A method for the rapid establishment of normal adult mammalian colonic epithelial cell cultures

Summary Normal colonic epithelial cell cultures of mammalian origin are required to facilitate the study of both normal cellular functions as well as pathogenesis of certain (human) colonic diseases. To date, little information is available regarding the growth requirements of colonic epithelial cells in culture of eitehr animal or human origin. Such data would enable the development of a long-term culture system for these cells. In this study, we present methodology that results in the establishment of homogeneous cultures of adult rabbit colonic epithelial reproducibly, quickly, and in quantity. The epithelial nature of the cultures is unambiguously established by intermediate filament typing using antikeratin antibodies. Such culutres can now be used for a variety of functional studies as well as to investigate the growth requirements of colonic epithelial in culture. This work was supported by the Blinder Foundation for Crohn’s Disease Research, Harbor UCLA IBD Center (AM 36200) and grant AM 27806 from the National Institutes of Health, Bethesda, MD.     

Selection intensities and consanguineity in the Yadava and Vadabalija of Visakhapatnam, Andhra Pradesh, India.

The opportunity for intensity of selection is studied in two backward caste groups with different occupations, namely Yadava (pastoral) and Vadabalija (fishing) of Bheemunipatnam Taluk of Visakhapatnam District, Andhra Pradesh. The amount of selection intensity is found to be higher in Vadabalija (0.8583) than Yadava (0.5827). The contribution of mortality to the total index of selection is greater in Vadabalija (0.6095) than in Yadava (0.3326). When consanguineity is considered, the fertility components of selection intensity (0.1885) are lower in the consanguineous group of Yadava than in the nonconsanguineous group (0.2845), whereas in Vadabalija they are higher in the consanguineous group (0.5261) than in the nonconsanguineous group (0.2223).     

Continuous biosorption of cadmium by Moringa olefera in a packed column

The biosorption of Cd(II) by Moringa oleifera using a batch system and a continuous up flow mode in a fixed bed column was studied. Batch adsorption experiments were performed as a function of pH, biosorbent dose, contact time, volume of the solution, and initial metal concentration. The adsorption isotherms obtained fitted well into the Freundlich and Langmuir isotherms. The dynamic removal of cadmium by powdered seed of the Moringa oleifera was studied in a packed column. The effect of bed height (4 and 8 cm) and flow rate (2 and 5mL/min) on biosorption process was investigated and the experimental breakthrough curves were obtained. Results showed that by increasing the bed height and decreasing the flow rate, the breakthrough and exhaustion times increased. The break-through time was considered as a measure of the column performance. The maximum break-through time of 320 min was achieved at the operating condition of 2 mL/min influent flow rate and bed height of 8 cm.     

Structural,conformational and thermodynamic aspects of groove-directed-intercalation of flavopiridol into DNA

Certain plant-derived alkaloids and flavonoids have shown propitious cytotoxic acitvity against different types of cancer, having deoxyribose nucleic acid (DNA) as their main cellular target. Flavopiridol, a semi-synthetic derivative of rohitukine (a natural compound isolated from Dysoxylum binectariferum plant), has attained much attention owing to its anticancer potential against various haematological malignancies and solid tumours. This work focuses on investigating interaction between flavopiridol and DNA at molecular level in order to decipher its underlying mechanism of action, which is not well understood. To define direct influence of flavopiridol on the structural, conformational and thermodynamic aspects of DNA, various spectroscopic and calorimetric techniques have been used. ATR-FTIR and SERS spectral outcomes indicate a novel insight into groove-directed-intercalation of flavopiridol into DNA via direct binding with nitrogenous bases guanine (C6=O6) and thymine (C2=O2) in DNA groove together with slight external binding to its sugar–phosphate backbone. Circular dichroism spectral analysis of flavopiridol–DNA complexes suggests perturbation in native B-conformation of DNA and its transition into C-form, which may be localized up to a few base pairs of DNA. UV–visible spectroscopic results illustrate dual binding mode of flavopiridol when interacts with DNA having association constant, K_a = 1.18 × 10⁴ M^?1. This suggests moderate type of interaction between flavopiridol and DNA. Further, UV melting analysis also supports spectroscopic outcomes. Thermodynamically, flavopiridol–DNA complexation is an enthalpy-driven exothermic process. These conclusions drawn from this study could be helpful in unveiling mechanism of cytoxicity induced by flavopiridol that can be further applied in the development of flavonoid-based new chemotherapeutics with more specificity and better efficacy.     

Purification and characterization of flavokinase from Neurospora crassa

The ATP-dependent phosphorylation of riboflavin to FMN by flavokinase is the key step in flavin biosynthesis. Flavokinase has been purified from a fungal source for the first time. The enzyme purified from a cell wall lacking mutant of Neurospora crassa, slime, is a monomer of M(r) 35.5 kDa with maximal activity at alkaline pH and high temperature (55 degrees C). The K(m) for both substrates is the lowest reported for flavokinase from any source so far (120 nM for riboflavin and 210 nM for MgATP2-). The enzyme exhibits preference for Mg2+ over Zn2+ as the essential activator and is also significantly activated by several cations. Activation by orthophosphate may be physiologically relevant for the intracellular regulation of flavokinase.     

