Structural Organization of Virulence-Associated Plasmids of Yersinia pestis

The complete nucleotide sequence and gene organization of the three virulence plasmids from Yersinia pestis KIM5 were determined. Plasmid pPCP1 (9,610 bp) has a GC content of 45.3% and encodes two previously known virulence factors, an associated protein, and a single copy of IS100. Plasmid pCD1 (70,504 bp) has a GC content of 44.8%. It is known to encode a number of essential virulence determinants, regulatory functions, and a multiprotein secretory system comprising the low-calcium response stimulation that is shared with the other two Yersinia species pathogenic for humans (Y. pseudotuberculosis and Y. enterocolitica). A new pseudogene, which occurs as an intact gene in the Y. enterocolitica and Y. pseudotuberculosis-derived analogues, was found in pCD1. It corresponds to that encoding the lipoprotein YlpA. Several intact and partial insertion sequences and/or transposons were also found in pCD1, as well as six putative structural genes with high homology to proteins of unknown function in other yersiniae. The sequences of the genes involved in the replication of pCD1 are highly homologous to those of the cognate plasmids in Y. pseudotuberculosis and Y. enterocolitica, but their localization within the plasmid differs markedly from those of the latter. Plasmid pMT1 (100,984 bp) has a GC content of 50.2%. It possesses two copies of IS100, which are located 25 kb apart and in opposite orientations. Adjacent to one of these IS100 inserts is a partial copy of IS285. A single copy of an IS200-like element (recently named IS1541) was also located in pMT1. In addition to 5 previously described genes, such as murine toxin, capsule antigen, capsule anchoring protein, etc., 30 homologues to genes of several bacterial species were found in this plasmid, and another 44 open reading frames without homology to any known or hypothetical protein in the databases were predicted.     

Candidemia by Species of the Candida parapsilosis Complex in Children’s Hospital: Prevalence, Biofilm Production and Antifungal Susceptibility

Opportunistic infections are an increasingly common problem in hospitals, and the yeast Candida parapsilosis has emerged as an important nosocomial pathogen. The aims of this study were to determine and compare (i) the prevalence rate among C. parapsilosis complex organisms isolated from blood in a public children’s hospital in São Paulo state, (ii) the ability of the complex C. parapsilosis species identified to produce biofilm and (iii) the antifungal susceptibility profiles. Forty-nine (49) specimens of isolated blood yeast were analyzed, previously identified as C. parapsilosis by conventional methods. After the molecular analysis, the isolates were characterized as C. parapsilosis sensu stricto (83.7 %), C. orthopsilosis (10.2 %) and C. metapsilosis (6.1 %). All species were able to form biofilm. The species with the highest biofilm production was C. parapsilosis sensu stricto, followed by C. orthopsilosis and further by C. metapsilosis. All of the strains have demonstrated similar susceptibility to fluconazole, caspofungin, voriconazole, cetoconazole and 5-flucytosine. Only one strain of C. parapsilosis was resistant to amphotericin B. Regarding itraconazole, 66.6 and 43.9 % isolates of C. metapsilosis and C. parapsilosis, respectively, have demonstrated to be susceptible dose-dependent, with one isolate of the latter species resistant to the drug. Candida parapsilosis sensu stricto has demonstrated to be the less susceptible, mainly to amphotericin B, caspofungin and “azoles” such as fluconazole. Therefore, C. metapsilosis and C. orthopsilosis are still involved in a restricted number of infections, but these data have become essential for there are very few studies of these species in Latin America.     

Effect of lipid supplements on the production and glycosylation of recombinant interferon-γ expressed in CHO cells

The effects of lipids on the glycosylation of recombinant human interferon- expressed in a Chinese Hamster Ovary cell line were investigated in batch culture. Lipids form an essential part of the N-glycosylation pathway, and have been shown to improve cell viability. In control (serum-free) medium the proportion of fully-glycosylated interferon- deteriorated reproducibly with time in batch culture, but the lipoprotein supplement ExCyte was shown to minimise this trend. Partially substituting the bovine serum albumin content of the medium with a fatty-acid free preparation also improved interferon- glycosylation, possibly indicating that oxidised lipids carried on Cohn fraction V albumin may damage the glycosylation process.Abbreviations BSA bovine serum albumin - CHO chinese hamster ovary - DHFR dihydrofolate reductase - FCS foetal calf serum - IFN- human interferon-gamma - q _IFN specific interferon production rate - specific growth rate - 2N doubly-gycosylated - 1N singly-glycosylated - ON non-glycosylated     

Autonomic control of heart rate is virtually independent of temperature but seems related to the neuroanatomy of the efferent vagal supply to the heart in the bullfrog, Lithobathes catesbeianus

Bullfrogs, Lithobathes catesbeianus, bearing a femoral artery cannula were held at 3 temperatures (10, 20 and 30 °C) for 24 h. Changes in heart rate were recorded before and after injection of cholinergic and adrenergic antagonists. Normal heart rate doubled for each temperature increment. Adrenergic tone on the heart varied around 20% at all 3 temperatures but cholinergic tone increased from −5% to 10% between 10 and 30 °C. In contrast, cholinergic tone increased from 75% at 5 °C to 329% at 25 °C in Xenopus laevis. Injection of the neural tracer True Blue into the cervical vagus of the bullfrog revealed a single location for vagal preganglionic neurons (VPN) in the dorsal vagal motor nucleus (DVN), while Xenopus had 30% of its VPN in a ventro-lateral group outside the DVN. Broader comparative studies have suggested that differences in the extent of vagal tone may relate to the location of VPN in the brainstem and this may be the case in these amphibians.     

