Identification of species of Brucella using Fourier transform infrared spectroscopy

Fourier transform infrared spectroscopy (FTIR) is a technique that has been used over the years in chemical analysis for the identification of substances and is one that may be applied to the characterisation of microorganisms. The marked tendency of Brucella towards variation in the smooth rough phase, together with the laboriousness and risk involved in the methods used in their identification, make their classification difficult. We studied the type strains of the different species and biovars of Brucella and 11 isolates of human origin of Brucella melitensis, six corresponding to biovar 1, one to biovar 2 and five to biovar 3. The results of linear discriminant analysis performed using the data provide an above 95% likelihood of correct classification, over half of which are in fact above 99% for the vast majority of Brucella strains. Only one case of B. melitensis biovar 1 has been incorrectly classified. The rest of the microorganisms studied (Staphylococcus aureus, Strteptococcus pyogenes, Enterococcus faecalis, Corynebacterium pseudodiphtheriticum, Clostridium perfringens, Escherichia coli, Acinetobacter calcoaceticus and Pseudomonas aeruginosa) have been classified correctly in all cases to a likelihood of over 80%. In the graphic representation of the analysis, a grouping of these can be seen in clusters, which include the different species. One of these comprises B. melitensis, another Brucella abortus, and another wider one is made up of Brucella suis. The Brucella canis, Brucella ovis and Brucella neotomae strains appear separate from the previously described groups.     

Partial cloning and expression of the cyclin B gene in the ovary of the bullseye puffer (Sphoeroides annulatus)

The bullseye puffer is a marine fish species with great potential for aquaculture in Mexico, and the understanding of its reproductive physiology at every level of biological organization is essential in order to succeed. Several molecules orchestrate the complex process of oocyte maturation and spawning. One of these molecules is cyclin B, which is the regulatory subunit of the maturation-promoting factor. In this study, a fragment of the cyclin B gene was isolated from the ovary of the bullseye puffer using an RT-PCR approach. The gene fragment was homologous to the cyclin B2 gene of other vertebrate species. Similar levels of cyclin B gene expression were detected in ovaries at different developmental stages, except for atretic ovaries from captive fish which did not spawn. However, cyclin B gene expression was maintained in captive fish treated with LHRH-a to induce spawning, and appeared to be similar to the pattern observed in wild fish. It is possible that the reduced expression of cyclin B in atretic ovaries is the result of mRNA degradation during atresia. Alternatively, reduced gene expression could be a controlling factor in the process of oocyte reabsorption since cyclin B is required for final oocyte maturation and ovulation.     

Breakthrough performance of stacks of dye-cellulosic fabric in affinity chromatography of lysozyme

The breakthrough performance of stacks of dye-cellulosic fabric in affinity chromatography of lysozyme was investigated in batch and flow experiments. Breakthrough curves were significantly affected by flow rate and were not dependent on the feed solution concentration. System dispersion curves could not explain the flow-rate dependence. Breakthrough curves were analyzed by coupling the kinetic model for pore mass transfer as the only controlling resistance and a system dispersion model. From the analysis, pore film mass transfer resistance was found to be the leading rate-limiting factor when the residence time in the column is greater than 5 min. The model was used to predict the operating and design parameters needed to obtain sharp breakthrough curves. Selectivity studies using lysozyme and bovine serum albumin mixtures showed a high system selectivity for lysozyme.     

Purification of human plasma haptoglobin by hemoglobin-affinity column chromatography

Haptoglobin (Hp) is an acute-phase protein; its plasma levels increase consistently in response to infection and inflammation. The concentration of human plasma Hp is ranged between 1 and 1.5 mg/ml. Similar to blood type, individual human Hp is classified as Hp 1-1, 2-1, or 2-2. The structural and functional analysis of the Hp, however, has not been studied in detail due to its difficult isolation procedure. Previously, we reported a single step for the purification of porcine Hp. In this study, we established a purification method using a high capacity hemoglobin-affinity column. Briefly, DEAE-purified human hemoglobin was first coupled to Sepharose 4B to prepare an affinity column in a 15-ml bed volume. Following a flow through of human plasma and an extensive wash, the bound material was eluted with a solution of 0.15 M NaCl, pH 11 (adjusted by ammonium), to remove low-affinity bound proteins. The high-affinity bound Hp was then eluted with 0.15 M NaCl containing 5 M urea, pH 11, and collected in tubes containing 100 microl of 1 M Tris buffer, pH 7.0. The biological activity of dialyzed Hp was retained as it formed a complex with hemoglobin on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Using this procedure, approximately 10 mg of Hp 1-1, with homogeneity greater than 96%, was obtained from 15 ml of human plasma. Affinity purified Hp 2-1 or 2-2, however, contained trace amounts of apoA-I with the similar approach. The Hp could be further purified by HPLC using a Superose 12 gel-permeation chromatography, if desired, to achieve 100% purity. All the phenotypes of purified Hp consisted of alpha and beta chains on SDS-PAGE in the presence of a reducing reagent, further confirmed by a Western blot analysis. We conclude that human hemoglobin-affinity column was most suitable for the isolation of Hp 1-1 in large quantities. Whereas, one additional step using a gel-permeation was necessary for that of Hp 2-1 and 2-2.     

