Apoptosis as a mediator of hyperplastic recovery in human prostate lesions: cytochemical and immunocytochemical evaluation

The present study was carried out to investigate the occurrence of apoptosis in human prostatic lesions with emphasis on nodular hyperplasia and adenocarcinomas, using cytochemistry and immunocytochemistry. The results showed that apoptosis is a common event on nodular hyperplasia but not in adenocarcinomas. This led to the hypothesis that apoptosis may represent an important factor on the localized recovery response of the hyperplastic acini.

Anatomy of smooth muscle cells in nonmalignant and malignant human prostate tissue

Differently graded areas of human prostate adenocarcinoma were examined after Masson's trichrome staining or immunohistochemistry for smooth muscle alpha-actin, type IV collagen and laminin. In addition, the ultrastructure of the prostatic smooth muscle cells (SMC) during glandular proliferation and epithelial invasion in selected tumors was studied. The SMC formed a thick layer below the epithelial structures in unaffected areas and were closely associated with each other in homotypic interactions. As the tumor grade increased, the SMC gradually lost interactions with each other and became atrophic. With the growth of the epithelial compartment, the SMC initially segregated to the tumor periphery and the intercellular spaces increased. In high grade tumors, the epithelial cancer cells invaded the spaces between the SMC. Immunohistochemical analysis of the basal membrane revealed increased disruption of the usually thick basal membrane, which became thinner and faintly stained with each of the antibodies used. We conclude that most SMC become atrophic following epithelial invasion in human tumors and that degradation of the basal membrane is an important factor in this process. At the ultrastructural level, different SMC phenotypes occur in prostatic tissues during epithelial invasion. Interconversion between these phenotypes is suggested and a probable relationship among them is proposed.

Response of human alveolar bone-derived cells to a novel poly(vinylidene fluoride-trifluoroethylene)/barium titanate membrane

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Alchornea glandulosa Ethyl Acetate Fraction Exhibits Antiangiogenic Activity: Preliminary Findings from In Vitro Assays Using Human Umbilical Vein Endothelial Cells

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Opposite effects of bFGF and TGF-beta on collagen metabolism by human periodontal ligament fibroblasts

This study evaluated the effects of bFGF and TGF-beta, individually and combined, on cell proliferation and collagen metabolism. Primary human periodontal ligament cells were stimulated with two concentrations (I and 10 ng/ml) of each growth factor, both individually and combined. Proliferation was determined by a commercial biochemical assay. Real time RT-PCR determined gene expression of NMP-1 and -2, collagen types I and III, TIMP-1, -2 and -3. Autocrine effects on synthesis of bFGF and TGF-beta were evaluated by ELISA. Only TGF-beta, either isolated or associated with bFGF, significantly increased cell proliferation. TGF-beta had anabolic effects, increasing expression of type I and III collagen as well as of TIMPs, whereas bFGF had opposite effects. When bFGF and TGF-beta were associated, the anabolic effects prevailed. Synthesis of TGF-beta was induced only by the association of lower concentrations of the growth factors, whereas there was a dose-dependent production of bFGF. It is concluded that bFGF had a predominantly catabolic effect, and TGF-beta exerted an anabolic effect on hPDL cells. (c) 2007 Elsevier Ltd. All rights reserved.

Structure of human uropepsin at 2.45 angstrom resolution

The molecular structure of human uropepsin, an aspartic proteinase from the urine produced in the form of pepsinogen A in the gastric mucosa, has been determined by molecular replacement using human pepsin as the search model. Crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 50.99, b = 75.56, c = 89.90 Angstrom. Crystallographic refinement led to an R factor of 0.161 at 2.45 Angstrom resolution. The positions of 2437 non-H protein atoms in 326 residues have been determined and the model contains 143 water molecules. The structure is bilobal, consisting of two predominantly beta -sheet lobes which, as observed in other aspartic proteinases, are related by a pseudo-twofold axis. A model of the uropepsin-pepstatin complex has been constructed based on the high-resolution crystal structure of pepsin complexed with pepstatin.

Granulocyte colony-stimulating factor expression from transduced vascular smooth muscle cells provides sustained neutrophil increases in rats

Granulocyte colony-stimulating factor (G-CSF) regulates granulocyte precursor cell proliferation, neutrophil survival, and activation. Cyclic hematopoiesis, a disease that occurs both in humans and grey collie dogs is characterized by cyclical variations in blood neutrophils. Although the underlying molecular defect is not known, long-term daily administration of recombinant G-CSF eliminates the severe recurrent neutropenia, indicating that expression of G-CSF by gene therapy would be beneficial. As a prelude to preclinical studies in affected collie dogs, we monitored hematopoiesis in rats receiving vascular smooth muscle cells transduced to express G-CSF. Cells transduced with LrGSN, a retrovirus expressing rat G-CSF, were implanted in the carotid artery and control animals received cells transduced with LASN, a retrovirus expressing human adenosine deaminase (ADA). Test animals showed significant increases in neutrophil counts for at least 7 weeks, with mean values of 3,670 +/- 740 cells/mu l in comparison to 1,870 +/- 460 cells/mu l in controls (p < 0.001). Thus, in rats G-CSF gene transfer targeted at vascular smooth muscle cells initiated sustained production of 1,800 neutrophils/mu l, a cell number that would provide clinical benefit to patients. Lymphocytes, red cells and platelets were not different between control and test animals (p > 0.05). These studies indicate that retrovirally transduced vascular smooth muscle cells can provide sustained clinically useful levels of neutrophils in vivo.

