Notes and Queries : a Medium of Intercommunication for Literary Men, etc.

5 (Second Series)

Notes and Queries : a Medium of Intercommunication for Literary Men, etc.

7

Notes and Queries : a Medium of Intercommunication for Literary Men, etc.

8

Notes and Queries : a Medium of Intercommunication for Literary Men, etc.

11

Notes and Queries : a Medium of Intercommunication for Literary Men, etc.

9

Notes and Queries : a Medium of Intercommunication for Literary Men, etc.

1sr Series Index

Abundance of Conepatus chinga (Carnivora, Mephitidae) and other medium-sized mammals in grasslands of southern Brazil

Between January 2007 and December 2010, the abundance of medium-sized mammals was studied, with special focus on the Molina's hog-nosed skunk, Conepatus chinga (Molina, 1782), at four locations in southern Brazil. In this study, transect line methodology was used to obtain data for Distance Analyses. Transects were traveled by car at night, searching with spotlights along the edges of secondary roads in agricultural landscapes. Along 1,811 km, we obtained 620 observations of 20 mammal species. The most common species was the exotic European hare, Lepus europaeus (Pallas, 1778); the highest abundance estimated for South America was observed in one of the study areas, where its density was estimated as 32 individuals/km². Carnivores were the most commonly recorded mammals, represented by 10 species and comprising 51% of all observations. Molina's hog-nosed skunk occurred in all study areas, but occurred in sufficient numbers to obtain density estimates in only two of the areas. We estimated 1.4 to 3.8 individuals/km², in the first density estimate made by the transect method for a member of Conepatus in the Neotropics. These values are similar to those estimated for North American species of Mephitidae. In Brazil, C. chinga is apparently more abundant in the Pampa biome than in the grasslands of the Atlantic Forest. For two other carnivores, Lycalopex gymnocercus (Fisher, 1814) and Cerdocyon thous (Linnaeus, 1766), we estimated preliminary densities that were similar to those previously cited for different regions.

Diversity and habitat preference of medium and large-sized mammals in an urban forest fragment of southwestern Amazon

We assessed the species composition and abundance of medium and large-sized mammals in an urban forest fragment in the Brazilian Amazon, and recorded the preference of some species for particular phytophysiognomies. We placed nine transects with 20 sand plots each in three phytophysiognomies: open rainforest with a dominance of bamboos (OFB), open rainforest with palm trees (OFP), and dense rainforest (DF). We calculated species abundance as the number of records/plot.day, in a total of 2,700 plots.day. We recorded twelve mammal species; Sylvilagus brasiliensis (Linnaeus, 1758) and Dasyprocta fuliginosa (Wagler, 1831) were the most abundant. The results differed among phytophysiognomies: DF presented the highest mammal diversity, whereas the species composition of OFP was less similar than that of other phytophysiognomies. Rodents showed higher preference for OFP and Sylvilagus brasiliensis was more abundant in OFB. The study area showed high species richness, with the occurrence of mesopredators, but there was a predominance of common species adaptable to disturbed environments, which reflects the severe isolation degree of the forest fragment and the hunting pressure that is still present.

A selective medium for the isolation of the acinetobacter genus bacteria

A selective and differencial medium was developed for the isolation of Acinetobacter genus bacteria. This Acinobacter Agar Medium (p.H + 7.4) contains in grams per litre: thiotone, 10; yeast extract, 3; naC1, 5; saccharose, 10; mannitol, 10; sodium citrate, 0.5; sodium desoxycholate, 0.1; crystal violet, 0.00025; phenol red, 0.04 and agar-agar 15. This medium has the advantage of inhibiting the growth of cocci and Gram-positive bacilli, by the use of sodium citrate and sodium desoxycholate associated with the crystal violet; and of differentiating the Gram-negative bacilli from the Enterobacteriaceae, through the fermentative activity upon the saccharose and/or mannitol, contrasting with the complete inactivity of the Acinetobacter genus bacteria over those substances.

Liquid nitrogen cryopreservation of Bracoccidioides brasiliensis in Fava's Netto medium

The aplicability of Fava's Netto medium in the liquid nitrogen cryopreservation technique of Paracoccidioides brasiliensis cells was demonstrated.

Results of An International Study of Quantitative BCR/ABL Assays Using Defined RNA Samples

