Influence of Bacillus spp. strains on seedling growth and physiological parameters of sorghum under moisture stress conditions.

Microorganisms isolated from stressed ecosystem may prove as ideal candidates for development of bio-inoculants for stressed agricultural production systems. In the present study, moisture stress tolerant rhizobacteria were isolated from the rhizosphere of sorghum, pigeonpea, and cowpea grown under semiarid conditions in India. Four isolates KB122, KB129, KB133, and KB142 from sorghum rhizosphere exhibited plant growth promoting traits and tolerance to salinity, high temperature, and moisture stress. These isolates were identified as Bacillus spp. by 16S rDNA sequence analysis. The strains were evaluated for growth promotion of sorghum seedlings under two different moisture stress conditions (set-I, continuous 50% soil water holding capacity (WHC) throughout the experiment and set-II, 75% soil WHC for 27 days followed by no irrigation for 5 days) under greenhouse conditions. Plate count and scanning electron microscope studies indicated successful root surface colonization by inoculated bacteria. Plants inoculated with Bacillus spp. strains showed better growth in terms of shoot length and root biomass with dark greenish leaves due to high chlorophyll content while un-inoculated plants showed rolling of the leaves, stunted appearance, and wilting under both stress conditions. Inoculation also improved leaf relative water content and soil moisture content. However, variation in proline and sugar content in the different treatments under two stress conditions indicated differential effect of microbial treatments on plant physiological parameters under stress conditions.

[Leishmania donovani: structural insignt in the recognition of C-methylated analogues of spermidine as natural polyamine].

The ability of alpha-, beta-, gamma- and omega-methylated spermidine analogues to restore the growth of L. donovani promastigotes that were depleted of putrescine and spermidine was investigated. Only beta-methylated spermidine, like natural spermidine was capable of restoring the growth of L. donovani, while the remaining three analogues turned out to be inactive. Considering that alpha-methylated spermidine is a functionally active spermidine surrogate both in vivo and in vitro, this analogue can be considered as an antidote in the host-parasite system, especially in cases where inhibitors of polyamine biosynthesis are used for the therapy of leishmaniasis.

Sodium sulphite enhances RNA isolation and sensitivity of Cucumber mosaic virus detection by RT-PCR in black pepper.

Isolation of intact high quality RNA suitable for RT-PCR from black pepper is greatly hindered by the presence of polyphenols and polysaccharides. These compounds adversely affect the sensitivity of virus detection by RT-PCR. The present study evaluated the effect of sodium sulphite in enhancing RNA yield and quality in a modified acid guanidium thiocyanate-phenol-chloroform (AGPC) protocol. The results were compared with the standard AGPC method and RNeasy Plant Mini Kit (Qiagen) for detection of Cucumber mosaic virus through RT-PCR. The addition of sodium sulphite in the extraction buffer increased the sensitivity of virus detection. Higher sensitivity of detection (than obtained from the kit) was seen when sodium sulphite was used at 0.5%. Similar levels of sensitivity were also observed for the detection of Cucumber mosaic virus from Piper longum.

Increased expression of LD1 genes transcribed by RNA polymerase I in Leishmania donovani as a result of duplication into the rRNA gene locus.

Eukaryotic protein-coding genes are generally transcribed by RNA polymerase II (Pol II), which has a lower transcription rate than that of Pol I. We report here the duplication of two LD1 genes into the rRNA locus and their resultant transcription by Pol I. The multigenic LD1 locus is present in a 2.2-Mb chromosome in all stocks of Leishmania spp. and is also present in multicopy 200- to 450-kb linear chromosomes or multicopy circular DNAs in over 15% of stocks examined. Genomic rearrangement in Leishmania donovani LSB-51.1 resulted in duplication of a 3.9-kb segment of LD1 containing two genes (orfF and orfG) and of a 1.3-kb segment from approximately 10 kb downstream into the rRNA gene repeat region of the 1.2-Mb chromosome. Short sequences (12 or 13 bp) common to the 2.2-Mb LD1 and 1.2-Mb rRNA loci suggest that this gene conversion occurred by homologous recombination. Transcription of the duplicated genes is alpha-amanitin resistant, indicating transcription by Pol I, in contrast to the alpha-amanitin-sensitive (Pol II) transcription of the genes in the 2.2-Mb LD1 locus. This results in higher transcript abundance than expected from the gene copy number in LSB-51.1 and in elevated expression of at least the orfF gene product.