Arabidopsis ALF4 encodes a nuclear-localized protein required for lateral root formation

Lateral root formation, the primary way plants increase their root mass, displays developmental plasticity in response to environmental changes. The aberrant lateral root formation (alf)4-1 mutation blocks the initiation of lateral roots, thus greatly altering root system architecture. We have positionally cloned the ALF4 gene and have further characterized its phenotype. The encoded ALF4 protein is conserved among plants and has no similarities to proteins from other kingdoms. The gene is present in a single copy in Arabidopsis. Using translational reporters for ALF4 gene expression, we have determined that the ALF4 protein is nuclear localized and that the gene is expressed in most plant tissues; however, ALF4 expression and ALF4's subcellular location are not regulated by auxin. These findings taken together with further genetic and phenotypic characterization of the alf4-1 mutant suggest that ALF4 functions independent from auxin signaling and instead functions in maintaining the pericycle in the mitotically competent state needed for lateral root formation. Our results provide genetic evidence that the pericycle shares properties with meristems and that this tissue plays a central role in creating the developmental plasticity needed for root system development.     

Differential contribution of dorsal and ventral lateral plate mesoderm to hemopoiesis during Rana pipiens embryogenesis

Data obtained from studies on the origin and development of hemopoietic cells in several classes of vertebrate embryos argue for two distinct sources of hemopoietic cells, the intraembryonic dorsal lateral plate and the extraembryonic ventral blood island/yolk sac. In the present study, a stage by stage comparison of the hemopoietic potential of both of these regions was made during development of the frog, Rana pipiens. Either dorsal lateral plate or ventral blood island mesoderm was reciprocally transplanted between cytogenetically labeled triploid and diploid embryos. The ratio of donor-derived cells to host-derived cells (labeling index) was determined from Feulgen-stained DNA measurements of cells harvested from hemopoietic organs of young larvae. Blood island transplants consistently resulted in larvae with positive labeling of the circulating blood. Transplanted dorsal mesoderm supplied mesonephric granulocytes and thymocytes, but not circulating erythrocytes to larvae. However, the contribution of dorsal mesoderm to larval hemopoiesis fluctuated with respect to embryonic stage at transplantation.     

Postnatal exposure to finasteride causes different effects on the prostate of male and female gerbils

The development and maintenance of prostate function depend on a fine balance between oestrogen and androgen levels. Finasteride inhibits 5α‐reductase, which is responsible for the conversion of testosterone into its most active form, dihydrotestosterone. Enzymes that metabolize these hormones have a highly relevant role in both the normal prostate metabolism and in the occurrence of pathological conditions. There are few studies on the impact of finasteride on male prostate development and fewer studies on the female prostate and possible intersexual differences. Therefore, we treated male and female gerbils from 7 to 14 days in postnatal life with a high dose of finasteride (500 μg/kg/day); the prostate complexes were then removed and submitted to immunohistochemistry, immunofluorescence and three‐dimensional reconstruction. In addition, hormonal serum dosages were administered. Treatment with finasteride resulted in an increased thickness of the periductal smooth musculature in the prostate of both male and female gerbils, such as well as a reduction in the thickness of developing prostate alveoli in both sexes. In addition, intersexual differences were observed as increased epithelial proliferation and decreases in the number of developing alveoli in females. Together, the data indicate that postnatal exposure to finasteride causes greater changes in the female gerbil prostate than in the male.     

Leprosy in a prison population: A new active search strategy and a prospective clinical analysis

BackgroundThis study evaluates an active search strategy for leprosy diagnosis based on responses to a Leprosy Suspicion Questionnaire (LSQ), and analyzing the clinical, immunoepidemiological and follow-up aspects for individuals living in a prison population.MethodsA cross-sectional study based on a questionnaire posing 14 questions about leprosy symptoms and signs that was distributed to 1,400 prisoners. This was followed by dermatoneurological examination, anti-PGL-I serology and RLEP-PCR. Those without leprosy were placed in the Non-leprosy Group (NLG, n = 1,216) and those diagnosed with clinical symptoms of leprosy were placed in the Leprosy Group (LG, n = 34).FindingsIn total, 896 LSQ were returned (64%), and 187 (20.9%) of the responses were deemed as positive for signs/symptoms, answering 2.7 questions on average. Clinically, 1,250 (89.3%) of the prisoners were evaluated resulting in the diagnosis of 34 new cases (LG), based on well-accepted clinical signs and symptoms, a new case detection rate of 2.7% within this population, while the NLG were comprised of 1,216 individuals. The confinement time medians were 39 months in the LG while it was 36 months in the NLG (p>0.05). The 31 leprosy cases who responded to the questionnaire (LSQ+) had an average of 1.5 responses. The symptoms “anesthetized skin area” and “pain in nerves” were most commonly mentioned in the LG while “tingling, numbness in the hands/feet”, “sensation of pricks and needles”, “pain in nerves” and “spots on the skin” responses were found in more than 30% of questionnaires in the NLG. Clinically, 88.2% had dysesthetic macular skin lesions and 97.1% presented some peripheral nerve impairment, 71.9% with some degree of disability. All cases were multibacillary, confirming a late diagnosis. Anti-PGL-I results in the LG were higher than in the NLG (p<0.0001), while the RLEP-PCR was positive in 11.8% of the patients.InterpretationOur findings within the penitentiary demonstrated a hidden prevalence of leprosy, although the individuals diagnosed were likely infected while living in their former communities and not as a result of exposure in the prison. The LSQ proved to be an important screening tool to help identify leprosy cases in prisons.