Structure of horse carbonmonoxyhaemoglobin

The structure is based on a difference Fourier synthesis at 2.8 Å resolution, using observed structure amplitudes and calculated phases, derived from a refinement of horse methaemoglobin at 2.0 Å resolution. Carbonmonoxyhaemoglobin has the same quaternary structure as methaemoglobin, but differs from it by slight changes in tertiary structure in the immediate vicinity of the haems. On transition from met- to carbonmonoxyhaemoglobin the distal histidines move away from the haem ligands towards the molecular surface, and both the haems and F-helices rotate slightly and shift towards the distal side. In methaemoglobin the sulphydryl group of cysteine F9(93)β is in equilibrium between two alternative positions: one external and the other half-buried in the “tyrosine pocket” between helices F and H. In carbonmonoxyhaemoglobin all the electron density for the sulphydryl group is in the half-buried position, so that the side chain of tyrosine HC2(145)β is completely displaced from its pocket. The difference map shows that the CO oxygen lies off the haem axis in both subunits, but the carbon cannot be seen as it coincides with the water molecule in methaemoglobin. A preliminary refinement of carbonmonoxyhaemoglobin suggests that the carbon may be displaced from the haem axis in the same direction as the oxygen. The haem pocket is so constructed that it fits an oxygen molecule in the bent conformation, but not a CO molecule which has its axis normal to the haem plane, because of steric hindrance by N_? of the distal histidine and by C_γ2 of the distal valine. These two side chains apparently push the CO oxygen off the haem axis. The difference map indicates that in methaemoglobin the α-haem is ruffled and that on transition from met- to carbonmonoxyhaemoglobin it becomes flattened; in the β-haem the iron appears to move towards the porphyrin plane. The resolution is not sufficient to determine the exact position of the iron atoms and the proximal histidines relative to the porphyrins.     

Structural basis of thermostability. Analysis of stabilizing mutations in subtilisin BPN'

The crystal structures of two thermally stabilized subtilisin BPN' variants, S63 and S88, are reported here at 1.8 and 1.9 A resolution, respectively. The micromolar affinity calcium binding site (site A) has been deleted (Delta75-83) in these variants, enabling the activity and thermostability measurements in chelating conditions. Each of the variants includes mutations known previously to increase the thermostability of calcium-independent subtilisin in addition to new stabilizing mutations. S63 has eight amino acid replacements: D41A, M50F, A73L, Q206W, Y217K, N218S, S221C, and Q271E. S63 has 75-fold greater stability than wild type subtilisin in chelating conditions (10 mm EDTA). The other variant, S88, has ten site-specific changes: Q2K, S3C, P5S, K43N, M50F, A73L, Q206C, Y217K, N218S, and Q271E. The two new cysteines form a disulfide bond, and S88 has 1000 times greater stability than wild type subtilisin in chelating conditions. Comparisons of the two new crystal structures (S63 in space group P2(1) with A cell constants 41.2, 78.1, 36.7, and beta = 114.6 degrees and S88 in space group P2(1)2(1)2(1) with cell constants 54.2, 60.4, and 82.7) with previous structures of subtilisin BPN' reveal that the principal changes are in the N-terminal region. The structural bases of the stabilization effects of the new mutations Q2K, S3C, P5S, D41A, Q206C, and Q206W are generally apparent. The effects are attributed to the new disulfide cross-link and to improved hydrophobic packing, new hydrogen bonds, and other rearrangements in the N-terminal region.     

Hip joint replacement surgery for idiopathic osteoarthritis aggregates in families

In order to determine whether there is a genetic component to hip or knee joint failure due to idiopathic osteoarthritis (OA), we invited patients (probands) undergoing hip or knee arthroplasty for management of idiopathic OA to provide detailed family histories regarding the prevalence of idiopathic OA requiring joint replacement in their siblings. We also invited their spouses to provide detailed family histories about their siblings to serve as a control group. In the probands, we confirmed the diagnosis of idiopathic OA using American College of Rheumatology criteria. The cohorts included the siblings of 635 probands undergoing total hip replacement, the siblings of 486 probands undergoing total knee replacement, and the siblings of 787 spouses. We compared the prevalence of arthroplasty for idiopathic OA among the siblings of the probands with that among the siblings of the spouses, and we used logistic regression to identify independent risk factors for hip and knee arthroplasty in the siblings. Familial aggregation for hip arthroplasty, but not for knee arthroplasty, was observed after controlling for age and sex, suggesting a genetic contribution to end-stage hip OA but not to end-stage knee OA. We conclude that attempts to identify genes that predispose to idiopathic OA resulting in joint failure are more likely to be successful in patients with hip OA than in those with knee OA.     

