ROOTING MINI-CUTTINGS OF Paulownia fortunei var. mikado DERIVED FROM CLONAL MINI-GARDEN1

ABSTRACTWe aimed to evaluate the technical efficiency of mini-cuttings technique on vegetative propagation of Paulownia fortunei (Seem.) Hemsl. var. Mikado, as well as the possible existence of anatomical barriers to its rooting. Therefore, plants originated from cuttings formed the mini-stumps and, consequently the clonal mini-garden, which was conducted in semi-hydroponic system. We evaluated the survival of mini-stumps and sprouts production for five successive collects, the percentage of mini-cuttings rooting and their anatomical description. The mini-cuttings were prepared with about 6 to 8 cm in length and two leaves reduced by about 50% in the upper third, being remained an area of, approximately 78 cm2 (10 cm diameter). The mini-cuttings were placed in tubes of 53 cm3, with substrate formed with fine vermiculite and carbonized rice hulls (1:1 v/v) and rooted in acclimatized greenhouse. After 30 days we evaluated the percentage of rooted mini-cuttings, radicial vigor (number and length of roots / mini-cutting), callus formation, emission of new shoots and maintenance of the original leaves. The mini-stumps showed 100% survival after five collects and an average production of 76-114 mini-cuttings/m2/month and rooting ranged from 70 to 90%. Mini-cuttings technique is efficient in to propagate adult propagules of the species and there are not anatomical barriers preventing roots emission.

The spindle assembly checkpoint as a drug target - Novel small-molecule inhibitors of Aurora kinases

Cell division (mitosis) is a fundamental process in the life cycle of a cell. Equal distribution of chromosomes between the daughter cells is essential for the viability and well-being of an organism: loss of fidelity of cell division is a contributing factor in human cancer and also gives rise to miscarriages and genetic birth defects. For maintaining the proper chromosome number, a cell must carefully monitor cell division in order to detect and correct mistakes before they are translated into chromosomal imbalance. For this purpose an evolutionarily conserved mechanism termed the spindle assembly checkpoint (SAC) has evolved. The SAC comprises a complex network of proteins that relay and amplify mitosis-regulating signals created by assemblages called kinetochores (KTs). Importantly, minor defects in SAC signaling can cause loss or gain of individual chromosomes (aneuploidy) which promotes tumorigenesis while complete failure of SAC results in cell death. The latter event has raised interest in discovery of low molecular weight (LMW) compounds targeting the SAC that could be developed into new anti-cancer therapeutics. In this study, we performed a cell-based, phenotypic high-throughput screen (HTS) to identify novel LMW compounds that inhibit SAC function and result in loss of cancer cell viability. Altogether, we screened 65 000 compounds and identified eight that forced the cells prematurely out of mitosis. The flavonoids fisetin and eupatorin, as well as the synthetic compounds termed SACi2 and SACi4, were characterized in more detail utilizing versatile cell-based and biochemical assays. To identify the molecular targets of these SAC-suppressing compounds, we investigated the conditions in which SAC activity became abrogated. Eupatorin, SACi2 and SACi4 preferentially abolished the tensionsensitive arm of the SAC, whereas fisetin lowered also the SAC activity evoked by lack of attachments between microtubules (MTs) and KTs. Consistent with the abrogation of SAC in response to low tension, our data indicate that all four compounds inhibited the activity of Aurora B kinase. This essential mitotic protein is required for correction of erratic MT-KT attachments, normal SAC signaling and execution of cytokinesis. Furthermore, eupatorin, SACi2 and SACi4 also inhibited Aurora A kinase that controls the centrosome maturation and separation and formation of the mitotic spindle apparatus. In line with the established profound mitotic roles of Aurora kinases, these small compounds perturbed SAC function, caused spindle abnormalities, such as multi- and monopolarity and fragmentation of centrosomes, and resulted in polyploidy due to defects in cytokinesis. Moreover, the compounds dramatically reduced viability of cancer cells. Taken together, using a cell-based HTS we were able to identify new LMW compounds targeting the SAC. We demonstrated for the first time a novel function for flavonoids as cellular inhibitors of Aurora kinases. Collectively, our data support the concept that loss of mitotic fidelity due to a non-functional SAC can reduce the viability of cancer cells, a phenomenon that may possess therapeutic value and fuel development of new anti-cancer drugs.

