Cyclic GMP-dependent protein kinase activates cloned BKCa channels expressed in mammalian cells by direct phosphorylation at serine 1072

NO-induced activation of cGMP-dependent protein kinase (PKG) increases the open probability of large conductance Ca2+-activated K+ channels and results in smooth muscle relaxation. However, the molecular mechanism of channel regulation by the NO-PKG pathway has not been determined on cloned channels. The present study was designed to clarify PKG-mediated modulation of channels at the molecular level. The cDNA encoding the alpha-subunit of the large conductance Ca2+-activated K+ channel, cslo-alpha, was expressed in HEK293 cells. Whole cell and single channel characteristics of cslo-alpha exhibited functional features of native large conductance Ca2+-activated K+ channels in smooth muscle cells. The NO-donor sodium nitroprusside increased outward current 2.3-fold in whole cell recordings. In cell-attached patches, sodium nitroprusside increased the channel open probability (NPo) of cslo-alpha channels 3.3-fold without affecting unitary conductance. The stimulatory effect of sodium nitroprusside was inhibited by the PKG-inhibitor KT5823. Direct application of PKG-Ialpha to the cytosolic surface of inside-out patches increased NPo 3.2-fold only in the presence of ATP and cGMP without affecting unitary conductance. A point mutation of cslo-alpha in which Ser-1072 (the only optimal consensus sequence for PKG phosphorylation) was replaced by Ala abolished the PKG effect on NPo in inside-out patches and the effect of SNP in cell attached patches. These results indicate that PKG activates cslo-alpha by direct phosphorylation at serine 1072.     

Identification and characterization of a mandelamide hydrolase and an NAD(P)+-dependent benzaldehyde dehydrogenase from Pseudomonas putida ATCC 12633

The enzymes of the mandelate metabolic pathway permit Pseudomonas putida ATCC 12633 to utilize either or both enantiomers of mandelate as the sole carbon source. The genes encoding the mandelate pathway were found to lie on a single 10.5-kb restriction fragment. Part of that fragment was shown to contain the genes coding for mandelate racemase, mandelate dehydrogenase, and benzoylformate decarboxylase arranged in an operon. Here we report the sequencing of the remainder of the restriction fragment, which revealed three further open reading frames, denoted mdlX, mdlY, and mdlD. All were transcribed in the opposite direction from the genes of the mdlABC operon. Sequence alignments suggested that the open reading frames encoded a regulatory protein (mdlX), a member of the amidase signature family (mdlY), and an NAD(P)(+)-dependent dehydrogenase (mdlD). The mdlY and mdlD genes were isolated and expressed in Escherichia coli, and the purified gene products were characterized as a mandelamide hydrolase and an NAD(P)(+)-dependent benzaldehyde dehydrogenase, respectively.     

Mechanism-based inactivation of thioredoxin reductase from Plasmodium falciparum by Mannich bases. Implication for cytotoxicity

Thioredoxin reductase (TrxR) is the homodimeric flavoenzyme that catalyzes reduction of thioredoxin disulfide (Trx). For Plasmodium falciparum, a causative agent of tropical malaria, TrxR is an essential protein which has been validated as a drug target. The high-throughput screening of 350000 compounds has identified Mannich bases as a new class of TrxR mechanism-based inhibitors. During catalysis, TrxR conducts reducing equivalents from the NADPH-reduced flavin to Trx via the two redox-active cysteine pairs, Cys88-Cys93 and Cys535'-Cys540', referred to as N-terminal and C-terminal cysteine pairs. The structures of unsaturated Mannich bases suggested that they could act as bisalkylating agents leading to a macrocycle that involves both C-terminal cysteines of TrxR. To confirm this hypothesis, different Mannich bases possessing one or two electrophilic centers were synthesized and first studied in detail using glutathione as a model thiol. Michael addition of glutathione to the double bond of an unsaturated Mannich base (3a) occurs readily at physiological pH. Elimination of the amino group, promoted by base-catalyzed enolization of the ketone, is followed by addition of a second nucleophile. The intermediate formed in this reaction is an alpha,beta-unsaturated ketone that can react rapidly with a second thiol. When studying TrxR as a target of Mannich bases, we took advantage of the fact that the charge-transfer complex formed between the thiolate of Cys88 and the flavin in the reduced enzyme can be observed spectroscopically. The data show that it is the C-terminal Cys 535'-Cys540' pair rather than the N-terminal Cys88-Cys93 pair that is modified by the inhibitor. Although alkylated TrxR is unable to turn over its natural substrate Trx, it can reduce low M(r) electron acceptors such as methyl methanethiolsulfonate by using its unmodified N-terminal thiols. On the basis of results with chemically distinct Mannich bases, a detailed mechanism for the inactivation of TrxR is proposed.     

