Site-directed mutagenesis of cys148 in the lac carrier protein of Escherichia coli

The lac y gene of Escherichia coli which encodes the lac carrier protein has been modified by oligonucleotide-directed, site-specific mutagenesis such that cys₁₄₈ is converted to a glycine residue. Cells bearing the mutated lac y gene exhibit initial rates of lactose transport that are about 4-fold lower than cells bearing the wild type gene on a recombinant plasmid. Furthermore, transport activity is less sensitive to inactivation by N-ethylmaleimide, and strikingly, galactosyl 1-thio-β-D-galactopyranoside affords no protection against inactivation. The findings suggest that although cys₁₄₈ is essential for substrate protection against sulfhydryl inactivation, it is not obligatory for lactose:proton symport and that another sulfhydryl group elsewhere within the lac carrier protein may be required for full activity.     

The structures of garcinones a,b and c: Three new xanthones from Garcinia mangostana

Three new tetraoxygenated xanthones (garcinones A, B and C), each disubstituted with C₅-units, have been isolated from the chloroform extract of the fruit-hulls of Garcinia mangostana. Their structures were established by a combination of spectral interpretation and chemical correlation.     

AtHsp101 research sets course of action for the genetic improvement of crops against heat stress

Journal of Plant Biochemistry and Biotechnology - Caseinolytic protease (Clp)/Hsp100 proteins are members of the AAA+ (ATPase associated with a variety of cellular activities) family of proteins...     

Novel Changes in NF-κB Activity during Progression and Regression Phases of Hyperplasia: ROLE OF MEK,ERK, AND p38*

Utilizing the Citrobacter rodentium-induced transmissible murine colonic hyperplasia (TMCH) model, we measured hyperplasia and NF-κB activation during progression (days 6 and 12 post-infection) and regression (days 20–34 post-infection) phases of TMCH. NF-κB activity increased at progression in conjunction with bacterial attachment and translocation to the colonic crypts and decreased 40% by day 20. NF-κB activity at days 27 and 34, however, remained 2–3-fold higher than uninfected control. Expression of the downstream target gene CXCL-1/KC in the crypts correlated with NF-κB activation kinetics. Phosphorylation of cellular IκBα kinase (IKK)α/β (Ser^176/180) was elevated during progression and regression of TMCH. Phosphorylation (Ser^32/36) and degradation of IκBα, however, contributed to NF-κB activation only from days 6 to 20 but not at later time points. Phosphorylation of MEK1/2 (Ser^217/221), ERK1/2 (Thr²⁰²/Tyr²⁰⁴), and p38 (Thr¹⁸⁰/Tyr¹⁸²) paralleled IKKα/β kinetics at days 6 and 12 without declining with regressing hyperplasia. siRNAs to MEK, ERK, and p38 significantly blocked NF-κB activity in vitro, whereas MEK1/2-inhibitor (PD98059) also blocked increases in MEK1/2, ERK1/2, and IKKα/β thereby inhibiting NF-κB activity in vivo. Cellular and nuclear levels of Ser⁵³⁶-phosphorylated (p65⁵³⁶) and Lys³¹⁰-acetylated p65 subunit accompanied functional NF-κB activation during TMCH. RSK-1 phosphorylation at Thr³⁵⁹/Ser³⁶³ in cellular/nuclear extracts and co-immunoprecipitation with cellular p65-NF-κB overlapped with p65⁵³⁶ kinetics. Dietary pectin (6%) blocked NF-κB activity by blocking increases in p65 abundance and nuclear translocation thereby down-regulating CXCL-1/KC expression in the crypts. Thus, NF-κB activation persisted despite the lack of bacterial attachment to colonic mucosa beyond peak hyperplasia. The MEK/ERK/p38 pathway therefore seems to modulate sustained activation of NF-κB in colonic crypts in response to C. rodentium infection.     

