Selection of membrane protein targets for crystallization using PFO-PAGE electrophoresis

A method to rapidly assess the oligomeric composition of multimeric proteins is notably absent from reported schemes for high throughput production and crystallization of membrane proteins. In this report we have investigated the suitability of PFO-PAGE electrophoresis for this purpose and present examples where it proves highly informative in selecting conditions favouring the functional oligomeric state of the target protein. Features such as the ability to analyze several samples in parallel, including crude membrane extracts, suggest it will be highly adaptable to high throughput analysis of membrane proteins.     

The use of an arbitrarily primed PCR product for the specific detection of Brucella

A 1.3 kb Brucella-specific DNA fragment produced through the use of arbitrarily primed polymerase chain reaction (AP-PCR) was tested for its specificity by DNA–DNA hybridization to Brucella and non-Brucella bacteria. The digoxigenin (DIG)-labelled 1.3 kb DNA fragment hybridized with Brucella abortus and Brucella melitensis but did not hybridize with other non-Brucella bacteria tested. The sensitivity of the reaction was determined; as little as 150 fg DNA or 30 Brucella cells could be detected. The specificity and sensitivity of the 1.3 kb DNA fragment combined with the simplicity and speed of the technique suggest the potential of this fragment as a DNA probe for the quick and reliable detection of Brucella organisms.     

Effect of herbizid and touchdown herbicides on soil fungi and on production of some extracellular enzymes

Glucophilic and cellulose-decomposing fungi were significantly reduced in soil samples treated with 0.019-0.152 mg a.i./kg soil of the herbicides Herbizid and Touchdown. The decrease was regularly correlated with the doses of the two herbicides and persisted till the end of the experiment (12 weeks). The isolated fungi were found to be able to produce hydrolytic extracellular enzymes in solid media but with variable capabilities. The ability to produce enzymes was adversily affected by the incorporation of herbicides in culture media. Lower doses of herbicides were occasionally promotive to enzyme production and mycelial growth of some fungi. Incorporation of 50 ppm of Herbizid and Touchdown significantly activated amylase production and mycelial dry weight in cultures of Fusarium oxysporum, Mucor hiemalis and Penicillium chrysogenum. There was a significant increase in C1-cellulase produced by F. oxysporum and P. aurantiogriseum when cultures were treated with 50, 100 and 200 ppm of Herbizid which induced also more Cx-cellulase production by P. chrysogenum. Lipase and protease production was always lower in treated than in control fungal cultures.     

The PcrA3 mutant binds DNA and interacts with the RepC initiator protein of plasmid pT181 but is defective in its DNA helicase and unwinding activities

Plasmid rolling-circle replication initiates by covalent extension of a nick generated at the plasmid double-strand origin (dso) by the initiator protein. The RepC initiator protein binds to the plasmid pT181 dso in a sequence-specific manner and recruits the PcrA helicase through a protein-protein interaction. Subsequently, PcrA unwinds DNA at the nick site followed by replication by DNA polymerase III. The pcrA3 mutant of Staphylococcus aureus has previously been shown to be defective in plasmid pT181 replication. Suppressor mutations in the repC initiator gene have been isolated that allow pT181 replication in the pcrA3 mutant. One such suppressor mutant contains a D57Y change in the RepC protein. To identify the nature of the defect in PcrA3, we have purified this mutant protein and studied its biochemical activities. Our results show that while PcrA3 retains its DNA binding activity, it is defective in its helicase and RepC-dependent pT181 DNA unwinding activities. We have also purified the RepC D57Y mutant and shown that it is similar in its biochemical activities to wild-type RepC. RepC D57Y supported plasmid pT181 replication in cell-free extracts made from wild-type S. aureus but not from the pcrA3 mutant. We also demonstrate that both wild-type RepC and its D57Y mutant are capable of a direct physical interaction with both wild-type PcrA and the PcrA3 mutant. Our results suggest that the inability of PcrA3 to support pT181 replication is unlikely to be due to its inability to interact with RepC. Rather, it is likely that a defect in its helicase activity is responsible for its inability to replicate the pT181 plasmid.     