Genetic damage in human peripheral lymphocytes exposed to antimicrobial endodontic agents

Objective. Formocresol, paramonochlorophenol, or calcium hydroxide have been widely used in dental practice to eradicate bacteria and consequently to produce root canal disinfection. Taking into consideration strong evidence for a relationship between DNA damage and carcinogenesis, the purpose of the present study was to evaluate the genotoxic effects of antimicrobial endodontic compounds in human peripheral lymphocytes by single-cell gel ( comet) assay. This technique detects DNA strand breaks in individual cells.Study design. A total of 10 mu L of the tested substance solution (formocreso1, paramonochlorofeno1, and calcium hydroxide at 100-mu g/mL concentration) was added to human peripheral lymphocytes from 10 volunteers for 1 hour at 37 degrees C. The negative control group was treated with vehicle control (PBS) for 1 hour at 37 degrees C, as well. For the positive control group, lymphocytes were exposed to hydrogen peroxide at 100 mu M during 5 minutes on ice.Results. No DNA breakage was detected after a treatment of peripheral lymphocytes by formocresol, paramonochlorophenol, or calcium hydroxide at 100 mu g/mL.Conclusions. In summary, our results indicate that exposure to formocresol, paramonochlorophenol, or calcium hydroxide may not be a factor that increases the level of DNA lesions in human peripheral lymphocytes as detected by single-cell gel (comet) assay.

Mutational analyses of human eIF5A-1-identification of amino acid residues critical for eIF5A activity and hypusine modification

The eukaryotic translation initiation factor 5A (eIF5A) is the only protein that contains hypusine [N-epsilon-(4-amino-2-hydroxybutyl)lysine], which is required for its activity. Hypusine is formed by post-translational modification of one specific lysine (Lys50 for human eIF5A) by deoxyhypusine synthase and deoxyhypusine hydroxylase. To investigate the features of eIF5A required for its activity, we generated 49 mutations in human eIF5A-1, with a single amino acid substitution at the highly conserved residues or with N-terminal or C-terminal truncations, and tested mutant proteins in complementing the growth of a Saccharomyces cerevisiae eIF5A null strain. Growth-supporting activity was abolished in only a few mutant eIF5As (K47D, G49A, K50A, K50D, K50I, K50R, G52A and K55A), with substitutions at or near the hypusine modification site or with truncation of 21 amino acids from either the N-terminus or C-terminus. The inactivity of the Lys50 substitution proteins is obviously due to lack of deoxyhypusine modification. In contrast, K47D and G49A were effective substrates for deoxyhypusine synthase, yet failed to support growth, suggesting critical roles of Lys47 and Gly49 in eIF5A activity, possibly in its interaction with effector(s). By use of a UBHY-R strain harboring genetically engineered unstable eIF5A, we present evidence for the primary function of eIF5A in protein synthesis. When selected eIF5A mutant proteins were tested for their activity in protein synthesis, a close correlation was observed between their ability to enhance protein synthesis and growth, lending further support for a central role of eIF5A in translation.

Prevalence and risk factors for human toxoplasmosis in a rural community

Toxoplasma gondii infection may lead to important pathological questions, especially in rural areas, where several sources of infection exist. Therefore, it is important to determine risk factors in order to establish adequate prophylactic measures. The present study aimed to assess the prevalence and risk factors involved in human toxoplasmosis infection in a rural community, in Eldorado, Mato Grosso do Sul State, Brazil. This community was composed of 185 farms - with 671 inhabitants - from which 20 were randomly chosen. In these farms, blood samples were collected from rural workers, who also answered a risk factor questionnaire. Serum samples were analyzed by means of direct agglutination test for the detection of anti-Toxoplasma gondii antibodies. From 73 samples collected, 79.45% were positive. None of the studied variables was significantly associated with the prevalence of the infection. However, among the individuals who reported eyesight impairments, 94.4% had anti-T. gondii antibodies, compared with 74.0% who did not report eyesight changes (p = 0.0594). Moreover, most individuals in the study (68.20%) were older than 18 years and presented 84.44% positivity, compared with 66.67% of positive individuals younger than 18 years old. We were able to conclude that a high prevalence of antibodies did not imply significant associations with the risk factors studied.

Expression of beta-human chorionic gonadotropin (beta-hCG) in non-trophoblastic elements of transitional cell carcinoma of the bladder: possible relationship with the prognosis.