Coordinating proliferation and tissue specification to promote regional identity in the Drosophila head

The Decapentaplegic and Notch signaling pathways are thought to direct regional specification in the Drosophila eye-antennal epithelium by controlling the expression of selector genes for the eye (Eyeless/Pax6, Eyes absent) and/or antenna (Distal-less). Here, we investigate the function of these signaling pathways in this process. We find that organ primordia formation is indeed controlled at the level of Decapentaplegic expression but critical steps in regional specification occur earlier than previously proposed. Contrary to previous findings, Notch does not specify eye field identity by promoting Eyeless expression but it influences eye primordium formation through its control of proliferation. Our analysis of Notch function reveals an important connection between proliferation, field size, and regional specification. We propose that field size modulates the interaction between the Decapentaplegic and Wingless pathways, thereby linking proliferation and patterning in eye primordium development.     

Susceptibility to ozone-induced acute lung injury in iNOS-deficient mice

Mice deficient in inducible nitric oxide synthase (iNOS; C57Bl/6Ai-[KO]NOS2 N5) or wild-type C57Bl/6 mice were exposed to 1 part/million of ozone 8 h/night or to filtered air for three consecutive nights. Endpoints measured included lavagable total protein, macrophage inflammatory protein (MIP)-2, matrix metalloproteinase (MMP)-9, cell content, and tyrosine nitration of whole lung proteins. Ozone exposure caused acute edema and an inflammatory response in the lungs of wild-type mice, as indicated by significant increases in lavage protein content, MIP-2 and MMP-9 content, and polymorphonuclear leukocytes. The iNOS knockout mice showed significantly greater levels of lung injury by all of these criteria than did the wild-type mice. We conclude that iNOS knockout mice are more susceptible to acute lung damage induced by exposure to ozone than are wild-type C57Bl/6 mice and that protein nitration is associated with the degree of inflammation and not dependent on iNOS-derived nitric oxide.     

Defining the mandate of proteomics in the post-genomics era: workshop report

Research in proteomics is the next step after genomics in understanding life processes at the molecular level. In the largest sense proteomics encompasses knowledge of the structure, function and expression of all proteins in the biochemical or biological contexts of all organisms. Since that is an impossible goal to achieve, at least in our lifetimes, it is appropriate to set more realistic, achievable goals for the field. Up to now, primarily for reasons of feasibility, scientists have tended to concentrate on accumulating information about the nature of proteins and their absolute and relative levels of expression in cells (the primary tools for this have been 2D gel electrophoresis and mass spectrometry). Although these data have been useful and will continue to be so, the information inherent in the broader definition of proteomics must also be obtained if the true promise of the growing field is to be realized. Acquiring this knowledge is the challenge for researchers in proteomics and the means to support these endeavors need to be provided. An attempt has been made to present the major issues confronting the field of proteomics and two clear messages come through in this report. The first is that the mandate of proteomics is and should be much broader than is frequently recognized. The second is that proteomics is much more complicated than sequencing genomes. This will require new technologies but it is highly likely that many of these will be developed. Looking back 10 to 20 years from now, the question is: Will we have done the job wisely or wastefully? This report summarizes the presentations made at a symposium at the National Academy of Sciences on February 25, 2002.     

Homeokinesis and short-term variability of human airway caliber.

We hypothesized that short-term variation in airway caliber could be quantified by frequency distributions of respiratory impedance (Zrs) measured at high frequency. We measured Zrs at 6 Hz by forced oscillations during quiet breathing for 15 min in 10 seated asthmatic patients and 6 normal subjects in upright and supine positions before and after methacholine (MCh). We plotted frequency distributions of Zrs and calculated means, skewness, kurtosis, and significance of differences between normal and log-normal frequency distributions. The data were close to, but usually significantly different from, a log-normal frequency distribution. Mean lnZrs in upright and supine positions was significantly less in normal subjects than in asthmatic patients, but not after MCh and MCh in the supine position. The lnZrs SD (a measure of variation), in the upright position and after MCh was significantly less in normal subjects than in asthmatic patients, but not in normal subjects in the supine position and after MCh in the supine position. We conclude that 1) the configuration of the normal tracheobronchial tree is continuously changing and that this change is exaggerated in asthma, 2) in normal lungs, control of airway caliber is homeokinetic, maintaining variation within acceptable limits, 3) normal airway smooth muscle (ASM) when activated and unloaded closely mimics asthmatic ASM, 4) in asthma, generalized airway narrowing results primarily from ASM activation, whereas ASM unloading by increasing shortening velocity allows faster caliber fluctuations, 5) activation moves ASM farther from thermodynamic equilibrium, and 6) asthma may be a low-entropy disease exhibiting not only generalized airway narrowing but also an increased appearance of statistically unlikely airway configurations.