Immunofluorescent visualization of DNA replication sites within nuclei of Chinese hamster ovary cells

Summary The spatial distribution of replication sites was studied by a sensitive method in cells cultured in vitro. Exponentially growing Chinese hamster ovary cells were permeabilized and pulse labeled in the presence of deoxyribonucleoside triphosphates, dTTP being replaced by biotin-11-dUTP as a substrate for DNA replication. The distribution of replication sites was visualized in isolated nuclei by fluorescent microscopy of samples taken periodically after short-term (2 min) in vitro labeling and pulse-chase experiments. Propidium iodide and 4,6-diamino-2-pheny-lindole served as fluorescent probes for total cellular DNA. Avidin-fluorescein isothiocyanate and biotinylated goat antiavidin antibody were used in an amplification procedure to fluorescently label the incorporated biotin-11-dUTP. Similar experiments using synchronized cells showed the distribution of replicons at different stages of S phase.     

Absolute reaction rate and kinetics of protein adsorption at solid-liquid interfaces.

Extents of adsorption of bovine serum albumin from aqueous solution to the surface of alumina, silica, carbon and chromium powder have been studied as function of time for various values of bulk protein concentration, pH, ionic strength and temperature. The rates of adsorption in all cases have been observed to fit in the first order rate equation with two different rate constants Ka1 and Ka2. Effects of addition of SDS, CTAB and neutral salts on values of Ka1 and Ka2 have also been studied. Using Arrhenius equation the activation energy values Ea1 and Ea2 have been evaluated from the values of Ka1 and Ka2 at three different temperatures, respectively. The corresponding values of enthalpy of activation (delta H*), entropy of activation (delta S*), and free energy of activation (delta G*) have been evaluated using Eyring's equation of absolute reaction rate. The mechanism of protein adsorption has been discussed in the light of basic principles of absolute reaction rate. It has been found that for Ka1 the delta H*1 greater than T delta S*1 and for Ka2 T delta S*2 greater than H*2, i.e. the anchorage and binding of protein to the surface are enthalpy controlled processes whereas the surface denaturation as well as rearrangement and folding is an entropy controlled process. The role of diffusion on rate of adsorption has also been discussed.     

Stimulation of tubulin synthesis by thyroid hormone in the developing rat brain

The effect of triiodothyronine on the biogenesis of tubulin has been studied in the developing rat brain. In organ cultures of brains from newborn rats, the hormone stimulates the incorporation of [14C]leucine into tubulin by 60-80% within 2 h in the absence of any significant change in total protein synthesis. This stimulation is strictly age-dependent (only brains from late fetal or newborn rats are sensitive), dose-dependent (stimulation increases progressively and reaches a maximum level with physiological dose of the hormone) and displays tissue specificity. The temporal correspondence of the sensitivity of the rat brain to triiodothyronine with the period of normal rise in the level of tubulin and that of the maximal level of nuclear triiodothyronine receptors in the brain strongly suggests the involvement of the hormone in regulating the biogenesis of tubulin during the differentiation and maturation of neonatal rat brain.     

Generation of Novel Plasmids in Escherichia coli S17-1(pSUP106)

When the highly metal-resistant acidophilic heterotrophic strain, Acidiphilium symbioticum KM2, was incubated with two Escherichia coli strains, viz. S17-1 (pSUP106) and K12, on a medium that supported growth of these two divergent species of different habitats, E. coli transconjugants were isolated that contained novel plasmids and were resistant to Zn²⁺ (48 mM), Cu²⁺ (12 mM), Ni²⁺ (12 mM), chloramphenicol (50 μg/ml), and tetracycline (25 μg/ml). The transconjugant plasmids did not hybridize with any of the A. symbioticum KM2 plasmids. After curing of the plasmids, the transconjugants became sensitive to 12 mM Zn²⁺, 12 mM Cu²⁺, and 12 mM Ni²⁺, but remained chloramphenicol and tetracycline resistant—the phenotypic markers that were originally present in pSUP106. That a part of pSUP106 was integrated into the chromosome of the transconjugants was evident from the hybridization of pSUP106 with chromosomal DNA of the cured derivatives of the transconjugants. Further, the transconjugant plasmids hybridized only with the chromosomal DNA of E. coli S17-1 and not with the chromosomal DNA of A. symbioticum KM2 or E. coli K12, suggesting their host chromosomal origin. Thus, the present study describes a unique event of genetic rearrangements in the E. coli strain S17-1 (pSUP106), resulting in the formation of novel plasmids conferring metal-resistance phenotypes in the cell. Received: 5 April 2002 / Accepted: 5 July 2002     

Geochemical and organic carbon isotope studies across the continental Permo-Triassic boundary of Raniganj Basin, eastern India

Organic carbon isotope and geochemical changes across a continental interior Permo-Triassic boundary section from the Raniganj Basin, India, indicate a ∼9‰ drop in organic carbon δ¹³C in the Early Triassic and synchroneity of this event throughout the Pangea. The study demands a common causative mechanism for the perturbation of the global carbon reservoir and not a combination of multiple causes. A global sea-level fall and oxidation of marine gas hydrates possibly increased the ¹²CO₂ input in the ocean-atmosphere system which caused a climatic shift from humid to warm semi-arid type and consequent extinction of land plants. Simultaneous increase in erosion from near-barren lands, change in the erosional base level and provenance deposited the boundary sandstone with positive europium anomaly and debris flow type matrix rich conglomerate. No extraterrestrial source, therefore, is needed to explain this Eu anomaly. The response of the terrestrial plant community to this perturbation of the carbon reservoir was, however, sluggish and the δ¹³C drop took place slowly, being maximal in the Early Triassic only.     

