A critical importance of polyamine site in NMDA receptors for neurite outgrowth and fasciculation at early stages of P19 neuronal differentiation

We have investigated the role of N-methyl-d-aspartate receptors (NMDARs) and γ-aminobutyric acid receptors type A (GABA_ARs) at an early stage of P19 neuronal differentiation. The subunit expression was profiled in 24-hour intervals with RT-PCR and functionality of the receptors was verified via fluo-3 imaging of Ca²⁺ dynamics in the immature P19 neurons showing that both NMDA and GABA excite neuronal bodies, but only polyamine-site sensitive NMDAR stimulation leads to enhanced Ca²⁺ signaling in the growth cones. Inhibition of NR1/NR2B NMDARs by 1 μM ifenprodil severely impaired P19 neurite extension and fasciculation, and this negative effect was fully reversible by polyamine addition. In contrast, GABA_AR antagonism by a high dose of 200 μM bicuculline had no observable effect on P19 neuronal differentiation and fasciculation. Except for the differential NMDAR and GABA_AR profiles of Ca²⁺ signaling within the immature P19 neurons, we have also shown that inhibition of NR1/NR2B NMDARs strongly decreased mRNA level of NCAM-180, which has been previously implicated as a regulator of neuronal growth cone protrusion and neurite extension. Our data thus suggest a critical role of NR1/NR2B NMDARs during the process of neuritogenesis and fasciculation of P19 neurons via differential control of local growth cone Ca²⁺ surges and NCAM-180 signaling.     

Involvement of Linear Plasmids in Aerobic Biodegradation of Vinyl Chloride

Pseudomonas putida strain AJ and Ochrobactrum strain TD were isolated from hazardous waste sites based on their ability to use vinyl chloride (VC) as the sole source of carbon and energy under aerobic conditions. Strains AJ and TD also use ethene and ethylene oxide as growth substrates. Strain AJ contained a linear megaplasmid (approximately 260 kb) when grown on VC or ethene, but it contained no circular plasmids. While strain AJ was growing on ethylene oxide, it was observed to contain a 100-kb linear plasmid, and its ability to use VC as a substrate was retained. The linear plasmids in strain AJ were cured, and the ability of strain AJ to consume VC, ethene, and ethylene oxide was lost following growth on a rich substrate (Luria-Bertani broth) through at least three transfers. Strain TD contained three linear plasmids, ranging in size from approximately 90 kb to 320 kb, when growing on VC or ethene. As with strain AJ, the linear plasmids in strain TD were cured following growth on Luria-Bertani broth and its ability to consume VC and ethene was lost. Further analysis of these linear plasmids may help reveal the pathway for VC biodegradation in strains AJ and TD and explain why this process occurs at many but not all sites where groundwater is contaminated with chloroethenes. Metabolism of VC and ethene by strains AJ and TD is initiated by an alkene monooxygenase. Their yields during growth on VC (0.15 to 0.20 mg of total suspended solids per mg of VC) are similar to the yields reported for other isolates (i.e., Mycobacterium sp., Nocardioides sp., and Pseudomonas sp.).     

Antineoplastic effects of clove buds (Syzygium aromaticum L.) in the model of breast carcinoma

It is supposed that plant functional foods, rich in phytochemicals, may potentially have preventive effects in carcinogenesis. In this study, the anticancer effects of cloves in the in vivo and in vitro mammary carcinoma model were assessed. Dried flower buds of cloves (CLOs) were used at two concentrations of 0.1% and 1% through diet during 13 weeks after the application of chemocarcinogen. After autopsy, histopathological and immunohistochemical analyses of rat mammary carcinomas were performed. Moreover, in vitro evaluation using MCF‐7 cells was carried out. Dietary administered CLO caused the dose‐dependent decrease in tumour frequency by 47.5% and 58.5% when compared to control. Analysis of carcinoma cells in animals showed bcl‐2, Ki67, VEGFA, CD24 and CD44 expression decrease and Bax, caspase‐3 and ALDH1 expression increase after high‐dose CLO administration. MDA levels were substantially decreased in rat carcinomas in both CLO groups. The evaluation of histone modifications revealed increase in lysine trimethylations and acetylations (H4K20me3, H4K16ac) in carcinomas after CLO administration. TIMP3 promoter methylation levels of CpG3, CpG4, CpG5 islands were altered in treated cancer cells. An increase in total RASSF1A promoter methylation (three CpG sites) in CLO 1 group was found. In vitro studies showed antiproliferative and pro‐apoptotic effects of CLO extract in MCF‐7 cells (analyses of cytotoxicity, Brdu, cell cycle, annexin V/PI, caspase‐7, Bcl‐2 and mitochondrial membrane potential). This study showed a significant anticancer effect of clove buds in the mammary carcinoma model in vivo and in vitro.     

