Survival of civilian and prisoner drug-sensitive, multi- and extensive drug- resistant tuberculosis cohorts prospectively followed in Russia

Objective and Methods

A long-term observational study was conducted in Samara, Russia to assess the survival and risk factors for death of a cohort of non-multidrug resistant tuberculosis (non-MDRTB) and multidrug resistant tuberculosis (MDRTB) civilian and prison patients and a civilian extensive drug-resistant tuberculosis (XDRTB) cohort.

Results

MDRTB and XDRTB rates of 54.8% and 11.1% were identified in the region. Half (50%) of MDRTB patients and the majority of non-MDRTB patients (71%) were still alive at 5 years. Over half (58%) of the patients died within two years of establishing a diagnosis of XDRTB. In the multivariate analysis, retreatment (HR = 1.61, 95%CI 1.04, 2.49) and MDRTB (HR = 1.67, 95%CI 1.17, 2.39) were significantly associated with death within the non-MDR/MDRTB cohort. The effect of age on survival was relatively small (HR = 1.01, 95%CI 1.00, 1.02). No specific factor affected survival of XDRTB patients although median survival time for HIV-infected versus HIV-negative patients from this group was shorter (185 versus 496 days). The majority of MDRTB and XDRTB strains (84% and 92% respectively) strains belonged to the Beijing family. Mutations in the rpoB (codon 531 in 81/92; 88.8%), katG (mutation S315T in 91/92, 98.9%) and inhA genes accounted for most rifampin and isoniazid resistance respectively, mutations in the QRDR region of gyrA for most fluroquinolone resistance (68/92; 73.5%).

Conclusions

Alarmingly high rates of XDRTB exist. Previous TB treatment cycles and MDR were significant risk factors for mortality. XDRTB patients'' survival is short especially for HIV-infected patients. Beijing family strains comprise the majority of drug-resistant strains.     

Resolution of two substrate-binding sites in an engineered cytochrome P450eryF bearing a fluorescent probe

To elucidate the mechanisms of cooperativity of cytochrome P450eryF an SH-reactive fluorescent probe was introduced close to the substrate-binding site. Cys-154, the only accessible cysteine, was eliminated by site-directed mutagenesis, and a novel cysteine was substituted for Ser-93 in the B'/C loop. S93C, C154A, C154S, S93C/C154A, and S93C/S154C were characterized in terms of affinity for 1-pyrenebutanol (1-PB), cooperativity, and ionic-strength dependence of the 1-PB-induced spin shift. S93C/C154S retains the key functional properties of the wild-type, and modification by three different SH-reactive probes had little effect on the characteristics of the enzyme. The labeled proteins exhibited fluorescence resonance energy transfer from 1-PB to the label, which allowed us to resolve two substrate-binding events, and to determine the corresponding KD values (KD1 = 1.2 +/- 0.2 microM, KD2 = 9.4 +/- 0.8 microM). Using these values for analysis of the substrate-induced spin transition, we demonstrate that the interactions of P450eryF with 1-PB are consistent with a sequential binding mechanism, where substrate interactions at a higher-affinity site cause a conformational transition crucial for the binding of the second substrate molecule and subsequent spin shift. This transition is apparently associated with an important rearrangement of the system of salt links in the proximity of Cys-154.     

Development of Microsatellite Markers for Japanese Scallop (Mizuhopecten yessoensis) and Their Application to a Population Genetic Study

Abstract The Japanese scallop (Mizuhopecten yessoensis) is one of the main fishery products in Japan, but with the expansion of culture operations of the Japanese scallop, various problems have been encountered including high mortality, poor growth, poor seed production, and so on. Moreover, there is concern that many years of cultivation may have affected the genetic structure of the scallop population. To approach these problems and concerns, we developed microsatellite markers as a molecular tool for population genetic studies. By using 4 microsatellite markers as well as a mitochondrial marker, we investigated the genetic structure of samples from the islands of Hokkaido (14 populations) and Honshu (Tohoku, 3 populations) in Japan, and south Primorye (4 populations) in Russia. All the populations sampled had high genetic diversity (average expected heterozygosity, 0.7011 to 0.7622; haplotype diversity, 0.6090 to 0.8848), and almost all showed a tendency of homozygote excess, which was significant in 2 populations. Hierarchical analysis of molecular variance tests based on the microsatellite and mitochondrial markers indicated that the 3 geographic regions were genetically divergent from one another, with little evidence of divergence within regions. Homogeneity in allele frequency distributions between natural and cultured scallops and allele frequency stability over a period of 2 decades indicated that the culturing operations have probably not had a substantial effect on the genetic structure of the populations.     