A cortical evoked potential study of afferents mediating human esophageal sensation

The aim of this study was to compare the characteristics of esophageal cortical evoked potentials (CEP) following electrical and mechanical stimulation in healthy subjects to evaluate the afferents involved in mediating esophageal sensation. Similarities in morphology and interpeak latencies of the CEP to electrical and mechanical stimulation suggest that they are mediated via similar pathways. Conduction velocity of CEP to either electrical or mechanical stimulation was 7.9-8.6 m/s, suggesting mediation via thinly myelinated Adelta-fibers. Amplitudes of CEP components to mechanical stimulation were significantly smaller than to electrical stimulation at the same levels of perception, implying that electrical stimulation activates a larger number of afferents. The latency delay of approximately 50 ms for each mechanical CEP component compared with the corresponding electrical CEP component is consistent with the time delay for the mechanical stimulus to distend the esophageal wall sufficiently to trigger the afferent volley. In conclusion, because the mechanical and electrical stimulation intensities needed to obtain esophageal CEP are similar and clearly perceived, it is likely that both spinal and vagal pathways mediate esophageal CEP. Esophageal CEP to both modalities of stimulation are mediated by myelinated Adelta-fibers and produce equally robust CEP responses. Both techniques may have important roles in the assessment of esophageal sensory processing in health and disease.     

Is the sarcolemmal or mitochondrial KATP channel activation important in the antiarrhythmic and cardioprotective effects during acute ischemia/reperfusion in the intact anesthetized rabbit model?

The relative contributions of cardiomyocyte sarcolemmal ATP-sensitive K(+) (K(ATP)) and mitochondrial K(ATP) channels in the cardioprotection and antiarrhythmic activity induced by K(ATP) channel openers remain obscure, though the mitochondrial K(ATP) channels have been proposed to be involved as a subcellular mediator in cardioprotection afforded by ischemic preconditioning. In the present study, we sought to investigate the effects of administration of ATP-sensitive K(+) channel (K(ATP)) openers (nicorandil and minoxidil), a specific mitochondrial K(ATP) channel blocker (5-hydroxydecanoate (5-HD)) and a specific sarcolemmal K(ATP) channel blocker (HMR 1883; (1-[5-[2-(5-chloro-o-anisamido)ethyl]-2-methoxyphenyl]sulfonyl-3-methylthiourea) prior to coronary occlusion as well as prior to post-ischemic reperfusion on survival rate, ischemia-induced and reperfusion-induced arrhythmias and myocardial infarct size in anesthetized albino rabbits. The thorax was opened in the left 4th intercostal space and after pericardiotomy the heart was exposed. In Group I (n=88), occlusion of the left main coronary artery and hence, myocardial ischemia-induced arrhythmias was achieved by tightening a previously placed loose silk ligature for 30 min. In Group II (n=206), arrhythmias were induced by reperfusion following a 20-min ligation of the left main coronary artery. Both in Group I and Group II, intravenous (i.v.) administration of nicorandil (0.47 mg/kg), minoxidil (0.5 mg/kg), HMR 1883 (3 mg/kg)/nicorandil and HMR 1883 (3 mg/kg)/minoxidil before coronary artery occlusion increased survival rate (86%, 75%, 75% and 86% vs. 55% in the control subgroup in Group I; 75%, 67%, 67% and 75% vs. 46% in the control subgroup in Group II), significantly decreased the incidence and severity of life-threatening arrhythmias. In Group II, i.v. administration of nicorandil and minoxidil before coronary artery occlusion significantly decreased myocardial infarct size. However, i.v. administration of nicorandil or minoxidil before reperfusion did neither increase survival rate nor confer any antiarrhythmic or cardioprotective effects. The antiarrhythmic and cardioprotective effects of both nicorandil and minoxidil were abolished by pretreating the rabbits with 5-HD (5 mg/kg, i.v. bolus), a selective mitochondrial K(ATP) channel blocker but not by HMR 1883 (3 mg/kg). In the present study, higher levels of malondialdehyde (MDA) and lower levels of reduced glutathione (GSH) and superoxide dismutase (SOD) in necrotic zone of myocardium in all the 16 subgroups in Group II suggest little anti-free radical property of nicorandil and minoxidil. We conclude that intervention by intravenous administration of nicorandil and minoxidil (through the selective activation of mitochondrial K(ATP) channels) increased survival rate and exhibited antiarrhythmic and cardioprotective effects during coronary occlusion and reperfusion in anesthetized rabbits when administered prior to coronary occlusion. The cardiomyocyte mitochondrial K(ATP) channel may be a pharmacologically modulable target of cardioprotection and antiarrhythmic activity.     

