Role of vitamin d on the inhibition of gastrin production after Cisplatin treatment.

In rats cisplatin induces hypocalcemia, bloating of the stomach, and ulceration ameliorated through calcium supplements. This study was undertaken to test the role of calcium on the gastrin mRNA production in vitro and in vivo. RIN B6 cells were cultured in medium with calcium (1.8, 3.6 and 7.2 mM) and the active form of vitamin D (calcijex). Cisplatin was added (10 mug/ml) for 12 hrs and cells were harvested for RNA from various treatment groups. Male Wistar rats were treated with cisplatin (9 mg/kg), before and after vitamin D (0.3 mg/100g/week). The rats were killed and stomach tissues excised on 1, 6, 10 and 15 days after cisplatin treatment. RNA from the stomach was analyzed using the northern blot technique. Gastrin mRNA was suppressed after cisplatin treatment both in vitro and in vivo. In vitro calcium but not vitamin D additions partially prevented the gastrin mRNA. In vivo, however, vitamin D and calcium were equally effective in preventing gastrin mRNA loss.

Expression of hepatocyte nuclear factor-3alpha in rat prostate, seminal vesicle, and bladder.

Hepatocyte nuclear factor-3alpha (HNF-3alpha), a member of the hepatocyte-forkhead-homolog family of transcription factors, regulates gene expression in the endoderm-derived liver and lung. To determine if HNF-3alpha might also play a role in endodermal derivatives of the urogenital sinus, the expression of HNF-3alpha in male accessory sex organs was assessed by Northern blotting, in situ hybridization, and electrophoretic mobility shift analysis. RNA from the dorsolateral prostate (DP), ventral prostate (VP), anterior prostate (AP), seminal vesicle (SV), and bladder was compared with RNA from the liver and spleen as positive and negative controls, respectively. HNF-3alpha mRNA levels in the DP, VP, AP, and bladder were 20, 14, 5, and 6 times higher than the SV equivalent in the liver. HNF-3alpha mRNA was detected in 8 of 10 prostate epithelial cell lines (rat NRP 152 and 154, mouse Pr14, and human DU-145, PC3, LNCaP, ND-1, and BPH-1) but not in rat Dunning epithelial or mouse Pr12 cells. Addition of testosterone to castrated rats was found to prevent a drastic loss of HNF-3alpha mRNA in the VP. This result suggests that HNF-3alpha mRNA levels are at least indirectly regulated by testosterone. The HNF-3alpha mRNA is expressed in epithelial cells of the urogenital sinus derivatives VP, AP, DP, and bladder and Wolffian duct derivative, the SV. To confirm that functional HNF-3alpha protein is produced in the VP, electrophoretic mobility shift assays were performed with whole-cell extracts and high-affinity oligonucleotide (TTR-S) from the transthyretin promoter. Binding to TTR-S was disrupted when the extract was incubated with HNF-3alpha, but not with HNF-3beta, antibody. Taken together, the results using VP, AP, DP, SV, and bladder suggest that HNF-3alpha may play an important role in development and maintenance of urogenital tract epithelial cells.

Molecular cloning of, and phylogenetic analysis of, an actin in Naegleria fowleri.

We cloned and sequenced an intronless actin gene from the amoebo-flagellate Naegleria fowleri, LEE strain, an opportunistic pathogen of man. Codon usage and third-position-codon nucleotide frequency were significantly different from Acanthamoeba, another amoeba genus which also includes opportunistic pathogens of man. Between the two amoebae, actin peptide sequences were 92.8% similar, while nucleotide sequences were only 70% similar. A phylogenetic reconstruction of actin amino acid sequences, using a distance method, placed Naegleria in a cluster with Plasmodium and Entamoeba.

A simple, efficient method to create a cDNA library.

The simplified "All In One Tube" protocol for constructing a cDNA library combines the advantages of the "Classic method" and the Okayama-Berg method while overcoming some of their drawbacks. In this method, adding adapters, linkers or enzyme digestion steps are no longer necessary after cDNA synthesis, thus making it quicker and especially useful when dealing with small samples of mRNA.

Cloning and characterization of transcripts showing virulence-related gene expression in Naegleria fowleri.

