Deletion analysis of sucrose metabolic genes from a Salmonella plasmid cloned in Escherichia coli K12

The sucrose operon from pUR400, a 78-kbp conjugative Salmonella plasmid, was cloned in Escherichia coli K12. The operon was located in a 5.7-kbp SalI restriction fragment and was subcloned, in each of two possible orientations, using the expression vector pUC18. The insert DNA was restriction mapped and duplicate restriction sites in the insert and in the polylinker of the vector were used to create various deletions promoter distal in the operon sequence. Additional deletions were made with the restriction exonuclease Bal31. Cells containing hybrid plasmids with specified deletions lacked the ability to transport sucrose or were constitutive for hydrolase and/or uptake activities. The scrA (enzyme IIScr) and scrR (regulatory) genes resided within 2900-bp SmaI-SalI DNA fragment and were assigned the order scrB, scrA, scrR. An amplified sucrose-inducible gene product, Mr 68,000, was detected only in the membrane fraction from recombinant cells that contained plasmid with the intact operon sequence. This protein represented 11% of the total membrane protein and was resistant to extraction with 0.5 M sodium chloride, 2% Triton X-100, and 0.5% sodium deoxycholate. The protein did not appear to be the product of either the scrA, scrB, or scrR gene and may therefore represent a previously unidentified membrane-bound sucrose protein. A new gene, scrC, is proposed. In addition, the cloned 5.7-kbp SalI and 2.5-kbp SmaI-SalI DNA fragments failed to hybridize to chromosomal DNA from Bacillus subtilis, Streptococcus lactis, Streptococcus mutans, and Lactobacillus acidophilus as well as to DNA from a sucrose plasmid from Salmonella tennessee. However, the probes showed weak homology with a 20-kbp EcoRI restriction fragment from Klebsiella pneumoniae.     

Mutants of H-ras that interfere with RAS effector function in Saccharomyces cerevisiae.

We report a class of interfering mutants of the human H-ras gene capable of inhibiting phenotypes arising from the expression of the activated RAS2 gene, RAS2val19, in the yeast Saccharomyces cerevisiae. All these mutants encode unprocessed H-ras proteins that remain in the cytoplasm. One of the mutants, H-rasarg186, was examined in detail. H-rasarg186 protein is a competitive inhibitor of RAS2val19 protein. It does not interfere with processing and membrane localization of RAS2val19, nor does it appear to compete with RAS protein for its proposed regulator, the CDC25 protein. By several criteria the RAS2val19 adenylate cyclase interaction is unaffected by H-rasarg186. We infer from our results that H-rasarg186 protein interferes with an alternative function of RAS2val19.     

Nutrient budgets in a dry heathland watershed in northeastern Spain

Dissolved nutrient inputs in bulk precipitation and outputs in streamwater were measured during 3 years of contrasting hydrological conditions in a 6.3-ha, grazed heathland watershed on schists in the Montseny mountains (NE Spain), drained by an intermittent stream. On average, 39% of the precipitation became streamflow. Bulk precipitation delivered positive net alkalinity (mean 0.22 keq/ha/yr), sulphate input was moderate (9.0 kg SO₄-S/ha/yr), and the mean input of inorganic N was not exceptionally high (6.6 kg/ha/yr). Ion concentrations were relatively low in streamwater; SO₄ ^2- was the dominant anion. Most concentrations in streamwater varied seasonally, with maxima in late summer or early autumn and minima in spring. This pattern probably resulted from increased availability of ions for leaching due to decomposition of organic matter and chemical weathering during the warm period. Nitrate concentrations were relatively high in winter and dropped sharply in early spring, probably because of biological uptake. Annual element outputs in streamwater varied between years and seemed to be controlled by both the amount of annual streamflow and its seasonal distribution. Annual inputs exceeded outputs for dissolved inorganic N. The watershed accumulated H⁺ and Ca²⁺, had net losses of Na⁺ and Mg²⁺, and was close to steady state for K⁺, SO₄ ^2-, Cl^- and alkalinity. The chloride budgets gave no evidence of substantial dry deposition in this system. The cationic denudation rate was negative (-0.14 keq/ha/yr) because Ca²⁺ retention was higher than net exports of Na⁺ and Mg²⁺ from silicate weathering. Low nutrient export and little production of alkalinity suggest that this watershed has a low buffering capacity.     