SWAP-70 regulates c-kit-induced mast cell activation, cell-cell adhesion, and migration

SWAP-70, an unusual phosphatidylinositol-3-kinase-dependent protein that interacts with the RhoGTPase Rac, is highly expressed in mast cells. Cultured bone marrow mast cells (BMMC) from SWAP-70(-/-) mice are reduced in FcepsilonRI-triggered degranulation. This report describes the hitherto-unknown role of SWAP-70 in c-kit receptor signaling, a key proliferation and differentiation pathway in mast cells. Consistent with the role of Rac in cell motility and regulation of the actin cytoskeleton, mutant cells show abnormal actin rearrangements and are deficient in migration in vitro and in vivo. SWAP-70(-/-) BMMC are impaired in calcium flux, in proper translocation and activity of Akt kinase (required for mast cell activation and survival), and in translocation of Rac1 and Rac2 upon c-kit stimulation. Adhesion to fibronectin is reduced, but homotypic cell association induced through c-kit is strongly increased in SWAP-70(-/-) BMMC. Homotypic association requires extracellular Ca(2+) and depends on the integrin alpha(L)beta(2) (LFA-1). ERK is hyperactivated upon c-kit signaling in adherent and dispersed mutant cells. Together, we suggest that SWAP-70 is an important regulator of specific effector pathways in c-kit signaling, including mast cell activation, migration, and cell adhesion.     

Attenuation of dengue virus infection by adeno-associated virus-mediated siRNA delivery

BACKGROUND: The need for safe and effective treatment of dengue virus (DEN), a class A agent that causes dengue hemorrhagic fever/dengue shock syndrome, has been a critical global priority. An effective vaccine for DEN is not yet available. In this study the possibility of attenuating DEN infection using adeno-associated virus (AAV)-encoded short interfering RNAs (siRNA) was examined in Vero cells and human dendritic cells (DCs). METHODS: A cassette encoding siRNA targeted to a 3' untranslated sequence common to all DEN serotypes was designed and tested for its ability to attenuate DEN infection by use of AAV delivery. RESULTS: Vero cells or DCs infected with AAV-siRNA showed a significant, dose-dependent reduction in DEN infection. Treatment of DCs with AAV-siRNA also decreased the DEN-induced apoptosis of DCs and did not induce significant inflammation. CONCLUSION: These results demonstrate that AAV-mediated siRNA delivery is capable of reducing DEN infection in cells and may be useful in decreasing DEN replication in humans.     

The significance of alpha 5 beta 1 integrin dependent and independent actin cytoskelton organization in cell transformation and survival

Cell-matrix and cell-cell interactions are important physiological determinants of cell growth, survival and transformation. Cell adhesion to the extra cellular matrix (ECM) via integrins also crucially influences the organization of the cytoskeleton. It triggers a cascade of intracellular biochemical events, which regulate cell viability and growth. We have studied the relationship between cell attachment to the substratum and cytoskeletal organization and cell survival and transformation. Our results demonstrate that in the absence of attachment to the substratum, adhesion-dependent fibroblasts exhibit rapid loss of viability. However, a small percentage of cells survive even after remaining non-adherent for 16h. The adherent and non-adherent cells differ from one another both morphologically and physiologically. The latter show a loss of alpha5beta1 integrin expression on their surface and bind non-specifically to the substratum and ECM, thereby activating certain pathways more efficiently than adherent cells. We have also shown that non-adherent cells grow faster and have worse cytoskeletal organization after attachment to the substratum, and do not form focal adhesions or actin stress fibres. Hence, our data suggests that rat fibroblasts in prolonged suspension exhibit some properties that are comparable to cells undergoing transformation, by adapting integrin-dependent or independent signalling pathways for their survival.     

Prion Protein Does Not Confer Resistance to Hippocampus-Derived Zpl Cells against the Toxic Effects of Cu2+, Mn2+, Zn2+ and Co2+ Not Supporting a General Protective Role for PrP in Transition Metal Induced Toxicity

The interactions of transition metals with the prion protein (PrP) are well-documented and characterized, however, there is no consensus on their role in either the physiology of PrP or PrP-related neurodegenerative disorders. PrP has been reported to protect cells from the toxic stimuli of metals. By employing a cell viability assay, we examined the effects of various concentrations of Cu²⁺, Zn²⁺, Mn²⁺, and Co²⁺ on Zpl (Prnp ^-/-) and ZW (Prnp ^+/+) hippocampus-derived mouse neuronal cells. Prnp ^-/- Zpl cells were more sensitive to all four metals than PrP-expressing Zw cells. However, when we introduced PrP or only the empty vector into Zpl cells, we could not discern any protective effect associated with the presence of PrP. This observation was further corroborated when assessing the toxic effect of metals by propidium-iodide staining and fluorescence activated cell sorting analysis. Thus, our results on this mouse cell culture model do not seem to support a strong protective role for PrP against transition metal toxicity and also emphasize the necessity of extreme care when comparing cells derived from PrP knock-out and wild type mice.     

Iodine-catalyzed conjugate addition of indoles onto en-1,4-dione: a novel synthesis of 3-(1-(1H-indol-3-yl)-2-oxo-2-phenylethyl)indolin-2-ones as antibacterial and antifungal agents

Indole and its derivatives undergo smooth conjugate addition onto en-1,4-dione derived from isatin and acetophenone, in the presence of a catalytic amount of molecular iodine in acetonitrile under mild conditions to afford a novel class of 3-(1-(1H-indol-3-yl)-2-oxo-2-phenylethyl)indolin-2-one derivatives in good yields with high degree of 1,4-selectivity. Some of these compounds are found to exhibit modest antibacterial and antifungal properties.