Reverse transformation of Chinese hamster ovary cells by methyl xanthines : Structure-function relationships

Using a number of drugs that increase cellular cAMP levels, alterations in the amount of cell surface fibronectin and other transformation parameters were studied in Chinese hamster ovary (CHO) cells. The drugs include db-cAMP, different methylxanthines (theophylline, aminophylline, methyl isobutyl xanthine (MIX), caffeine and theobromine), papaverine and cholera toxin. Methylxanthines that have a methyl group at the seventh position lack reverse transforming potential; those that lack a methyl group at the seventh position induced reverse transformation in CHO cells, causing an increase in surface fibronectin, cell substratum adhesive strength and anchorage dependence for growth. Further, as methyl xanthines are substituted in other positions different from the seventh position, the more efficient they become in restoring normal phenotypic properties; the later agents also induced low saturation density via a cytostatic state causing accumulation of cells in the S and G2 phases of the cycle in contrast to the G1 arrest of normal cells at low saturation density. db-cAMP and cholera toxin induced cell elongation but like caffeine and theobromine, did not induce surface fibronectin. The non-methylxanthine phosphodiesterase inhibitor papaverine induced neither cell elongation nor surface fibronectin but produced a cytostatic effect similar to aminophylline and MIX. These studies suggest that the reverse transformation properties fall into two groups: (a) Differentiation-related properties including cell morphology, parallel alignment and surface matrix fibronectin, etc.; (b) cell cycle-related properties-low saturation density, cell arrest at G1 phase and anchorage-dependent growth. Phosphodiesterase inhibitors reversibly eliminate indefinite division potential of CHO cells by inducing a cytostatic situation and not by inducing a G1-specific arrest.     

Purification of proteins from polyacrylamide gels, free of detergent or dye

A two step procedure recovers proteins from sodium dodecyl sulfate polyacrylamide gels. The proteins are eluted by electrophoretic dialysis. The eluent is then passed through an Amberlite CG-400 anion-exchange resin. The recovery of protein is nearly total. The recovered proteins have no detectable sodium dodecyl sulfate contamination. With gels that have been stained with Coomassie Brilliant Blue R, the procedure recovers the proteins free of the dye. We have used this procedure successfully during the purification of epidermal glycoproteins.     

Nerve Growth Factor as a Mitogen for a Pancreatic Carcinoid Cell Line

Abstract: Carcinoid tumors are a group of neuroendocrine neoplasms distributed widely throughout the body but most commonly occurring in the gut. These tumors retain many characteristics of their neural crest origin, including secretion of neuroactive peptides and responsiveness to neurotrophic substances. Nerve growth factor (NGF), a neurotrophic protein involved in maintenance and differentiation of peripheral sympathetic and sensory neurons, regulates growth of several neural tumor cells by inducing a differentiated phenotype and subsequent inhibition of cell growth rate. We examined the actions of NGF in a functioning human pancreatic carcinoid cell line (termed BON). NGF has no effect on the cytoarchitecture or constitutive secretion of bioamines in this carcinoid cell line. NGF, however, stimulates the in vitro cellular proliferation of BON cells. BON cells possess mRNA for the NGF receptors (p75^LNGFR and p140^trkA) and membrane-associated tyrosine kinase activity is increased in response to NGF. Both the mitogenic activity of NGF, as well as the receptor-linked tyrosine kinase activity, can be abrogated in BON cells by the trkA inhibitor K-252a and specific anti-NGF antibody. Our studies demonstrate that NGF is a mitogen for this carcinoid cell line without effect on cellular phenotype or cytoarchitecture. NGF may play a role in the development and progression of human carcinoid tumors.     

Purification and characterization of a milk clotting protease from Rhizomucor miehei

Benzamidine, an inhibitor of serine proteases, was used as an affinity ligand for the purification of aspartyl protease from culture filtrate of Rhizomucor miehei. The two step purification protocol (ion-exchange and affinity chromatography) resulted in a homogenous enzyme preparation with seven-fold purification and a final recovery of 22%. The purified enzyme was free of brown pigmentation, a factor inherently associated with the enzyme; it was stable and active at acidic pH (optimum pH 4.1 for proteolytic activity and 5.6 for milk clotting activity). The significant positive characteristic of the enzyme is its comparatively lower thermostability; the enzyme was comparable to calf rennet in its properties of thermostability, milk-clotting to proteolytic activity ratio and sensitivity to CaCl2. Limited protease digestion of the purified enzyme with proteinase K yielded a 20kDa fragment as shown by SDS–PAGE. Native gel electrophoresis of the digest showed an additional peak of activity corresponding to the 20kDa fragment on SDS–PAGE, this fragment retained both milk-clotting and proteolytic activities. It was also inhibited by pepstatin A and hence it is presumed that this fragment contained the active site of the enzyme.     

Macrophage migration stimulation factor, migration inhibition factor and the role of suppressor cells in their production.