Properties of L1210 cells resistant to alpha-difluoromethylornithine.

L1210 cells were selected for resistance to the ornithine decarboxylase (ODC) inhibitor, alpha-difluoromethylornithine. When grown in the absence of the inhibitor, these cells possessed very high ornithine decarboxylase levels. These represented about 1 part in 300 of the soluble protein, which is several hundred times greater than the maximal value found in the original L1210 cells. The resistant cells contained at least 100-fold higher levels of ODC mRNA but the half-life of ODC (about 45 min) was not altered significantly. The resistant cells had much higher putrescine and cadaverine levels than control cells, but there was no significant difference in cellular spermidine or spermine content or in production of 5'-methylthioadenosine, which is a measure of polyamine synthesis. Addition of putrescine to the control or resistant cells had no effect on their content of spermidine and spermine but addition of decarboxylated S-adenosylmethionine increased the content of spermidine and spermine. These results indicate that ornithine decarboxylase is not the rate-limiting step in polyamine synthesis in these L1210 cells. The growth of the alpha-difluoromethylornithine-resistant L1210 cells was inhibited when their ability to synthesize spermidine and spermine was blocked by the addition of the S-adenosylmethionine decarboxylase inhibitor, 5'-deoxy-5'-[N-methyl-N-(3-hydrazinopropyl)]aminoadenosine. Treatment with this compound produced a reduction of more than 85% in the production of 5'-methylthioadenosine and led to a large increase in the content of putrescine and a substantial decline in the content of spermidine and spermine. These results indicate the potential value of S-adenosylmethionine decarboxylase inhibitors as therapeutic agents in conditions where ODC inhibitors are ineffective.

Occurrence of Cucumber mosaic virus on vanilla (Vanilla planifolia Andrews) in India.

Cucumber mosaic virus (CMV) causing mosaic, leaf distortion and stunting of vanilla (Vanilla planifolia Andrews) in India was characterized on the basis of biological and coat protein (CP) nucleotide sequence properties. In mechanical inoculation tests, the virus was found to infect members of Chenopodiaceae, Cucurbitaceae, Fabaceae and Solanaceae. Nicotiana benthamiana was found to be a suitable host for the propagation of CMV. The virus was purified from inoculated N. benthamiana plants and negatively stained purified preparations contained isometric particles of about 28 nm in diameter. The molecular weight of the viral coat protein subunits was found to be 25.0 kDa. Polyclonal antiserum was produced in New Zealand white rabbit, immunoglobulin G (IgG) was purified and conjugated with alkaline phosphatase enzyme. Double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA) method was standardized for the detection of CMV infection in vanilla plants. CP gene of the virus was amplified using reverse transcriptase-polymerase chain reaction (RT-PCR), cloned and sequenced. Sequenced region contained a single open reading frame of 657 nucleotides potentially coding for 218 amino acids. Sequence analyses with other CMV isolates revealed the greatest identity with black pepper isolate of CMV (99%) and the phylogram clearly showed that CMV infecting vanilla belongs to subgroup IB. This is the first report of occurrence of CMV on V. planifolia from India.

Antileishmanial activity of berenil and methylglyoxal bis (guanylhydrazone) and its correlation with S-adenosylmethionine decarboxylase and polyamines.

Leishmania donovani S-adenosyl-L-methionine (AdoMet) decarboxylase was found to show a growth related pattern. Methylglyoxal bis (guanylhydrazone) (MGBG) and Berenil inhibited the growth of Leishmania donovani promastigotes (strain UR6) in a dose dependent manner. The concentrations of MGBG and Berenil required for 50% inhibition of rate of growth were 67 and 47 microM, respectively. The growth inhibition of MGBG was partially reversed by spermidine (100 microM) and spermine (100 microM). Berenil inhibition of promastigote growth was partially reversed by 100 microM spermidine whereas 100 microM spermine did not result in any reversal of growth. The reduction in parasitemia in vitro by these inhibitors was accompanied by inhibition of AdoMet decarboxylase activity and spermidine levels.