Clinico-hormonal correlation of oligospermic patients in the below sea level environment (Jordan Valley)

A correlation between serum levels of luteinizing hormone (LH), total testosterone (T), free T and sex-hormone binding globulin (SHBG) in normospermic and in oligospermic male people was done. This study was designed to measure serum levels of these hormones and of SHBG in people living at different altitude environments relative to sea level: at 209-408 meters below (the Jordan Valley, JV) and at 620 meters above (Irbid city, IC). In addition, a clinical awareness study of oligospermia was done in the North of Jordan (IC). Seminal analysis in 287 male people (age range, 18 to 40 years old) during the period between 12/6/1999 and 12/2/2002 showed an oligospermia of 31.4%. Serum levels of LH, total T, free T and SHBG in normospermic subjects in IC were similar to those in normospermic of the JV (3.4 +/- 1.2 vs. 4.0 +/- 1.7 MIU/ml, 19.9 +/- 4.0 vs. 20.4 +/- 5.6 ng/ml, 53.9 +/- 15.6 vs. 47.9 +/- 10.7 pg/ml, 19.5 +/- 3.2 vs. 18.6 +/- 2.16 nmol/l, respectively). Oligospermia was associated with increase in total T at both IC (27.5 +/- 4.6 vs. 19.9 +/- 4.0 ng/ml) and the JV (30.7 +/- 3.4 vs. 20.5 +/- 5.6). The higher serum level of total T in oligospermic people in both IC and the JV was associated with higher levels of SHBG compared to those levels in normospermic subjects. On the other hand, oligospermic subjects have lower serum level of free T than in normospermic males (41.5 +/- 10.0 vs. 53.9 +/- 15.6) only in IC, while in the JV, serum free T level was similar (46.5 +/- 6.1 vs. 47.9 +/- 10.7). Taken together data for both locations, IC and the JV, suggest a clear correlation between total T and SHBG levels in both groups' normospermic and oligospermic subjects.     

Sphingosine kinase 2 is a nuclear protein and inhibits DNA synthesis

Sphingosine kinase-1 (SPHK1) is a key enzyme catalyzing the formation of an important bioactive lipid messenger, sphingosine 1-phosphate, and is implicated in the regulation of cell proliferation and antiapoptotic processes. Biological features of another isozyme SPHK2, however, remain unclear. The present studies were undertaken to characterize SPHK2 by comparison with SPHK1. When SPHK2 was transiently expressed in various cell lines, it was localized in the nuclei as well as in the cytosol, whereas SPHK1 was distributed in the cytosol but not in the nucleus. We have mapped a functional nuclear localization signal (NLS) to the N-terminal region of SPHK2. We have observed that the expression of SPHK2 in various cell types causes inhibition of DNA synthesis, resulting in the cell cycle arrest at G1/S phase. We have also demonstrated that an NLS mutant of SPHK2, SPHK2R93E/R94E, failed to enter the nucleus and to inhibit DNA synthesis. Moreover, a fusion protein, NLS-SPHK1, where SPHK1 was fused to the NLS sequence of SPHK2 acquired the ability to enter nuclei and inhibited DNA synthesis. These results indicate that SPHK2 localizes in the nuclei and causes inhibition of DNA synthesis, and this may affect subsequent cellular events.     

Kinetic and structural basis of reactivity of pentaerythritol tetranitrate reductase with NADPH, 2-cyclohexenone, nitroesters, and nitroaromatic explosives

