Effects of 1,25-dihydroxyvitamin D3 and its analogs on butyrate-induced differentiation of HT-29 human colonic carcinoma cells and on the reversal of the differentiated phenotype

1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) greatly enhances sodium butyrate (NaB)-induced enterocyte differentiation of HT-29 human colonic carcinoma cells while 1,25-(OH)2D3 alone induces growth restriction without associated differentiation. In the present study, the efficacies of various analogs of 1,25-(OH)2D3 to enhance NaB-induced HT-29 differentiation and to prolong the reversal of the differentiated phenotype under NaB-free growth conditions were subsequently examined. Extent of HT-29 differentiation was assessed by measurement of alkaline phosphatase (AP) activity, appearance of mucin-producing cells, changes in morphological characteristics, and expression of differentiation-associated cytokeratin proteins. Among active analogs of 1,25-(OH)2D3, 26,26,26,27,27,27-hexafluoro-1,25-(OH)2D3 (F6-1,25-(OH)2D3), 24,24-difluoro-24-homo-1,25-(OH)2D3, and 26,27-dimethyl-1,25-(OH)2D3 were 100-, 10-, and 5-fold, respectively, more effective than 1,25-(OH)2D3 in enhancing NaB-induced mucin production. Combined use of NaB and F6-1,25-(OH)2D3 (10(-9) M) also induced HT-29 cells to form highly differentiated goblet-like enterocytes, and increased both cellular AP enzymatic activity and tissue-type cytokeratin content. This differentiated state was qualitatively more advanced than that achieved by a combination of NaB and 10(-7) M 1,25-(OH)2D3. NaB-mediated HT-29 differentiation (in short-term inductions) was found to be reversible following a return to NaB-free medium. HT-29 cells differentiated by combined use of NaB and 1,25-(OH)2D3 or its analogs exhibited a significant prolonged reversal time relative to cells differentiated with NaB alone. The most prominent effect was achieved using cells differentiated with NaB and 10(-9) M F6-1,25-(OH)2D3 which exhibited a 7-fold prolonged reversal time over colonocytes differentiated by NaB alone. Our data suggest that a combined use of NaB and 1,25-(OH)2D3 or its derivatives may provide a convenient in vitro model system to probe molecular events associated with steroid-target tissue interactions in a differentiating cell system as commonly occurs in vivo. Such an analysis might lend itself to design of a rational combination differentiation-based therapy for the clinical management of colon cancer.     

The human ryanodine receptor gene: Its mapping to 19q13.1, placement in a chromosome 19 linkage group, and exclusion as the gene causing myotonic dystrophy

The recent cloning of cDNA encoding the Ca++ release channel (ryanodine receptor) of human sarcoplasmic reticulum has enabled us to use somatic cell hybrids to localize the ryanodine receptor gene (RYR) to the proximal long arm of human chromosome 19. Studies with additional hybrids containing deletions or translocations in chromosome 19 enabled us to localize RYR to 19q13.1 in a region distal to GPI/MAG and proximal to D19S18/DNF11. On the basis that the myotonic dystrophy (DM) locus maps near this region and that myotonia could result from a defect in the ryanodine receptor, we examined the linkage between the DM locus and RYR. Our results, showing several DM-RYR recombinants, rule out an RYR defect as the cause of DM. However, localization of RYR to a region of human chromosome 19 which is syntenic to an area of pig chromosome 6 containing the HAL gene responsible for porcine malignant hyperthermia supports the candidacy of RYR for this disorder.     

Host fruit chemical stimuli eliciting distinct ovipositional responses from sibling species of Rhagoletis fruit flies

Laboratory experiments tested whether two economically-important sibling species of tephritid fruit flies have evolved distinct egg-laying responses to chemical stimuli on the fruits of their respective hostplants. The egg-laying preferences displayed by apple maggot flies, R. pomonella, and blueberry maggot flies, R. mendax, on artificial fruits treated with apple and blueberry extract paralleled their egg-laying responses to whole apples and blueberries. R. pomonella flies laid more eggs than R. mendax flies in artificial fruits treated with extract from ripe McIntosh apples, and vice versa for artificial fruits treated with extract from ripe Bluehaven blueberries. Furthermore, both species laid more eggs in artificial fruits treated with extract from their respective host fruits than control artificial fruits which were not treated with fruit extract. Prior electroantennogram recordings from R. mendax and R. pomonella flies exposed to volatiles from pentane extracts of apples and blueberries indicate that the antennal sensitivity of both species is selectively tuned to their respective host fruit odors. This differentiation in their olfactory responses to fruit odors could be important in mediating their distinct ovipositional responses to blueberry and apple fruits. Extract from unripe McIntosh apples also elicited egg laying by R. pomonella flies, however, artificial fruits treated with unripe apple extract received 1.9 times fewer eggs than those treated with ripe apple extract. Moreover, the numbers of R. pomonella ovipositor punctures and eggs placed in wax artificial fruits were increased when the artificial fruits were treated with a blend of 7 identified apple esters. Black coloration on these artificial fruits and the presence of apple esters had a synergistic effect on the egg-laying behavior of R. pomonella flies, which caused them to lay substantially more eggs per black fruit than white fruit treated with the same concentration of apple esters. In summary, our results indicate that the egg-laying responses of R. pomonella flies are mediated by the integration of information from fruit chemical and visual cues, and that R. mendax and R. pomonella flies have evolved divergent egg-laying responses to chemical stimuli on the fruits of their respective hostplants. These findings are discussed in the context of other studies on plant compounds which influence the ovipositional behavior of phytophagous Diptera.

