Light and auxin responsive cytochrome P450s from Withania somnifera Dunal: cloning, expression and molecular modelling of two pairs of homologue genes with differential regulation.

Cytochrome P450s (CYPs) catalyse a wide variety of oxygenation/hydroxylation reactions that facilitate diverse metabolic functions in plants. Specific CYP families are essential for the biosynthesis of species-specialized metabolites. Therefore, we investigated the role of different CYPs related to secondary metabolism in Withania somnifera, a medicinally important plant of the Indian subcontinent. In this study, complete complementary DNAs (cDNAs) of four different CYP genes were isolated and christened as WSCYP93Id, WSCYP93Sm, WSCYP734B and WSCYP734R. These cDNAs encoded polypeptides comprising of 498, 496, 522 and 550 amino acid residues with their deduced molecular mass of 56.7, 56.9, 59.4 and 62.2 kDa, respectively. Phylogenetic study and molecular modelling analysis of the four cloned WSCYPs revealed their categorization into two CYP families (CYP83B1 and CYP734A1) belonging to CYP71 and CYP72 clans, respectively. BLASTp searches showed similarity of 75 and 56 %, respectively, between the two CYP members of CYP83B1 and CYP734A1 with major variances exhibited in their N-terminal regions. The two pairs of homologues exhibited differential expression profiles in the leaf tissues of selected chemotypes of W. somnifera as well as in response to treatments such as methyl jasmonate, wounding, light and auxin. Light and auxin regulated two pairs of WSCYP homologues in a developing seedling in an interesting differential manner. Their lesser resemblance and homology with other CYP sequences suggested these genes to be more specialized and distinct ones. The results on chemotype-specific expression patterns of the four genes strongly suggested their key/specialized involvement of the CYPs in the biosynthesis of chemotype-specific metabolites, though their further biochemical characterization would reveal the specificity in more detail. It is revealed that WSCYP93Id and WSCYP93Sm may be broadly involved in the oxygenation reactions in the plant and, thereby, control various pathways involving such metabolic reactions in the plant. As a representative experimental validation of this notion, WSCYP93Id was heterologouly expressed in Escherichia coli and catalytic capabilities of the recombinant WSCYP93Id protein were evaluated using withanolides as substrates. Optimized assays with some major withanolides (withanone, withaferin A and withanolide A) involving spectrophotometric as well as high-pressure liquid chromatography (HPLC)-based evaluation (product detection) of the reactions showed conversion of withaferin A to a hydroxylated product. The genes belonging to other CYP group are possibly involved in some specialised synthesis such as that of brassinosteroids.

Zantholic acid, a new monoterpenoid from Zanthoxylum zanthoxyloides.

From the extract of Zanthoxylum zanthoxyloides fruits, a new menthane monoterpenoid, (4R,7R)-8-carboxy-7,9-dihydroxy-trans-menthane, tentatively, named as zantholic acid along with several known compounds was isolated. Its structure was determined using spectroscopic methods. The cytotoxic activity of zantholic acid against a panel of cancer cell lines was evaluated using the MTT assay. The results show that zantholic acid possesses a selective cytotoxic activity towards breast cancer cell lines.

Antibacterial, cytotoxic activities and chemical composition of fruits of two Cameroonian Zanthoxylum species.

ETHNOPHARMACOLOGICAL RELEVANCE: Zanthoxylum leprieurii (Syn. Fagara leprieurii) is a plant extensively used by traditional healers in Cameroon for the treatment of stomach disorders, gonorrhea, intestinal parasites and sterility. The infusion from the fruit is taken in Cameroon for the treatment of sickle cell anemia, as well. Similarly, the roots of Zanthoxylum zanthoxyloides (Syn. Fagara zanthoxyloides) are also used for wound dressing, sickle cell anemia and as pain reliever, in addition to the uses mentioned for Zanthoxylum leprieurii. AIM OF THE STUDY: To scientifically validate their traditional use, the in vitro antimicrobial and anticancer assays were performed. In addition, chemical compositions of their fruit extracts and essential oils were also studied. MATERIAL AND METHODS: The chemical compositions of their essential oils and methanol extracts were studied. GC and GC-MS were used for analyzing the essential oils while various chromatographic methods were used to isolate the pure compounds from methanol extracts. MTT assay was used for the cytotoxic studies of methanol extracts, their fractions and some pure compounds. Antimicrobial activity of methanol extracts, some isolated compounds as well as essential oils was tested against a panel of human pathogenic bacteria by diffusion and dilution methods. RESULTS: The principal constituents of the essential oil of Zanthoxylum zanthoxyloides were identified as citronellol and geraniol whereas Zanthoxylum leprieurii was rich in E-ß-ocimene. Furthermore, we report for the first time, the presence of 7,8-dimethoxycoumarin and sinapic acid in the methanol extract of Zanthoxylum leprieurii. In vitro bioassays reveal that the methanol extracts, essential oil and some of the isolated compounds from fruits of both the plants exhibit moderate anticancer and antimicrobial activities. CONCLUSION: Altogether, the present findings justify the traditional use of Zanthoxylum zanthoxyloides and Zanthoxylum leprieurii in the treatment of stomach disorders and cancer disease. The antimicrobial properties in the essential oil of Zanthoxylum zanthoxyloides are of additional significance.