Clonal study of avian Escherichia coli strains by fliC conserved-DNA-sequence regions analysis

The clonal relationship among avian Escherichia coli strains and their genetic proximity with human pathogenic E. coli, Salmonela enterica, Yersinia enterocolitica and Proteus mirabilis, was determined by the DNA sequencing of the conserved 5' and 3'regions fliC gene (flagellin encoded gene). Among 30 commensal avian E. coli strains and 49 pathogenic avian E. coli strains (APEC), 24 commensal and 39 APEC strains harbored fliC gene with fragments size varying from 670bp to 1,900bp. The comparative analysis of these regions allowed the construction of a dendrogram of similarity possessing two main clusters: one compounded mainly by APEC strains and by H-antigens from human E. coli, and another one compounded by commensal avian E. coli strains, S. enterica, and by other H-antigens from human E. coli. Overall, this work demonstrated that fliC conserved regions may be associated with pathogenic clones of APEC strains, and also shows a great similarity among APEC and H-antigens of E. coli strains isolated from humans. These data, can add evidence that APEC strains can exhibit a zoonotic risk.

Análise fenotípica e genotípica da virulência de Staphylococcus spp. e de sua dispersão clonal como contribuição ao estudo da mastite bovina

A mastite é uma inflamação da glândula mamária causada principalmente por bactérias, dentre as quais o gênero Staphylococcus ocupa um papel importante. Bactérias pertencentes a este gênero são caracterizadas por expressar fatores de virulência que permitem sua persistência e disseminação no hospedeiro. O presente trabalho teve por objetivo avaliar fenogenotipicamente os fatores de virulência de isolados de Staphylococcus spp. a partir de casos de mastite bovina. Foram analisadas 272 amostras de leite provenientes de oito propriedades da região Sul-Fluminense do Estado do Rio de Janeiro. Após identificação, obteve-se um total de 250 isolados de Staphylococcus spp. Estes foram submetidos às provas fenotípicas de detecção da produção de "slime" em microplaca e em ágar vermelho congo; produção de hemolisinas e sinergismo hemolítico; produção de caseinase e DNase. Posteriormente foram submetidos à técnica de PCR para detecção dos genes de produção de cápsula (cap5 e cap8), fibronectina (fnbA,e fnbB), "slime" (icaA e icaD) e hemolisinas (hla e hlb). Do total avaliado, 58% (145/250) foi identificado como Staphylococcus spp. coagulase-negativos e 42% (105/250) como Staphylococcus spp. coagulase-positivos, destes 36,2% (38/105) foram identificados como S. aureus, 11,4% (12/105) como S. intermedius e 3,8% (4/105) como pertencentes ao grupo SIG. Apenas 6,4% (16/250) dos isolados foram produtores de α-hemólise, 4,8% (12/250) de β-hemólise e, 1,6% (4/250) de α e β-hemólise. A produção de caseinase foi observada em 66,4% (166/250), e a produção de "slime" avaliada pela técnica da microplaca em 76,8% (192/250) dos isolados, respectivamente. A DNase foi detectada em ECNs (38/145) e S. aureus (14/38). Os marcadores genéticos avaliados para a produção de slime, icaA e icaD apresentaram nenhuma ou leve concordância com a produção fenotípica, respectivamente, utilizando o coeficiente Kappa. Tal dado parece indicar que outros marcadores genéticos podem estar envolvidos com a expressão desta característica. Os demais genes detectados com frequência de 4% (10/250) para cap5 e para cap8, 32,8% (82/250) para fnbA, 4,4% (11/250) para fnbB, 19,2% (48/250) para hla e 18% (45/250) para hlb. O perfil circulante nas propriedades foi o 1: isolado produtor de "slime" e caseinase. O gene spaA foi positivo em todos os S. aureus, apresentando amplicons de tamanhos variados, sendo o tamanho prevalente o de 300pb. A amplificação do gene coa apresentou nove tipos polimórficos distintos, sendo prevalente o amplicon de 600pb. O gene agr foi detectado em todos os S. aureus, com amplicon de 200pb. Foi observado que os genes de virulência estudados estavam distribuídos de modo aleatório entreos 6 distintos perfis eletroforéticos obtidos através da Eletroforese em Gel de Campo Pulsado (PFGE).