The pathogenic LEE strain of Naegleria fowleri isolated from human or mouse brain loses pathogenicity when cultured axenically in a nutrient broth. To identify genes differentially expressed in highly virulent versus weakly virulent amoebae, a cDNA library was constructed by using mRNA from amoebae recovered from a mouse brain. Two cDNA clones were isolated by differential screening of the library. The transcript homologous to clone Nf314 was preferentially expressed in highly virulent cells, whereas the transcript homologous to clone Nf435 was preferentially expressed in weakly virulent cells. Other clones showed negligible differential hybridization, but actin transcript levels were slightly elevated in the highly virulent cells. The Nf314 cDNA has an open reading frame for a 53-kDa protein 94% similar and 19% identical over 194 amino acid residues to serine carboxypeptidase from yeast cells, barley, and wheat. Southern blot analysis is consistent with a single copy of the Nf314 gene in the genome. Interestingly, the increased Nf314 transcript levels were present in cells fed on mouse brain, liver, or NIH 3T3 fibroblasts but not in cells fed on bacteria or in axenic culture. Thus, the inducer of the increased gene expression correlates with use of mammalian cells as a food source without regard to level of virulence. Since amoebae fed in culture on dissociated mouse brain were weakly virulent, as measured by their abilities to kill mice, the Nf314 gene may be required but is not sufficient for increased virulence.

Virulence-related protein synthesis in Naegleria fowleri.

Protein synthesis patterns of the low-virulence Naegleria fowleri LEE strain from axenic culture, the same strain after mouse brain passage to increase virulence, and the same strain after growth on bacteria were studied. Comparisons of accumulated proteins, in vivo-synthesized proteins, and in vitro-synthesized proteins translated from poly(A)+ mRNA were made. Differences between amoebae from the different treatments were noted. After 6 months in axenic culture, pathogenic protein synthesis patterns were lost and there was a decrease in virulence. Therefore, the increase in virulence is correlated with numerous specific changes in protein synthesis.

Glorin-regulated protein synthesis in Polyspondylium violaceum.

Protein synthesis by Polyspondylium violaceum in response to the chemoattractant glorin was analyzed by two-dimensional gel electrophoresis to determine if glorin can affect gene expression. When cells developing in a shaking suspension culture were given glorin, five proteins exhibited significantly increased and three proteins exhibited decreased incorporation of L-[35S]methionine. Glorin was active from 10-1000 nM, a concentration range within which cells are chemotactically active. The extent of the response was dependent on the concentration and the length of exposure to glorin. This evidence suggests that glorin may act in part to mediate changes in gene expression during development.

Regulatory signals affecting a selective loss of mRNA in Dictyostelium discoideum.

We identified signals that affect mRNA levels complementary to a gene that is highly expressed in vegetative Dictyostelium discoideum cells. This gene has been cloned as cDNA in the plasmid pcD-D2. The level of transcripts homologous to pcD-D2 fell dramatically in strain XP55 during the aggregation stage of development when cells differentiate on agar. The level, however, did not fall simply as a result of starvation or aggregation-specific cell contact. Rather, before the level is reduced cells must be deprived of amino acids and cyclic AMP administered in amounts and at intervals in pulses to mimic cyclic AMP signal-relay in aggregation. This effect can be blocked either with cyclic AMP-S (a non-hydrolysable cyclic AMP analogue) or adenosine, both of which prevent cyclic AMP binding to the cyclic AMP cell surface receptor. It is also blocked in 'frigid' aggregation-deficient mutants HC85 and HC112 known to be defective in a G alpha protein. We conclude that the transcript level is balanced by positive nutritional signals acting against negative signals transduced in part through a cell surface cyclic AMP receptor.

Genes selectively expressed in proliferating Dictyostelium amoebae.

