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 µM. 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

Plasmodium falciparum: detection and strain identification of Indian isolates by polymerase chain reaction

The polymerase chain reaction (PCR) was employed for detection and strain identification of P. falciparum in a comparative field study of Indian isolates. The primers were selected from highly conserved regions flanking the variable, tandemly repeated regions of highly polymorphic cell surface antigens, major merozoite surface antigen-1 (MSP-1), major surface antigen-2 (MSP-2), circumsporozoite surface antigen (CSP) and ring-infected erythrocyte surface antigen (RESA). Out of the 52 microscopically positive P. falciparum infected field samples, 47 samples were positive by PCR. Variation in the size of the amplified products was observed using MSP-1, MSP-2 specific primers respectively in different field isolates of P. falciparum, but CSP and RESA did not exhibit any variation in size of the amplified product. The multiplex PCR results demonstrated that amplified products from these surface antigens vary in size and there is a specific pattern for each strain and this could be utilized to identify a particular field isolate. One P. falciparum infected field sample detected by the above PCR method was found to be a mixed infection by two different strains. Five microscopically positive P. vivax infeced samples were also analyzed by PCR method using P. falciparum cell surface antigen (MSP-2) specific primers. PCR results showed one P. vivax infected sample was positive when P. falciparum specific primers were used, this could be due to inaccurate and reduced limit of detection of Plasmodial species by microscopic examination

Control of ornithine decarboxylase activity in α -difluoromethylornithine-resistant L1210 cells by polyamines and synthetic analogues

The regulation of ornithine decarboxylase (ODC) activity by the polyamine derivatives N1,N8-bis(ethyl)-spermidine and N1,N12-bis(ethyl)spermine was studied using a line of L1210 cells resistant to α -difluoromethylornithine (D-R cells), which contain very high levels of ODC, and a synthetic mRNA prepared from a plasmid containing an insert corresponding to ODC mRNA adjacent to an SP6 RNA polymerase promoter. Studies in which ODC protein was labeled in the D-R cells by exposure to [35S]methionine indicated that the polyamine derivatives and their physiological counterparts led to an increased rate of degradation of ODC and to a rapid reduction in ODC synthesis without affecting the content of ODC mRNA. Direct evidence that the polyamine derivatives act by inhibiting the translation of the ODC mRNA was obtained by studying their effects on the translation of ODC mRNA in reticulocyte lysates. This translation was strongly inhibited by the addition of N1,N8-bis(ethyl)spermidine, spermidine, N1,N12-bis(ethyl)spermine, or spermine but was not affected much by putrescine. The inhibition of the translation of ODC mRNA by either of the bis(ethyl) polyamine derivatives occurred at concentrations which stimulated total protein synthesis showing the selectivity of the reduction in ODC. The effects of polyamine derivatives and polyamines on translation of the plasmid-derived ODC mRNA were identical with those found with the D-R L1210 cell mRNA. This synthetic ODC mRNA lacks 261 bases of the 5'-leader sequences and 200 bases plus the poly(A) section from the 3'-nontranslated sequence. Therefore, these regions appear not to influence sensitivity of the ODC mRNA to inhibition of translation by polyamine derivatives

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

Assessing the safety and efficacy of dinoprostone vaginal insert in pregnancy

Background: Induction of labor (IOL) is a procedure used to achieve vaginal birth when the hazards of extending the pregnancy for either the mother or the infant outweigh the dangers of delivery. It is often used in high-risk pregnancies, although it can also be useful in low-risk groups, as demonstrated by A Randomized Trial of Induction Versus Expectant Management (ARRIVE) study. Methods: The cross-sectional study was conducted among 414 patients at Department of Obstetrics and Gynaecology tertiary care hospital. The study was conducted for one-year duration in pregnant women with maternal age >18 years, gestational week >37 weeks, and Bishop score <7 was included in the study with no signs of labor. Demographic details such as age, pregnancy history, and mode of delivery were recorded for comparison. Patients with no induction of labor were administered misoprostol and/or dinoprostone based on clinical conditions with further evaluation of maternal complications, delivery time, birthweight of the fetus, and fetal heart rate. Data were analyzed based on percentages and a chi-square test was used (p-value <0.05). Results: The mode of delivery did not significantly affect delivery outcomes (p=0.354), with assisted delivery being the most common (35.41%). Indication for induction was found to be significant (p=0.034), with non-progress of labor being the most common indication (55.2%). Maternal complications were not significantly associated with delivery outcomes (p=0.390), with 60 (14.49%) patients experiencing complications. The use of misoprostol reported a significant difference between modes of delivery with 74.93% of vaginal delivery, 19.47% with lower segment cesarean section (LSCS), and 5.60% with assisted delivery (p value <0.03). Conclusions: In low-risk pregnant women, the dinoprostone or misoprostol vaginal inserts are both safe and effective for inducing labor. Nulliparous individuals and those who did not get epidural analgesia during labor had a higher chance of caesarean section

