Development of new genomic microsatellite markers from robusta coffee (Coffea canephora Pierre ex A. Froehner) showing broad cross-species transferability and utility in genetic studies.

BACKGROUND: Species-specific microsatellite markers are desirable for genetic studies and to harness the potential of MAS-based breeding for genetic improvement. Limited availability of such markers for coffee, one of the most important beverage tree crops, warrants newer efforts to develop additional microsatellite markers that can be effectively deployed in genetic analysis and coffee improvement programs. The present study aimed to develop new coffee-specific SSR markers and validate their utility in analysis of genetic diversity, individualization, linkage mapping, and transferability for use in other related taxa. RESULTS: A small-insert partial genomic library of Coffea canephora, was probed for various SSR motifs following conventional approach of Southern hybridisation. Characterization of repeat positive clones revealed a very high abundance of DNRs (1/15 Kb) over TNRs (1/406 kb). The relative frequencies of different DNRs were found as AT >> AG > AC, whereas among TNRs, AGC was the most abundant repeat. The SSR positive sequences were used to design 58 primer pairs of which 44 pairs could be validated as single locus markers using a panel of arabica and robusta genotypes. The analysis revealed an average of 3.3 and 3.78 alleles and 0.49 and 0.62 PIC per marker for the tested arabicas and robustas, respectively. It also revealed a high cumulative PI over all the markers using both sib-based (10-6 and 10-12 for arabicas and robustas respectively) and unbiased corrected estimates (10-20 and 10-43 for arabicas and robustas respectively). The markers were tested for Hardy-Weinberg equilibrium, linkage dis-equilibrium, and were successfully used to ascertain generic diversity/affinities in the tested germplasm (cultivated as well as species). Nine markers could be mapped on robusta linkage map. Importantly, the markers showed ~92% transferability across related species/genera of coffee. CONCLUSION: The conventional approach of genomic library was successfully employed although with low efficiency to develop a set of 44 new genomic microsatellite markers of coffee. The characterization/validation of new markers demonstrated them to be highly informative, and useful for genetic studies namely, genetic diversity in coffee germplasm, individualization/bar-coding for germplasm protection, linkage mapping, taxonomic studies, and use as conserved orthologous sets across secondary genepool of coffee. Further, the relative frequency and distribution of different SSR motifs in coffee genome indicated coffee genome to be relatively poor in microsatellites compared to other plant species.

Nonclinical toxicology study of recombinant-plasmid DNA anti-rabies vaccines.

The absence of standard guidelines from National and International regulatory agencies for the safety evaluation of biotechnology products challenges the ingenuity of toxicologists. At present, the development of standard pre-clinical toxicology protocols for such products is on an individual case basis. The present investigation is an attempt to evaluate the safety profile of the first indigenously developed DNA based anti-rabies vaccine in India. The test compounds were DNA rabies vaccine [DRV (100 microg)] and combination rabies vaccine (CRV (100 microg DRV and 1/50 dose of cell culture vaccine)), intended for clinical use by intramuscular route on 1, 7, 14 and 28 day. As per the regular mandatory requirements, the study has been designed to undertake acute (single dose--10 days), sub-chronic (repeat dose--28 days) and chronic (intended clinical dose--120 days) toxicity tests using three dose levels viz. therapeutic, average (2 x therapeutic dose) and highest dose (10 x therapeutic dose) exposure in Swiss Albino mice. The selection of the rodent model viz. Swiss Albino mice is based on affinity and rapid higher antibody response during the efficacy studies. Apart from physical, physiological, clinical, hematological and histopathology profiles of all target organs, the tier-I immunotoxicity parameters have also been monitored. There were no observational adverse effects even at levels of 10x therapeutic dose administration of DRV and CRV. The procedure also emphasizes on the designing of protocols for the products developed by recombinant technique.

DNA polymerase-beta may be the main player for defective DNA repair in aging rat neurons.

A close relationship between the DNA repair potential of various organisms and their rate of aging has been long suspected. We have been looking into the steps of the DNA repair process in isolated neurons from rats of different ages. Unscheduled DNA synthesis (UDS) was low in aging neurons, and also the response of these cells to raise their DNA repair capacity against a mutagenic challenge was poor. Attempts to identify the possible defective locus in the overall DNA repair pathways indicated that the step involving DNA polymerase may be defective. The activity of DNA polymerase-beta, the most predominant DNA polymerase in neurons that is generally considered to be a short-patch repair enzyme, shows a significant decrease in aging neurons. Northern and Southern blotting and immunotitration experiments suggest that there may be an accumulation of inactive beta polymerase molecules in the aging rat brain. Most recent preliminary studies reveal significant 3'-5' exonuclease activity in rat neurons at all ages. However, extension of a primer in a synthetic oligo duplex, either with a mismatch or correct base pair at the 3' end of the primer, was low in neurons of any age and was very poor (almost undetectable) in older ones. Supplementation of neuronal extracts with pure polymerase enzyme revealed that only polymerase beta, but not polymerase alpha, was able to increase the primer extension activity significantly in old neurons. These findings are taken to indicate an age-dependent decline in the DNA repair capacity of neurons and that DNA polymerase beta is a key player in the DNA repair mechanisms of nerve cells.