Leishmania donovani: cellular control of ornithine decarboxylase in promastigotes.

Ornithine decarboxylase, a key enzyme in polyamine biosynthesis, is essential for normal cell growth and proliferation. Furthermore, the inhibition of this enzyme is a potential way of controlling such growth. In order to shed light on the role of ornithine decarboxylase in regulation of Leishmania growth we examined the activity of this enzyme during the life cycle of these organisms. Exponentially growing Leishmania promastigotes were resuspended at a density of 3 x 10(6) cells/ml. 2 x 10(7) cells were withdrawn 24 hr later at different time intervals for induction studies and ornithine decarboxylase activity was measured. Ornithine decarboxylase showed a growth related pattern in L. donovani promastigotes. Induction studies showed that ornithine decarboxylase activity rapidly increased in late log phase cells when resuspended in fresh medium. A biphasic induction curve was observed similar to that observed in mammalian cells. The first peak was observed at 6 hr and the second at 16 hr. Cycloheximide and Actinomycin D inhibited induction at 16 hr by 65-68%. Polyamines at a level not inhibitory to growth (10 microM) inhibited ornithine decarboxylase induction by 30-40% late in the induction period. Putrescine and spermidine both inhibited the first peak of induction. Putrescine suppressed ornithine decarboxylase activity by 39% at 16 hr whereas spermidine by only 29%. The half life of ornithine decarboxylase in promastigote forms grown in the presence of cycloheximide was >6 hr. These studies indicate that although the Leishmanial ornithine decarboxylase follows a similar induction pattern to that previously reported in the mammalian cells, it is less susceptible to exogenous polyamines and is comparatively stable. This lack of ornithine decarboxylase regulation and turnover may be exploitable in the development of various therapeutic agents to inhibit Leishmanial growth.

The Leishmania donovani LD1 locus gene ORFG encodes a biopterin transporter (BT1).

We have previously described two genes, ORFF and ORFG, from the LD1 locus near one telomere of chromosome 35, which are frequently amplified in Leishmania isolates. In Leishmania donovani LSB-51.1, gene conversion of the rRNA gene locus on chromosome 27 with these two genes resulted in their over-expression, because of their transcription by the RNA polymerase I-mediated rRNA promoter. The predicted ORFG protein has substantial sequence homology to the ESAG10 gene product from the Trypanosoma brucei VSG expression site and both are putative membrane proteins. Using successive rounds of gene replacement of the three ORFG genes in L. donovani LSB-51.1, ORFG null mutants were obtained. These mutant cell lines show a direct relationship between ORFG mRNA, protein expression levels and active transport of biopterin into the cells. Transformation of the null mutant with a plasmid containing ORFG restores biopterin transport activity. In addition, the null mutants are unable to grow in the absence of supplemental biopterin. Thus, ORFG encodes a biopterin transporter and has been renamed BTI.

Chemoprevention of DMBA-induced transplacental and translactational carcinogenesis in mice by oil from mustard seeds (Brassica spp.).

The present study reports the chemopreventive potential of the oil from mustard seed on 7,12-dimethylbenz[a]anthracene-induced transplacental and translactational carcinogenesis in Swiss albino mice. Gestating females were treated with mustard oil at dose levels of 0.05 and 0.10 ml per day from days 13 to 19 of gestation. In addition, they were given DMBA (3 mg/animal) on days 15-17 of gestation. The percentage of tumour incidence in the F1 progeny was reduced significantly at both dose levels from 65% in the control group to 29% and 16%, respectively, in the experimental groups. The mean number of tumours per effective F1 progeny was reduced from 1.56 in the control group to 0.93 and 0.41 in the animals treated with lower and higher doses of mustard oil, respectively. When lactating mothers were given the mustard oil at dose levels of 0.05 and 0.10 ml per day for the first 15 days of lactation in addition to DMBA given on days 3, 6, 9, 12 and 15 of lactation, the multiple site tumour incidence was brought down significantly from a control value of 70% to 32% and 18%, respectively, in lower and higher dose groups. The mean number of tumours in the F1 mouse was reduced from a control value of 1.71 to 0.96 at the lower dose level and to 0.34 at the higher dose level. From earlier studies done in our laboratory, it appears that mustard oil exerts its effect by inducing the enzymes of drug detoxification and also by changing the profile of the antioxidant defence system. The quantitative and qualitative nature of the active principles and their passage into the F1 progeny remains to be seen.