Structure and regulation of mammalian S-adenosylmethionine decarboxylase

In order to understand the structure and regulation of S-adenosylmethionine decarboxylase, cDNA clones encoding this enzyme have been isolated from rat prostate and human fibroblast cDNA libraries. The authenticity of the cDNAs was verified by: (a) transfecting the Chinese hamster ovary cells with the human cDNA in the pcD vector which resulted in a transient 10-20-fold increase in S-adenosylmethionine decarboxylase activity in recipient cells; and (b) translating the mRNA formed by transcription of the cDNA insert in a reticulocyte lysate and recording an increase in S-adenosylmethionine decarboxylase activity. The amino acid sequences deduced from the cDNAs indicate that the human proenzyme for this protein contains 334 amino acids and has a molecular weight of 38,331 whereas the rat proenzyme contains 333 amino acid residues. The human and rat enzymes are very similar having only 11 amino acid differences and the cDNAs are also closely related showing over 90% homology in the 1617-nucleotide overlap which was sequenced. A further indication of the highly conserved nature of mammalian S-adenosylmethionine decarboxylases is that the amino acid sequences deduced from the human and the rat cDNAs contained peptide sequences identical to those previously reported for the purified bovine enzyme. In vitro transcription/translation experiments showed that the proenzyme is converted to two polypeptides of molecular weights about 32,000 and 6,000 in a processing reaction which generates the prosthetic pyruvate group and that the final enzyme contains both polypeptides. Two forms of S-adenosylmethionine decarboxylase mRNA (2.1 and about 3.4-3.6 kilobases) are present in human and rodent tissues and may originate from the utilization of two different polyadenylation signals. Southern blots of rat genomic DNA indicated that the S-adenosylmethionine decarboxylase gene belongs to a multigene family. Depletion of cellular polyamines by inhibitors or ornithine decarboxylase or the aminopropyltransferases led to an increase in the content of S-adenosylmethionine decarboxylase protein and mRNA, but the elevation in the mRNA was not sufficient to account for all of the change in the enzyme level, particularly in cells in which spermine was depleted

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

Purification, production of antiserum and development of enzyme linked immunosorbent assay-based diagnosis for Cucumber mosaic virus infecting black pepper (Piper nigrum L.)

Cucumber mosaic virus (CMV) infecting black pepper (Piper nigrum) in India was propagated on Nicotiana benthamiana and N. glutinosa, and purified by differential and sucrose densitygradient centrifugation. The purified virus preparations showed the presence of typical isometric particles of about 28 nm diameter. Polyclonal antiserum against the virus was produced in New Zealand white rabbits. Immunoglobulin G was purified from the crude antiserum and coupled with the enzyme, alkaline phosphatase. 'Double antibody sandwich-enzyme linked immunosorbent assay method was standardized for detection of CMV in diseased black pepper samples collected from different regions of Karnataka, Kerala and Tamil Nadu. The virus infection was also detected on other Piper species such as P. chaba, P. colubrinum and P. longum and a few of the common weeds such as Ageratum conyzoides, Colacasia esculanta, Synedrella nodiflora, Cynodon dactylon and Sonchus oleraceus found in and around black pepper gardens. The utility of the method developed in diagnosis, epidemiology and management of the disease is discussed. &nbsp

Studies on pollen micro-morphology, pollen storage methods, and cross-compatibility among grape (Vitis spp.) genotypes

The knowledge of pollen morphology, suitable storage condition, and species compatibility is vital for a successful grapevine improvement programme. Ten grape genotypes from three different species, viz., Vitis vinifera L., Vitis parviflora Roxb., and Vitis champini Planc., were studied for their pollen structure and pollen storage with the objective of determining their utilization in grape rootstock improvement programs. Pollen morphology was examined through the use of a scanning electron microscope (SEM). The viability of the pollen was assessed using 2,3,5-triphenyltetrazolium chloride (TTC). In vitro pollen germination was investigated using the semi-solid medium with 10 % sucrose, 100 mg/L boric acid, and 300 mg/L calcium nitrate. The results revealed variations in pollen micro-morphology in 10 genotypes, with distinct pollen dimensions, shapes, and exine ornamentation. However, species-wise, no clear difference was found for these parameters. Pollen of V. parviflora Roxb. and Dogridge was acolporated and did not germinate. The remaining eight genotypes exhibited tricolporated pollen and showed satisfactory in vitro pollen germination. Storage temperature and duration interactions showed that, at room temperature, pollen of most of the grape genotypes can be stored for up to 1 day only with an acceptable pollen germination rate (>30 %). However, storage for up to 7 days was successfully achieved at 4 °C, except for ‘Pearl of Csaba’. The most effective storage conditions were found to be at −20 °C and −196 °C (in liquid N2), enabling pollen storage for a period of up to 30 days, and can be used for pollination to overcome the challenge of asynchronous flowering. Four interspecific combinations were studied for their compatibility, among which V. parviflora Roxb. × V. vinifera L. (Pusa Navrang) and V. parviflora Roxb. × V. champini Planc. (Salt Creek) showed high cross-compatibility, offering their potential use for grape rootstock breeding. However, V. parviflora Roxb. × V. vinifera L. (Male Hybrid) recorded the lowest compatibility index among studied crosses. In the case of self-pollinated flowers from V. parviflora Roxb. and V. parviflora Roxb. × V. champini Planc. (Dogridge), pollen failed to germinate on the stigma due to male sterility caused by acolporated pollen. As a result, the flowers of these genotypes functioned as females, which means they are ideal female parents for grape breeding without the need for the tedious process of emasculation