1 and PS-containing vesicles was investigated by fluorescent spectroscopy. Membranes containing pyrene-labeled lipid showed that binding of beta(2)GP1 induced a decrease in excimer/monomor ratios (E/M) of the target membrane. Although these membrane alterations occurred in isotonic buffer, the effects were greater in low ionic strength buffer and were coincident to membrane precipitation. In contrast, increases in membrane polarization were only seen in low ionic strength buffer. Analysis of beta(2)GP1 binding kinetics by resonance energy transfer between fluorescein-labeled beta(2)GP1 and rhodamine-containing PS vesicles revealed a two-component process: (1) a primary and rapid binding via the C-terminus that occurred <2 s in both isotonic and low ionic strength buffers, and (2) a sequential binding of the N-terminus that was approximately 100-fold slower in low ionic strength solution. Taken together, these data suggest that beta(2)GP1 alters the fluidity and membrane polarization of its target membrane, which in low ionic strength buffer is of sufficient magnitude to induce precipitation.     

Oxygen supply without gas-liquid film resistance to xanthomonas campestris cultivation

Alternative methods of oxygen supply are of crucial importance, especially in viscous fermentations and shear-sensitive fermentations. A method of oxygen supply that completely eliminates the gas-liquid transport resistance has been presented. The method involves a need-based liquid-phase decomposition of hydrogen peroxide to provide the necessary oxygen. When Xanthomonas campestris was cultivated (viscous cultivation) using this method of oxygen supply, dissolved oxygen (DO) levels were maintained above the setpoint of 50% throughout the cultivation, whereas the conventional cultivation was able to meet culture oxygen demand only for about 6 h in a 72-h fermentation. Furthermore, the maximum specific growth rate and xanthan yields in the novel cultivation were 89% and 169%, respectively, of those obtained in conventional cultivation. A mathematical model was also developed to simulate and predict results in fermentations employing the presented methodology. In addition, studies with HOCl pretreatments indicated that monofunctional catalase may be responsible for the decomposition of H2O2 supplied externally to cells; HOCl pretreatments also increased the tolerance of cells to H2O2. The decomposition kinetics of externally supplied H2O2 was Michaelis-Menten in nature with vmax = 1.196 x 10(-6) M s-1 and Km = 0.21 mM. The catalase concentration was estimated to be 3.4 x 10(-10) mol/g of cells. Copyright 1998 John Wiley & Sons, Inc.     

Breaking the Hydrophobicity of the MscL Pore: Insights into a Charge-Induced Gating Mechanism

The mechanosensitive channel of large conductance (MscL) is a protein that responds to membrane tension by opening a transient pore during osmotic downshock. Due to its large pore size and functional reconstitution into lipid membranes, MscL has been proposed as a promising artificial nanovalve suitable for biotechnological applications. For example, site-specific mutations and tailored chemical modifications have shown how MscL channel gating can be triggered in the absence of tension by introducing charged residues at the hydrophobic pore level. Recently, engineered MscL proteins responsive to stimuli like pH or light have been reported. Inspired by experiments, we present a thorough computational study aiming at describing, with atomistic detail, the artificial gating mechanism and the molecular transport properties of a light-actuated bacterial MscL channel, in which a charge-induced gating mechanism has been enabled through the selective cleavage of photo-sensitive alkylating agents. Properties such as structural transitions, pore dimension, ion flux and selectivity have been carefully analyzed. Besides, the effects of charge on alternative sites of the channel with respect to those already reported have been addressed. Overall, our results provide useful molecular insights into the structural events accompanying the engineered MscL channel gating and the interplay of electrostatic effects, channel opening and permeation properties. In addition, we describe how the experimentally observed ionic current in a single-subunit charged MscL mutant is obtained through a hydrophobicity breaking mechanism involving an asymmetric inter-subunit motion.     

Dasatinib Attenuates Pressure Overload Induced Cardiac Fibrosis in a Murine Transverse Aortic Constriction Model

Reactive cardiac fibrosis resulting from chronic pressure overload (PO) compromises ventricular function and contributes to congestive heart failure. We explored whether nonreceptor tyrosine kinases (NTKs) play a key role in fibrosis by activating cardiac fibroblasts (CFb), and could potentially serve as a target to reduce PO-induced cardiac fibrosis. Our studies were carried out in PO mouse myocardium induced by transverse aortic constriction (TAC). Administration of a tyrosine kinase inhibitor, dasatinib, via an intraperitoneally implanted mini-osmotic pump at 0.44 mg/kg/day reduced PO-induced accumulation of extracellular matrix (ECM) proteins and improved left ventricular geometry and function. Furthermore, dasatinib treatment inhibited NTK activation (primarily Pyk2 and Fak) and reduced the level of FSP1 positive cells in the PO myocardium. In vitro studies using cultured mouse CFb showed that dasatinib treatment at 50 nM reduced: (i) extracellular accumulation of both collagen and fibronectin, (ii) both basal and PDGF-stimulated activation of Pyk2, (iii) nuclear accumulation of Ki67, SKP2 and histone-H2B and (iv) PDGF-stimulated CFb proliferation and migration. However, dasatinib did not affect cardiomyocyte morphologies in either the ventricular tissue after in vivo administration or in isolated cells after in vitro treatment. Mass spectrometric quantification of dasatinib in cultured cells indicated that the uptake of dasatinib by CFb was greater that that taken up by cardiomyocytes. Dasatinib treatment primarily suppressed PDGF but not insulin-stimulated signaling (Erk versus Akt activation) in both CFb and cardiomyocytes. These data indicate that dasatinib treatment at lower doses than that used in chemotherapy has the capacity to reduce hypertrophy-associated fibrosis and improve ventricular function.     

What Do Pneumocystis Organisms Tell Us about the Phylogeography of Their Hosts? The Case of the Woodmouse Apodemus sylvaticus in Continental Europe and Western Mediterranean Islands

Pneumocystis fungi represent a highly diversified biological group with numerous species, which display a strong host-specificity suggesting a long co-speciation process. In the present study, the presence and genetic diversity of Pneumocystis organisms was investigated in 203 lung samples from woodmice (Apodemus sylvaticus) collected on western continental Europe and Mediterranean islands. The presence of Pneumocystis DNA was assessed by nested PCR at both large and small mitochondrial subunit (mtLSU and mtSSU) rRNA loci. Direct sequencing of nested PCR products demonstrated a very high variability among woodmouse-derived Pneumocystis organisms with a total number of 30 distinct combined mtLSU and mtSSU sequence types. However, the genetic divergence among these sequence types was very low (up to 3.87%) and the presence of several Pneumocystis species within Apodemus sylvaticus was considered unlikely. The analysis of the genetic structure of woodmouse-derived Pneumocystis revealed two distinct groups. The first one comprised Pneumocystis from woodmice collected in continental Spain, France and Balearic islands. The second one included Pneumocystis from woodmice collected in continental Italy, Corsica and Sicily. These two genetic groups were in accordance with the two lineages currently described within the host species Apodemus sylvaticus. Pneumocystis organisms are emerging as powerful tools for phylogeographic studies in mammals.