Effect of antioxidants on the growth and polyamine levels of Leishmania donovani.

Butylated hydroxyanisole (BHA), retinoic acid (RA), retinol acetate (RAc) and sodium selenite (Na2SeO3) inhibited the growth of Leishmania donovani promastigotes (strains UR6 and AG83). There is a dose dependent inhibition of promastigote growth in both the strains. The concentrations of BHA, RA/RAc and Na2SeO3 required for 50% inhibition of the rate of growth were 0.5 microgram/mL, 0.5 microM and 0.125 mM, respectively, for UR6. In the case of AG83, LD50 for BHA was 1 microgram/mL whereas LD50 for RA/RAc and Na2SeO3 were the same as that of UR6. In Leishmania spp., growth appears to be related to and dependent upon polyamine biosynthesis (Bachrach U et al., Exp Parasitol 48: 457-463, 1979). Experiments to test the possibility that these antileishmanial agents exert their inhibitory effect by blocking polyamine biosynthesis suggest that decrease in ornithine decarboxylase activity and the inhibition of polyamine levels could be a mechanism of inhibition of promastigote growth by BHA and RA.

Antileishmanial activity and modification of hepatic xenobiotic metabolizing enzymes in golden hamster by 2(3)-tert-butyl-4-hydroxyanisole following infection with Leishmania donovani.

Butylated hydroxyanisole (BHA) inhibited the growth of Leishmania donovani promastigotes (strain AG83) in vitro. The inhibition was dose dependent: the concentration of BHA required for 50% inhibition of the rate of growth was 1 microgram/mL. BHA also prevented growth of L. donovani in vivo in golden hamsters infected with L. donovani. In addition, the effect of BHA on several enzymes involved in the metabolism of xenobiotics both in uninfected animals and animals infected with L. donovani is reported.

Serodiagnosis of leishmaniasis with recombinant ORFF antigen.

The serodiagnostic potential of recombinant ORFF protein (rORFF) from Leishmania infantum was assessed by ELISA. Of 49 sera from confirmed cases of visceral leishmaniasis (VL), all were seropositive using 5 ng of rORFF and serum diluted 1:20, while only 38 were positive with 500 ng of soluble antigen (SA) and 44 were positive by a direct agglutination test. There was also a positive correlation between spleen size and level of seropositivity with rORFF or SA. The reciprocal endpoint titer with rORFF was 1,280 for sera from VL patients, but < 20 with sera from malaria, filariasis, and tuberculosis patients, as well as with sera from healthy individuals from endemic and non-endemic areas. Sera from 10 confirmed cutaneous leishmaniasis cases from Turkey were negative or only weakly positive with rORFF although 9 were positive with SA. Thus, rORFF protein appears useful as a sensitive reagent for the differential diagnosis of VL caused by the Leishmania donovani complex.

Detection of Leishmania causing visceral leishmaniasis in the Old and New Worlds by a polymerase chain reaction assay based on telomeric sequences.

We present a new polymerase chain reaction assay based on telomeric sequences of Leishmania donovani. When this assay was used in dilutions of purified L. donovani DNA, a strong amplification signal was observed with 1 fg of DNA. In a specificity test that used purified DNA from Old World and New World Leishmania, the assay recognized all parasites isolated from patients with visceral leishmaniasis, except for 2 isolates of Leishmania colombiensis from Venezuela and 1 isolate from Brazil. All Leishmania major and Leishmania tropica isolates tested were negative, except for one isolate in each species. We also used the assay on fresh and archive bone marrow samples recovered from Giemsa-stained slides and from dried blood stains.