Effects of calcium channel blocker, mibefradil, and potassium channel opener, pinacidil, on the contractile response of mid-pregnant goat myometrium

The present study was undertaken to investigate the in vitro influence of mibefradil, a calcium channel blocker, and pinacidil, a potassium channel opener, on pregnant goat myometrial spontaneous rhythmic contractility and contractions induced with the agonist, oxytocin. Longitudinal strips from the distal region of uterus, collected from goats at midgestation, were mounted in an organ bath for recording isometric contractions. Mibefradil (10(-8)-10(-4) M) or pinacidil (10(-10)-10(-4) M), added cumulatively to the bath at an increment of 1 log unit, caused concentration-dependent inhibition of the spontaneous rhythmic contractions of isolated uterine strips. The rhythmic contraction was, respectively, abolished at 100 and 10 microM concentrations of mibefradil and pinacidil. In a concentration-dependent manner, mibefradil (1 and 10 microM) antagonized the contractions elicited with oxytocin (10(-5)-10(-2) IU). Pretreatment of uterine strips with glibenclamide (10 microM), a selective KATP channel blocker, caused a rightward shift of the concentration-response curve of pinacidil with a concomitant decrease in its pD2 value. Pinacidil (0.3, 1 and 3 microM), in a concentration-related manner, antagonized the oxytocin (10(-5)-10(-2) IU)-induced contractile response. The inhibition of spontaneous rhythmic contractions and antagonism of oxytocin-induced contraction by mibefradil in the pregnant goat myometrium may be related to the antagonism of voltage-dependent Ca2+ channels, while by pinacidil suggests that KATP channel could be a therapeutic target for tocolysis.     

Interaction of piroxicam with micelles: effect of hydrophobic chain length on structural switchover

Molecules, whose pK(a) values can be easily fine-tuned by their microenvironment, are expected to be profoundly affected by the heterogeneous environments of membranes. Membrane parameters can have a strong effect in choosing a particular structural form of a molecule for incorporation/interaction. A case study has been presented for piroxicam, a non-steroidal anti-inflammatory drug of oxicam group, whose targets are cyclooxygenases, which are membrane active proteins. The structural dynamism of piroxicam is reflected in the ease with which it can switchover or convert from one prototropic form to the other guided by its environment. In this work we have studied the effect of varying hydrophobic chain length and surface charges in fine-tuning the interaction of piroxicam with micelles. Interaction of piroxicam with three types of micelles with identical negatively charged head groups and varying tail lengths viz., sodium dodecyl sulfate (S12S), sodium decyl sulfate (S10S) and sodium octyl sulfate (S8S) shows that there is a shift in the apparent pK(a) in the direction that favors the switchover or conversion from the anionic form to the global neutral form. The binding constants of piroxicam with three micelles show a linear dependence on chain length. Interaction was also studied with micelles having oppositely charged head groups and different chain lengths viz., dodecyl N,N,N-trimethyl ammonium bromide (DTAB) and cetyl N,N,N-trimethyl ammonium bromide (CTAB). For micelles having identical chain lengths but oppositely charged head groups viz., S12S and DTAB, pK(a) shifts in two opposite directions compared to that in the absence of any surfactant. This is expected when electrostatic force is the only driving force. This case study demonstrates the effect of hydrophobic chain length and surface charges in fine-tuning the equilibrium between different structural forms of piroxicam. Our results also imply that for structurally dynamic drugs like piroxicam the nature of the biomembranes, characterized by different membrane parameters, should play a crucial role in choosing a particular structural form of the drug that will be finally presented to their targets.     

Targeted comparative RNA interference analysis reveals differential requirement of genes essential for cell proliferation

Differences in the genetic and epigenetic make up of cell lines have been very useful for dissecting the roles of specific genes in the biology of a cell. Targeted comparative RNAi (TARCOR) analysis uses high throughput RNA interference (RNAi) against a targeted gene set and rigorous quantitation of the phenotype to identify genes with a differential requirement for proliferation between cell lines of different genetic backgrounds. To demonstrate the utility of such an analysis, we examined 257 growth-regulated genes in parallel in a breast epithelial cell line, MCF10A, and a prostate cancer cell line, PC3. Depletion of an unexpectedly high number of genes (25%) differentially affected proliferation of the two cell lines. Knockdown of many genes that spare PC3 (p53-) but inhibit MCF10A (p53+) proliferation induces p53 in MCF10A cells. EBNA1BP2, involved in ribosome biogenesis, is an example of such a gene, with its depletion arresting MCF10A at G1/S in a p53-dependent manner. TARCOR is thus useful for identifying cell type-specific genes and pathways involved in proliferation and also for exploring the heterogeneity of cell lines. In particular, our data emphasize the importance of considering the genetic status, when performing siRNA screens in mammalian cells.