Fluorescence spectroscopy: a rapid tool for assessing tetracycline resistance in Bifidobacterium longum

The tetracycline uptake kinetics of 35 Bifidobacterium longum strains isolated from the human gastrointestinal tract were examined by fluorescence spectroscopy, and the suitability of the technique as a screening tool of tetracycline resistance or susceptibility was determined. The strains were first grouped into three classes based on their corresponding minimum inhibitory concentrations (MICs) of tetracycline, as established by the microdilution method: susceptible (MICs or=32 microg mL(-1)). The kinetics of tetracycline uptake for the strains in each resistance group were then analyzed over a 20 min period by fluorescence spectroscopy (absorbance wavelength 524 nm, excitation wavelength 400 nm) in a buffer system containing 100 microg mL(-1) tetracycline. Principal component analysis and factorial discriminant analysis of the results showed excellent distinction among susceptible, semi-resistant, and resistant strains. The proposed method provides a powerful and convenient means of rapidly screening tetracycline resistance in B. longum.     

The development of an enzyme-linked immunosorbent assay for Trypanosoma vivax antibodies and its use in epidemiological surveys

There are data indicating that the distribution of Trypanosoma vivax in the Brazilian territory is expanding with potential to reach other areas, where the vectors are present. The detection of anti-trypanosomal antibodies in serum provides important information of the trypanosomal status in cattle herds. For this reason, an enzyme-linked immunosorbent assay (Tv-ELISA-Ab) with crude antigen from one Brazilian isolate of T. vivax was developed and evaluated. The sensitivity and specificity were respectively 97.6 and 96.9%. In the evaluation of cross-reactions, three calves inoculated with T. evansi trypimastigotes blood forms showed optical densities (OD) under the cut-off during the whole experimental period, except one at 45 days post-inoculation. With relation to Babesia bovis, B. bigemina, and Anaplasma marginale, which are endemic hemoparasites in the studied area, the cross-reactions were shown to be 5.7, 5.3, and 1.1%, respectively. The first serological survey of Pantanal and state of Pará showed that T. vivax is widespread, although regions within both areas had significantly different prevalences. Therefore, this Tv-ELISA-Ab may be a more appropriate test for epidemiological studies in developing countries because the diagnostic laboratories in most countries may be able to perform an ELISA, which is not true for polymerase chain reaction.     

Expression, purification, and structural analysis of (HIS)UBE2G2 (human ubiquitin-conjugating enzyme)

The ubiquitin system represents a selective mechanism for intracellular proteolysis in eukaryotic cells that involves the sequential activity of three enzymes, ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin-protein ligase (E3). The identification of these proteins and their cellular targets, as well as structural data, are essential to understanding how this system operates in the eukaryotic cell. In the present study, the open reading frame of the human ubiquitin-conjugating enzyme UBE2G2 was isolated from a human brain cDNA panel, cloned into pET28a vector and expressed in Escherichia coli. The His-tagged protein was then purified through nickel-affinity chromatography and subjected to structural and functional studies using circular dichroism (CD) and an in vitro ubiquitin-binding assay, respectively. Our results showed that the production of the HISUBE2G2 protein in bacteria, carried out with 0.1 mM of IPTG at 30 degrees C, was successfully achieved, rendering high concentrations of soluble, pure and stable enzyme after a single purification step. The recombinant protein was able to bind ubiquitin molecules when exposed to a HeLa cell extract during the ubiquitin assay. Moreover, the fact that HISUBE2G2 was expressed in its active form is supported by the typical alpha/beta secondary structure specific to other class I E2 enzymes displayed during the CD assay.     

Evaluation of tumour necrosis factor alpha, interleukin-2 soluble receptor, nitric oxide metabolites, and lipids as inflammatory markers in type 2 diabetes mellitus