Clonal growth and dispersal potential of Leiothrix flagellaris Ruhland (Eriocaulaceae) in the rocky grasslands of Southeastern Brazil

Leiothrix flagellaris is a small clonal plant that grows in sandy/rocky, nutrient poor soils in the rocky grasslands ("campos rupestres") of southeastern Brazil. In the rainy season most of their flower heads differentiate into small rosettes, produced by pseudovivipary, and connected to the mother-plant by flexible, elongated stalks that can reach up to 90cm. Most of these rosettes remain suspended over rocks or over the sparse herbaceous surrounding vegetation, while a few arch low enough to come into contact with the surrounding soil and take root. These suspended rosettes can reach diameters comparable to currently rooted and reproductively active rosettes produced during previous reproductive periods. As the rooted rosettes grow up, their potential to generate and disperse new pseudoviviparous rosettes increase rapidly. This unusual guerrilla strategy of L. flagellaris seems to congregate a suit of traits that promote a fast increase in photosynthetic area and improve recruitment, thereby helping to circumvent dispersal and establishment failures in its severe environment.

Chromosomal evolution and comparative gene mapping in the Drosophila repleta species group

A review of our recent work on the cromosomal evolution of the Drosophila repleta species group is presented. Most studies have focused on the buzzatii species complex, a monophyletic set of 12 species which inhabit the deserts of South America and the West Indies. A statistical analysis of the length and breakpoint distribution of the 86 paracentric inversions observed in this complex has shown that inversion length is a selected trait. Rare inversions are usually small while evolutionary successful inversions, fixed and polymorphic, are predominantly of medium size. There is also a negative correlation between length and number of inversions per species. Finally, the distribution of inversion breakpoints along chromosome 2 is non-random, with chromosomal regions which accumulate up to 8 breakpoints (putative "hot spots"). Comparative gene mapping has also been used to investigate the molecular organization and evolution of chromosomes. Using in situ hybridization, 26 genes have been precisely located on the salivary gland chromosomes of D. repleta and D. buzzatii; another nine have been tentatively identified. The results are fully consistent with the currently accepted chromosomal homologies between D. repleta and D. melanogaster, and no evidence for reciprocal translocations or pericentric inversions has been found. The comparison of the gene map of D. repleta chromosome 2 with that of the homologous chromosome 3R of D. melanogaster shows an extensive reorganization via paracentric inversions and allows to estimate an evolution rate of ~1 inversion fixed per million years for this chromosome

Chromosomal polymorphism in urban populations of Drosophila paulistorum

Drosophila paulistorum populations colonizing the urban area of Porto Alegre, southern Brazil, were studied with the objective of characterizing their chromosomal polymorphism in this new environment. Despite being geographically and ecologically marginal and the fact that the colonization of the urban area seems to be a recent event, the populations showed a large number of inversions on all chromosome arms. Differences regarding inversion frequencies and percentage of heterozygosis were found when we compared the samples with respect to geographical, microenvironmental and temporal aspects. Such differences, however, could be attributed to both selective and stochastic factors

Apolipoprotein and lipid abnormalities in chronic liver failure

Total serum lipids, as well as apolipoproteins A-I (apo A-I) and B (apo B), were determined in 74 patients with chronic liver failure without cholestasis and in 82 normal subjects. The VLDL, LDL and HDL lipid fractions were reduced in the liver failure group by 36%, 24% and 46%, respectively (P<0.001). Apolipoproteins A-I and B were also reduced by 26% and 25%, respectively (P<0.001). However, the reduction of HDL cholesterol (HDLc) was more pronounced than that of apo A-I and the HDLc:apo A-I ratio was significantly lower in the liver failure group. After separating these patients into groups with plasma albumin lower than 3.0, between 3.0 and 3.5, and higher than 3.5 g/dl, the HDLc:apo A-I ratio was proportional to plasma albumin, but the correlation was not statistically significant. When these patients were separated by the Child classification of liver function, there was a correlation between the HDLc:apo A-I ratio and liver function. The differences in the HDLc:apo A-I ratio between the Child groups B and C, and A and C were statistically significant (P<0.05). We conclude that there is a more pronounced reduction in HDL cholesterol than in apo A-I in liver failure patients. Therefore, the HDLc:apo A-I ratio is a marker of liver function, probably because there is a decreased lecithin-cholesterol acyltransferase production by the diseased liver

Autonomic abnormalities demonstrable in young normotensive subjects who are children of hypertensive parents

