Hormones andCuscuta development: In vitro induction of haustoria by cytokinin and its inhibition by other hormones

Cytokinins induced haustoria formation in excised 10-mm segments ofCuscuta vine, the subapical 25-to-50-mm region being most responsive, producing a mean of 4–6 haustoria per segment. The order of effectiveness of cytokinins continuously applied (72 h) was 6-benzylaminopurine (BA) ges isopentenyladenine (iP) Gt zeatin (Z). Ribosides of BA and Z were as effective as the bases, whereas riboside of iP ([9R]iP) was half as effective as iP. Haustoria induction was influenced by weather and seasonal conditions at the time of vine collection; materials obtained on warm, sunny days responded better than those obtained on rainy, cloudy, or cool days. Haustoria were induced equally well all around the segment, and no thigmostimulus was needed for induction. p ]A 10-min pulse of 100 mgrM BA induced half as many haustoria as a 60-min pulse or continuous application of BA. White light inhibited haustoria induction elicited by a short (30-min) pulse of BA, whereas a longer (120-min) BA application overcame this light inhibition. Auxins (IAA or NAA, 1–10 mgrM), gibberellin (GA3, 1–10 mgrM), ethylene (as ethrel, 10–100 mgrM), and abscisic acid (ABA, 100 mgrM) were individually inhibitory (60–80%) with respect to haustoria induction when given continuously with 50 mgrM BA. A 60-min pulse of auxins (10 mgrM), GA3 (100 mgrM), or ethrel (10 mgrM), given at various time intervals during or after a 60-min pulse of 100 mgrM BA, showed that inhibition was maximal (70–95%) between 4 and 16 h of BA application and negligible (GA3) or much reduced (auxin, ethrel) at 20 h, indicating a ldquocommitmentrdquo to haustoria formation by this time.

Inhibition of in Vitro Aminoacylation and Translation by RNA Reactive Antibodies

Antibodies were raised against guanosine-BSA, GMP-BSA and tRNA-mBSA conjugates separately in rabbits. Binding characteristics of these antibodies to various RNAs were studied using a sensitive avidin-biotin micro ELISA. These antibodies inhibited in vitro aminoacylation of tRNA in a dose dependent manner. This inhibition was reversed by the addition of the respective homologous haptens thereby showing the specificity of these antibodies. In vitro translation of endogenous mRNAs in rabbit reticulocyte lysate was also inhibited by these antibodies in a dose dependent manner.

Micromolar concentration of pentoxifylline improves development in vitro of hamster 8-cell embryos: conrmation of biological viability by embryo transfer

The inßuence of the sperm motility stimulant pentoxifylline (PF) on preimplantation embryo development in hamsters was evaluated. Eight-cell embryos were cultured in hamster embryo culture medium (HECM)-2, with or without PF (0· 0233·6 mM). There was 90%, 37% and 29% inhibition of blastocyst development by 3·6 (used for human sperm), 0·9 and 0 ·45 mM PF, respectively. However, 23 µM PF (exposed to hamster oocytes during IVF) signicantly (P < 0·05) improved blastocyst development (63· 6% v. 51· 8%); morulae development was, however, not curtailed by 0·45 mM or 0·9 mM PF (51·8%±6·0 or 50·5%±11·3, respectively). Post-implantation viability of PF-treated embryos was assessed by embryo transfer; 43% of 80 PF-treated embryos implanted compared with 40% of 79 control embryos. Of the 9 recipients, 6 females delivered pups (19, i.e. 16% of transferred embryos or 53% of implanted embryos). These data show that in hamsters, continuous presence of PF at 0·45-3·6 mM is detrimental to 8-cell embryo development whereas 23 µM PF improves the development of embryos to viable blastocysts which produce live offspring.