Temperature-dependent variations and intraspecies diversity of the structure of the lipopolysaccharide of Yersinia pestis

Yersinia pestis spread throughout the Americas in the early 20th century, and it occurs predominantly as a single clone within this part of the world. However, within Eurasia and parts of Africa there is significant diversity among Y. pestis strains, which can be classified into different biovars (bv.) and/or subspecies (ssp.), with bv. orientalis/ssp. pestis most closely related to the American clone. To determine one aspect of the relatedness of these different Y. pestis isolates, the structure of the lipopolysaccharide (LPS) of four wild-type and one LPS-mutant Eurasian/African strains of Y. pestis was determined, evaluating effects of growth at mammalian (37 degrees C) or flea (25 degrees C) temperatures on the structure and composition of the core oligosaccharide and lipid A. In the wild-type clones of ssp. pestis, a single major core glycoform was synthesized at 37 degrees C whereas multiple core oligosaccharide glycoforms were produced at 25 degrees C. Structural differences occurred primarily in the terminal monosaccharides. Only tetraacyl lipid A was made at 37 degrees C, whereas at 25 degrees C additional pentaacyl and hexaacyl lipid A structures were produced. 4-Amino-4-deoxyarabinose levels in lipid A increased with lower growth temperatures or when bacteria were cultured in the presence of polymyxin B. In Y. pestis ssp. caucasica, the LPS core lacked D-glycero-D-manno-heptose and the content of 4-amino-4-deoxyarabinose showed no dependence on growth temperature, whereas the degree of acylation of the lipid A and the structure of the oligosaccharide core were temperature dependent. A spontaneous deep-rough LPS mutant strain possessed only a disaccharide core and a slightly variant lipid A. The diversity and differences in the structure of the Y. pestis LPS suggest important contributions of these variations to the pathogenesis of this organism, potentially related to innate and acquired immune recognition of Y. pestis and epidemiologic means to detect, classify, control and respond to Y. pestis infections.     

Purification and physical-chemical properties of methanobactin: a chalkophore from Methylosinus trichosporium OB3b

Methanobactin is an extracellular, copper-binding chromopeptide from the methane-oxidizing bacterium, Methylosinus trichosporium OB3b, believed to be involved in copper detoxification, sequestration, and uptake. Although small (1217.2 Da), methanobactin possesses a complex three-dimensional macrocyclic structure with several unusual moieties. The molecule binds one copper and has the N-2-isopropylester-(4-thionyl-5-hydroxyimidazolate)-Gly(1)-Ser(2)-Cys(3)-Tyr(4)-pyrrolidine-(4-hydroxy-5-thionylimidazolate)-Ser(5)-Cys(6)-Met(7) sequence [Kim, H. J., et al. (2004) Science 305, 1612-1615]. We report methods for purifying methanobactin from M. trichosporium OB3b and present initial evidence of its physiological function. MALDI-TOF MS was used to systematically monitor samples for optimizing purification conditions, and for detecting and analyzing specific metal-methanobactin complexes. Purification was performed by first stabilizing the extracted compound with copper followed by separation using reversed-phase HPLC in neutral pH buffers. Purified methanobactin exhibited UV-visible maxima at 342 nm, a shoulder at 388 nm, and a broad peak at 282 nm. These features were lost upon CuCl(2) titration with appearance of new features at 335, 356, 290, and 255 nm. Furthermore, methanobactin contains two fluorescent moieties, which exhibit broad emissions at 440-460 nm (lambda(max)(ex) at 388 nm) and 390-430 nm (lambda(max)(ex) = 342 nm), respectively. Finally, methanobactin eliminates the growth lag in M. trichosporium OB3b and substantially increases growth rates when cultures are exposed to elevated copper levels.     

Brightness of yellow fluorescent protein from coral (zFP538) depends on aggregation

The yellow fluorescent protein from coral (zFP538) forms aggregates in water solutions. According to dynamic light scattering and gel filtration data, the aggregation number is approximately 1000-10000 at pH 8-9 and protein concentration 1 mg/mL. Gel filtration demonstrated that dissociation of the aggregates takes place upon dilution, and the molecular weight of the aggregates decreases with pH. Atomic force microscopy (AFM) and near-field scanning optical microscopy (NSOM) were used to obtain images of zFP538 in the solid state. It was shown that protein films are comprised of fluorescent ellipsoidal granules with a 50-300 nm major axis and a 30-130 nm minor axis. The dependence of zFP538 fluorescence on protein concentration between 1.2 x 10(-)(9) and 5.5 x 10(-)(7) M can be divided in two linear regions with different slopes indicating the existence of at least two different forms of zFP538. The fluorescence of zFP538 decreases with time upon acidification, and the decrease depends on pH and protein concentration. Between pH 3.5 and pH 5.5, relative residual fluorescence is higher for concentrated zFP538 solutions (about 10(-)(6) M) as compared with diluted ones (10(-)(7) M and below). Aggregation makes zFP538 more stable against fluorescence quenching upon acidification: the decrease in zFP538 fluorescence at protein concentration 1 mg/mL is completely reversible, unlike that observed for less concentrated solutions. This phenomenon may be due to the decrease in the freedom of chromophore mobility in zFP538 aggregates.     

Multidrug-resistant tumor cells with decreased malignancy: a role for integrin alphavbeta3

We studied whether acquisition of multidrug resistance (MDR) by tumor cells can alter their integrin profile and malignant behavior. Hamster fibroblast cell line HET-SR-2SC-LNM was selected for MDR, yielding the 2SC/20 subline. Compared with the parental cells, the 2SC/20 subline weakly adhered to denatured collagen (dCol) which correlated with decreased expression of alphavbeta3, a dCol receptor. Importantly, 2SC/20 subline demonstrated significantly decreased activity of collagenase MMP-2, lower ability to invade Matrigel, and attenuated metastasis in syngeneic animals. We provide evidence for the first time that selection for MDR can be associated with down-regulation of alphavbeta3 integrin, supporting our recent proof of the pro-apoptotic role of this integrin (Oncogene 20 (2001) 4710). Lack of alphavbeta3 expression may link cell survival under toxic conditions with decreased malignancy of the resulting drug resistant tumor.