Autonomously replicating plasmids carrying theAMA1 region inPenicillium chrysogenum

Plasmid vectors containing theAMA1 sequence transformed with high efficiency and replicated autonomously inPenicillium chrysogenum. The efficiency of transformation ofP. chrysogenum was related to the length of theAMA1 fragment used for constructing the different autonomously replicating plasmids. One of the two palindromic inverted repeats ofAMA1 (the 2.2-kbSalI-HindIII fragment) is sufficient to confer autonomous replication and a high transformation efficiency. Deletion of the 0.6-kb central fragment located between the inverted repeats did not affect either the ability of the plasmids to replicate autonomously or the efficiency of transformation, but did alter the mitotic stability and the plasmid copy number. Deletion of any fragment of the 2.2-kb repeat caused the loss of the ability to replicate autonomously and reduced the transformation efficiency. Most of the transformants retained the original plasmid configuration, as multimers and without reorganization, after several rounds of autonomous replication. TheAMA1 region works as an origin of replication inP. chrysogenum andA. nidulans but not apparently inAcremonium chrysogenum.     

The epidemiology of anotia and microtia.

We studied a large data set from three registries of congenital malformations (central-east France, Sweden, and California), a total of 954 cases, known chromosome anomalies excluded. The prevalence at birth varied significantly between programmes, probably to a large extent because of different ascertainment and inclusion criteria, from 0.76 per 10,000 births in the French programme to 2.35 in the Swedish. Within the California programme, there is a racial variability in prevalence with lower values among whites (and probably blacks) than in Hispanics and Asians. Also the proportion of anotia and microtia varies between races with the lowest proportion of anotia in whites. Anotia and microtia are equally often associated with other malformations and show other similar epidemiological characteristics. In unilateral cases, the right side is more frequently malformed than the left side, especially when the ear malformation is isolated. There is a male excess, most pronounced in isolated forms. Among associated malformations, facial clefts and cardiac defects are the most common ones (each about 30% of infants with associated malformations), followed by anophthalmia or microphthalmia (14%), limb reduction defects or severe renal malformations (11%), and holoprosencephaly (7%). There is a maternal parity effect seen, an increased risk at parity 4+ (standardised for maternal age), more pronounced for anotia than microtia.     

Experimental Staphylococcus aureus arthritis in mice.

Staphylococcus aureus arthritis is usually caused by bacteremia and is highly destructive. Controlled studies on septic arthritis in humans are difficult to perform, because the time of onset of the infection is unknown. Animal models of bacterial arthritis make it possible to control important variables in experimental studies. We present a mouse model of S. aureus arthritis in which the intravenous administration of 10(7) cells of S. aureus LS-1 induced arthritis or osteitis or both within 3 weeks in 80 to 90% of the mice. Signs of arthritis emerged within the first few days after the injection. An interesting finding was that the S. aureus strain used in this study binds bone sialoprotein, a glycoprotein known to be specifically localized to bone tissue. This new model of S. aureus arthritis enables the study of the kinetics of joint destruction and the host-bacterium relationship as well as therapeutical approaches to septic arthritis and osteomyelitis.     

Development of flow cytogenetics and physical genome mapping in chickpea (Cicer arietinum L.)

Procedures for flow cytometric analysis and sorting of mitotic chromosomes (flow cytogenetics) have been developed for chickpea (Cicer arietinum). Suspensions of intact chromosomes were prepared from root tips treated to achieve a high degree of metaphase synchrony. The optimal protocol consisted of a treatment of roots with 2mmol/L hydroxyurea for 18h, a 4.5-h recovery in hydroxyurea-free medium, 2h incubation with 10µmol/L oryzalin, and ice-water treatment overnight. This procedure resulted in an average metaphase index of 47%. Synchronized root tips were fixed in 2% formaldehyde for 20min, and chromosome suspensions prepared by mechanical homogenization of fixed root tips. More than 4×10⁵ morphologically intact chromosomes could be isolated from 15 root tips. Flow cytometric analysis of DAPI-stained chromosomes resulted in histograms of relative fluorescence intensity (flow karyotypes) containing eight peaks, representing individual chromosomes and/or groups of chromosomes with a similar relative DNA content. Five peaks could be assigned to individual chromosomes (A, B, C, G, H). The purity of sorted chromosome fractions was high, and chromosomes B and H could be sorted with 100% purity. PCR on flow-sorted chromosome fractions with primers for sequence-tagged microsatellite site (STMS) markers permitted assignment of the genetic linkage group LG8 to the smallest chickpea chromosome H. This study extends the number of legume species for which flow cytogenetics is available, and demonstrates the potential of flow cytogenetics for genome mapping in chickpea.     