Structure and function of the phenazine biosynthesis protein PhzF from Pseudomonas fluorescens 2-79

Phenazines, including pyocyanin and iodonin, are biologically active compounds that are believed to confer producing organisms with a competitive growth advantage, and also are thought to be virulence factors in certain diseases including cystic fibrosis. The basic, tricyclic phenazine ring system is synthesized in a series of poorly characterized steps by enzymes encoded in a seven-gene cistron in Pseudomonas and other organisms. Despite the biological importance of these compounds, and our understanding of their mode of action, the biochemistry and mechanisms of phenazine biosynthesis are not well resolved. Here we report the 1.8 A crystal structure of PhzF, a key enzyme in phenazine biosynthesis, solved by molecular replacement. PhzF is structurally similar to the lysine biosynthetic enzyme diaminopimelate epimerase, sharing an unusual fold consisting of two nearly identical domains with the active site located in an occluded cleft between the domains. Unlike diaminopimelate epimerase, PhzF is a dimer in solution. The two apparently independent active sites open toward opposite sides of the dimer and are occupied by sulfate ions in the structure. In vitro experiments using a mixture of purified PhzF, -A, -B, and -G confirm that phenazine-1-carboxylic acid (PCA) is readily produced from trans-2,3-dihydro-3-hydroxyanthranilic acid (DHHA) without aid of other cellular factors. PhzA, -B, and -G have no activity toward DHHA. However, in the presence of PhzF, individually or in combinations, they accelerate the formation of PCA from DHHA and therefore appear to function after the action of PhzF. Surprisingly, PhzF is itself capable of producing PCA, albeit slowly, from DHHA. These observations suggest that PhzF catalyzes the initial step in the conversion of DHHA to PCA, probably via a rearrangement reaction yielding the more reactive 3-oxo analogue of DHHA, and that subsequent steps can occur spontaneously. A hypothetical model for how DHHA binds to the PhzF active site suggests that Glu45 and Asp208 could act as general acid-base catalysts in a rearrangement reaction. Given that four reactions lie between DHHA and PCA, ketone formation, ring formation, decarboxylation, and oxidation, we hypothesize that the similar PhzA and -B proteins catalyze ring formation and thus may be more than noncatalytic accessory proteins. PhzG is almost certainly an oxidase and is predicted to catalyze the final oxidation/aromatization reaction.     

Monocyte adherence results in selective induction of novel genes sharing homology with mediators of inflammation and tissue repair

Adherence of monocytes to endothelial cells or extracellular matrices is likely to play a critical role in triggering monocyte activation in extravascular sites of infection, chronic inflammatory disorders, tissue damage and neoplastic growth. We have constructed a cDNA library from monocytes adhered for 30 min on plastic and have screened it by differential hybridization for mRNA rapidly induced by adherence. Two of the cDNA isolated have been identified as IL-1 beta and superoxide dismutase. Sequence data from three other adherence specific clones demonstrates the presence of ATTTA mRNA instability sequences in their 3' untranslated regions signifying inflammation-associated genes. The deduced amino acid sequences indicate the presence of open reading frames with sequence homologies to a family of host defense cytokines, one of them being identified as IL-8. Of the 14 clones initially identified, 4 have been analyzed for induction of mRNA expression. Although 3 of the 4 clones were equally induced by PMA and LPS under nonadherent conditions, all 4 cDNA showed distinct patterns of induction with adherence to extracellular matrix components or endothelial cells. The deduced amino acid sequence homologies indicate that we have isolated cDNA that code for three unique gene products. These cDNA belong to a gene family of early host defense cytokines involved in inflammation and cell growth, but which are differentially regulated by adherence to different surfaces.     

Endogenous chloride channels of insect sf9 cells. Evidence for coordinated activity of small elementary channel units

