Phylogenetic study of the species within the family Streptomycetaceae.

Species of the genus Streptomyces, which constitute the vast majority of taxa within the family Streptomycetaceae, are a predominant component of the microbial population in soils throughout the world and have been the subject of extensive isolation and screening efforts over the years because they are a major source of commercially and medically important secondary metabolites. Taxonomic characterization of Streptomyces strains has been a challenge due to the large number of described species, greater than any other microbial genus, resulting from academic and industrial activities. The methods used for characterization have evolved through several phases over the years from those based largely on morphological observations, to subsequent classifications based on numerical taxonomic analyses of standardized sets of phenotypic characters and, most recently, to the use of molecular phylogenetic analyses of gene sequences. The present phylogenetic study examines almost all described species (615 taxa) within the family Streptomycetaceae based on 16S rRNA gene sequences and illustrates the species diversity within this family, which is observed to contain 130 statistically supported clades, as well as many unsupported and single member clusters. Many of the observed clades are consistent with earlier morphological and numerical taxonomic studies, but it is apparent that insufficient variation is present in the 16S rRNA gene sequence within the species of this family to permit bootstrap-supported resolution of relationships between many of the individual clusters.

Cerebral granular cell tumor: immunohistochemical and electron microscopic study.

A rare intracerebral granular cell tumor (GCT) was studied by immunocytochemical and ultrastructural methods. The tumor was composed of two cell types--filament-rich and granular cells. Granular cells contained PAS-positive, diastase-resistant granules that ultrastructurally corresponded to autophagic cytosegresomes. Glial fibrillary acidic protein, the intermediate filament protein specific for astrocytes, was demonstrated in the filament-rich and, to a lesser extent, in the granular cells. Unlike noncerebral GCT, neither S-100 protein nor vimentin was detected in the tumor cells. On the other hand, both cerebral and noncerebral GCT were labeled immunocytochemically with peanut lectin (Arachis hypogaea). The results suggest that cerebral GCT share some features with noncerebral GCT, but differ in other respects. They further suggest that GCT may be derived from different cell types depending on the tissue of origin, and that cerebral GCT may be derived from astrocytes.

Enzyme-linked immunosorbent assay detection of prostate-specific antigen messenger ribonucleic acid in prostate cancer.

OBJECTIVES: To develop a rapid, sensitive, reverse transcriptase-polymerase chain reaction (RT-PCR) prostate-specific antigen (PSA) messenger ribonucleic acid (mRNA) detection method by applying colorimetric enzyme-linked immunosorbent assay (ELISA). METHODS: Total RNA was extracted from 16 urogenital cancer cells (including PSA-producing LNCaP cells) from pelvic and inguinal lymph node aspiration biopsy samples from patients with prostate, bladder, and penile cancer, as well as from blood samples of 500 patients with urogenital cancer. We used rTth polymerase for RT and PCR. The RNA target was amplified by RT-PCR with dinitrophenyl-labeled primer. The PCR product was denatured and hybridized on a PSA-specific probe-coated microwell plate. RESULTS: In 1 6 cancer cell lines, only LNCaP cells expressed especially high PSA mRNA values, with an optical density (OD) greater than 3. In other cell lines, two testicular cells had relatively high ODs, 1.909 and 0.987, respectively. A high PSA mRNA value was obtained by fine needle aspiration from pelvic lymph node specimens of cytologically positive lymph nodes from patients with prostate cancer but not from patients with cytologically proved bladder or penile cancer. Sensitivity and specificity of fine needle aspiration samples were 70% and 100%, respectively. Blood tests obtained from patients with prostate cancer demonstrated high PSA mRNA values. CONCLUSIONS: The PSA mRNA RT-PCR ELISA method provides a sensitive photometric enzyme immunoassay for the detection of PSA mRNA, using nonradioactive techniques.

Two patients with N3 bladder cancer successfully treated by internal iliac arterial infusion chemotherapy and irradiation: case reports.

The prognosis of patients with bladder cancer with pelvic lymph node metastasis is poor, and only 30% of them have been reported to achieve 5- and 10-year survival rates. Prognosis of the patients with pelvic lymph node metastasis larger than 5 cm (N3) is especially poor. and no patient has been reported to have survived more than 3 years. The authors report the successful treatment of two patients with pelvic N3 bladder cancer by internal iliac arterial infusion chemotherapy combined with whole-pelvis irradiation.

[Impaired insulin secretion in isolated islets of Goto-Kakizaki rats, an animal model of non obese type 2 diabetes, is a primary event]. / Deficiência primária da secreção de insulina de ilhéus isolados de ratos Goto-Kakizaki. Um modelo animal de diabetes tipo 2 não obesa.