Somatostatin affects morphology and secretion of pituitary luteinizing hormone (LH) cells in male rats

The somatostatin peptides (SRIH-14, SRIH-28) and their multiple receptors are generally associated with anti-proliferative and anti-secretory actions. This study compared, using standard morphometric measurements and terminal serum LH concentrations, effects of intracerebroventricular (icv) SRIH-14 and SRIH-28 in nanomolar amounts on immunohistochemically identified LH cells in pituitary glands of male rats. Rats received l microg/5 microl of SRIH-14 or SRIH-28 icv on days 1,3, and 5, whereas control rats received only icv saline. Animals were killed 5 days later for serum LH assays. Pituitarys were harvested for PAP immunohistochemistry and morphometry. Morphometric measurements were made by an observer blinded to the treatment group. Histochemically identified LH cells from both SRIH groups appeared smaller, often pycnotic and darkly stained compared to those from saline-treated rats. Both SRIH treatments reduced (p < 0.05) the quantitative morphometric measurements for cell volume, nuclear volume, and relative volume density. Both SRIH treatments also reduced serum LH concentration (p < 0.05), supporting the hypothesis that systemic physiology was altered. Collectively, the data support the opinion that nanomolar amounts of either SRIH peptide, acting on receptors reached from cerebrospinal fluid, exert an anti-secretory effect on LH cells of male rats. Modifications of central SRIH receptors may provide an approach for treatment of male sexual dysfunction and/or be of pathophysiologic significance in these disturbances.     

Distinct natures of beryllium fluoride-bound, aluminum fluoride-bound, and magnesium fluoride-bound stable analogues of an ADP-insensitive phosphoenzyme intermediate of sarcoplasmic reticulum Ca2+-ATPase: changes in catalytic and transport sites during phosphoenzyme hydrolysis

The structural natures of stable analogues for the ADP-insensitive phosphoenzyme (E2P) of Ca(2+)-ATPase formed in sarcoplasmic reticulum vesicles, i.e. the enzymes with bound beryllium fluoride (BeF.E2), bound aluminum fluoride (AlF.E2), and bound magnesium fluoride (MgF.E2), were explored and compared with those of actual E2P formed from P(i) without Ca(2+). Changes in trinitrophenyl-AMP fluorescence revealed that the catalytic site is strongly hydrophobic in BeF.E2 as in E2P but hydrophilic in MgF.E2 and AlF.E2; yet, the three cytoplasmic domains are compactly organized in these states. Thapsigargin, which was shown in the crystal structure to fix the transmembrane helices and, thus, the postulated Ca(2+) release pathway to lumen in a closed state, largely reduced the tryptophan fluorescence in BeF.E2 as in E2P, but only very slightly (hence, the release pathway is likely closed without thapsigargin) in MgF.E2 and AlF.E2 as in dephosphorylated enzyme. Consistently, the completely suppressed Ca(2+)-ATPase activity in BeF-treated vesicles was rapidly restored in the presence of ionophore A23187 but not in its absence by incubation with Ca(2+) (over several millimolar concentrations) at pH 6, and, therefore, lumenal Ca(2+) is accessible to reactivate the enzyme. In contrast, no or only very slow restoration was observed with vesicles treated with MgF and AlF even with A23187. BeF.E2 thus has the features very similar to those characteristic of the E2P ground state, although AlF.E2 and MgF.E2 most likely mimic the transition or product state for the E2P hydrolysis, during which the hydrophobic nature around the phosphorylation site is lost and the Ca(2+) release pathway is closed. The change in hydrophobic nature is probably associated with the change in phosphate geometry from the covalently bound tetrahedral ground state (BeF(3)(-)) to trigonal bipyramidal transition state (AlF(3) or AlF(4)(-)) and further to tetrahedral product state (MgF(4)(2-)), and such change likely rearranges transmembrane helices to prevent access and leakage of lumenal Ca(2+).     