Diel Migrations of Microorganisms within a Benthic, Hypersaline Mat Community

We studied the diel migrations of several species of microorganisms in a hypersaline, layered microbial mat. The migrations were quantified by repeated coring of the mat with glass capillary tubes. The resulting minicores were microscopically analyzed by using bright-field and epifluorescence (visible and infrared) microscopy to determine depths of coherent layers and were later dissected to determine direct microscopic counts of microorganisms. Microelectrode measurements of oxygen concentration, fiber optic microprobe measurements of light penetration within the mat, and incident irradiance measurements accompanied the minicore sampling. In addition, pigment content, photosynthesis and irradiance responses, the capacity for anoxygenic photosynthesis, and gliding speeds were determined for the migrating cyanobacteria. Heavily pigmented Oscillatoria sp. and Spirulina cf. subsalsa migrated downward into the mat during the early morning and remained deep until dusk, when upward migration occurred. The mean depth of the migration (not more than 0.4 to 0.5 mm) was directly correlated with the incident irradiance over the mat surface. We estimated that light intensity at the upper boundary of the migrating cyanobacteria was attenuated to such an extent that photoinhibition was effectively avoided but that intensities which saturated photosynthesis were maintained through most of the daylight hours. Light was a cue of paramount importance in triggering and modulating the migration of the cyanobacteria, even though the migrating phenomenon could not be explained solely in terms of a light response. We failed to detect diel migration patterns for other cyanobacterial species and filamentous anoxyphotobacteria. The sulfide-oxidizing bacterium Beggiatoa sp. migrated as a band that followed low oxygen concentrations within the mat during daylight hours. During the nighttime, part of this population migrated toward the mat surface, but a significant proportion remained deep.     

Replication of the promiscuous plasmid pLSI: a region encompassing the minus origin of replication is associated with stable plasmid inheritance

Deletion of a region of the promiscuous plasmid pLS1 encompassing the initiation signals for the synthesis of the plasmid lagging strand led to plasmid instability in Streptococcus pneumoniae and Bacillus subtilis. This defect could not be alleviated by increasing the number of copies (measured as double-stranded plasmid DNA) to levels similar to those of the wild-type plasmid pLS1. Our results indicate that in the vicinity of, or associated with the single-stranded origin region of pLS1 there is a plasmid component involved in its stable inheritance. Homology was found between the DNA gyrase binding site within the par region of plasmid pSC101 and the pLS1 specific recombination site RS_R.     

CHARACTERIZATION AND BIOLOGICAL IMPLICATIONS OF SCYTONEMIN,A CYANOBACTERIAL SHEATH PIGMENT1

Scytonemin, the yellow-brown pigment of cyanobacterial (blue-green algal) extracellular sheaths, was found in species thriving in habitats exposed to intense solar radiation. Scytonemin occurred predominantly in sheaths of the outermost parts or top layers of cyanobacterial mats, crusts, or colonies. Scytonemin appears to be a single compound identified in more than 30 species of cyanobacteria from cultures and natural populations. It is lipid soluble and has a prominent absorption maximum in the near-ultraviolet region of the spectrum (384 nm in acetone; ca. 370 nm in vivo) with a long tail extending to the infrared region. Microspectrophotometric measurements of the transmittance of pigmented sheaths and the quenching of ultraviolet excitation of phycocyanin fluorescence demonstrate that the pigment was effective in shielding the cells from incoming near-ultraviolet-blue radiation, but not from green or red light. High light intensity (between 99 and 250 μmol photon · m^?2· S^?1, depending on species) promoted the synthesis of scytonemin in cultures of cyanobacteria. In cultures, high light intensity caused reduction in the specific content of Chl a and phycobilins, increase in the ratio of total carotenoids to Chl a, and scytonemin increase. UV-A (320–400 nm) radiation was very effective in eliciting scytonemin synthesis. Scytonemin production was physiological and not due to a mere photochemical conversion. These results strongly suggest that scytonemin production constitutes an adaptive strategy of photoprotection against short-wavelength solar irradiance.     

