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PubMed2005

Virulence insights from the Paracoccidioides brasiliensis transcriptome.

Paracoccidioides brasiliensis, the etiologic agent of paracoccidioidomycosis, is a dimorphic fungus, which is found as mycelia at 22-26 degrees C and as yeasts at 37 degrees C. A remarkable feature common to several pathogenic fungi is their ability to differentiate from mycelium to yeast morphologies, or vice-versa. Although P. brasiliensis is a recognized pathogen for humans, little is known about its virulence genes. In this sense, we performed a search for putative virulence genes in the P. brasiliensis transcriptome. BLAST comparative analyses were done among P. brasilienses assembled expressed sequence tags (PbAESTs) and the sequences deposited in GenBank. As a result, the putative virulence PbAESTs were grouped into five classes, metabolism-, cell wall-, detoxification-related, secreted factors, and other determinants. Among these, we have identified orthologs of the glyoxylate cycle enzymes, a metabolic pathway involved in the virulence of bacteria and fungi. Besides the previously described alpha- and beta-glucan synthases, orthologs to chitin synthase and mannosyl transferases, also important in cell wall synthesis and stabilization, were identified. With respect to the enzymes involved in the intracellular survival of P. brasiliensis, orthologs to superoxide dismutase, thiol peroxidase and an alternative oxidase were also found. Among the secreted factors, we were able to find phospholipase and urease orthologs in P. brasiliensis transcriptome. Collectively, our results suggest that this organism may possess a vast arsenal of putative virulence genes, allowing the survival in the different host environments.

Animals↗
PubMed2005

[Relationship between ascorbic acid accumulation and related enzyme activities in fruit of Rosa roxburghii Tratt].

Ascorbic acid (AsA) and sugar levels, together with activities of L-galactono-1,4-lactone dehydrogenase (GalLDH, a key enzyme in AsA biosynthesis in higher plant), AAO, AAP, MDAR and DHAR in fruit of Rosa roxburghii Tratt were measured during development. The result showed that AsA accumulated continually but with a slow, fast and slow accumulating rate in the R. roxburghii fruit during its development, in which the period from the end of June to the early of August was the most important period of AsA accumulation, since the AsA accumulated in this stage accounting for approximately 90% of the final level (Fig.1B). Changes in GalLDH activity coincided with AsA accumulating rate during fruit development (Fig.2B). The extremely significant positive correlation existed between the GalLDH activity and AsA accumulating rate (r(2)=0.783**) (Fig.3). Transitory and low activities of AAO and AAP in the fruit were detected in the initial stage of development (Table 1), suggesting that little AsA degraded by the activities of the two oxidative enzymes during the whole development. This data elucidated, at least in part, the reason for high accumulation of AsA in the R. roxburghii fruit. Furthermore, no activity of MDAR or DHAR in the fruit was detected during the whole development, implying that the two enzymes are not key factors for contribution of AsA level. No correlation was found between AsA content and sugar contents in the R. roxburghii fruit.

Ascorbate Oxidase↗
PubMedDate not supplied

[Electron structure of the heme of reduced cytochrome P450 and P420 from the data of low-temperature magnetic circular dichroism].

Magnetic circular dichroism spectra (MCD) of reduced cytochromes P450 and P420 from rabbit liver microsomes have been recorded and analyzed for the 350-600 nm spectral region in the temperature interval from 2 to 290 K. The shape, intensity and temperature dependence of the MCD of reduced P450 in the Soret region are quite different from that of other high-spin ferrous hemoproteins, whose heme iron is coordinated to the imidazole of histidine (deoxymyoglobin, deoxyhemoglobin, reduced peroxidase and cytochrome c oxidase). Assuming that in the reduced P450 as well as in its CO-complex the protein-derived ligand is mercaptide (RS-) the differences can be explained by the existence of two electronic transitions in the Soret region: the common for hemoproteins pi----pi porphyrin transition and sulfur to porphyrin charge-transfer transition, p+(Sp)----eg (pi). The unusual spectral characteristics of the CO-complex of P450 have been ascribed earlier to strong configurational interaction of these two transitions. From the similarities of the Soret MCD and their temperature dependences for the reduced P420 and for other high-spin ferrous hemoproteins one can conclude that heme iron of the reduced P420 is high-spin and is coordinated to the imidazole of histidine. The zero-field splitting parameter D of the spin Hamiltonian has been estimated from the MCD temperature dependences. The obtained splitting of approximately 30 cm-1 for P450 and of approximately 10 cm-1 for P420 exceeds that for myoglobin and hemoglobin (approximately 5 cm-1).

Animals↗
PubMed1978

Automated enzymic measurement of total cholesterol in serum.

We describe a completely automated enzymic system for measuring total cholesterol in serum. All reagents are contained in an analytical test pack and the test is performed on Du Pont's Automatic Clinical Analyzer (aca), which mixes the sample (20 microliter) and reagents and performs the necessary absorbance measurements and calculations. In the procedure, cholesterol oxidase oxidizes free cholesterol. The oxidation step produces cholest-4-en-3-one and hydrochloride peroxidesterase hydrolyzes cholesterol esters and cholesterol in direct proportion to the amount of cholesterol present. N,N-Diethylaniline hydrochloride and 4-aminoantipyrine react with the hydrogen peroxide to produce a quinoneimine dye (lambda max = 553 nm). Interacting reagents have been optimized simultaneously (coöptimization) utilizing response surface designs coupled with computer analysis of the data. Reagent efficiency is high and analytical performance reliable.

Autoanalysis↗