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Stimuli chimiques des pommes et des myrtilles induisant la ponte des espèces jumelles, Rhagoletis pomonella et R. mendax

Résumé Des fruits artificiels en cire traités avec des extraits de fruits ont provoqué chez les espèces jumelles de R. mendax (Curran) et R. pomonella (Walsh) des réactions de ponte différentes suivant les stimulations chimiques par les fruits. Le comportement de ponte sur des fruits artificiels traités avec des extraits au pentane des myrtilles mûres (Vaccinium corymbosum L.) et de pommes mûres (Malus pumila Miller = Pyrus malus L.), est le même que sur des fruits naturels, ce qui montre que la réponse aux stimulations chimiques provenant du fruit constitue un aspect important de la reconnaissance de l'hôte. R. pomonella pond plus d'ufs que R. mendax sur les fruits artificiels traités à l'extrait de pommes mûres; c'est l'inverse pour les fruits traités aux extraits de myrtille. Les fruits artificiels traités avec des pommes ou des myrtilles provoquent la ponte de R. pomonella, tandis que les myrtilles mûres seules provoquent la ponte de R. mendax. Les extraits de pommes vertes stimulent la ponte de R. pomonella mais elle est alors 2 fois plus faible qu'avec des extraits de pommes mûres. Un mélange de 7 esters identifiés dans l'extrait de pomme induit aussi la ponte de R. pomonella. Le nombre de piqûres de tarièresfli dans les fruits artificiels en cire et le nombre d'ufs par fruit ont été augmentés par addition d'esters de pommes à des fruits blancs ou noirs. La couleur des fruits artificiels influence aussi la réaction de ponte de R. pomonella; la fréquence des piqûres de tarière contenant un uf et le nombre d'ufs par fruit étaient significativement plus élevés sur les fruits noirs que sur les fruits blancs traités avec la même concentration d'esters de pomme. Les fruits artificiels noirs traités avec la concentration la plus stimulante d'esters de pommes ont reçu 2, 3 fois plus d'ufs que les fruits blancs avec les mêmes concentrations en esters. Ces résultats montrent que les esters de pomme et la couleur noire stimulent synergiquement la ponte de R. pomonella sur des fruits artificiels.

     

Quinine and quinidine production by cinchona leaf,root and unorganized cultures

Serially propagated Cinchona ledgeriana and C. succirubra (Rubiaceae) leaf, root and unorganized suspension cultures established from germinated seeds were studied for quinine and quinidine production. Leaf organ cultures were grown and subcultured in Murashige and Skoog's Revised Tobacco Medium supplemented with benzyladenine; root organ cultures were grown on the same medium supplemented with indolebutyric acid; and unorganized suspension cultures were grown on the same medium supplemented with 2,4-dichlorophenoxyacetic acid and benzyladenine. On a dry weight basis, leaf organ cultures of C. ledgeriana contained 0.06 % quinine and 0.05 % quinidine and of C. succirubra contained 0.04 % quinine and 0.04 % quinidine. No quinine and quinidine were detected in either root organ or unorganized suspension cultures.     

Response of Guanosine 5′-Triphosphate Concentration to Nutritional Changes and Its Significance for Bacillus subtilis Sporulation