Effect of the microtubule stabilising agent taxol on leishmanial protozoan parasites in vitro.

Taxol, a mitotic spindle toxin, was found to selectively inhibit the proliferation of Leishmania donovani in vitro at nanomolar concentrations with an IC50 of 35 nM. Concentrations of taxol as high as 50 nM, however, did not affect J774A.1 murine macrophages. Taxol (30 nM) also inhibited amastigote multiplication within a J774A.1 macrophage cell line when used in a 10-day experiment. It resulted in the in vitro assembly of L. donovani microtubules in a dose-dependent manner. When promastigotes were exposed to different concentrations of taxol for 24 h, cells were largely blocked in the G2-M phase of the cell cycle and there was a marked reduction in the percentage of cells in the S phase. The selective nature of taxol action against the parasite and its effectiveness in controlling amastigote multiplication emphasise its use as a promising chemotherapeutic against kala-azar.

Isolation of a taxol-resistant Leishmania donovani promastigote mutant that exhibits a multidrug-resistant phenotype.

We raised a strain of Leishmania donovani in the laboratory that was resistant to 500 nM taxol. The IC50 of the wild-type strain for taxol was 35 nM and that of the taxol-resistant strain (T-500) was 1 microM. The T-500 strain exhibited a Mdr phenotype; it was also resistant to other unrelated drugs like vinblastine, adriamycin and the commonly used antimonial drugs pentostam and glucantime. Verapamil (20 nM), a calcium channel blocker, was found to reverse the resistance of T-500 to taxol. Acquired resistance to taxol has been reported to be mediated by alterations involving tubulin in cancer cells. Thus polymerisation assays with tubulin fractions in wild-type versus taxol-resistant cells (T-500) were performed in vitro. The tubulin fraction from T-500 was more resistant to in vitro polymerisation than the tubulin isolated from the wild-type, suggesting that this is one means by which the parasite may acquire resistance to taxol.

Regulation of mammalian S-adenosylmethionine decarboxylase.

S-Adenosylmethionine decarboxylase is a key enzyme in the biosynthesis of polyamines that is the rate limiting step in the formation of spermidine and spermine. The activity of S-adenosylmethionine decarboxylase is known to be regulated negatively by these polyamines and positively by their precursor, putrescine. A specific antiserum to S-adenosylmethionine decarboxylase was raised by immunizing rabbits with the homogeneous enzyme purified from rat prostate and a specific radioimmunoassay for the protein was set up. Using this radioimmunoassay it was found that a number of inhibitors of other steps in the polyamine biosynthetic pathway lead to increases in the amount of S-adenosylmethionine decarboxylase protein. These changes were caused by both a decreased rate of degradation and an increased rate of synthesis of the protein. The increased synthesis was due to two factors; a rise in the amount of translatable mRNA and an enhanced translation efficiency. The mRNA content of the prostate was substantially increased by treatment for 3 days with alpha-difluoromethylornithine (2% in drinking water). The translation of mRNA for S-adenosylmethionine decarboxylase was studied using a polyamine-depleted reticulocyte lysate supplemented with mRNA from rat prostate and the antiserum to precipitate the proteins corresponding to S-adenosylmethionine decarboxylase. These studies indicated that the enzyme was synthesized as an inactive precursor of Mr 37,000 which was converted to the enzyme sub-unit of Mr 32,000. The conversion of the precursor to the active sub-unit in vitro was increased by putrescine. The precursor could also be detected by immunoblotting of extracts from prostates of rats depleted of putrescine by treatment with the ornithine decarboxylase inhibitor, alpha-difluoromethylornithine. The translation of the S-adenosylmethionine decarboxylase mRNA in the reticulocyte lysates was strongly inhibited by the addition of spermidine or spermine demonstrating that polyamines directly inhibit the synthesis of S-adenosylmethionine decarboxylase. cDNA clones corresponding to S-adenosylmethionine decarboxylase were isolated using prostatic mRNA from polysomes enriched in S-adenosylmethionine decarboxylase by immunopurification. The use of these probes showed that rat ventral prostate contains two S-adenosylmethionine decarboxylase mRNA species of approximately 3.4 and 2.1 kb which differ in the 3' non-translated sequence. The sequence of these cDNAs will enable the amino acid sequence of the precursor to be obtained. This will provide evidence on the origin of the pyruvate prosthetic group of S-adenosylmethionine decarboxylase.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibition of ornithine decarboxylase activity by follicle stimulating hormone in primary culture of rat Sertoli cells.