Down‐regulation of uPA and uPAR by 3,3′‐diindolylmethane contributes to the inhibition of cell growth and migration of breast cancer cells

3,3′‐Diindolylmethane (DIM) is a known anti‐tumor agent against breast and other cancers; however, its exact mechanism of action remains unclear. The urokinase plasminogen activator (uPA) and its receptor (uPAR) system are involved in the degradation of basement membrane and extracellular matrix, leading to tumor cell invasion and metastasis. Since uPA‐uPAR system is highly activated in aggressive breast cancer, we hypothesized that the biological activity of B‐DIM could be mediated via inactivation of uPA‐uPAR system. We found that B‐DIM treatment as well as silencing of uPA‐uPAR led to the inhibition of cell growth and motility of MDA‐MB‐231 cells, which was in part due to inhibition of VEGF and MMP‐9. Moreover, silencing of uPA‐uPAR led to decreased sensitivity of these cells to B‐DIM indicating an important role of uPA‐uPAR in B‐DIM‐mediated inhibition of cell growth and migration. We also found similar effects of B‐DIM on MCF‐7, cells expressing low levels of uPA‐uPAR, which was due to direct down‐regulation of MMP‐9 and VEGF, independent of uPA‐uPAR system. Interestingly, over‐expression of uPA‐uPAR in MCF‐7 cells attenuated the inhibitory effects of B‐DIM. Our results, therefore, suggest that B‐DIM down‐regulates uPA‐uPAR in aggressive breast cancers but in the absence of uPA‐uPAR, B‐DIM can directly inhibit VEGF and MMP‐9 leading to the inhibition of cell growth and migration of breast cancer cells. J. Cell. Biochem. 108: 916–925, 2009. © 2009 Wiley‐Liss, Inc.     

Gender differences in psychotic bipolar mania

Objective: This study examined gender differences in the prevalence and types of psychotic symptoms in bipolar mania.Methods: Participants were drawn from consecutive admissions to the psychiatric clinic in Chemnitz, Germany, in 2005. The diagnosis of bipolar disorder, manic episode was made within 24 hours of admission, and the severity of mania was assessed using the Young Mania Rating Scale (YMRS) and the German version of the Altman Self-Rating Mania Scale. Data collected for each patient included age at the onset of bipolar illness, number of previous episodes, social functioning between episodes, and duration of hospitalization for the index episode. Based on the Task Force for Methods and Documentation in Psychiatry system, psychotic symptoms were classified as hallucinations (visual, auditory, olfactory, tactile, acousma, somatic); delusions (paranoid, reference, guilt, grandeur, religious, erotomania, hypochondriac, poverty, jealousy); and ego disorder (thought control, thought broadcasting).Results: One hundred thirty-seven women and 109 men met the criteria for an acute manic episode, of whom 93 women and 62 men had psychotic symptoms. Compared with psychotic men, psychotic women had more delusions and hallucinations, both overall and per patient, and more delusions of reference and paranoid delusions. Psychotic women had more mixed states compared with psychotic men. Psychotic women differed from both psychotic men and nonpsychotic women on a number of clinical and social variables: they had higher YMRS scores and more previous episodes of depression despite an earlier onset of illness.Conclusion: Women with bipolar mania exhibited a specific pattern of psychotic symptoms that appeared to be associated with greater severity of the acute episode, more mixed states, and a more severe course of illness.     

Balanced nitrogen economy as a flexible strategy on yield stabilizing and quality of aquatic food crops in wetland ecosystem

In wetland ecosystem, nitrogen along with other elements and its management is most imperative for the production of so many aquatic food, non-food and beneficial medicinal plants and for the improvement of soil and water characteristics. With great significant importance of INM (integrated nutrient management) as sources, emphasizing on management on nitrogen as a key element and its divergence, a case study was undertaken on such aquatic food crops (starch and protein-rich, most popular and remunerative) in the farmers' field of low-lying 'Tal' situation of New Alluvial Zone of Indian subtropics. The study was designed in factorial randomized block design, where, three important aquatic food crops (water chestnut (Trapa bispinosa Roxb.), makhana (Euryale ferox Salisb.) and water lily (Nymphaea spp.) as major factor and eleven combinations of organic and inorganic sources of nutrients as sub-factor was considered in the experiment. It revealed from the results that the production of fresh kernels or nuts of water chestnut (8.571 ha-1), matured nut yield of makhana (3.06 t ha-1) and flower stalks of water-lily as vegetables (6.38 t ha-1) including its nutritional quality (starch, protein, sugar and minerals) was remarkably influenced with the application of both organic (neem oilcake @ 0.2 t ha-1) and inorganic sources (NPK @ 30:20:20 kg ha-1 along with spraying of NPK @ 0.5% each over crop canopy at 20 days interval after transplanting) than the other INM combinations applied to the crops. Among the crops, highest WCYE (water chestnut yield equivalence) exhibited in makhana due to its high price of popped-form in the country, which is being exported to other countries at now. Sole application of both (organic and inorganic sources) with lower range did not produce any significant outcome from the study and exhibited lower value for all the crops. Besides production of food crops, INM also greatly influenced the soil and water characterization and it was favourably reflected in this study. The physico-chemical characteristics of soil (textural class, pH, organic carbon, organic matter, ammoniacal nitrogen, nitrate nitrogen, available nitrogen, phosphorus and potassium) are most important and contributed a significant improvement due to cultivation of these aquatic crops. Analysis of such wet bodies represented the water characteristics (pH, BOD, COD, CO3 =, HCO3-, NO3- N, SO4-S and Cl-) were most responsive, adaptable and quite favourable for the cultivation of these crops in this vast waste unused wetlands for the mankind without any environmental degradation.