Guinea pig lymph node lymphocytes and human peripheral blood lymphocytes when stimulated by specific antigen or mitogen will release factors that affect in vitro macrophage migration. Migration inhibition factor production appears to be under the control of suppressor cells which are T lymphocytes. When suppressor cells are generated by stimulation with Con A for 4 days, migration stimulation factor (M.St.F.) activity is found. In other situations where M.St.F. is found this is thought to be due to increased suppressor cell activity. For example, young adults produce this lymphokine when stimulated with Con A, whereas aged individuals produce MIF. Concanavalin A appears to be the mitogen of choice for M.St.F. production, and phytohemagglutinin for MIF production. The release of this putative factor M.St.F. from suppressor T cells helps to explain some of the difficulties that have existed in studies of macrophage migration inhibition.     

Effect of Carica papaya on beta catenin and Wnt mRNA expression in human colon cancer (HT-29) cells in vitro

Colon cancer is the third most frequent cancer in humans. Carica papaya leaves are vegetable foods consumed by most people around the world; it has potential as an anticancer. Therefore it is of interest to investigate the effect of Carica papaya on beta catenin and Wnt mRNA expression in human colon cancer (HT-29) cells in vitro. Human Colon cancer cell line (HT-29) was purchased from the National Centre for Cell Sciences, Pune, India. Cell viability test was done by MTT assay. Gene expression analysis was done by Real Time-PCR. The obtained data were analyzed statistically by one-way analysis of variance and Duncan''s multiple range test with Graph Pad Prism version 5 to analyze the significance of individual variations among the control and experimental groups. The significance was considered at p<0.05 level in Duncan''s test. Carica papaya caused a marked increase in cell death in a dose dependent manner. At the end of 48 hours, maximum inhibition was at 300 and 400 µg/ml. Carica papaya has significantly reduced the mRNA expression of Wnt and beta catenin (p<0.05). Data showed that Carica papaya leaf extract has anticancer activity on Colon cancer cell lines (HT-29).     

Molecular docking analysis of stachydrine and sakuranetin with IL-6 and TNF-α in the context of inflammation

Inflammation is a process triggered by pro-inflammatory cytokines and anti-inflammatory molecules. Therefore, it is of interest to document the anti-inflammatory activity of Stachydrine and Sakuranetin against the inflammatory target proteins IL-6 and TNF-α by using molecular docking analysis. Both compounds showed good binding features with the selected target proteins. Compared to Sakuranetin, the Stachydrine have low binding energy and good hydrogen bond interactions. Hence, data show that Stachydrine possessed high and specific inhibitory activity on tumor necrosis factor-α and interleukin-6.     

Characterization of Chromosome Stability in Diploid,Polyploid and Hybrid Yeast Cells

Chromosome instability is a key component of cancer progression and many heritable diseases. Understanding why some chromosomes are more unstable than others could provide insight into understanding genome integrity. Here we systematically investigate the spontaneous chromosome loss for all sixteen chromosomes in Saccharomyces cerevisiae in order to elucidate the mechanisms underlying chromosome instability. We observed that the stability of different chromosomes varied more than 100-fold. Consistent with previous studies on artificial chromosomes, chromosome loss frequency was negatively correlated to chromosome length in S. cerevisiae diploids, triploids and S. cerevisiae-S. bayanus hybrids. Chromosome III, an equivalent of sex chromosomes in budding yeast, was found to be the most unstable chromosome among all cases examined. Moreover, similar instability was observed in chromosome III of S. bayanus, a species that diverged from S. cerevisiae about 20 million years ago, suggesting that the instability is caused by a conserved mechanism. Chromosome III was found to have a highly relaxed spindle checkpoint response in the genome. Using a plasmid stability assay, we found that differences in the centromeric sequence may explain certain aspects of chromosome instability. Our results reveal that even under normal conditions, individual chromosomes in a genome are subject to different levels of pressure in chromosome loss (or gain).     

Interaction of human recombinant alphaA- and alphaB-crystallins with early and late unfolding intermediates of citrate synthase on its thermal denaturation

We have investigated the role of recombinant human alphaA- and alphaB-crystallins in the heat-induced inactivation and aggregation of citrate synthase. Homo-multimers of both alphaA- and alphaB-crystallins confer protection against heat-induced inactivation in a concentration-dependent manner and also prevent aggregation. Interaction of crystallins with early unfolding intermediates of citrate synthase reduces their partitioning into aggregation-prone intermediates. This appears to result in enhanced population of early unfolding intermediates that can be reactivated by its substrate, oxaloacetate. Both these homo-multimers do not form a stable complex with the early unfolding intermediates. However, they can form a soluble, stable complex with aggregation-prone late unfolding intermediates. This soluble complex formation prevents aggregation. Thus, it appears that the chaperone activity of alpha-crystallin involves both transient and stable interactions depending on the nature of intermediates on the unfolding pathway; one leads to reactivation of the enzyme activity while the other prevents aggregation.