Differences in virulence factors among clinical isolates of Escherichia coli causing cystitis and pyelonephritis in women and prostatitis in men

Differences in the presence of nine urovirulence factors among clinical isolates of Escherichia coli causing cystitis and pyelonephritis in women and prostatitis in men have been studied. Hemolysin and necrotizing factor type 1 occur significantly more frequently among isolates causing prostatitis than among those causing cystitis (P < 0.0001) or pyelonephritis (P < 0.005). Moreover, the papGIII gene occurred more frequently in E. coli isolates associated with prostatitis (27%) than in those associated with pyelonephritis (9%) (P < 0.05). Genes encoding aerobactin and PapC occurred significantly less frequently in isolates causing cystitis than in those causing prostatitis (P < 0.01 and P < 0.0001, respectively) and pyelonephritis (P < 0.01 and P < 0.0001, respectively). No differences in the presence of Sat or type 1 fimbriae were found. Finally, AAFII and Bfp fimbriae are no longer considered uropathogenic virulence factors since they were not found in any of the strains analyzed. Overall, the results showed that clinical isolates producing prostatitis need greater virulence than isolates producing pyelonephritis in women or, in particular, cystitis in women (P < 0.05). Overall, the results suggest that clinical isolates producing prostatitis are more virulent that those producing pyelonephritis or cystitis in women.     

Differential effects of cancer rehabilitation depending on diagnosis and patients' cognitive coping style

OBJECTIVE: The major aim was to explore the extent to which the Miller Behavioral Style Scale (MBSS) can be used to differentiate cancer patients who are likely to benefit from rehabilitation efforts with a strong information component from those who are not. METHODS: Newly diagnosed patients with breast, gastrointestinal, or prostate cancer (N = 442) were included in a randomized, prospective study of the effects (on anxiety, depression, intrusion, avoidance) of rehabilitation approximately 4 months after diagnosis as compared with control patients. Patients were classified as "monitors" or "blunters" on the basis of the MBSS (368 patients, 83%, completed the MBSS). RESULTS: The expected interaction at postintervention between coping style and experimental condition (ie, rehabilitation or control) was found only for avoidance among breast and prostate cancer patients. Assignment to the rehabilitation or control condition was of no importance for outcome among blunters. Among monitors, the response pattern differed between breast and prostate cancer patients. Prostate cancer monitors seemed to benefit from rehabilitation on all outcome measures, whereas intrusion and avoidance were reduced among breast cancer patients in the control condition. This interaction of diagnosis with condition (rehabilitation or control) among monitors is suggested to be due to demands for diagnosis-specific information during diagnostic work, in the period just after diagnosis, and before treatment decision. CONCLUSIONS: Only the monitor concept seems useful for predicting response to cancer rehabilitation with a strong information component. However, whether rehabilitation is of benefit depends also on other factors.     

INK4a/ARF locus alterations in human non-Hodgkin's lymphomas mainly occur in tumors with wild-type p53 gene

