Alterations in osteoclast morphology following long-term 17beta-estradiol administration in the mouse

Background  

Although the role of the osteoclast in bone resorption is becoming better understood, much remains to be learned about osteoclastogenesis and the exact mechanism of action of anti-resorbing agents such as 17β-estradiol. This study investigated bone and morphologic osteoclast alterations following long-term estrogen administration to the B6D2F1 mouse. B6D2F1 mice aged 4-5 weeks were exposed to high levels of estrogen via implanted silastic tubing for at least 12 weeks; controls received empty tubing. Femurs of control and treated mice were assessed with radiology, quantitative histomorphometry and transmission electron microscopy.     

Extracellular hyaline material in association with other cytologic features in aspirates from collagenous spherulosis and adenoid cystic carcinoma of the breast

OBJECTIVE: To reevaluate breast aspirates showing extracellular hyaline material (EHM) and globules to assess if clinicoradiologic and cytologic features could help in differentiating between benign and malignant lesions, especially collagenous spherulosis (CS) and adenoid cystic carcinoma (ACC). STUDY DESIGN: Fine needle aspiration was performed on 884 patients with breast lumps. The cytomorphologic features of 6 cases showing EHM, including classic hyaline globules (HGs), were analyzed in detail. Three cases also had hemorrhagic nipple discharge. Tissue diagnosis (4) and mammography (6) were available. RESULTS: Aspirate smears revealed high cellularity composed of monolayers: clusters of uniform, small cells; EHM; and HGs surrounded by similar cells. Benign naked nuclei and stromal fragments (4), nuclear pleomorphism (3), apocrine cells (2), foam cells (2), naked HGs (2) and spindle cells in proximity to HGs were also seen (4). Nipple discharge smears showed foam cells, erythrocytes (3) and epithelial cell clusters with hyaline material (1). The cytologic diagnosis was CS (4) and ACC (2). Histopathology confirmed the diagnosis of CS (2) and ACC (1). CONCLUSION: There may be a morphologic overlap between the cytomorphologic features of CS and ACC, leading to diagnostic errors. The presence of EHM and HGs in association with bland cellular features should be interpreted with caution to avoid erroneous diagnoses. Histopathology is mandatory in these cases because of their different prognostic implications.     

Oxidant and SDS-stable alkaline protease from Bacillus clausii I-52: production and some properties

AIMS: An investigation was carried out on an oxidative and SDS-stable alkaline protease secreted by Bacillus clausii of industrial significance. METHODS AND RESULTS: Maximum enzyme activity was produced when the bacterium was grown in the medium containing (g l-1): soyabean meal, 15; wheat flour, 10; liquid maltose, 25; K2HPO4, 4; Na2HPO4, 1; MgSO4.7H2O, 0.1; Na2CO3, 6. The enzyme has an optimum pH of around 11 and optimum temperature of 60 degrees C. The alkaline protease showed extreme stability towards SDS and oxidizing agents, which retained its activity above 75 and 110% on treatment for 72 h with 5% SDS and 10% H2O2, respectively. Inhibition profile exhibited by phenylmethylsulphonyl fluoride suggested that the protease from B. clausii belongs to the family of serine proteases. CONCLUSIONS: Bacillus clausii produced high levels of an extracellular protease having high stability towards SDS and H2O2. SIGNIFICANCE AND IMPACT OF THE STUDY: The alkaline protease from B. clausii I-52 is significant for an industrial perspective because of its ability to function in broad pH and temperature ranges in addition to its tolerance and stability in presence of an anionic surfactant, like SDS and oxidants like peroxides and perborates. The enzymatic properties of the protease also suggest its suitable application as additive in detergent formulations.     

Carnosic acid: A potent chemopreventive agent against oral carcinogenesis

The present study was aimed to investigate the chemopreventive potential of carnosic acid in 7,12-dimethylbenz(a)anthracene (DMBA)-induced hamster buccal pouch carcinogenesis. The chemopreventive potential was assessed by analyzing the tumor incidence, tumor volume and burden as well as by measuring the status of lipid peroxidation, non-enzymatic and enzymatic antioxidants and phase I and phase II detoxification enzymes. Oral squamous cell carcinoma was developed in the buccal pouch of golden Syrian hamsters by painting with 0.5% DMBA in liquid paraffin three times a week for 14 weeks. In the present study, 100% tumor formation was observed in hamsters treated with DMBA alone. Also, the status of lipid peroxidation, antioxidants and phase I and phase II detoxification enzymes were significantly altered during DMBA-induced oral carcinogenesis. Oral administration of carnosic acid at a dose of 10 mg/kg body weight/day to DMBA-treated animals completely prevented the tumor formation in the hamsters’ buccal pouches. Also, carnosic acid exerted potent anti-lipid peroxidative function and stimulated the detoxification cascade during DMBA-induced hamster buccal pouch carcinogenesis. The results of the present study suggest that the chemopreventive potential of carnosic acid is probably due to its anti-lipid peroxidative potential and modulating effect on carcinogen detoxification enzymes during DMBA-induced oral carcinogenesis.     

