Use of on-line liquid chromatography-nuclear magnetic resonance spectroscopy for the rapid investigation of flavonoids from Sorocea bomplandii

This paper reports the identification of di- and triglycosylated flavonoids from Sorocea bomplandii (Moraceae) by liquid chromatography coupled on-line to nuclear magnetic resonance (LC-NMR). These glycosylated flavonoids may be used as a taxonomic marker in future work. (C) 2002 Elsevier B.V. B.V All rights reserved.

Identification of bacteria in endodontic infections by sequence analysis of 16S rDNA clone libraries

A significant proportion of oral bacteria are unable to undergo cultivation by existing techniques. In this regard, the microbiota from root canals still requires complementary characterization. The present study aimed at the identification of bacteria by sequence analysis of 16S rDNA clone libraries from seven endodontically infected teeth. Samples were collected from the root canals, subjected to the PCR with universal 16S rDNA primers, cloned and partially sequenced. Clones were clustered into groups of closely related sequences (phylotypes) and identification to the species level was performed by comparative analysis with the GenBank, EMBL and DDBJ databases, according to a 98 % minimum identity. All samples were positive for bacteria and the number of phylotypes detected per subject varied from two to 14. The majority of taxa (65(.)2 %) belonged to the phylum Firmicutes of the Gram-positive bacteria, followed by Proteobacteria (10(.)9 %), Spirochaetes (4(.)3 %), Bacteroidetes (6(.)5 %), Actinobacteria (2(.)2 %) and Deferribacteres (2(.)2 %). A total of 46 distinct taxonomic units was identified. Four clones with low similarity to sequences previously deposited in the databases were sequenced to nearly full extent and were classified taxonomically as novel representatives of the order Clostridiales, including a putative novel species of Mogibacterium. The identification of novel phylotypes associated with endodontic infections suggests that the endodontium may still harbour a relevant proportion of uncharacterized taxa.

Molecular analysis of the rDNA ITS-1 intergenic spacer in Drosophila mulleri, D-arizonae, and their hybrids

We analyzed the ITS-1 spacer region of the rDNA in Drosophila mulleri and D. arizonae, two sibling species belonging to the mulleri complex (repleta group) and in hybrids obtained in both cross directions. In spite of several previous studies showing the incompatibility of crosses involving D. arizonae females and D. mulleri males, we were able to obtain hybrids in this direction. Complete ITS-1 region was amplified using primers with homology at the 3'-end of the 18S rDNA and the 5'-end of the 5.8S rDNA genes. Our data demonstrated that D. mulleri and D. arizonae can be differentiated as they present a difference in length for the ITS-1 region. The amplified fragment for this region in D. mulleri has a length of 600 bp, whereas in D. arizonae this fragment is about 500 bp. It was also observed that male and female hybrids obtained in both cross directions present two amplified fragments, confirming the location of the ribosomal cistrons in the X chromosomes and microchromosomes of both parental species.

rDNA-based characterization of a new binucleate Rhizoctonia spp. causing root rot on kale in Brazil

In this paper we present the first report of the occurrence of a binucleate Rhizoctonia spp. causing hypocotyl and root rot in kale in Brazil. Rhizoctonia spp. were isolated from kale (Brassica oleracea var. acephala) with symptoms of hypocotyl and root rot. The isolates, characterized as binucleate Rhizoctonia spp., did not show an anastomosis reaction with any of the binucleate Rhizoctonia spp. testers used. The pathogenicity of the isolates was tested under greenhouse conditions; all isolates were pathogenic and showed different symptom severities on kale. The ITS-5.8S rDNA sequences of kale isolates and 50 testers (25 binucleate Rhizoctonia spp. and 25 Rhizoctonia solani) were compared in order to characterize the genetic identity of Rhizoctonia spp. infecting kale. The kale isolates showed genetic identities ranging from 99.3 to 99.8% and were phylogenetically closely related to CAG 7 (AF354084), with identities of 98.5 and 98.7%. It is suggested that the binucleate Rhizoctonia spp. causing hypocotyl and root rot on kale Brazil comprises a new AG not yet described.

P53 PROTEIN EXPRESSION AND NUCLEAR-DNA CONTENT IN BREAST INTRADUCTAL PROLIFERATIONS

Immunohistochemical analysis of the p53 gene protein and cytometric assessment of nuclear DNA were performed in a series of 51 cases of intraductal breast proliferation. The series included 22 cases of intraductal hyperplasia without atypia, 6 cases of intraductal hyperplasia with atypia, and 23 cases of pure intraductal carcinoma. Expression of p53 protein was detected in one case of intraductal hyperplasia without atypia (4.5 per cent), one case of intraductal hyperplasia with atypia (16.6 per cent) and six cases of intraductal carcinoma (26.0 per cent). No significant correlation was observed between p53 expression and histological subtype of intraductal carcinoma. Aneuploidy was demonstrated in two cases of intraductal hyperplasia with atypia (33.3 per cent) and in 18 cases of intraductal carcinoma (78.2 per cent). All cases of intraductal hyperplasia without atypia were euploid. No significant association was observed between p53 protein expression and ploidy in intraductal hyperplasia. The only case of intraductal hyperplasia without atypia positive for p53 was euploid, whereas the only p53-positive case of intraductal hyperplasia with atypia was aneuploid. Among the intraductal carcinomas, only the aneuploid cases showed positivity for p53, regardless of histological subtype. The results suggest that some of the changes observed in invasive breast carcinoma, such as p53 expression and aneuploidy, are already present in breast intraductal proliferation, especially in areas with atypia and in intraductal carcinoma. The expression of p53 in breast intraductal proliferation may reflect the acquisition of p53 gene mutations in cells unable adequately to repair DNA damage, with genomic instability which would lead to clonal expansion and putative evolution to invasive disease.