Molecular monitoring of BCR/ABL transcripts by real time quantitative reverse transcription PCR (qRT-PCR) is an essential technique for clinical management of patients with BCR/ABL-positive CML and ALL. Though quantitative BCR/ABL assays are performed in hundreds of laboratories worldwide, results among these laboratories cannot be reliably compared due to heterogeneity in test methods, data analysis, reporting, and lack of quantitative standards. Recent efforts towards standardization have been limited in scope. Aliquots of RNA were sent to clinical test centers worldwide in order to evaluate methods and reporting for e1a2, b2a2, and b3a2 transcript levels using their own qRT-PCR assays. Total RNA was isolated from tissue culture cells that expressed each of the different BCR/ABL transcripts. Serial log dilutions were prepared, ranging from 100 to 10-5, in RNA isolated from HL60 cells. Laboratories performed 5 independent qRT-PCR reactions for each sample type at each dilution. In addition, 15 qRT-PCR reactions of the 10-3 b3a2 RNA dilution were run to assess reproducibility within and between laboratories. Participants were asked to run the samples following their standard protocols and to report cycle threshold (Ct), quantitative values for BCR/ABL and housekeeping genes, and ratios of BCR/ABL to housekeeping genes for each sample RNA. Thirty-seven (n=37) participants have submitted qRT-PCR results for analysis (36, 37, and 34 labs generated data for b2a2, b3a2, and e1a2, respectively). The limit of detection for this study was defined as the lowest dilution that a Ct value could be detected for all 5 replicates. For b2a2, 15, 16, 4, and 1 lab(s) showed a limit of detection at the 10-5, 10-4, 10-3, and 10-2 dilutions, respectively. For b3a2, 20, 13, and 4 labs showed a limit of detection at the 10-5, 10-4, and 10-3 dilutions, respectively. For e1a2, 10, 21, 2, and 1 lab(s) showed a limit of detection at the 10-5, 10-4, 10-3, and 10-2 dilutions, respectively. Log %BCR/ABL ratio values provided a method for comparing results between the different laboratories for each BCR/ABL dilution series. Linear regression analysis revealed concordance among the majority of participant data over the 10-1 to 10-4 dilutions. The overall slope values showed comparable results among the majority of b2a2 (mean=0.939; median=0.9627; range (0.399 - 1.1872)), b3a2 (mean=0.925; median=0.922; range (0.625 - 1.140)), and e1a2 (mean=0.897; median=0.909; range (0.5174 - 1.138)) laboratory results (Fig. 1-3)). Thirty-four (n=34) out of the 37 laboratories reported Ct values for all 15 replicates and only those with a complete data set were included in the inter-lab calculations. Eleven laboratories either did not report their copy number data or used other reporting units such as nanograms or cell numbers; therefore, only 26 laboratories were included in the overall analysis of copy numbers. The median copy number was 348.4, with a range from 15.6 to 547,000 copies (approximately a 4.5 log difference); the median intra-lab %CV was 19.2% with a range from 4.2% to 82.6%. While our international performance evaluation using serially diluted RNA samples has reinforced the fact that heterogeneity exists among clinical laboratories, it has also demonstrated that performance within a laboratory is overall very consistent. Accordingly, the availability of defined BCR/ABL RNAs may facilitate the validation of all phases of quantitative BCR/ABL analysis and may be extremely useful as a tool for monitoring assay performance. Ongoing analyses of these materials, along with the development of additional control materials, may solidify consensus around their application in routine laboratory testing and possible integration in worldwide efforts to standardize quantitative BCR/ABL testing.

Melanoma patients respond to a cytotoxic T lymphocyte-defined self-peptide with diverse and nonoverlapping T-cell receptor repertoires.

HLA-A2+ melanoma patients develop naturally a strong CD8+ T cell response to a self-peptide derived from Melan-A. Here, we have used HLA-A2/peptide tetramers to isolate Melan-A-specific T cells from tumor-infiltrated lymph nodes of two HLA-A2+ melanoma patients and analyzed their TCR beta chain V segment and complementarity determining region 3 length and sequence. We found a broad diversity in Melan-A-specific immune T-cell receptor (TCR) repertoires in terms of both TCR beta chain variable gene segment usage and clonal composition. In addition, immune TCR repertoires selected in the patients were not overlapping. In contrast to previously characterized CD8+ T-cell responses to viral infections, this study provides evidence against usage of highly restricted TCR repertoire in the natural response to a self-differentiation tumor antigen.

Transport of methotrexate by the in vitro isolated rabbit proximal tubule.

The tubular transport of [3H]methotrexate was studied in isolated nonperfused and perfused superficial proximal tubular segments of rabbit kidneys. Reabsorption represented only 5% of perfused methotrexate, and appeared to be mostly of passive nature inasmuch as it was not modified by reducing the temperature or by ouabain. Cellular accumulation in nonperfused segments and secretion in perfused tubules were highest in the S2 segment and lower in the S3 and S1 segments. Secretion against a bath-to-lumen concentration gradient was observed only in S2 segments (with a maximum methotrexate secretory rate of 478 +/- 48 fmol/mm.min and an apparent Km of transport of 363 +/- 32 microM), and was inhibited by probenecid and folate. The low capacity for methotrexate secretion may be explained by a low capacity of transport across the basolateral membrane of the proximal cell as methotrexate was accumulated only to a low extent in nonperfused tubules (tissue water to medium concentration ratio of 8.2 +/- 1 in S2 segments). During secretion a small amount of methotrexate was metabolized; the nature of the metabolite(s) remains to be defined.

Synthesis of medium-chain-length polyhydroxyalkanoates in arabidopsis thaliana using intermediates of peroxisomal fatty acid beta-oxidation.

Polyhydroxyalkanoate (PHA) is a family of polymers composed primarily of R-3-hydroxyalkanoic acids. These polymers have properties of biodegradable thermoplastics and elastomers. Medium-chain-length PHAs (MCL-PHAs) are synthesized in bacteria by using intermediates of the beta-oxidation of alkanoic acids. To assess the feasibility of producing MCL-PHAs in plants, Arabidopsis thaliana was transformed with the PhaC1 synthase from Pseudomonas aeruginosa modified for peroxisome targeting by addition of the carboxyl 34 amino acids from the Brassica napus isocitrate lyase. Immunocytochemistry demonstrated that the modified PHA synthase was appropriately targeted to leaf-type peroxisomes in light-grown plants and glyoxysomes in dark-grown plants. Plants expressing the PHA synthase accumulated electron-lucent inclusions in the glyoxysomes and leaf-type peroxisomes, as well as in the vacuole. These inclusions were similar to bacterial PHA inclusions. Analysis of plant extracts by GC and mass spectrometry demonstrated the presence of MCL-PHA in transgenic plants to approximately 4 mg per g of dry weight. The plant PHA contained saturated and unsaturated 3-hydroxyalkanoic acids ranging from six to 16 carbons with 41% of the monomers being 3-hydroxyoctanoic acid and 3-hydroxyoctenoic acid. These results indicate that the beta-oxidation of plant fatty acids can generate a broad range of R-3-hydroxyacyl-CoA intermediates that can be used to synthesize MCL-PHAs.