Acute toxicity of methyl isocyanate in rabbit: In vitro and in vivo effects on rabbit erythrocyte membrane

Methyl isocyanate (MIC) interaction with the rabbit erythrocyte membrane increased the fluidity of the membrane and decreased the osmotic fragility of erythrocytes both in vitro and in vivo in rabbits intoxicated with MIC subcutaneously. MIC inhibited both acetylcholinesterase (AChE) and adenosine triphosphatase (ATPase) activities of erythrocytes dose-dependently in vitro, while in vivo a decreased trend in ATPase activity with unaltered AChE activity was observed. MIC also caused significant decrease in plasma sodium level with corresponding increase in potassium level in rabbits. The observed effects are due to MIC, per se, as the hydrolysis products of MIC, methylamine and N,Nprime-dimethylurea did not affect the erythrocyte fluidity and enzymes activities both in vitro and in vivo while they increased the osmotic fragility of erythrocytes in vivo in rabbits administered subcutaneously in equimolar concentration to MIC dosage. Inhibition of Na+-K+-dependent ATPase with altered permeability to cations and also probably water transport of plasma membrane due to MIC interaction are envisaged.

In vitro and in vivo effects of methyl isocyanate on rat brain mitochondrial respiration

The present study deals with the in vitro and in vivo effects of methyl isocyanate (MIC) on rat brain mitochondrial function. Addition of MIC to tightly coupled brain mitochondria in vitro resulted in a mild stimulation of state 4 respiration, abolition of respiratory control, decrease in ADP/0 ratio, and inhibition of state 3 oxidation. The oxidation of NAD+-linked substrates (glutamate + malate) was more sensitive (fourfold) to the inhibitory action of MIC than succinate while cytochrome oxidase was unaffected. Administration of MIC subcutaneously at a lethal dose affected respiration only with glutamate + malate as the substrate (site I) and caused a 20% decrease in state 3 oxidation leading to a significant decrease in respiratory control index while state 4 respiration and ADP/O ratio remained unaffected. As both the malondialdehyde and iron contents of brain mitochondria were not altered, it may be inferred that the observed in vivo inhibition of state 3 oxidation is induced by MIC through systemic stagnant hypoxia leading to ischemia of brain, which further contributes to the cerebral hypoxia.

In vitro and in vivo effect of methyl isocyanate on rat liver mitochondrial respiration

Previous work has shown that irrespective of the route of exposure methyl isocyanate (MIC) caused acute lactic acidosis in rats (Jeevaratnam et al., Arch. Environ. Contam. Toxicol. 19, 314�319, 1990) and the hypoxia was of stagnant type due to tissue hypoperfusion resulting from hypovolemic hypotension in rabbits administered MIC subcutaneously (Jeevarathinam et al., Toxicology 51, 223�240, 1988). The present study was designed to investigate whether MIC could induce histotoxic hypoxia through its effects on mitochondrial respiration. Male Wistar rats were used for liver mitochondrial and submitochondrial particle (SMP) preparation. Addition of MIC to tightly coupled mitochondria in vitro resulted in stimulation of state 4 respiration, abolition of respiratory control, decrease in ADP/O ratio, and inhibition of state 3 oxidation. The oxidation of NAD+-linked substrates (glutamate + malate) was more sensitive (fiveto sixfold) to the inhibitory action of MIC than succinate while cytochrome oxidase remained unaffected. MIC induced twofold delay in the onset of anerobiosis, and cytochrome b reduction in SMP with NADH in vitro confirms inhibition of electron transport at complex I region. MIC also stimulated the ATPase activity in tightly coupled mitochondria while lipid peroxidation remained unaffected. As its hydrolysis products, methylamine and N,N?-dimethylurea failed to elicit any change in vitro; these effects reveal that MIC per se acts as an inhibitor of electron transport and a weak uncoupler. Administration of MIC sc at lethal dose caused a similar change only with NAD+-linked substrates, reflecting impairment of mitochondrial respiration at complex I region and thereby induction of histotoxic hypoxia in vivo.