Transitional cell carcinoma (TCC) of the bladder is a neoplasm with variability in its clinical behavior. Although there are several studies correlating stage and ABO isoantigen expression with invasiveness, there is no single predictor factor to assess the potential invasiveness, especially in the low grade, non-invasive TCC. In the present study we evaluated the correlation of histological grade plus stage and the expression of beta human chorionic gonadotropin (beta-hCG), in 100 cases of TCC, with the clinical behavior. These features were correlated with tumor progression in patients with at least two years of follow up. We observed more aggressiveness in G4 group (high grade and invasive) (93% had tumor progression) when compared to G1 group (low grade and superficial) (11% had tumor progression). However in 25.5% of the TCC cases (groups G2: low grade and invasive and G3: high grade and superficial) the clinical behavior was intermediate, showing some limitation in using grading and staging only, as a predictive factor. There was an expression of beta-hCG in 21.4% of the cases in up to 25% of the tumor cells without any trophoblastic morphology. These beta-hCG producing TCC had a strong correlation with aggressiveness: 39.1% and 12.8% of the TCC expressed beta-hCG with and without tumor progression, respectively.

Structures of the noncovalent complexes of human and bovine prothrombin fragment 2 with human PPACK-thrombin

Both human and bovine prothrombin fragment 2 (the second kringle) have been cocrystallized separately with human PPACK (D-Phe-Pro-Arg)-thrombin, and the structures of these noncovalent complexes have been determined and refined (R = 0.155 and 0.157, respectively) at 3.3-Å resolution using X-ray crystallographic methods. The kringles interact with thrombin at a site that has previously been proposed to be the heparin binding region. The latter is a highly electropositive surface near the C-terminal helix of thrombin abundant in arginine and lysine residues. These form salt bridges with acidic side chains of kringle 2. Somewhat unexpectedly, the negative groups of the kringle correspond to an enlarged anionic center of the lysine binding site of lysine binding kringles such as plasminogens K1 and K4 and TPA K2. The anionic motif is DGDEE in prothrombin kringle 2. The corresponding cationic center of the lysine binding site region has an unfavorable Arg70Asp substitution, but Lys35 is conserved. However, the folding of fragment 2 is different from that of prothrombin kringle 1 and other kringles: the second outer loop possesses a distorted two-turn helix, and the hairpin β-turn of the second inner loop pivots at Val64 and Asp70 by 60°. Lys35 is located on a turn of the helix, which causes it to project into solvent space in the fragment 2-thrombin complex, thereby devastating any vestige of the cationic center of the lysine binding site. Since fragment 2 has not been reported to bind lysine, it most likely has a different inherent folding conformation for the second outer loop, as has also been observed to be the case with TPA K2 and the urokinase kringle. The movement of the Val64-Asp70 β-turn is most likely a conformational change accompanying complexation, which reveals a new heretofore unsuspected flexibility in kringles. The fragment 2-thrombin complex is only the second cassette module-catalytic domain structure to be determined for a multidomain blood protein and only the third domain-domain interaction to be described among such proteins, the others being factor Xa without a Gla domain and Ca2+ prothrombin fragment 1 with a Gla domain and a kringle. © 1993 American Chemical Society.

Cloning and expression of the cDNA encoding rat granulocyte colony-stimulating factor

Granulocyte colony-stimulating factor (G-CSF) acts on precursor hematopoietic cells to control the production and maintenance of neutrophils. Recombinant G-CSF (re-G-CSF)is used clinically to treat patients with neutropenia and has greatly reduced the infection risk associated with bone marrow transplantation. Cyclic hematopoiesis, a stem cell defect characterized by severe recurrent neutropenia, occurs in man and grey collie dogs, and can be treated by administration of re-G-CSF. Availability of the rat G-CSF cDNA would benefit the use of rats as models of gene therapy for the treatment of cyclic hematopoiesis. In preliminary rat experiments, retroviral-mediated expression of canine G-CSF caused neutralizing antibody formation which precluded long-term increases in neutrophil counts. To overcome this problem we cloned the rat G-CSF cDNA from RNA isolated from skin fibroblasts. The rat G-CSF sequence shared a high degree of identity in both the coding and non-coding regions with both the murine G-CSF (85%) and human G-CSF (74%). The signal peptides of murine and human G-CSF both contained 30 amino acids (aa), whereas the deduced signal sequence for rat G-CSF possessed 21 aa. A retrovirus encoding the rat G-CSF cDNA synthesized bioactive G-CSF from transduced vascular smooth muscle cells.

Intrapulpar temperature during continuous CO2 laser irradiation in human molars: An in vitro study

To establish safety parameters, we in vitro studied the increase in intrapulpal temperature caused by the use of a cw CO2 laser. A thermistor was implanted in the inner part of the pulpal chamber of 25 human lower third molars to measure the intrapulpal temperature produced by laser powers between 2-10 W and exposure times of 0.5-25.0 s. The Pearson linear correlation factor applied to the measured values showed there is a direct relationship between the independent variable and the applied power. A variance analysis produced the linear regression equation: T=1.10+(0.127)E where T is the temperature and E the energy. The results showed that, with a power of 4 W and maximum exposure time of 2.5 s (10 J) and a power density of 12738.85 W cm-2, there will be no damaging reactions affecting the pulpal tissues.