The development of type 2 diabetes is associated with the impairment of insulin secretion. To evaluate the evolution of the secretory response, a chronological study comparing normal Wistar (W) vs Goto-Kakizaki (GK) rats, an animal model of non obese type 2 diabetes, was done. Glucose and arginine were tested in collagenase isolated islets of Langerhans with perfusion and ELISA immunoassay techniques. Fasting glycaemia and insulinemia and glucose tolerance were also evaluated. We have seen, in W rats, a mild glucose intolerance in the first two weeks of age. GK rats were always glucose intolerant with hyperglycaemia and hyperinsulinemia at fasten after one month old. Wistar islets had a characteristic biphasic response to glucose after the first two weeks of age. GK islets were always glucose irresponsive. Arginine induced an increase in insulin secretion in both animal models, independent of age, although GK rats had always a smaller response when compared to W rats. We concluded that 1) in W rats, a biphasic insulin secretion in response to glucose is observed after the first two weeks of age, simultaneously with glycaemia stabilization 2) in GK rats, both first and second phases of glucose-induced insulin release are significantly reduced and a smaller reduction in response to arginine is observed. This beta-cell disfunction is a primary event in this model of type 2 diabetes, preceding fasting hyperglycaemia and hyperinsulinemia.

Distribution of beta-lactamases in actinomycetes.

The distribution of beta-lactamase activities in a collection of actinomycete strains was surveyed. Six of 127 strains were found to produce beta-lactamase. This low frequency was in contrast to the case with Streptomyces species. The producing strains were not related phylogenetically. MICs of benzylpenicillin did not correlate with beta-lactamase production.

Trans-urethral whole layer core biopsy for detection of residual tumor after neoadjuvant therapy in invasive bladder cancer.

The most essential information necessary for the treatment of bladder cancer is to know its exact staging. We have developed a percutaneous whole layer core biopsy (PC-WLCB) of the bladder tumor and applied it successfully since April 1985 for the staging and evaluation of neoadjuvant therapy in locally invasive bladder cancer. We report here a modified method, the trans-urethral WLCB (TU-WLCB) and present its clinical results. Methods: A 20 F. rigid nephroscope was introduced trans-urethrally and an 18 gauge, 350mm-long biopsy needle or newly developed 450mm-long biopsy needle was advanced to the tumor through the nephroscope. Biopsy was performed under trans-abdominal ultrasound guidance. Results: Specimens of all 20 TU-WLCB cases included the muscle layer and adipose tissue, and demonstrated small focus of residual cancers after neoadjuvant therapy. Serious complications were not observed so far. Conclusion: TU-WLCB may prove to be a reliable method to stage and evaluate neoadjuvant therapy for invasive bladder cancer.

Ballooned neurons in select neurodegenerative diseases contain phosphorylated neurofilament epitopes.

Ballooned neurons are histological features of several neurodegenerative diseases of the central nervous system. We describe the immunocytochemical staining of ballooned neurons in Pick's disease, unclassified dementia, corticonigral degeneration, pigment-spheroid degeneration and Alzheimer's disease. In all of these conditions the ballooned neurons contain phosphorylated epitopes recognized by monoclonal antibodies to neurofilaments, but not epitopes unique to Alzheimer neurofibrillary tangles and Pick bodies. The morphological features and immunohistochemical properties of ballooned neurons in these disorders bear resemblance to swollen neurons produced by neurotoxins that impair axoplasmic transport of neurofilaments. This finding, by analogy, suggests that impaired axoplasmic transport of neurofilaments may be a common mechanism in various dementing neurodegenerative diseases.

Diagnosis and treatment of pelvic lymph node metastasis in bladder control.

BACKGROUND AND METHODS: Bipedal lymphography and percutaneous fine needle aspiration biopsy (FNAB) of pelvic lymph node was done in 200 patients with bladder cancer. RESULTS: A diagnosis of metastasis to the pelvic lymph node was obtained by this method in 34 patients. Of these 34 patients, only 12 (35%) were positive or suspected of having pelvic lymph node metastasis by computed tomography. Sixteen patients (47%) had unequivocally positive or highly suspicious lymphogram and 18 patients (53%) had normal lymphogram. Seventy-eight cases, including eight FNAB-positive cases, were treated by radical cystectomy and regional lymph node dissection. Sensitivity, specificity, positive predictive value and negative predictive value of FNAB were 57, 100, 100 and 91%, respectively. Thirteen FNAB-positive M0 patients could be treated by multimodality therapy and evaluated after neoadjuvant therapy by repeated lymphography and FNAB. All were changed to N0 by FNAB after neoadjuvant therapy. In eight patients treated by total cystectomy and lymph node dissection, four had down staged to pN0 but four had one positive node. In five follow-up cases without surgery, one had recurrent pelvic lymph node metastasis 35 months later. Cause-specific survival of these patients was 76% at 5 years and 57% at 8 years. CONCLUSIONS: Pretreatment diagnosis of pelvic lymph node metastasis and multimodality therapy may improve the prognosis of regional lymph node-positive bladder cancer. Because it is difficult to diagnose pN1 using FNAB, radical operation should be performed even if neoadjuvant therapy is effective and down staging is obtained.