In Vitro and in Vivo Anticancer Activity of Copper Bis(thiosemicarbazone) Complexes

Neutral and cationic copper bis(thiosemicarbazone) complexes bearing methyl, phenyl, and hydrogen, on the diketo-backbone of the ligand have been synthesized. All of them were characterized by spectroscopic methods and in three cases by X-ray crystallography. In vitro cytotoxicity studies revealed that they are cytotoxic unlike the corresponding zinc complexes. Copper complexes Cu(GTSC) and Cu(GTSCHCl) derived from glyoxal-bis(4-methyl-4-phenyl-3-thiosemicarbazone) (GTSCH(2)) are the most cytotoxic complexes against various human cancer cell lines, with a potency similar to that of the anticancer drug adriamycin and up to 1000 fold higher than that of the corresponding zinc complex. Tritiated thymidine incorporation assay revealed that Cu(GTSC) and Cu(GTSCHCl) inhibit DNA synthesis substantially. Cell cycle analyses showed that Cu(GTSC) and Cu(GTSCHCl) induce apoptosis in HCT116 cells. The Cu(GTSCHCl) complex caused distinct DNA cleavage and Topo II alpha inhibition unlike that for Cu(GTSC). In vivo administration of Cu(GTSC) significantly inhibits tumor growth in HCT116 xenografts in nude mice.

In vitro and in silico biological studies of novel thiazolo3,2-a]pyrimidine-6-carboxylate derivatives

In the present study, we report the synthesis, characterization of new series of thiazolo3,2-a]pyrimidine-6-carboxylate derivatives 3a-f and 4a-f. The newly synthesized compounds were screened for in vitro antimicrobial and antiviral activities. The probable mode of action of these active compounds was determined through in silico docking study by docking the receptor methionyl-tRNA synthetase and human inosine-5'-monophosphate dehydrogenase (IMPDH) for antibacterial and antiviral activities, respectively. Among the compounds, 4c exhibited excellent in vitro antimicrobial activity against all tested strains with binding and docking energies -35.6 and -12.4 kcal/mol, respectively. The antiviral studies were carried out for the selected compounds in which 4a exhibited 73.69 and 54.42 % of inhibition of buffalopox and camelpox viruses, respectively. Furthermore, compound 4a showed minimum docking and binding energy along with the maximum hydrogen/hydrophobic interaction with IMPDH. The study contributes towards identification and screening of potential antimicrobial and antiviral agent's against the pathogens.

In vitro propagation of Eucalyptus grandis L. by tissue culture

Multiple shoots were induced from nodal segments of five year old trees of Eucalyptus grandis L. on solid medium containing Murashige and Skoog's (MS) Basal medium supplemented with additional thiamine, BAP and NAA. Rooting could be achieved from shoot culture on half strength MS salts or white's medium supplemented with low auxins like IAA, IBA and NAA.

Synthesis and in vitro cytotoxic evaluation of novel diazaspiro bicyclo hydantoin derivatives in human leukemia cells: A SAR study

To study the structure activity relationship (SAR) on the cytotoxic activity and probe the structural requirement for the potent antitumor activity, a series of novel diazaspiro bicyclo hydantoin derivatives were designed and synthesized. Their structures were confirmed by H-1 NMR, LCMS and IR analyses. The antiproliferative effect of these compounds were determined against human leukemia, K562 (chronic myelogenous leukemia) and CEM (T-cell leukemia) cells using trypan blue and MTT assay, and the SAR associated with the position of N-terminal substituents in diazaspiro bicyclo hydantoin have also been discussed. It has been observed that these compounds displayed strong, moderate and weak cytotoxic activities. Interestingly, compounds having electron withdrawing groups at third and fourth position of the phenyl ring displayed selectively cytotoxic activities to both the cell lines tested with IC50 value lower than 50 mu M. In addition, the cytotoxic activities of the compounds 7(a-o) bearing the substituents at N-3 position of diazaspiro bicyclo hydantoin increases in the order alkene > ester > ether and plays an important role in determining their antitumor activities. The position and number of substituents in benzyl group attached to N-8 of diazaspiro bicyclo hydantoin nucleus interacted selectively with specific targets leading to the difference of biochemical and pharmacological effects.

