L-thiocitrulline. A stereospecific, heme-binding inhibitor of nitric-oxide synthases

Nitric-oxide synthase (NOS) catalyzes the oxidation of L-arginine to citrulline and nitric oxide (NO). The enzyme is inhibited by a variety of N omega-monosubstituted L-arginine analogs, and some of these compounds are useful in reversing pathologies associated with the overproduction of NO (e.g. the hypotension of septic shock). We report here that L-thiocitrulline (gamma-thioureido-L-norvaline) is a potent, stereospecific inhibitor of the constitutive brain and endothelial isoforms of NOS as well as the isoform induced in vascular smooth muscle cells by lipopolysaccharide and interferon-gamma. Steady state kinetic studies show L-thiocitrulline inhibition is competitive with L-arginine (Ki approximately 4-20% of KArgm), indicating that initial binding is as a substrate/product analog. In contrast to L-arginine and N omega-methyl-L-arginine, the prototypic NOS inhibitor, L-thiocitrulline binding elicits a "Type II" difference spectrum, indicating a high spin to low spin transition of the iron in the heme cofactor. This finding suggests that L-thiocitrulline is contributing the sixth ligand to heme iron, probably through the thioureido sulfur. Such interaction with heme iron neither stimulates nor inhibits the direct flavin-mediated cytochrome c reduction activity of the enzyme, but it does inhibit heme-dependent superoxide formation. In vivo, L-thiocitrulline is a potent pressor agent in both normal and endotoxemic rats, the latter finding suggesting utility in treating the hypotension of septic shock.     

Synergistic cooperation between phorbol ester and IFN-gamma for induction of nitric oxide synthesis in murine peritoneal macrophages

The role of protein kinase C (PKC) in the induction of nitric oxide (NO) synthesis in murine peritoneal macrophages was examined. Phorbol ester, a PKC activator, had no effect on NO synthesis by itself, whereas IFN-gamma alone had modest activity. When phorbol ester was used in combination with IFN-gamma, there was a marked cooperative induction of NO synthesis in a dose-dependent manner. This increase in NO synthesis was reflected as increased amount of inducible NO synthase (iNOS) mRNA, as determined by Northern blotting. The optimal effect of phorbol ester was shown at 6 h after treatment with IFN-gamma. Phorbol ester also induced the release of NO to the incubation medium by bacillus Calmette-Guerin-infected peritoneal macrophages. Prolonged incubation of cells with phorbol ester, which down-regulates PKC activity, abolished the synergistic cooperative effect on NO production with IFN-gamma. In addition, such PKC inhibitors as staurosporin or polymyxin B reduced NO production induced by IFN-gamma plus phorbol ester. When the cells were treated with both actinomycin D and phorbol ester after IFN-gamma stimulation, more NO was produced and more iNOS mRNA was expressed than in the cells treated with actinomycin D alone. On the basis of these observations, we conclude that PKC might not be directly involved in the expression of NO synthase, but, instead, might be involved in the stabilization of the iNOS mRNA already expressed by the treatment of IFN-gamma.     

Truncating the putative membrane association region circumvents the difficulty of expressing hepatitis C virus protein E1 in Escherichia coli

The putative E1 of hepatitis C virus (HCV) was expressed in Escherichia coli using a glutathione-S-transferase (GST) fusion protein system. The full length E1 protein is difficult to express. A series of E1 DNA fragments was generated and used for expression vector construction. Fusion proteins containing the E1 C-terminal region could not be expressed. When this region was truncated, the fusion proteins were synthesized to high levels. The possibility of this C-terminal region hampering the production of fusion protein was further explored. A construct with this segment directly fused to the C-terminus of GST indeed generated no detectable recombinant protein. According to the predicted structure of E1, this region may have membrane-associating properties. The expression results suggest a general approach to facilitate the production of viral membrane proteins in prokaryotes. Furthermore, these recombinant E1 proteins generated as antigens were used for Western blotting with sera from HCV-infected individuals. It was found that E1 is antigenic during HCV natural infection.     

Crevicular fluid enzymes from endosseous dental implants and natural teeth

Several neutrophil-derived enzymes that are present in the gingival crevicular fluid have been evaluated for use as risk markers for periodontal disease progression. However, very little information is available about the presence of these enzymes in peri-implant tissues. The purpose of this cross-sectional study was to compare levels of enzymes in gingival crevicular fluid between natural teeth and endosseous dental implants and between well-integrated and failing implants. Scores of plaque and gingivitis were recorded for 68 integrated implants, five failing implants, and 34 natural teeth in 12 completely edentulous and 18 partially edentulous subjects. Samples of gingival crevicular fluid were obtained from these sites using filter paper strips and were assayed for levels of neutral protease, neutrophil elastase, myeloperoxidase, and beta-glucuronidase. Neutral protease levels were higher (P = .066) at moderately to severely inflamed implant sites (Gingival Index of 2, 3) compared to mildly or noninflamed sites (Gingival Index of = 0, 1). Despite the small number (n = 5) of failing implants evaluated in this study, levels of neutrophil elastase, myeloperoxidase, and beta-glucuronidase were significantly higher (P < or = .001) around failing implants compared to successful implants. Neutral protease levels were also elevated around failing implants, but the difference was not statistically significant. Results of this study indicate that neutrophil elastase, myeloperoxidase, and beta-glucuronidase levels in GCF appear to be good candidates for study as risk markers of implant failure.     