The study of PSA gene expression on urogenital cell lines.

PURPOSE: Reverse transcriptase-polymerase chain reaction (RT-PCR) offers a potentially more sensitive assay for detecting cells expressing prostate-specific antigen (PSA) mRNA in peripheral circulation. But the sensitivity and specificity are variable depending on the position of the PSA amplification. To increase sensitivity and specificity, the whole PSA cDNA (1466 bp) was separated into eight different parts. METHODS: We examined RT-PCR on 12 urogenital cell lines, including three prostate cancer (LNCaP, PC3, DU145), five human renal cell carcinoma (SMKT-R3, TOS-1, TOS-2, R4, ACHN), two urinary bladder cancer (YTS-1, KK-47) and two testicular cancer (NEC8, NEC14) cell lines. The sizes of the eight fragmented PSA used in the experiment were PSA-1 (1-257bp), PSA-2 (1-322bp), PSA-3 ( 172-507bp), PSA-4 (172-851 bp), PSA-5 (595-1347 bp), PSA-6 (682 967 bp), PSA-7 (682-1347 bp) and PSA-8 (863-1466 bp). RESULTS: All cell lines had positive signals from PSA-6, PSA-7 and PSA-8. The positive signals from PSA-1, PSA-2 and PSA-3 were detected in some other cell lines in addition to the three prostate cancer cell lines. Only LNCaP which produces the PSA protein had a positive signal from PSA-5. PC3 and DU145 (which do not produce PSA) and LNCaP had a positive signal from PSA-4. Therefore, the inner primer PSA-4' (578-782 bp) used to increase sensitivity and specificity. Nested RT-PCR on the 12 cell lines, using the PSA-4 and 4' primers, detected more clear bands in the three prostate cancer cells. CONCLUSION: Nested RT-PCR using PSA-4 (outer primer) and PSA-4' (inner primer) may be useful for detecting prostate cancer cells in the peripheral blood.

[Morphological changes of islet of Langerhans in an animal model of type 2 diabetes]. / Alterações da morfologia do ilhéu de Langerhans na evolução da síndrome diabética num modelo animal de diabetes tipo 2.

The impairment of insulin secretion, a major feature of type 2 diabetes, is caused by beta-cell mass reduction and functional failure. Pancreatic beta-cell mass reduction is variable in humans, not exceeding 50%, and has been associated with amyloid deposits. In the present study, we have chronologically compared the endocrine pancreas morphology of Wistar control rats (W) and Goto-Kakizaki (GK) rats, an animal model of non obese type 2 diabetes. We have also characterised and compared their body weight, glycaemia (fasting and after oral glucose load) as well as other biochemical parameters. GK rats were always glucose intolerant and fasting hyperglycaemia arised at four week of age. Wistar rats had mild glucose intolerance in their first two weeks of life. GK rats had a total beta-cell mass always decreased when compared to controls, but above 40%. In adult GK rats (12 weeks old) alterations in the architecture of a sub-population of islets occurred which displayed signs of prominent fibrosis, with cluster of beta-cells widely separated by strands of connective tissue and deposits of PAS positive material. Our findings demonstrate that, using GK rats from the Coimbra colony, beta-cell mass reduction is one of the primary features in the pathological sequence leading to diabetes. Structural lesions of the islets, that will further increase beta-cell mass reduction and compromise beta-cell function, will appear latter mainly due to hyperglycaemia.

Novel malonamide derivatives as alpha v beta 3 antagonists. Syntheses and evaluation of 3-(3-indolin-1-yl-3-oxopropanoyl)aminopropanoic acids on vitronectin interaction with alpha v beta 3.

In attempt to find novel integrin alphavbeta3 antagonists, we selected SC65811 and its guanidine analogue (1) as lead compounds. Modification of the glycine part of SC65811 led to a new series of malonamide derivatives that exhibited alphavbeta3 inhibitory activity. Among them, (R,S)-3-[3-[6-(3-benzylureido)indolin-1-yl]-3-oxopropanoylamino]-3- (pyridin-3-yl)propanoic acid (43a) showed not only potent activity with an IC50 value of 3.0 nM but also good selectivity for alphavbeta3 relative to alphaIIbbeta3, alpha5beta1, and alphavbeta5 with IC50 values of 19,000, 11,000, and 14 nM, respectively. Furthermore, optimization of 43a led to the most potent alphavbeta3 antagonist, (R,S)-3-(3-[6-[(4,5-dihydro-1H-imidazol-2-yl)amino]indolin-1-yl]-3-oxopropanoylamino)-3-(quinolin-3-yl)propanoic acid (431) with an IC50 value of 0.42 nM. The synthesis and the structure-activity relationships of these malonamide derivatives are presented.