Artifact production in the assay of anhydroecgonine methyl ester in serum using gas chromatography-mass spectrometry

The detection of the pyrolysis product anhydroecgonine methyl ester (AEME, methylecgonidine) after cocaine smoking using gas chromatography-mass spectrometry is hampered by the artifactual production of AEME. The amount of AEME increases with the amount of cocaine used producing false positive values in authentic samples. A method for the correction of quantitative values was established using calibration of pyrolysis and estimation of the artifactual AEME. Authentic AEME in serum was differentiated from the artifact above 3.5 microg/l, 99% prediction limits of the quantitation were +/-3.1 microg/l. In 16 serum samples and five postmortem blood samples, cocaine and AEME were detected, but after application of the correction method only ten were truly positive for AEME.     

Pathogenomics

The genomes described this month reflect the overall historical bias of microbial genomics towards pathogenic bacteria. Although the balance is now being redressed to some extent, especially through the study of extremophiles, it is still the case that the opportunities provided by genomic studies are primarily taken up by those who study bacterial pathogenicity. This part of the field is, however, being broadened by including the study of pathogens of animals, insects and plants alongside those that afflict humans.     

Transferrin modifications and lipid peroxidation: implications in diabetes mellitus

Free iron is capable of stimulating the production of free radicals which cause oxidative damage such as lipid peroxidation. One of the most important mechanisms of antioxidant defense is thus the sequestration of iron in a redox-inactive form by transferrin. In diabetes mellitus, increased oxidative stress and lipid peroxidation contribute to chronic complications but it is not known if this is related to abnormalities in transferrin function. In this study we investigated the role of transferrin concentration and glycation. The antioxidant capacity of apotransferrin to inhibit lipid peroxidation by iron-binding decreased in a concentration-dependent manner from 89% at > or = 2 mg/ml to 42% at 0.5 mg/ml. Pre-incubation of apotransferrin with glucose for 14 days resulted in a concentration-dependent increase of glycation: 1, 5 and 13 micromol fructosamine/g transferrin at 0, 5.6 and 33.3 mmol/l glucose respectively, p < 0.001. This was accompanied by a decrease in the iron-binding antioxidant capacity of apotransferrin. In contrast, transferrin glycation by up to 33.3 mmol/l glucose did not affect chemiluminescence-quenching antioxidant capacity, which is iron-independent. Colorimetric evaluation of total iron binding capacity in the presence of an excess of iron (iron/transferrin molar ratio = 2.4) also decreased from 0.726 to 0.696 and 0.585mg/g transferrin after 0, 5.6 and 33.3 mmol/l glucose, respectively, p < 0.01. In conclusion, these results suggest that lower transferrin concentration and its glycation can, by enhancing the pro-oxidant effects of iron, contribute to the increased lipid peroxidation observed in diabetes.     

Engineering increased vitamin C levels in plants by overexpression of a D-galacturonic acid reductase

L-Ascorbic acid (vitamin C) in fruits and vegetables is an essential component of human nutrition. Surprisingly, only limited information is available about the pathway(s) leading to its biosynthesis in plants. Here, we report the isolation and characterization of GalUR, a gene from strawberry that encodes an NADPH-dependent D-galacturonate reductase. We provide evidence that the biosynthesis of L-ascorbic acid in strawberry fruit occurs through D-galacturonic acid, a principal component of cell wall pectins. Expression of GalUR correlated with changing ascorbic acid content in strawberry fruit during ripening and with variations in ascorbic acid content in fruit of different species of the genus Fragaria. Reduced pectin solubilization in cell walls of transgenic strawberry fruit with decreased expression of an endogenous pectate lyase gene resulted in lower ascorbic acid content. Overexpression of GalUR in Arabidopsis thaliana enhanced vitamin C content two- to threefold, demonstrating the feasibility of engineering increased vitamin C levels in plants using this gene.