Effect of slice thickness and blanching time on different quality attributes of instant ginger candy.

Fresh ginger (Zingiber officinale Rosc.) suffers from weight loss, shrinkage, sprouting and rotting during storage after 3-4 weeks. This spoilage may be overcome by processing fresh produce to some value added products. An attempt was made to optimize the protocol for production of instant ginger candy. The experimental parameters considered were slice thickness (5.0-25.0 mm) and blanching duration (10-30 min) followed by dipping in 40°B and 75°B sugar solutions containing 2.0% citric acid respectively, for 1 and 2 h at 95 °C and dried at 60 °C for 1 h. RSM design was considered for this experiment and final products were evaluated for their textural properties, TSS, acidity, TSS: acid ratio, taste score and overall acceptability. The optimum product qualities in terms of hardness (2.08 kg), TSS (73.4%), acidity (1.31%), TSS: acid ratio (56.3), taste score (7.98) and overall acceptability (8.07) were obtained for slice thickness of 10.9 mm and blanching time of 24.9 min.

Extension of shelf life of pear fruits using different packaging materials.

An experiment was conducted on pear fruit (cv. 'Lagoon') to extend the shelf life by using different packaging materials. Fruits were packed in low density polyethylene (LDPE, 0.025 mm), polypropylene (PP, 0.025 mm), linear low density polyethylene (LLDPE, 0.0125 mm) and high density polyethylene (HDPE, 0.025 mm) with or without perforation and stored at ambient condition (25 ± 2 °C and 65.0 ± 5% RH). Periodical observations were recorded on CO2 & O2 concentration (%), physiological loss in weight (PLW, %), decay loss (%), firmness (kgf), colour value (colour difference and colour index), total soluble solid (TSS, °Brix), acidity (mg of malic acid/g), and ascorbic acid loss (%) at 3 days interval. Reduced rate of PLW and decay losses was recorded in pear fruits packed in PP non-perforated (8.04%) and PP perforated (12.5%), respectively as compared to other treatments. The maximum firmness (5.18 kgf) and minimum ascorbic acid loss (49.97%) were also recorded in PP non-perforated up to 12 and 15 days of storage, respectively. It could be inferred that the, PP non-perforated (0.025 mm) was the most suitable packaging materials for extending the shelf life of pear fruits up to 15 days at ambient condition.

Immune responses of goats against foot-and-mouth disease quadrivalent vaccine: comparison of double oil emulsion and aluminium hydroxide gel vaccines in eliciting immunity.

The epidemiological role of small ruminants in foot-and-mouth disease (FMD) outbreaks has been generally neglected. Although, the disease in these species is sub-clinical in nature, their role as virus carriers represents a reservoir for further infection and spread of disease. Data on the usefulness of polyvalent FMD vaccine (FMDV) in goats is scant. Thus, the present study was undertaken to evaluate the benefits of a highly potent polyvalent FMDV in goats. In the present investigations, FMDV quadrivalent double oil emulsion (Montanide ISA 206) vaccines were tested in goats at reduced doses of 2 ml per animal (antigen payload 3.5 microg per serotype per dose). The oil adjuvant elicited superior immune response at any given period than aluminium hydroxide gel (AGS) vaccine and the rapidity of development of response was quicker. The duration of immunity also appeared to be maintained for long period. The differences in immune response between two adjuvant groups were statistically significant (P<0.05). The differences were apparent even in kinetics of immune response. Unlike cattle, goats were found to be late responders for oil-adjuvanted vaccine. Our results indicate possible universal usage of double oil emulsion vaccines for disease control programs irrespective of species of animals.

Early antibody responses of cattle for foot-and-mouth disease quadrivalent double oil emulsion vaccine.

Foot-and-mouth disease (FMD) is an economically important disease of cloven-hoofed animals. The multiplicity of FMDV serotypes in animals poses a central problem in the policy of vaccination and is of much concern to health authorities. Hence it is the practice of vaccination with polyvalent vaccine for prophylactic measure. In the present report, we analysed the early antibody responses elicited by FMDV quadrivalent (FMDV O, A, C and Asia 1 serotypes) double emulsion (Montanide ISA 206) vaccines in cattle. We observed variations between various viral serotypes in eliciting early antibody response although neutralizing antibody response against all the four serotypes were detected as early as fourth day following vaccination. The duration of immunity also appeared to maintain for long period. The neutralizing antibody titres were maintained well above 2log(10) even after 6 months of vaccination irrespective of serotypes. Thus, allows the possibilities of two vaccinations per year for the maintenance of herd immunity.