Estimation of growth fraction in fine needle aspirates from non-Hodgkin's lymphoma using anti-proliferating cell nuclear antigen - Correlation with the Kiel classification

OBJECTIVE: This study was undertaken to assess whether fine needle aspirates from non-Hodgkins lymphoma (NHL) could be used for growth fraction analysis with proliferating cell nuclear antigen (PCNA) staining and if there was a relationship between the growth fraction and cytomorphologic classification according to the Kiel classification.STUDY DESIGN: the study group consisted of 40 patients with NHL diagnosed by fine needle aspiration (FNA) cytology. The cytologic classification of the lymphomas was made by two cytopathologists on May-Grunwald-Giemsa-stained slides using the Kiel classification. There were 27 cases of low and 13 of high grade lymphoma. The estimation of the growth fraction was made by PCNA immunoreactivity. The PCNA index was quantitated in smears by counting an average of 1,000 cells, and the count teas correlated with the cytomorphologic classification.RESULTS: There was It strong correlation between the PCNA index and lymphoma grading. High grade lymphomas exhibited a mean PCNA positivity of 74.0%, which was significantly higher (P <.001) than that of low grade lymphomas (17.6%).CONCLUSION: Our study showed that PCNA evalua tion is suitable for smears obtained by FNA on NHL, correlates with increasing grades of lymphoma according to the Kiel classification and may offer a method of monitoring treatment of lymphoma.

Use of strontium in the activation of bovine oocytes reconstructed by somatic cell nuclear transfer

As an important step in the nuclear transfer (NT) procedure, we evaluated the effect of three different treatments for oocyte activation on the in vitro and in vivo developmental capacity of bovine reconstructed embryos: (1) strontium, which has been successfully used in mice but not yet tested in cattle; (2) ionomycin and 6-dimethylammopurine (6-DMAP), a standard treatment used in cattle; (3) ionomycin and strontium, in place of 6-DMAP. As regards NT blastocyst development, no difference was observed when strontium (20.1%) or ionomycin/6-DMAP (14.4%) were used. However, when 6-DMAP was substituted by strontium (3), the blastocyst rate (34.8%) was superior to that in the other activation groups (p < 0.05). Results of in vivo development showed the possibility of pregnancies when NT embryos activated in strontium were transferred to recipient cows (16.6%). A live female calf was obtained when ionomycin/strontium were used, but it died 30 days after birth. Our findings show that strontium can be used as an activation agent in bovine cloning procedures and that activation with a combination of strontium and ionomycin increased the in vitro developmental capacity of reconstructed embryos. This is the first report of a calf produced by adult somatic cell NT in Latin America.

Role of roscovitine and IBMX on kinetics of nuclear and cytoplasmic maturation of bovine oocytes in vitro

The 3-isobutyl-1-methylxanthine (IBMX) is able to prevent resumption of meiosis by maintaining elevated cyclic AMP (cAMP) concentrations in the oocyte, and roscovitine, a purine known to specifically inhibit MPF kinase activity, maintains bovine oocytes at the germinal vesicle (GV) stage. The present study was conducted to analyze whether cytoplasmic maturation (examined by the pattern of cortical granule (CG) distribution) of bovine oocytes is improved during meiotic arrest with IBMX and roscovitine. Oocytes were matured in vitro in a 10% Knockout(SR) supplemented TCM-199 medium (Control) with either 0.5 mM IBMX or 25 mu M roscovitine (ROSC). Oocytes were stained with fluorescein isothiocyanate conjugated Lens culinaris agglutinin (FITC-LCA) for CG evaluation and with Hoechst 33342 for nuclear stage assessment. At 16 h of culture, the percentage of oocytes remaining in the GV stage was higher (P < 0.05) in the ROSC group (32.41%) compared with the Control and IBMX groups (8.61% and 9.73%, respectively). At 24h of culture, progression of meiosis to M II stage was retarded (P < 0.05) in the ROSC group (24.05%) compared to the Control (60.20%), whereas the IBMX group (33.88%) showed no significant difference to the other two groups. At 16h of maturation, the proportion of oocytes with CG in clusters (immature cytoplasm) was similar between the groups, as was the percentage of peripheral CG (mature) at 24h of maturation. The results of the present study demonstrated that the meiotic inhibitors IBMX and roscovitine delay the progression of nuclear maturation without affecting cytoplasmic maturation, assessed by the analysis of CG repositioning. (c) 2006 Elsevier B.V. All rights reserved.

The proliferating cell nuclear antigen index in breast carcinomas does not correlate with mitotic index and estrogen receptor immunoreactivity

To investigate the expression of a marker of cell proliferation (PCNA/Cyclin) and its putative relationship with histological grading, mitotic index and estrogen receptor immunoreactivity, we studied twenty-seven cases of invasive breast carcinoma in formalin-fixed, paraffin-embedded tissue sections. The PCNA and estrogen receptor were detected by the PC10 and H222 monoclonal antibodies respectively, using an avidin-biotin-pernxidase method. The median value of PCNA index was 20.9% with a range from 1.4 to 84.2%. We did not find any significant relationship between PCNA index anti the histological grading, mitotic index and estrogen receptor immunoreactivity. We conclude that PCNA detected by the monoclonal antibody PC10 in formalin-fixed material looks at present unrealiable as a proliferation marker in breast carcinoma.