Few eukaryotic genes are expressed only during cell growth and division. We found that the slime mold Dictyostelium discoideum is unusual in that it expresses many genes only during proliferation. Thirty-two percent (304/950) of the sequences in a cDNA library made from vegetative mRNA were homologous to RNAs that are present at high levels during growth but at low or undetectable levels during differentiation when no cell growth occurs. In vitro translation assays confirmed that one-third of the vegetative cell mRNAs decreased in steady-state levels during differentiation. These vegetative cell-specific transcripts identified a diverse coordinately regulated class of genes: (i) 9 of the 10 cDNAs tested hybridized to unique small transcripts ranging from 400 to 620 bases long; (ii) the sequences showed various degrees of homology to related species; (iii) transcript levels synchronously fell by a factor of greater than 20 during development and synchronously increased during germination. This class of genes may play important roles in normal cell proliferation.

Selective induction of stalk-cell-specific proteins in Dictyostelium.

We compared the proteins synthesized and accumulated by Dictyostelium discoideum amoebae in response to the morphogenetic factor termed differentiation-inducing factor (DIF) to assess the proposed ability of DIF to regulate the choice of differentiation pathway. When amoebae of a mutant strain with low endogenous DIF levels were given DIF, they dramatically increased the expression of 21 of 23 proteins preferentially found in stalk cells, but drastically repressed 4 major spore-specific proteins. Most of the induced proteins were also expressed in amoebae of a developmentally competent strain developing at low cell densities and exposed to DIF, low extracellular pH, or the proton pump inhibitor diethylstilbestrol; this suggests that an intracellular acidification may be a key part of the mechanism of DIF action. We conclude from the similar morphology and extensive homology of proteins of DIF-induced and stalk cells that most stalk-pathway functions are DIF dependent.

Dictyostelium mutants lacking DIF, a putative morphogen.

DIF is an endogenous extracellular signal that may control differentiation of D. discoideum cells. It is a dialyzable, lipid-like factor that induces stalk cell formation among isolated amebae incubated in vitro with cAMP. To examine the consequences of DIF deprivation, we have isolated several mutant strains that are impaired in DIF accumulation, and whose inability to make stalk cells in vitro and during normal development on agar can be corrected by the addition of exogenous DIF. Little DIF is made by the mutants, and morphological development on agar stops after the cells have aggregated, but before a slug forms. In these DIF-deprived conditions, prespore cells can differentiate, but prestalk cells cannot.

Size regulation in Dictyostelium.

The division of large aggregate centres into separate slugs was examined in two strains of Dictyostelium discoideum which differ in size. The evidence is consistent with the hypothesis that the size of the slugs is determined by two factors; one is the ability of a tip to inhibit surrounding cells from forming an independent, rival tip; the other is the ability of the surrounding cells to resist being subjected to the inhibition of the newly arisen tip. If the surrounding cells are easily inhibited, then the slugs produced will be large; if they are resistant to inhibition the resulting slugs will be correspondingly small. An assay for tip inhibition was developed which was used to estimate the volume and distance over which inhibition occurs, the time over which it acts and the effect of tip size and cell mass shape on size regulation. The measurements and the results of experiments which showed inhibition across a thin agar layer are consistent with the hypothesis that an inhibitor spreads out from the tip by simple diffusion. In further studies it was found that although inhibition strength varies with the size of the tip, the ability to inhibit was the same in both strains whereas the resistance to inhibition was greater in the smaller strain.

Cell contact-induced inhibition of division in Dictyostelium.

The developmental stage at which Dictyostelium discoideum strain v12/M2 cells was inhibited in resuming cell division was monitored by a Coulter counter. Neither vegetative nor pre-aggregating v12/M2 amoebae used as inocula in liquid growth cultures exhibited a delay in recommencing cell division. Inocula prepared from aggregates were delayed for 3 h before the onset of division. Although cyclic AMP had no effect upon vegetative or preaggregating stages the aggregating amoebae were inhibited for 9 h before resuming division. Following the onset of division by both controls and cyclic AMP-treated cells the normal growth rate of vegetative amoebae (3.2 h/generation) was attained. Aggregateless mutant vegetative amoebae and those of comparable ages to vas/M2 aggregates were not inhibited in the rate of cell division by cyclic AMP. Cyclic AMP sustains the delay in cell division only among amoebae derived from aggregates. Cyclic AMP induces structural components in the plasma membranes which may be necessary in cell adhesion and interactions. Consequently interactions between the membranes of apposing cells may transmit stimuli intracellularly to inhibit cell division.