Fatty acids and sterols from Cymbopogon martinii var. motia roots.

The hexane and methanol extracts of the roots of Cymbopogon martinii var. motia have been investigated to afford mainly fatty acids and common sterols. A new hydroxy unsaturated fatty acid, namely, 16-hydroxypentacos-14(z)-enoic acid, has also been isolated.

Triterpenoids, essential oil and photo-oxidative 28 --> 13-lactonization of oleanolic acid from Lantana camara.

Two novel triterpenoids have been isolated from the roots of Lantana camara L.: 3beta,19alpha dihydroxy ursan-28-oic acid and 21,22beta-epoxy-3beta-hydroxy olean-12-en-28-oic acid in its methyl ester form. Its leaves have yielded an essential oil which is rich in sesquiterpenes. Oleanolic acid, which is thought to be a hepatoprotective compound, was isolated from L. camara roots and converted into its 28 --> 13beta lactone by a facile photo-oxidation reaction.

Risperidone in the treatment of choreiform movements and aggressiveness in a child with "PANDAS".

The acronym 'PANDAS' (pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections) is used to describe neuropsychiatric symptoms putatively resulting from autoimmune responses to streptococcal infection in vulnerable children. A case of 'PANDAS' is presented to increase physician awareness of this disorder and to document effectiveness of risperidone in chorea and treatment-resistant disruptive behavior associated with this syndrome. To our knowledge, this is the first case report on risperidone in pediatric chorea, although studies on effectiveness of this agent in Tourette's disorders have been previously published.

Protective immune response to 16 kDa immunoreactive recombinant protein encoding the C-terminal VP1 portion of Foot and Mouth Disease Virus type Asia 1.

Recombinant protein of Foot and Mouth Disease Virus (FMDV) type Asia 1 corresponding to the C-terminal half of VP1 was expressed in Escherichia coli. As an alternative to the synthetic peptide, this selected C-terminal region was used as a protein vaccine in guinea pigs in order to study the immune response with various adjuvant formulations: immune stimulatory complexes (ISCOMs), Montanide ISA 206, Freund's incomplete adjuvant (FIA), lipopolysaccharide (LPS) and cytokine mixture. A primary dose of 40 microg/animal followed by a booster of the same dose was injected after a 21-day interval. The sera were collected at intervals of 21, 42 and 63 days after the booster. The humoral response to vaccine was monitored by sandwich enzyme-linked immunosorbent assay (ELISA) and a serum neutralization test (SNT). The guinea pig sera showed high titers both in ELISA and SNT, which could be protective. Further, irrespective of the adjuvant preparation used, the vaccine conferred protection against the challenge virus 105 days post-vaccination in 13 of 15 animals (86%). The results indicated that a combination of recombinant protein ISCOMs and Montanide ISA 206 would be a better choice for achieving early protective titers and longer lasting immunity and that the C-terminal half of the VP1 protein may be tried as a safe vaccine for secondary immunization.

Cystic fibrosis carrier screening practices in an ethnically diverse region: experience of the Genetic Network of the Empire State, Puerto Rico, and the U.S. Virgin Islands.

We surveyed clinical genetics centers to assess current cystic fibrosis (CF) screening practices with regard to clinical and laboratory aspects. The survey was developed by the CF committee of the Genetics Network of the Empire State, Puerto Rico, and the U.S. Virgin Islands (GENES) to gauge changes in trends following the April, 1997, NIH Consensus Statement recommending the offering of CF carrier screening to all pregnant patients. Thirty-five of 45 Centers (78%) returned the survey, which was mailed in June, 1998. Sixteen centers currently offer population-based screening, whereas 19 centers do not. Reasons cited for not offering testing included the low risk for CF in ethnic groups served, lack of data about test sensitivity in the populations served, and the absence of CF screening policies in the current standards of care. Approximately half (56%) of genetics centers that are offering testing altered their screening policy following the NIH Consensus statement, either by offering screening to patients of higher-risk ethnicities or by offering it to all patients. Less than half of the Centers that offer routine carrier screening offer screening to all patients regardless of ethnicity. This report is an initial step in documenting and understanding the current service practices regarding CF carrier testing in a diverse region. Our conclusions: (1) Screening practices vary widely among genetic centers in the region. (2) The decision to offer routine CF carrier screening is largely based on ethnicity of the patient population served. (3) Methods used to screen pregnant women and their partners in this part of the country reflect the diversity of models employed throughout the United States. (4) CF screening practices in the GENES region have changed significantly following the April, 1997, NIH consensus statement.