The reaction of pentaerythritol tetranitrate reductase with reducing and oxidizing substrates has been studied by stopped-flow spectrophotometry, redox potentiometry, and X-ray crystallography. We show in the reductive half-reaction of pentaerythritol tetranitrate (PETN) reductase that NADPH binds to form an enzyme-NADPH charge transfer intermediate prior to hydride transfer from the nicotinamide coenzyme to FMN. In the oxidative half-reaction, the two-electron-reduced enzyme reacts with several substrates including nitroester explosives (glycerol trinitrate and PETN), nitroaromatic explosives (trinitrotoluene (TNT) and picric acid), and alpha,beta-unsaturated carbonyl compounds (2-cyclohexenone). Oxidation of the flavin by the nitroaromatic substrate TNT is kinetically indistinguishable from formation of its hydride-Meisenheimer complex, consistent with a mechanism involving direct nucleophilic attack by hydride from the flavin N5 atom at the electron-deficient aromatic nucleus of the substrate. The crystal structures of complexes of the oxidized enzyme bound to picric acid and TNT are consistent with direct hydride transfer from the reduced flavin to nitroaromatic substrates. The mode of binding the inhibitor 2,4-dinitrophenol (2,4-DNP) is similar to that observed with picric acid and TNT. In this position, however, the aromatic nucleus is not activated for hydride transfer from the flavin N5 atom, thus accounting for the lack of reactivity with 2,4-DNP. Our work with PETN reductase establishes further a close relationship to the Old Yellow Enzyme family of proteins but at the same time highlights important differences compared with the reactivity of Old Yellow Enzyme. Our studies provide a structural and mechanistic rationale for the ability of PETN reductase to react with the nitroaromatic explosive compounds TNT and picric acid and for the inhibition of enzyme activity with 2,4-DNP.     

Purification and characterization of the PcrA helicase of Bacillus anthracis

PcrA is an essential helicase in gram-positive bacteria, and a gene encoding this helicase has been identified in all such organisms whose genomes have been sequenced so far. The precise role of PcrA that makes it essential for cell growth is not known; however, PcrA does not appear to be necessary for chromosome replication. The pcrA gene was identified in the genome of Bacillus anthracis on the basis of its sequence homology to the corresponding genes of Bacillus subtilis and Staphylococcus aureus, with which it shares 76 and 72% similarity, respectively. The pcrA gene of B. anthracis was isolated by PCR amplification and cloning into Escherichia coli. The PcrA protein was overexpressed with a His6 fusion at its amino-terminal end. The purified His-PcrA protein showed ATPase activity that was stimulated in the presence of single-stranded (ss) DNA (ssDNA). Interestingly, PcrA showed robust 3'-->5' as well as 5'-->3' helicase activities, with substrates containing a duplex region and a 3' or 5' ss poly(dT) tail. PcrA also efficiently unwound oligonucleotides containing a duplex region and a 5' or 3' ss tail with the potential to form a secondary structure. DNA binding experiments showed that PcrA bound much more efficiently to oligonucleotides containing a duplex region and a 5' or 3' ss tail with a potential to form a secondary structure than to those with ssDNAs or duplex DNAs with ss poly(dT) tails. Our results suggest that specialized DNA structures and/or sequences represent natural substrates of PcrA in biochemical processes that are essential for the growth and survival of gram-positive organisms, including B. anthracis.     

Acute pancreatitis,expression of inducible nitric oxide synthase and defective insulin secretion

A high level of nitric oxide (NO) produced by inducible NO synthase (iNOS) is involved in pancreatic beta-cell dysfunction and apoptosis. In the present study, we examined whether iNOS is also expressed in beta cells after induction of acute pancreatitis (AP) in the rat. Pancreatic islets taken from AP animals and incubated for 60 min in the presence of 20.0 mmol/l glucose showed a decreased insulin secretory response to glucose. The basal insulin release at 1.0 mmol/l glucose was also moderately reduced. Experiments on the dynamics of insulin secretion from perfused pancreas revealed an impairment of both first and second phase of glucose-stimulated insulin release after the induction of AP. Confocal microscopy demonstrated that most of the beta cells in pancreas of rat with AP expressed strong immunoreactivity for iNOS. This was further confirmed by biochemical and Western blot analysis that showed a marked increase in iNOS protein expression and enzyme activity concomitant with a modest reduction in the cNOS protein and activity. Although the mechanisms underlying the defective insulin secretory response of beta cells seen during the early stage of AP are complex, the present finding suggests that the expression of iNOS and a marked iNOS-derived NO production in the beta cells may play at least a contributory role in the impairment of beta-cell function.This study was supported by the Swedish Medical Research Council (14X-4286), the Swedish Diabetes Association, and the Crafoord, Albert Påhlsson and Magnus Bergvall Foundations