Loss of base excision repair in aging rat neurons and its restoration by DNA polymerase beta.

Synthetic staggered oligodeoxynucleotide duplexes are formed by annealing a 5'-32P-labeled 14-mer with four different 21-mers. These duplexes have either a correct or mismatched base pair at 3'-end of the primer. With these model template primers the ability of neuronal extracts, obtained from rats of different ages, to extend the primer to the predicted length was tested. While the neuronal extracts of all ages were able to degrade the 14-mer to shorter lengths, extension of the primers in general and in particular, the mismatched, is achieved only feebly by the young and adult neuronal extracts and undetectable with old neuronal extracts. The possibility of restoring the lost activity by supplementing the neuronal extracts with pure DNA polymerases was examined. Of the three polymerases tested (calf thymus alpha polymerase, E. coli DNA polymerase I and rat liver DNA polymerase beta) only polymerase beta gave consistent and encouraging results although the extension was slow and distributive in nature and mismatched primers were extended much less efficiently than the correctly paired primer. However, significantly improved extension, including those of mismatched primers, was achieved by prior removal of mismatched bases in a preincubation with just the neuronal extracts (3'-5'exonuclease activity) followed by extension by the added polymerase beta and dNTPs in the presence of Mn(2+) instead of the usual Mg(2+). These results are taken to indicate that the activity of polymerase beta in brain cells is compromised with age and that this deficit can be corrected in vitro by the addition of pure recombinant rat liver polymerase beta under appropriate conditions.

Effect of two anti-estrogens, clomiphene citrate and tamoxifen, on cervical mucus and sperm-cervical mucus interaction.

OBJECTIVE: To compare the effect of two ovulation-inducing agents, clomiphene citrate and tamoxifen, on cervical mucus and sperm-cervical mucus interaction. SUBJECTS AND SETTING: Forty couples with unexplained infertility attending infertility clinic. METHODS: Cervical mucus scoring and postcoital test done using the Moghissi system in a spontaneous cycle (control cycle) and with clomiphene citrate or tamoxifen (study cycles). RESULTS: Clomiphene citrate significantly decreased cervical mucus production, whereas tamoxifen significantly improved the total score. CONCLUSION: Tamoxifen is a better drug than clomiphene for ovulation induction in women with poor cervical mucus quality.

A case study of nutrient intervention of oral precancerous lesions in India.

Tobacco chewing and/or smoking are strongly related to several cancers, mainly of the upper aerodigestive tract. Several studies on diet and cancer links suggest that micronutrients, particularly antioxidant vitamins and minerals, are risk modifiers of cancers of epithelial origin. This study looks at the impact of micronutrients such as vitamin A, riboflavin, zinc and selenium as intervention agents in subjects with and without precancerous lesions in a high risk group (reverse smokers of chutta-rolled tobacco leaf). Reverse smokers from four villages were enrolled in the study. 150 subjects were supplemented with four nutrients, namely vitamin A, riboflavin, zinc and selenium in the form of a capsule twice a week for 1 year. 148 controls received a placebo capsule containing lactose for the same period. Clinical history and anthropometric data were collected from all the subjects and a clinical photograph of the palate was taken. Micronutrients were estimated in random blood collected from a sub-sample before and after the study. Micronutrients improved the vitamin A, riboflavin and selenium nutriture in the supplemented group with a concomittant regression of precancerous lesions present on the palate. Clinically complete remission of white, red and combination lesions was seen in 57% of subjects on supplements whereas 8% on placebo showed a positive response. Further progression of these lesions was seen in 10% of the supplemented group compared with 47% in the placebo group (P < 0.001). In the non-lesion group, new lesions appeared in 12% on supplements while more than 38% on the placebo developed new lesions (P < 0.02).(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of turmeric and curcumin on BP-DNA adducts.