Tasa estimada de disposición de glucosa en pacientes menores de 18 años con diabetes mellitus tipo 1 y sobrepeso-obesidad

ObjectiveTo assess the estimated glucose disposal rate (eGDR), insulin dose, and lipoprotein profile in children with type 1 diabetes mellitus (T1DM) and overweight or obesity as compared to children with T1DM and normal weight.MethodsA total of 115 patients (aged 5-16 years) with T1DM on intensive insulin therapy were recruited. The following parameters were measured: weight, height, body mass index, waist and hip circumference, insulin dose, eGDR, glycosylated hemoglobin, blood pressure, and lipoprotein profile. Results were stratified by sex and age.ResultsNo significant differences were found in eGDR between children with normal weight, overweight, and obesity. However, obese children older than 11 years had lower eGDR values (9.3 ± 1.3 vs 10.1 ± 0.8 mg kg^-1min^-1; p < 0.01). Insulin dose was higher in overweight and obese children, especially in IU/m²/day (37.7 vs 36.1 vs 29.4 respectively; p < 0.01). Obese children had higher low-density lipoprotein cholesterol levels than children with overweight and normal weight (106.5 vs 91.7 vs 91.5 mg/dL respectively; p < 0.01). No correlation was found between waist circumference and the different markers of insulin resistance.ConclusionsValues of eGDR values were lower in obese children with T1DM older than 11 years, and this may therefore be considered a marker of insulin resistance. Insulin dose was higher in diabetic patients with overweight or obesity, specially in IU/m²/day. Obese children with T1DM had a lipoprotein profile of cardiovascular risk.     

Molecular Detection of Rickettsia typhi in Cats and Fleas

Background

Rickettsia typhi is the etiological agent of murine typhus (MT), a disease transmitted by two cycles: rat-flea-rat, and peridomestic cycle. Murine typhus is often misdiagnosed and underreported. A correct diagnosis is important because MT can cause severe illness and death. Our previous seroprevalence results pointed to presence of human R . typhi infection in our region; however, no clinical case has been reported. Although cats have been related to MT, no naturally infected cat has been described. The aim of the study is to confirm the existence of R . typhi in our location analyzing its presence in cats and fleas.

Methodology/Principal Findings

221 cats and 80 fleas were collected from Veterinary clinics, shelters, and the street (2001-2009). Variables surveyed were: date of collection, age, sex, municipality, living place, outdoor activities, demographic area, healthy status, contact with animals, and ectoparasite infestation. IgG against R . typhi were evaluated by indirect immunofluorescence assay. Molecular detection in cats and fleas was performed by real-time PCR. Cultures were performed in those cats with positive molecular detection. Statistical analysis was carried out using SPSS. A p < 0.05 was considered significant.Thirty-five (15.8%) cats were seropositive. There were no significant associations among seropositivity and any variables. R . typhi was detected in 5 blood and 2 cultures. High titres and molecular detection were observed in stray cats and pets, as well as in spring and winter. All fleas were Ctenocephalides felis. R . typhi was detected in 44 fleas (55%), from shelters and pets. Co-infection with R . felis was observed.

Conclusions

Although no clinical case has been described in this area, the presence of R . typhi in cats and fleas is demonstrated. Moreover, a considerable percentage of those animals lived in households. To our knowledge, this is the first time R . typhi is detected in naturally infected cats.     

Genome-wide association study for feed efficiency in collective cage-raised rabbits under full and restricted feeding

Feed efficiency (FE) is one of the most economically and environmentally relevant traits in the animal production sector. The objective of this study was to gain knowledge about the genetic control of FE in rabbits. To this end, GWASs were conducted for individual growth under two feeding regimes (full feeding and restricted) and FE traits collected from cage groups, using 114 604 autosome SNPs segregating in 438 rabbits. Two different models were implemented: (1) an animal model with a linear regression on each SNP allele for growth trait; and (2) a two-trait animal model, jointly fitting the performance trait and each SNP allele content, for FE traits. This last modeling strategy is a new tool applied to GWAS and allows information to be considered from non-genotyped individuals whose contribution is relevant in the group average traits. A total of 189 SNPs in 17 chromosomal regions were declared to be significantly associated with any of the five analyzed traits at a chromosome-wide level. In 12 of these regions, 20 candidate genes were proposed to explain the variation of the analyzed traits, including genes such as FTO, NDUFAF6 and CEBPA previously associated with growth and FE traits in monogastric species. Candidate genes associated with behavioral patterns were also identified. Overall, our results can be considered as the foundation for future functional research to unravel the actual causal mutations regulating growth and FE in rabbits.