Although a slightly elevated office blood pressure (BP) has been reported in several studies, little is known about the prolonged resting blood pressure, heart rate (HR) and baroreflex sensitivity (BRS) of prehypertensive subjects with a family history of hypertension. Office blood pressure, prolonged resting (1 h) BP and HR were measured in 25 young normotensives with a positive family history of hypertension (FH+) and 25 young normotensives with a negative family history of hypertension (FH-), matched for age, sex, and body mass index. After BP and HR measurements, blood samples were collected for the determination of norepinephrine, plasma renin activity and aldosterone levels, and baroreflex sensitivity was then tested. Casual BP, prolonged resting BP and heart rate were significantly higher in the FH+ group (119.9 ± 11.7/78.5 ± 8.6 mmHg, 137.3 ± 12.3/74.4 ± 7.9 mmHg, 68.5 ± 8.4 bpm) compared to the FH- group (112.9 ± 11.4/71.2 ± 8.3 mmHg, 128.0 ± 11.8/66.5 ± 7.4 mmHg, 62.1 ± 6.0 bpm). Plasma norepinephrine level was significantly higher in the FH+ group (220.1 ± 104.5 pg/ml) than in the FH- group (169.1 ± 63.3 pg/ml). Baroreflex sensitivity to tachycardia (0.7 ± 0.3 vs 1.0 ± 0.5 bpm/mmHg) was depressed in the FH+ group (P<0.05). The FH+ group exhibited higher casual blood pressure, prolonged resting blood pressure, heart rate and plasma norepinephrine levels than the FH- group (P<0.05), suggesting an increased sympathetic tone in these subjects. The reflex tachycardia was depressed in the FH+ group.

Abnormalities of glucose metabolism in spontaneously hypertensive rats

Abnormalities in glucose metabolism and insulin action are frequently detected in patients with essential hypertension. Spontaneously hypertensive rats (SHR) have been used as an experimental model to understand this pathological condition. The objective of the present study was to assess glucose metabolism and insulin action in SHR and Wistar rats under fed and fasting conditions. Peripheral glucose utilization was estimated by kinetic studies with [6-³H]-glucose and gluconeogenetic activity was measured during continuous [14C]-bicarbonate infusion. Plasma glucose levels were higher in the SHR group. Plasma insulin levels in the fed state were higher in the SHR group (99.8 ± 6.5 µM) than in the control group (70.4 ± 3.6 µM). Muscle glycogen content was reduced in SHR compared to control under the various experimental conditions. Peripheral glucose utilization was slightly lower in the SHR group in the fed state (8.72 ± 0.55 vs 9.52 ± 0.80 mg kg-1 min-1 in controls). Serum free fatty acid levels, hepatic glycogen levels, hepatic phosphoenolpyruvate carboxykinase activity and gluconeogenetic activity were similar in the two groups. The presence of hyperglycemia and hyperinsulinemia and the slightly reduced peripheral glucose utilization suggest the presence of resistance to the action of insulin in peripheral tissues of SHR. Hepatic gluconeogenesis does not seem to contribute to the metabolic alterations detected in these animals.

Cytogenetic description of breast fibroadenomas: alterations related solely to proliferation?

Twelve breast fibroadenomas were analyzed cytogenetically and only four were found to have clonal alterations. The presence of chromosomal alterations in fibroadenomas must be the consequence of the proliferating process and must not be related to the etiology of this type of lesion. In contrast, the few fibroadenomas that exhibit chromosomal alterations are likely to be those presenting a risk of neoplastic transformation. Clonal numerical alterations involved chromosomes 8, 18, 19, and 21. Of the chromosomal alterations found in the present study, only monosomy of chromosomes 19 and 21 has been reported in breast fibroadenomas. The loss of chromosome 21 was the most frequent alteration found in our sample. The study of benign proliferations and their comparison with chromosome alterations in their malignant counterparts ought to result in a better understanding of the genes acting on cell proliferation alone, and of the genes that cause these cells to exhibit varied behaviors such as recurrences, spontaneous regression and fast growth.

Pulmonary function, cholinergic bronchomotor tone, and cardiac autonomic abnormalities in type 2 diabetic patients

This prospective study analyzed the involvement of the autonomic nervous system in pulmonary and cardiac function by evaluating cardiovascular reflex and its correlation with pulmonary function abnormalities of type 2 diabetic patients. Diabetic patients (N = 17) and healthy subjects (N = 17) were evaluated by 1) pulmonary function tests including spirometry, He-dilution method, N2 washout test, and specific airway conductance (SGaw) determined by plethysmography before and after aerosol administration of atropine sulfate, and 2) autonomic cardiovascular activity by the passive tilting test and the magnitude of respiratory sinus arrhythmia (RSA). Basal heart rate was higher in the diabetic group (87.8 ± 11.2 bpm; mean ± SD) than in the control group (72.9 ± 7.8 bpm, P<0.05). The increase of heart rate at 5 s of tilting was 11.8 ± 6.5 bpm in diabetic patients and 17.6 ± 6.2 bpm in the control group (P<0.05). Systemic arterial pressure and RSA analysis did not reveal significant differences between groups. Diabetes intragroup analysis revealed two behaviors: 10 patients with close to normal findings and 7 with significant abnormalities in terms of RSA, with the latter subgroup presenting one or more abnormalities in other tests and clear evidence of cardiovascular autonomic dysfunction. End-expiratory flows were significantly lower in diabetic patients than in the control group (P<0.05). Pulmonary function tests before and after atropine administration demonstrated comparable responses by both groups. Type 2 diabetic patients have cardiac autonomic dysfunction that is not associated with bronchomotor tone alterations, probably reflecting a less severe impairment than that of type 1 diabetes mellitus. Yet, a reduction of end-expiratory flow was detected.