The effect of follicle stimulating hormone on the activity of ornithine decarboxylase (ODC) was determined in primary culture of rat Sertoli cells. Three different FSH preparations (NIH oFSH-S-15, S-16, and eFSH) inhibited ODC activity in rat Sertoli cells under different media conditions. The inhibition was both time- and dose-dependent. The mechanism of the FSH inhibitory effect was studied using dibutyryl cyclic adenosine monophosphate (dbcAMP), 1-methyl-3-isobutylxanthine (MIX), forskolin, and isoproterenol. All of these agents, known to elevate cellular cAMP levels, inhibited ODC activity in cultured rat Sertoli cells. The combined effect of each of these substances plus FSH was either greater than, or equal to, that of FSH alone, and was not additive. Dibutyryl cyclic guanosine monophosphate had no effect on the ODC activity. These findings suggest that FSH inhibition of ODC activity in the rat Sertoli cell may be mediated by cAMP.

Difluoromethylornithine antagonizes taxol cytotoxicity in MCF-7 human breast cancer cells.

Taxol is a naturally occurring anticancer agent. We studied the combined effects of taxol with 0.1 mM of the ornithine decarboxylase inhibitor alpha-difluoromethylornithine (DFMO) in the MCF-7 human breast adenocarcinoma cell line. The effects of taxol on MCF-7 cells were evident at 0.05-1 microM and the half-maximum inhibition was calculated to be 0.05 microM. Although the cells in the control group continued to proliferate during an 8-day growth period, cells in the taxol-treated group showed approximately 78% inhibition on day 6 and approximately 92% inhibition on day 8. The combined effects of different concentrations of taxol with 0.1 mM DFMO for 48 h showed that DFMO reversed the cytotoxicity of taxol. The combined effects of 0.5 microM taxol and 0.1 mM DFMO over an 8-day period resulted in the reversal of taxol cytotoxicity by 74% on the sixth day of culture. Pretreatment and posttreatment with 0.1 mM DFMO protected the MCF-7 human breast adenocarcinoma cells from the cytotoxic effect of taxol. Polyamine levels were inhibited in cells treated with DFMO for 24 h. In a separate experiment, we verified that the addition of exogenous putrescine along with taxol and DFMO to cultures for 48 h restored the cytotoxic effects of taxol. Following exposure to 0.5 microM taxol, over 59% of MCF-7 cells were in G2/M phase. DFMO (0.1 mM) showed only a slight increase in the G1 phase of the cell cycle. However, in cells treated with taxol and DFMO, there was no change in the percent of cells in the G2/M phase compared to taxol-treated cells. Therefore, depletion of cellular polyamines may not interfere with cell cycle changes induced by taxol. Treatment of MCF-7 cells with 0.5 microM taxol resulted in the fragmentation of genomic DNA, indicating apoptosis, whereas the combined effects of taxol with DFMO inhibited DNA fragmentation.

Desensitization of ornithine decarboxylase activity to norepinephrine in the testis of rat.

Injection of norepinephrine (NE) at a dose of 10 micrograms per testis caused the testis refractory in terms of ornithine decarboxylase (ODC) activity at 24 h. This desensitization was found to be both time and dose dependent. Injection with follicle stimulating hormone, luteinizing hormone, prostaglandin F2 alpha, cyclic AMP or epinephrine to norepinephrine desensitized testis caused stimulation of ODC activity. This indicates that the refractoriness caused by norepinephrine is specific to this agent alone.