Suppression of cell proliferation, DNA protein cross-links, and induction of apoptosis by vanadium in chemical rat mammary carcinogenesis

Vanadium, a dietary micronutrient, has recently been considered as an important pharmacological agent. The present investigation was carried out to ascertain its anticarcinogenic potential against an experimental rat mammary carcinogenesis. Female Sprague-Dawley rats were treated with 7,12dimethylbenz(alpha)anthracene (DMBA) (0.5 mg/100 g body weight) by a single tail vein injection in an oil emulsion. Vanadium (ammonium monovanadate) at a concentration of 0.5 ppm (4.27 micromol/L) was supplemented in drinking water and given ad libitum to the experimental group. The present study was an attempt to assess the effect of vanadium (ammonium monovanadate) on cell proliferation, apoptosis and histopathology in the mammary tissue. We also have examined DNA fragmentation and DNA protein cross-links (DPC) in the liver of rats as well. Immunohistochemical analysis indicated that early neoplasia in mammary tissue proceeds by a decrease in apoptotic cell death (ACD), which was also examined with TUNEL assay, rather than an increase in cell proliferation (P<0.01). DPC in liver were reduced by vanadium treatment (ANOVA, F=13.7, P<0.01). Agarose gel electrophoresis revealed DNA fragmentation in the vanadium-treated group, confirming apoptosis further. Results of the study indicate that the mammary preneoplasia is sensitive to vanadium intervention whereas normal proliferating cells are not.     

Binding of nucleotide triphosphates to cardiotoxin analogue II from the Taiwan cobra venom (Naja naja atra). Elucidation of the structural interactions in the dATP-cardiotoxin analogue ii complex.

Snake venom cardiotoxins have been recently shown to block the enzymatic activity of phospholipid protein kinase and Na+,K+-ATPase. To understand the molecular basis for the inhibitory effects of cardiotoxin on the action of these enzymes, the nucleotide triphosphate binding ability of cardiotoxin analogue II (CTX II) from the Taiwan cobra (Naja naja atra) venom is investigated using a variety of spectroscopic techniques such as fluorescence, circular dichroism, and two-dimensional NMR. CTX II is found to bind to all the four nucleotide triphosphates (ATP, UTP, GTP, and CTP) with similar affinity. Detailed studies of the binding of dATP to CTX II indicated that the toxin molecule is significantly stabilized in the presence of the nucleotide. Molecular modeling, based on the NOEs observed for the dATP.CTX II complex, reveals that dATP binds to the CTX II molecule at the groove enclosed between the N- and C-terminal ends of the toxin molecule. Based on the results obtained in the present study, a molecular mechanism to account for the inhibition of the enzymatic activity of the phospholipid-sensitive protein kinase and Na+,K+-ATPase is also proposed.     

Multiplex PCR assay for the detection of enterotoxic <Emphasis Type="Italic">Bacillus cereus</Emphasis> group strains and its application in food matrices

Bacillus cereus, Bacillus thuringiensis and Bacillus anthracis are the major concerns for the food safety in terms of frequency and/or seriousness of the disease. Being members of the same group and sharing DNA homology to a larger extent, they do create problems when their specific detection/identification is attempted from different food and environmental sources. Numerous individual polymerase chain reaction (PCR) and few multiplex PCR (mPCR) methods have been employed to detect these organisms by targeting toxin genes but with lack of internal amplification control (IAC). Therefore, we attempted a mPCR with IAC for the detection of enterotoxic B. cereus group strains by selecting hbl A, nhe A and cyt K genes from B. cereus, indicative of the diarrheal potential and cry I A and pag genes, the plasmid borne phenotypic markers specific to B. thuringiensis and B. anthracis strains, respectively. Multiplex PCR assay validation was performed by simultaneous comparison with the results of single-target PCR assays and correlated to the classical conventional and biochemical identification of the organisms. The mPCR was able to detect as low as 10¹–10² organisms per ml following overnight enrichment of spiked food samples (vegetable biriyani and milk) in buffered peptone water (BPW). The presence of these organisms could also be detected by mPCR in naturally contaminated samples of rice based dishes and milk. The high throughput and cost-effective mPCR method described could provide a powerful tool for simultaneous, rapid and reliable detection of enterotoxic B. cereus group organisms.     

Vibrio cholerae Cytolysin Causes an Inflammatory Response in Human Intestinal Epithelial Cells That Is Modulated by the PrtV Protease

Background

Vibrio cholerae is the causal intestinal pathogen of the diarrheal disease cholera. It secretes the protease PrtV, which protects the bacterium from invertebrate predators but reduces the ability of Vibrio-secreted factor(s) to induce interleukin-8 (IL-8) production by human intestinal epithelial cells. The aim was to identify the secreted component(s) of V. cholerae that induces an epithelial inflammatory response and to define whether it is a substrate for PrtV.

Methodology/Principal Findings

Culture supernatants of wild type V. cholerae O1 strain C6706, its derivatives and pure V. cholerae cytolysin (VCC) were analyzed for the capacity to induce changes in cytokine mRNA expression levels, IL-8 and tumor necrosis factor-α (TNF-α) secretion, permeability and cell viability when added to the apical side of polarized tight monolayer T84 cells used as an in vitro model for human intestinal epithelium. Culture supernatants were also analyzed for hemolytic activity and for the presence of PrtV and VCC by immunoblot analysis.

Conclusions/Significance

We suggest that VCC is capable of causing an inflammatory response characterized by increased permeability and production of IL-8 and TNF-α in tight monolayers. Pure VCC at a concentration of 160 ng/ml caused an inflammatory response that reached the magnitude of that caused by Vibrio-secreted factors, while higher concentrations caused epithelial cell death. The inflammatory response was totally abolished by treatment with PrtV. The findings suggest that low doses of VCC initiate a local immune defense reaction while high doses lead to intestinal epithelial lesions. Furthermore, VCC is indeed a substrate for PrtV and PrtV seems to execute an environment-dependent modulation of the activity of VCC that may be the cause of V. cholerae reactogenicity.