The apical control of lateral bud development in excised shoot tips of Cuscuta reflexa cultured in vitro

Excised shoot tips of Cuscuta reflexa Roxb. (dodder), a rootless and leafless angiospermic plant parasite, were cultured in vitro for the study of the control of lateral bud development by the apex. In a chemically defined medium lacking hormones, the basal bud alone developed into a shoot. The addition of coconut milk to the growth medium induced the activation of multiple lateral buds, but only a single bud developed further into a shoot. The decapitation of this shoot induced the development of another shoot and the process could be repeated. This showed the controlling effect of the apex in correlative control of bud development. Application of indole-3-acetic acid to the shoot tip explant delayed the development of the lateral bud. Gibberellic acid A3 induced a marked elongation growth of the explant and reinforced apical dominance. The direct application of cytokinin to an inhibited bud relieved it from apical dominance. A basipetally decreasing concentration gradient of auxin may prevail at the nodes. Bud outgrowth is probably stimulated by cytokinin produced locally in the bud.

A hexokinase effect in the inhibition of oxidative phosphorylation in heart muscle mitochondria by Adriamycin

The inhibitory action of the anticancer antibiotic, Adriamycin, on succinate-dependent oxidative phosphorylation in heart mitochondria was markedly potentiated by the presence of hexokinase in the reaction medium. This 'hexokinase effect' was not observed in the oxidation of NAD+-linked substrates, or when liver or kidney mitochondria were used in place of heart mitochondria. These results offer a biochemical explanation for the extreme cardiac toxicity of the drug.

Interaction of rat testis protein, TP, with nucleic acids in vitro. Fluorescence quenching, UV absorption, and thermal denaturation studies

The nucleic acid binding properties of the testis protein, TP, were studied with the help of physical techniques, namely, fluorescence quenching, UV difference absorption spectroscopy, and thermal melting. Results of quenching of tyrosine fluorescence of TP upon its binding to double-stranded and denatured rat liver nucleosome core DNA and poly(rA) suggest that the tyrosine residues of TP interact/intercalate with the bases of these nucleic acids. From the fluorescence quenching data, obtained at 50 mM NaCl concentration, the apparent association constants for binding of TP to native and denatured DNA and poly(rA) were calculated to be 4.4 X 10(3) M-1, 2.86 X 10(4) M-1, and 8.5 X 10(4) M-1, respectively. UV difference absorption spectra upon TP binding to poly(rA) and rat liver core DNA showed a TP-induced hyperchromicity at 260 nm which is suggestive of local melting of poly(rA) and DNA. The results from thermal melting studies of binding of TP to calf thymus DNA at 1 mM NaCl as well as 50 mM NaCl showed that although at 1 mM NaCl TP brings about a slight stabilization of the DNA against thermal melting, a destabilization of the DNA was observed at 50 mM NaCl. From these results it is concluded that TP, having a higher affinity for single-stranded nucleic acids, destabilizes double- stranded DNA, thus behaving like a DNA-melting protein.

Studies on the effects of in vitro methyiation on aminoacylation of transfer RNA

In vitro methyiation of Escherichia coli transfer ribonucleic acid by cell free extracts of Mycobacterium smegmatis leads exclusively to the formation of 1-methyl adenine [Vani, B. R., Ramakrishnan, T., Taya, Y., Noguchi, S., Yamaiuzumi, Z. and Nishimura, S.(1978) J. Bact., 137,1085]. We have studied the effect of this modification on aminoacylation of Escherichia coli tRNA by mycobacterial enzymes. Aminoacylation with total algal protein hydrolysate as well as several individual aminoacids like methionine, valine, tyrosine, aspartic acid and lysine were monitored. In all the cases methyiation had a positive effect on the extent of aminoacylation by mycobacterial enzymes. Decreased aminoacylation in vitro was observed when hypomethylated transfer RNA from ethionine treated cells was used as the substrate for aminoacylation.