Quetiapine: a new atypical antipsychotic.

Quetiapine has recently been approved for treatment of psychotic disorders. In short term (6 weeks) trials this atypical antipsychotic was shown to be as efficacious as the standard antipsychotics for the treatment of the positive symptoms of schizophrenia without causing any extrapyramidal symptoms or increase in the prolactin levels. Its efficacy for treating the negative symptoms was variable. Preliminary observations suggest its potential to improve the cognitive deficits of schizophrenia. It is metabolized by the p450 CYP 3A4 system with an estimated elimination half life of 6 hours. The optimal treatment is 300 mg to 400 mg/day in two to three divided oral doses. The most common side effects include dizziness, hypotension, somnolence and weight gain. Changes in the ECG, the thyroid hormone and hepatic enzymes levels appear to be clinically insignificant. Quetiapine interacts with phenytoin, carbamazepine, barbiturates, rifampin and glucocorticoids; and coadministration with these drugs may require dosage adjustment. Doses need not be adjusted when fluoxetine, imipramine, haloperidol and resperidone are coadministered. Quetiapine may enhance the effects of antihypertensive agents and may antagonize those of levodopa and dopamine. Long term efficacy of quetiapine has not been determined. Also undetermined are its effectiveness for treating the first episode and treatment-refractory schizophrenia. Data suggest that quetiapine may be used for the management of psychotic disorders in patients who may not tolerate the side effects of the typical antipsychotics and clozapine. It may also be helpful in patients whose psychotic manifestations did not adequately respond to risperidone and olanzapine.

The foot and mouth disease virus type O outbreak of 1992 is not related to vaccine strain (O/R2/75).

Vaccination is the only pragmatic approach to control foot and mouth disease in India. Strict quality control measures are essential to supply potent vaccine to the field application, in addition to monitoring the performance of the vaccine in the field. During the process of monitoring, an outbreak of FMD in vaccinated animals caused by type "O" virus in Tanjavur district of Tamil Nadu and a type "O" virus from unvaccinated herd of Karnataka were studied. Field isolates and vaccine virus were sequenced and analyzed. Data indicated that the virus from the outbreak in vaccinated cattle was a variant which could escape neutralization by antibodies against vaccine virus.

An alkenol and C-methylated flavones from Callistemon lanceolatus leaves.

Callistemon lanceolatus leaves after extraction with a combination of organic solvents have afforded 3-methyltetradec-2-en-7-ol and the rare C-methylated flavones: 5-hydroxy-7,4'-dimethoxy-6,8-dimethylflavone and 5-hydroxy-7,4'-dimethoxy-6-methylflavone. Structures have been established by spectroscopic methods. Isolation of C-methylated flavones from C. lanceolatus supported the view that they are characteristic to Myrtaceae.

Trazodone is only slightly faster than fluoxetine in relieving insomnia in adolescents with depressive disorders.

This retrospective chart review examined the relative effectiveness of fluoxetine and trazodone in relieving insomnia associated with depressive disorders in adolescents (aged 13-17 years). We reviewed the hospital charts of consecutively admitted adolescents with a depressive disorder and insomnia, who received one of three treatments: fluoxetine (20 +/- 2.2 mg), trazodone (71 +/- 32 mg), or a fluoxetine-trazodone combination (fluoxetine 29 +/- 2.2 mg, trazodone 68 +/- 29 mg). Each treatment was examined in 20 patients. Insomnia was defined as a change in sleep patterns characterized by decreased total sleep time that was sufficient to cause clinical concern, and insomnia resolution was defined as sleep starting by midnight and lasting 6 hours. Mean time to resolution of insomnia was significantly faster in adolescents treated with trazodone rather than fluoxetine (2.5 vs. 5.1 days, p < 0.05). Trazodone seemed to save only about 3 days and insomnia resolved in all subjects by the 11th day of antidepressant treatment. Median time to insomnia resolution was 2 days (range 1-5 days) in the trazodone group and 4 days (range 1-11 days) in the fluoxetine group. This difference between trazodone and fluoxetine, although statistically significant, was generally not clinically significant in the management of insomnia associated with depressive disorders in adolescents. The resolution of insomnia was not faster for treatment with a combination of fluoxetine and trazodone in comparison to fluoxetine monotherapy. Insomnia resolution was slightly later in older children. These clinical findings await confirmation by a controlled study. Both drugs seemed effective in ameliorating sleep symptoms in this sample, although it is likely that they produced these changes by different mechanisms.