Involvement of linear plasmids in aerobic biodegradation of vinyl chloride

Pseudomonas putida strain AJ and Ochrobactrum strain TD were isolated from hazardous waste sites based on their ability to use vinyl chloride (VC) as the sole source of carbon and energy under aerobic conditions. Strains AJ and TD also use ethene and ethylene oxide as growth substrates. Strain AJ contained a linear megaplasmid (approximately 260 kb) when grown on VC or ethene, but it contained no circular plasmids. While strain AJ was growing on ethylene oxide, it was observed to contain a 100-kb linear plasmid, and its ability to use VC as a substrate was retained. The linear plasmids in strain AJ were cured, and the ability of strain AJ to consume VC, ethene, and ethylene oxide was lost following growth on a rich substrate (Luria-Bertani broth) through at least three transfers. Strain TD contained three linear plasmids, ranging in size from approximately 90 kb to 320 kb, when growing on VC or ethene. As with strain AJ, the linear plasmids in strain TD were cured following growth on Luria-Bertani broth and its ability to consume VC and ethene was lost. Further analysis of these linear plasmids may help reveal the pathway for VC biodegradation in strains AJ and TD and explain why this process occurs at many but not all sites where groundwater is contaminated with chloroethenes. Metabolism of VC and ethene by strains AJ and TD is initiated by an alkene monooxygenase. Their yields during growth on VC (0.15 to 0.20 mg of total suspended solids per mg of VC) are similar to the yields reported for other isolates (i.e., Mycobacterium sp., Nocardioides sp., and Pseudomonas sp.).     

The histopathological characterization of oralCandida leukoplakias

Sixty four biopsy specimens of oralCandida leukoplakias were examined. Histological sections were stained with periodic acid-Schiff (PAS) reagent and by Grocott’s silver method.C. albicans isolates were cultivated from all the patients but fungal hyphae were proved histologically only in 23 of them. PAS-positive and Grocott-positive elements were exclusively observed in the superficial epithelial layers,e.g., the cornified layer and the stratum grnulosum. The inflammatory reaction was characterized by an early phase during which polymorphonuclear cells predominated. The skeletal muscle fascicles immediately adjacent to the infected epithelium showed striking degeneration and atrophy associated with a marked infiltrate of chronic inflammatory cells.     

Yeast-like microorganisms in eye infections

The proportion of yeast species involved in eye infections in 11 patients was examined. The presence of yeast organisms as causative agents of endophthalmitis was found in corneal smears (n=4), conjunctival swabs (4), and vitreous fluid (3). Altogether 5 strains ofCandida albicans, 2 strains ofC. krusei and one strain each ofC. guilliermondii, C. parapsilosis, C. tropicalis andCryptococcus neoformans were isolated from the clinical material. The hematogenic origin of endophthalmitis was proved in 7 cases on the basis of positive blood samples and in 2 cases by the isolation of yeasts from the tip of an intravenous catheter. Endophthalmitis-supporting risk factors such as indwelling intravenous catheters, prolonged use of broad-spectrum antibiotics and chemotherapy, surgical intervention, diabetes mellitus, and malignancy were observed in the patients.     

Electrophoretic karyotyping ofCandida albicans strains isolated from premature infants and hospital personnel in a neonatal intensive care unit

Electrophoretic karyotyping was used to compare DNA probes of yeasts isolated from blood of preterm neonates (n=66) in a neonatal intensive care unit (NICU) and from the hands of healthy hospital personnel (n=10). The yeasts were identified asCandida albicans using standard laboratory methods. DNA was extracted from yeasts and isolation of identical DNA strains from the pairs nurse-neonate suggested that one nurse transmitted one yeast strain by her hands to three neonates. Four neonates harbored two identical strains originating from two nurses,i.e. each nurse transmitted the same strain to two neonates. In the additional 7 cases transmission of 1 yeast strain by 1 nurse, to 1 neonate was observed. Our data suggest that nonperinatal nosocomial transmission ofC. albicans occurs in neonates. possiblyvia cross-contamination being transferred on hands of health care workers The importance of careful hand washing of staff (health care workers) and other infectioncontrol procedures (to prevent the nosocomial transmission of pathogens in the NICU environment) is emphasizeded.