Survivorship analysis of cemented total hip arthroplasty acetabular components implanted with second-generation techniques

The clinical and radiographic results of primary cemented total hip arthroplasty performed by a single surgeon, with particular emphasis on the performance of acetabular components implanted with so-called second-generation cement techniques, were studied. Seventy hips with 48 metal-backed and 22 polyethylene acetabular components were followed for a mean of 9 years (range, 5-11.5 years). The clinical results were evaluated using a recognized hip score. The fixation status of the cemented acetabular component was evaluated using two methods of measuring radiolucent lines at 5 years and at the last evaluation. Acetabular component loosening was defined as a circumferential radiolucent line, component migration, or revision for loosening. This study was unable to confirm the findings of others that demonstrated higher failure rates with cemented metal-backed components when compared with all-polyethylene components. The survival of cemented acetabular components with 28-mm head femoral prostheses was worse than the survival of cemented acetabular components with 22-mm femoral heads in other published reports, despite advances in cement techniques. Because of the high rate of loosening of cemented 28-mm-inner-diameter acetabular components at 5 and 10 years, the authors no longer use these cemented components for acetabular reconstruction.     

Importance of participation rate in sampling of data in population based studies, with special reference to bone mass in Sweden

OBJECTIVE: To study the effects of participation rate in sampling on normative bone mass data. DESIGN: This was a comparison between two randomly selected samples from the same population. The participation rates in the two samples were 61.9% and 83.6%. Measurements were made of bone mass at different skeletal sites and of muscle strength, as well as an assessment of physical activity. SETTING: Malm?, Sweden. SUBJECTS: There were 230 subjects (117 men, 113 women), aged 21 to 42 years. RESULTS: Many subjects participated in both studies (163). Those who took part only in the study with the higher participation rate (67) almost invariably had higher values for bone mass density at the sites measured (up to 7.6% for men) than participants in the study with the lower participation rate. No differences in muscle strength were recorded. CONCLUSION: A high degree of compliance is important to achieve a reliable result in determining normal values in population based studies.     

The catalytic core of RNase P

A deletion mutant of the catalytic RNA component of Escherichia coli RNase P missing residues 87-241 retains the ability to interact with the protein component to form a functional catalyst. The deletion of this phylogenetically conserved region significantly increases the Km, indicating that the deleted structures may be important for binding to the precursor tRNA substrate but not for the cleavage reaction. Under some reaction conditions, this RNase P deletion mutant can become a relatively non-specific nuclease, indicating that this RNA's catalytic center may be more exposed. The catalytic core of the RNase P is formed by less than one third of the 377 residues of the RNase P RNA.     

Effects of Na+ and Mg2+ on the structures of supercoiled DNAs: comparison of simulations with experiments

Recent cryo-electron microscopy (cryo-EM) results suggest that sufficient NaCl concentration (> or approximately 0.1 M) and superhelix density (> or approximately-0.05) cause circular DNAs to adopt highly extended, tightly interwound configurations, in which the strands are laterally contiguous along almost their entire length. Millimolar levels of MgCl2 reportedly act synergistically with NaCl to produce similar conformations. However, Monte Carlo simulations with purely repulsive interduplex forces failed to reproduce such structures. In the present work, solution measurements of particular physical properties were performed both to characterize the effects of Na+ and Mg2+ on DNA structure and to provide quantitative tests of Monte Carlo simulations of circular DNAs. Supercoiled p30 delta DNAs in 10 mM Tris plus 0, 0.122, and 0.1 M NaCl, and 0.1 M NaCl plus 4 mM Mg2+ were examined by static and dynamic light scattering (LS and DLS), time-resolved fluorescence polarization anisotropy (FPA) of intercalated ethidium, and circular dichroism (CD) spectroscopy. Upon addition of 0.122 M NaCl, the radius of gyration (Rg) decreased substantially, which indicates that p30 delta adopts a more compact structure. This contradicts the cryo-EM studies, where molecular extension and Rg both increase upon adding 0.1 M NaCl. In 0.1 M NaCl, the torsion constant measured by FPA is practically invariant to superhelix density, and the plateau diffusion coefficient at large scattering vector (Dplat) is likewise nearly the same at both relaxed and native superhelix densities. Such invariance is difficult to reconcile with any transition from relaxed circles to tightly interwound structures with laterally contiguous strands. Metropolis Monte Carlo simulations were performed to generate canonically distributed sets of structures, from which average Do values and scattered intensity ratios, [symbol: see text]I (zero) [symbol: see text]/[symbol: see text] l(k) [symbol: see text], were calculated. Agreement between simulations and experiments in regard to [symbol: see text] I(O) [symbol: see text] /[symbol: see text] I(k) [symbol: see text], D(zero) and the supercoiling free energy, delta Gsc (delta l), is remarkably good for the most extensively studied p30 delta samples. The simulated structures exhibit no sign of very tight interwinding with extensive lateral contacts, but instead exhibit most probable superhelix diameters of 85 to 90 A. When 4 mM Mg2+ was added to native supercoiled p30 delta in 0.1 M NaCl, Rg decreased, D(zero) increased, and the longest internal relaxation rate (1/tau 2(zero)) increased, all of which indicate a further overall contraction of the molecular envelope. The torsion constant exhibited a slight increase that is hardly statistically significant. In this case, agreement between the simulations and experiments was only semi-quantitative for most samples investigated, although the predicted contraction was exhibited by all five samples of p30 delta and one of pBR322 DNA. The simulated structures in 0.1 M NaCl plus 4 mM Mg2+ again showed no sign of extensive lateral contacts. A plausible explanation is proposed for the highly extended, tightly interwound structures seen in cryo-EM, and explicitly tested by Monte Carlo simulations of a 1000 bp circular DNA at +25 and -50 degrees C. Structures identical to those seen in cryo-EM are in fact the equilibrium structures in the simulations at -50 degrees C, and the estimated time for equilibration (2.3 x 10(-6) second) is much smaller than the estimated time for vitrification (1 x 10(-4) second).