INK4a/ARF locus codes for two different proteins, p16(INK4a) and p14(ARF), involved in cell cycle regulation. p14(ARF) is considered an upstream regulator of p53 function. To determine the role of these genes in the pathogenesis of human non-Hodgkin's lymphomas we have analyzed exon 1beta, 1alpha, and 2 of the INK4a/ARF locus and p53 gene aberrations in 97 tumors previously characterized for p16(INK4a) alterations. p53 alterations were detected in four of 51 (8%) indolent lymphomas but in 15 of 46 (33%) aggressive tumors. Inactivation of p14(ARF) was always associated with p16(INK4a) alterations. Exon 1beta was concomitantly deleted with exon 1alpha and 2 in eight tumors. One additional lymphoblastic lymphoma showed deletion of exon 1alpha and 2 but retained exon 1beta. No mutations were detected in exon 1alpha and 1beta in any case. Two of the three mutations detected in exon 2 caused a nonsense mutation in the p16(INK4a) reading frame and a missense mutation in the ARF reading frame involving the nucleolar transport domain of the protein. The third mutation was a missense mutation in the p16(INK4a) reading frame, but it was outside the coding region of p14(ARF). Aggressive lymphomas with p14(ARF) inactivation and p53 wild type showed a significantly lower p53 protein expression than tumors with no alteration in any of these genes. In this series of tumors, inactivation of the INK4a/ARF locus mainly occurred in tumors with a wild-type p53 gene because only two lymphomas showed simultaneous aberrations in these genes. Tumors with concomitant alterations of p16(INK4a) and p14(ARF)/p53 genes seem to exhibit a worse clinical behavior than lymphomas with no alterations or isolated inactivation of any of these genes. These findings indicate that p14(ARF) genetic alterations occur in a subset of aggressive NHLs, but they are always associated with p16(INK4a) aberrations. Concomitant disruption of p16(INK4a) and p14(ARF)/p53 regulatory pathways may have a cooperative effect in the progression of these tumors.     

Granulocyte turnover in the feline intestine

The objective of this study was to determine the turnover rate of the extravascular pool of granulocytes in different regions of the feline gastrointestinal tract. Leukocyte emigration from the vasculature was prevented over a 48-h period by repeated intravenous injections of a monoclonal antibody (MAb IB₄) directed against the leukocyte adhesion glycoprotein complex CD11/CD18. Tissue-associated myeloperoxidase (MPO) activity was used to monitor the total tissue granulocyte pool at 0.5, 12, 24, and 48 h after MAb IB₄ administration. The mucosal layer of the duodenum, jejunum, ileum, and colon exhibited different kinetics of granulocyte clearance, with average life-spans (t1/2) ranging between 6.9 (colon) and 10.4 h (duodenum). Granulocyte clearance rates of 0.5 × 10⁶ and 2.4 x 10⁶ cells/h/g tissue were estimated (from measured values oft1/2 and tissue granulocyte pool) for the small bowel and colonie mucosae, respectively. The submucosal layer of the intestine exhibited a biphasic reduction in tissue MPO activity following immunoneutralization of CD11/CD18, with an initialt1/2 0.5 h followed by at1/2 of 36–60 h. The initial rapid decline in tissue MPO suggests that a significant fraction of granulocytes in the submucosa is localized in a readily exchangeable pool (e.g., marginated cells within the vasculature). The results of this study indicate that the average life-span of resident granulocytes varies significantly between different regions of the gastrointestinal tract, with the intestinal mucosa exhibiting at1/2 comparable to that previously reported for circulating feline neutrophils (R 8 h).     

Cell type determines the relative proportions of (-) and (+) strand RNA during poliovirus replication.

To examine the influence of the host cell type on poliovirus RNA synthesis we compared the levels of (-) and (+) strand poliovirus RNA during infection of epithelial (HeLa and HEp-2), leukocytic (U-937, HL-60 and K-562) and nerve (IMR-32) cells. The levels of (-) strand RNA were higher in IMR-32, U-937, K-562 or HL-60 cells than those in HeLa or HEp-2 cells. By contrast, (+) strand RNA content was greater in HeLa or HEp-2 cells. Although significant levels of (+) strand RNA were detected in U-937, K-562 and HL-60 cells, no viral protein synthesis was detected by polyacrylamide gel electrophoretic analysis of metabolically labelled proteins. The molar ratio of poliovirus (-) and (+) RNAs was 2-3 fold higher in IMR-32, U-937 and K-562 cells than in HeLa or HL-60 cells and 5-6 fold higher than in HEp-2 cells. Differentiation of HL-60 cells with a variety of inducers produced differential effects on poliovirus (-) and (+) RNA content and modified the molar ratio of (-)/(+) strand RNAs. These findings indicate that host cell components play a critical role in the regulation of the amount of poliovirus (-) and (+) strand RNAs synthesized during infection.     

Percepciones estudiantiles sobre las propiedades del examen clínico objetivo estructurado en Uruguay. Disponiendo un cuestionario válido para analizar la factibilidad del nuevo formato durante la transición curricular

Introduction

Although an objective structured clinical examination (OSCE) format has been applied in Uruguay since 2004, and providing reliable performance measures, perceptions of it properties and level of student satisfaction have not been determined.