The endogenous Cl- conductance of Spodoptera frugiperda (Sf9) cells was studied 20-35 h after plating out of either uninfected cells or cells infected by a baculovirus vector carrying the cloned beta-galactosidase gene (beta-Gal cells). With the cation Tris+ in the pipette and Na+ in the bath, the reversal potential of whole-cell currents was governed by the prevailing Cl- equilibrium potential and could be fitted by the Goldman-Hodgkin-Katz equation with similar permeabilities for uninfected and beta-Gal cells. In the frequency range 0.12 < f < 300 Hz, the power density spectrum of whole-cell Cl- currents could be fitted by three Lorentzians. Independent of membrane potential, >50% of the total variance of whole-cell current fluctuations was accounted for by the low frequency Lorentzian (fc = 0.40 +/- 0.03 Hz, n = 6). Single-Cl- channels showed complex gating kinetics with long lasting (seconds) openings interrupted by similar long closures. In the open state, channels exhibited fast burst-like closures. Since the patches normally contained more than a single channel, it was not possible to measure open and closed dwell-time distributions for comparing single-Cl- channel activity with the kinetic features of whole-cell currents. However, the power density spectrum of Cl- currents of cell-attached and excised outside-out patches contained both high and low frequency Lorentzian components, with the corner frequency of the slow component (fc = 0.40 +/- 0.02 Hz, n = 4) similar to that of whole-cell current fluctuations. Chloride channels exhibited multiple conductance states with similar Goldman-Hodgkin-Katz-type rectification. Single-channel permeabilities covered the range from approximately 0.6.10(-14) cm5/s to approximately 6.10(-14) cm3/s, corresponding to a limiting conductance (gamma 150/150) of approximately 3.5 pS and approximately 35 pS, respectively. All states reversed near the same membrane potential, and they exhibited similar halide ion selectivity, P1 > PCl approximately PBr. Accordingly, Cl- current amplitudes larger than current flow through the smallest channel unit resolved seem to result from simultaneous open/shut events of two or more channel units.     

Characterization of the genetic diversity of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> in São Paulo city,Brazil

Background

Tuberculosis is a major health problem in São Paulo, Brazil, which is the most populous and one of the most cosmopolitan cities in South America. To characterize the genetic diversity of Mycobacterium tuberculosis in the population of this city, the genotyping techniques of spoligotyping and MIRU were applied to 93 isolates collected in two consecutive years from 93 different tuberculosis patients residing in São Paulo city and attending the Clemente Ferreira Institute (the reference clinic for the treatment of tuberculosis).

Findings

Spoligotyping generated 53 different spoligotype patterns. Fifty-one isolates (54.8%) were grouped into 13 spoligotyping clusters. Seventy- two strains (77.4%) showed spoligotypes described in the international databases (SpolDB4, SITVIT), and 21 (22.6%) showed unidentified patterns. The most frequent spoligotype families were Latin American Mediterranean (LAM) (26 isolates), followed by the T family (24 isolates) and Haarlem (H) (11 isolates), which together accounted for 65.4% of all the isolates. These three families represent the major genotypes found in Africa, Central America, South America and Europe. Six Spoligo-International-types (designated SITs by the database) comprised 51.8% (37/72) of all the identified spoligotypes (SIT53, SIT50, SIT42, SIT60, SIT17 and SIT1). Other SITs found in this study indicated the great genetic diversity of M. tuberculosis, reflecting the remarkable ethnic diversity of São Paulo city inhabitants. The MIRU technique was more discriminatory and did not identify any genetic clusters with 100% similarity among the 93 isolates. The allelic analysis showed that MIRU loci 26, 40, 23 and 10 were the most discriminatory. When MIRU and spoligotyping techniques were combined, all isolates grouped in the 13 spoligotyping clusters were separated.

Conclusions

Our data indicated the genomic stability of over 50% of spoligotypes identified in São Paulo and the great genetic diversity of M. tuberculosis isolates in the remaining SITs, reflecting the large ethnic mix of the São Paulo city inhabitants. The results also indicated that in this city, M. tuberculosis isolates acquired drug resistance independently of genotype and that resistance was more dependent on the selective pressure of treatment failure and the environmental circumstances of patients.

     

Informing the Historical Record of Experimental Nonhuman Primate Infections with Ebola Virus: Genomic Characterization of USAMRIID Ebola Virus/H.sapiens-tc/COD/1995/Kikwit-9510621 Challenge Stock “R4368” and Its Replacement “R4415”

The creation of licensed medical countermeasures against Select Agents such as Ebola virus (EBOV) is critically dependent on the use of standardized reagents, assays, and animal models. We performed full genome reconstruction, population genomics, contaminant analysis, and characterization of the glycoprotein gene editing site of historical United States Army Medical Research Institute of Infectious Diseases (USAMRIID) nonhuman-primate challenge stock Ebola virus Kikwit “R4368” and its 2014 replacement “R4415.” We also provide characterization of the master stock used to create “R4415.” The obtained data are essential to understanding the quality of the seed stock reagents used in pivotal animal studies that have been used to inform medical countermeasure development. Furthermore, these data might add to the understanding of the influence of EBOV variant populations on pathogenesis and disease outcome and inform attempts to avoid the evolution of EBOV escape mutants in response to current therapeutics. Finally, as the primary challenge stocks have changed over time, these data will provide a baseline for understanding and correlating past and future animal study results.