Abnormalities of hippocampal signal intensity in patients with familial mesial temporal lobe epilepsy

Mesial temporal lobe epilepsy (MTLE) is associated with hippocampal atrophy and hippocampal signal abnormalities. In our series of familial MTLE (FMTLE), we found a high proportion of hippocampal abnormalities. To quantify signal abnormalities in patients with FMTLE we studied 152 individuals (46 of them asymptomatic) with FMTLE. We used NIH-Image® for volumetry and signal quantification in coronal T1 inversion recovery and T2 for all cross-sections of the hippocampus. Values diverging by 2 or more SD from the control mean were considered abnormal. T2 hippocampal signal abnormalities were found in 52% of all individuals: 54% of affected subjects and 48% of asymptomatic subjects. T1 hippocampal signal changes were found in 34% of all individuals: 42.5% of affected subjects and 15% of asymptomatic subjects. Analysis of the hippocampal head (first three slices) revealed T2 abnormalities in 73% of all individuals (74% of affected subjects and 72% of asymptomatic subjects) and T1 abnormalities in 59% (67% of affected subjects and 41% of asymptomatic subjects). Affected individuals had smaller volumes than controls (P < 0.0001). There was no difference in hippocampal volumes between asymptomatic subjects and controls, although 39% of asymptomatic patients had hippocampal atrophy. Patients with an abnormal hippocampal signal (133 individuals) had smaller ipsilateral volume, but no linear correlation could be determined. Hippocampal signal abnormalities in FMTLE were more frequently found in the hippocampal head in both affected and asymptomatic family members, including those with normal volumes. These results indicate that subtle abnormalities leading to an abnormal hippocampal signal in FMTLE are not necessarily related to seizures and may be determined by genetic factors.

Clonal dissemination of VanA-type glycopeptide-resistant Enterococcus faecalis between hospitals of two cities located 100 km apart

Nosocomial dissemination of glycopeptide-resistant enterococci represents a major problem in hospitals worldwide. In Brazil, the dissemination among hospitals in the city of São Paulo of polyclonal DNA profiles was previously described for vancomycin-resistant Enterococcus faecium. We describe here the dissemination of VanA phenotype E. faecalis between two hospitals located in different cities in the State of São Paulo. The index outbreak occurred in a tertiary care university hospital (HCUSP) in the city of São Paulo and three years later a cluster caused by the same strain was recognized in two patients hospitalized in a private tertiary care hospital (CMC) located 100 km away in the interior of the state. From May to July 1999, 10 strains of vancomycin-resistant E. faecalis were isolated from 10 patients hospitalized in the HCUSP. The DNA genotyping using pulsed-field gel electrophoresis (PFGE) showed that all isolates were originated from the same clone, suggesting nosocomial dissemination. From May to July 2002, three strains of vancomycin-resistant E. faecalis were isolated from two patients hospitalized in CMC and both patients were colonized by the vancomycin-resistant Enterococcus in skin lesions. All isolates from CMC and HCUSP were highly resistant to vancomycin and teicoplanin. The three strains from CMC had minimum inhibitory concentration >256 µg/ml for vancomycin, and 64 (CMC 1 and CMC 2) and 96 µg/ml (CMC 3) for teicoplanin, characterizing a profile of VanA resistance to glycopeptides. All strains had the presence of the transposon Tn1546 detected by PCR and were closely related when typed by PFGE. The dissemination of the E. faecalis VanA phenotype among hospitals located in different cities is of great concern because E. faecalis commonly colonizes the gastrointestinal tract of patients and healthy persons for periods varying from weeks to years, which, together with the persistence of vancomycin-resistant Enterococcus in hospital rooms after standard cleaning procedures, increases the risk of the dissemination and reservoir of the bacteria.