We have investigated the changes in the guanosine 5'-triphosphate (GTP) and P-ribosyl-PP pools in stringent and relaxed strains of Bacillus subtilis under conditions frequently used to initiate sporulation. After a shift-down from a Casamino Acids-glutamate to a glutamate medium (Sterlini-Mandelstam shift-down), the pools of adenosine 5'-triphosphate and P-ribosyl-PP increased in both strains; in the stringent strain, ppGpp and pppGpp increased and GTP decreased rapidly, whereas in the relaxed strain, ppGpp and pppGpp increased only slightly and GTP decreased only slowly and less extensively. The stringent strain sporulated well, whereas the relaxed strain sporulated late and poorly. Addition of decoyinine, an inhibitor of guanosine 5'-monophosphate synthetase, caused a further decrease of GTP and initiated good sporulation of the relaxed strain. After a shift-down from a glucose-lactate to a lactate medium (Ramaley-Burden shift-down) the pool of P-ribosyl-PP (and GTP) decreased in both strains, indicating a shortage of purine precursors. This shift-down also caused a stringent response which prevented the consumption of nucleotides, as shown by the maintenance of adenosine 5'-triphosphate at a high concentration in the stringent strain but not in the relaxed strain. After a delay, the relaxed strain, in which GTP decreased as fast as in the stringent strain, sporulated also as efficiently. In nutrient sporulation medium the stringent strain and, less effectively, the relaxed strain accumulated ppGpp and pppGpp transiently towards the end of exponential growth. Eventually, the P-ribosyl-PP pool decreased drastically in both strains. In all cases the initiation of sporulation was correlated with a significant decrease of GTP. Granaticin, an antibiotic which prevents the charging of leucyl-transfer ribonucleic acid, was used to show that the stringent response inhibited the formation of xanthosine monophosphate from inosine monophosphate. It prevented the accumulation of xanthosine monophosphate in decoyinine-treated cultures of the stringent strain but not in those of the relaxed strain.     

Evaluation of Bone Strength During Aflatoxicosis and Ochratoxicosis

Young chickens were fed graded levels of aflatoxin (0, 0.625, 1.25, 2.5, 5.0, and 10.0 μg/g of diet) or ochratoxin (0, 0.5, 1.0, 2.0, 4.0, and 8.0 μg/g of diet), and the breaking strength, displacement before failure, and diameter of their tibias were determined. Breaking strength was decreased at growth inhibitory levels of aflatoxin (2.5 μg/g) and ochratoxin (2 μg/g), whereas a reduction in diameter required higher levels (5.0 and 4.0 μg/g, respectively). Bones from birds with ochratoxicosis selected to have diameters equal to control bones had lower breaking strength. In an attempt to negate mathematically the effect of decreased diameter and bias in any selection process, stress at time of failure of the bones was calculated and found to be decreased by feeding aflatoxin but not ochratoxin. Total displacement of bones before breaking was increased significantly (P < 0.05) by both toxins at the highest levels administered, but this increase was primarily the result of an increase in displacement from the start of failure to complete failure. Increased displacement associated with both toxicoses was equal in bones selected to be of equal diameter or in bones from the same treatment but of different diameters. However, calculation of modulus of elasticity which is corrected for diameter revealed aflatoxin had no effect whereas ochratoxin tripled the effect. These data indicate that the material properties of bones can be altered during mycotoxicoses and suggest yet another way in which mycotoxins are detrimental to animal health.     

TRANSMITTER METABOLIZING ENZYMES AND FREE AMINO ACID LEVELS IN SENSORY AND MOTOR NERVES AND GANGLIA OF THE SHORE CRAB (CARCINUS MAENAS)

—The distribution of ChAT (choline acetyltransferase), GAD (glutamate decarboxylase) and acetylcholinesterase in some sensory and motor nerves of the shore crab, Carcinus maenas, has been investigated using micro-assay techniques. ChAT was concentrated in the afferent nerve fibres of the thoracic-coxal muscle receptor as well as in the coxo-basal chordotonal receptor nerve and other leg sensory fibres. GAD was found in leg motor nerves including the promotor and remotor muscle nerves, being undetectable in the sensory nerves. Acetylcholinesterase was found in similar levels in both sensory and motor nerves assayed. Amino acid analysis using a micro-dansylation technique showed that sensory nerves had low GABA levels, whereas the leg nerve including motor fibres had substantially higher GABA concentrations. GAD and GABA were also found in low amounts in the leg promoter mucle, which is consistent with GABA being a neuromuscular transmitter.     

Cooperative control of translational fidelity by ribosomal proteins in Escherichia coli

Summary Two spontaneous mutants of Escherichia coli strain KMBL-146 selected for resistance to the aminoglycoside antibiotic neamine show severe restriction of amber suppressors in vivo. Purified ribosomes from the mutant strains exhibit low neamine-induced misreading in vitro and a decreased affinity for the related antibiotic streptomycin.Biochemical analysis shows that the mutants each have two modified 30S ribosomal proteins, S12 and S5. In agreement with these results, genetic analysis shows that two mutations are present, neither of which confers resistance to neamine by itself; the mutation located in gene rpxL (the structural gene for protein S12) confers streptomycin dependence but this dependence is suppressed in the presence of the second mutation, located in gene rpxE (the structural gene for protein S5).