This study compared the results of tumour necrosis factor alpha (TNF-alpha), interleukin-2 soluble receptor (sIL-2R), nitric oxide metabolites (NO(x)), C-reactive protein (CRP), and lipids (total cholesterol, high-density lipoprotein (HDL-cholesterol), low-density lipoprotein (LDL-cholesterol), and triglycerides) between control group (nondiabetic subjects) and overweight type 2 DM subjects. To restrict the influence of variables that could interfere in the interpretation of data, subjects with obesity and/or acute or chronic inflammatory disease, haemoglobinopathies, recent use of antibiotics, antiinflammatory drugs, and trauma were excluded. Type 2 DM patients (n = 39; age 53.3 +/- 9.0 years; median glycated haemoglobin A(1c)< 8%) presented higher levels of TNF-alpha, triglycerides (P < .01), NO(x) and sIL-2R (P < .05) than control group (n = 28; age 39.7 +/- 14.1 years). CRP, LDL-cholesterol, total cholesterol, and HDL-cholesterol did not differ among groups. Diabetic women (n = 21) had higher levels of TNF-alpha, total cholesterol, LDL-cholesterol, and HDL-cholesterol than diabetic men (n = 18) (P < .05), but there were no differences among sexes in the control group. This study indicates that increased level of proinflammatory markers occurs in type 2 DM even in the absence of obesity and marked hyperglycaemia, confirming that the inflammation course of the atherosclerotic process is more severe in diabetic patients than in nondiabetic subjects.     

Growing seasons of Nordic mountain birch in northernmost Europe as indicated by long-term field studies and analyses of satellite images

The phenophases first greening (bud burst) and yellowing of Nordic mountain birch (Betula pubescens ssp.tortuosa, also called B. p. ssp. czerepanovii) were observed at three sites on the Kola Peninsula in northernmost Europe during the period 1964–2003, and at two sites in the trans-boundary Pasvik-Enare region during 1994–2003. The field observations were compared with satellite images based on the GIMMS-NDVI dataset covering 1982–2002 at the start and end of the growing season. A trend for a delay of first greening was observed at only one of the sites (Kandalaksha) over the 40 year period. This fits well with the delayed onset of the growing season for that site based on satellite images. No significant changes in time of greening at the other sites were found with either field observations or satellite analyses throughout the study period. These results differ from the earlier spring generally observed in other parts of Europe in recent decades. In the coldest regions of Europe, e.g. in northern high mountains and the northernmost continental areas, increased precipitation associated with the generally positive North Atlantic Oscillation in the last few decades has often fallen as snow. Increased snow may delay the time of onset of the growing season, although increased temperature generally causes earlier spring phenophases. Autumn yellowing of birch leaves tends towards an earlier date at all sites. Due to both later birch greening and earlier yellowing at the Kandalaksha site, the growing season there has also become significantly shorter during the years observed. The sites showing the most advanced yellowing in the field throughout the study period fit well with areas showing an earlier end of the growing season from satellite images covering 1982–2002. The earlier yellowing is highly correlated with a trend at the sites in autumn for earlier decreasing air temperature over the study period, indicating that this environmental factor is important also for autumn phenophases.     

Quantitative determination of 20-hydroxyecdysone in methanolic extract of twigs from Vitex polygama Cham

20-Hydroxyecdysone (20E) is effective in stimulating protein synthesis, therefore, it has been largely used as anabolic agent in several commercial formulas. Phytochemical study of methanolic extract of twigs from Vitex polygama, used in traditional Brazilian medicine as emenagogue, yielded a large quantity of 20E. This finding led us to developing and validating a simple and reliable method to determine 20E in the surveyed extract. Chromatographic separation of 20E was achieved on a phenyl-hexyl-based column using reversed elution mode. Extract was cleaned-up by solid phase extraction employing C(18) cartridge, and an absolute recovery of 97% was acquired. External standard and standard addition calibration graphs were obtained and good linearity was accomplished (r>0.999 for both curves). The limit of quantification and detection were determined. The results for accuracy fell within the -5 to +7% range.     

A new species of Mysella Angas, 1877 (Bivalvia: Galeommatoidea) from Admiralty Bay, King George Island, South Shetlands, Antarctica, with data on its biology and functional anatomy

Mysella narchii sp. nov. is described from the material collected in shallow-waters of Admiralty Bay at King George Island, South Shetlands, Antarctica. The species is characterized by shell features, biology and functional anatomy. The main shell features distinguishing M. narchii sp. nov. from all other Antarctic, Subantarctic and Magellanic Mysella spp. are provided, as are anatomical characteristics that separate this new species from M. charcoti (Lamy, 1906), its most similar congener and the first Antarctic species studied in its morpho-functional aspects. M. narchii sp. nov. is an infaunal, free-living, predominantly deposit-feeding bivalve; its creeping sole and the secretion of byssal threads allow it to crawl vertically and live sporadically on firm substrata.