Many human cancers that are widely prevalent today can be prevented through modifications in life-styles, of which diet appears to be an important agent. Several dietary constituents modulate the process of carcinogenesis and prevent genotoxicity. Many plant constituents including turmeric appear to be potent antimutagens and antioxidants. Therefore the modulatory effects of turmeric and curcumin on the levels of benzo[a]pyrene induced DNA adducts in the livers of rats were studied by the newly developed 32P-postlabelling assay method. Turmeric when fed at 0.1, 0.5 and 3% and the active principle of turmeric (curcumin) when fed at a level of 0.03% in the diet for 4 weeks significantly reduced the level of BP-DNA adducts including the major adduct dG-N2-BP, formed within 24 h in response to a single i.p. injection of benzo[a]pyrene. The significance of these effects in terms of the potential anticarcinogenic effects of turmeric is discussed. Further, these results strengthen the various other biological effects of turmeric which have direct relevance to anticarcinogenesis and chemoprevention.

Relationship between serum pseudocholinesterase and triglycerides in experimentally induced diabetes mellitus in rats.

This study was designed to understand the reasons for the increase in serum pseudocholinesterase activity in diabetes mellitus. Streptozotocin-induced diabetic rats were used for the study. Serum pseudocholinesterase activity increased with the induction of diabetes (381.5 units/l +/- 11.8) compared to the non-diabetic rats (243.1 units/l +/- 7.2). Serum triglycerides, total low density lipoprotein and glycerol also increased concurrently with the development of diabetes. Insulin treatment of the diabetic rats normalized serum glucose concomitant with the reduction of pseudocholinesterase activity, triglycerides, total low density lipoprotein and glycerol. Heparin injection appeared to activate lipoprotein lipase in the diabetic rats by showing a marked fall in serum triglyceride and total low density lipoprotein levels but not in pseudocholinesterase activity. Administration of tetraisopropylpyrophosphoramide a specific pseudocholinesterase inhibitor, inhibited serum and adipose tissue pseudocholinesterase activity by greater than 80% and liver greater than 50%. Concurrent with the inhibition of pseudocholinesterase activity serum triglyceride, low density lipoprotein and glycerol decreased significantly. In normal rats treatment with tetraisopropylpyrophosphoramide also reduced serum lipoproteins markedly, while glycerol only showed a marginal decrease. Glycerol was used as a marker of adipose tissue lipolysis and total low density lipoprotein which is defined as lipoproteins of density less than 1.063 (LDL + VLDL).(ABSTRACT TRUNCATED AT 250 WORDS)

Spirulina as a source of vitamin A.

Experiments were carried out to assess spirulina fusiformis-a blue green algae as a source of vitamin A in rats. In one experiment, the control rats were fed synthetic vitamin A and the experimental rats spirulina as the sole source of vitamin A. The liver vitamin A concentration of spirulina-fed rats of both sexes was found to be significantly higher than that of the control rats. In another experiment the absorption of carotenes from the solvent extract of spirulina and their availability (vitamin A value) as judged by the levels of vitamin A and carotene in plasma and liver were compared with those of synthetic beta-carotene or vitamin A in male rats. The absorption of beta-carotene from spirulina extract tended to be lower than that of crystalline beta-carotene at doses of 550 and 1100 micrograms of beta-carotene. The difference became insignificant at lower beta-carotene dose of 275 micrograms. Spirulina carotene-fed rats did not show a strict dose related increase in the liver or serum vitamin A concentration. The liver vitamin A storage and plasma levels of vitamin A of spirulina carotene-fed rats was much higher than expected. The results of the two studies reported suggest that the algae spirulina can be a valuable source of vitamin A.

Effects of female sex steroids on concanavalin A-mediated agglutination of hepatocytes from nonregenerating and regenerating rat liver and hepatic tumor marker enzymes.

Effect of treatment of female rats with an oral contraceptive agent (OCA), Ovulen-50, for 7 weeks on agglutination of hepatocytes with concanavalin A (con A) and activities of certain tumor marker enzymes were examined to find out if OCA treatment is related to preneoplastic or neoplastic processes. Hepatocytes from regenerating and nonregenerating livers of control female rats showed negligible agglutination with Con A, whereas hepatocytes from non regenerating but not from the regenerating livers of female rats treated with a combination of 5 micrograms ethinyl estradiol and 100 micrograms ethynodiol diacetate showed agglutination. Of the tumor marker enzymes such as hepatic glucose 6-phosphatase, gamma-glutamyl transpeptidase (gamma-GT), and arginase examined in the liver, only gamma-glutamyl transpeptidase showed a significant increase in activity in the steroid-treated rats. Plasma alkaline phosphatase activity was also higher in the treated animals. However, the magnitude of the changes observed was relatively small and perhaps unrelated to the neoplastic process.

Genetic factors in cephalo facial measurements at birth as revealed by new born-parent correlations.

Father-mother and newborn-parent correlations have been obtained for twelve selected cephalofacial measurements. Newborn-parent correlations were significant for almost all the twelve measurements, indicating a strong genetic component and were of the order 30 to 60 percent of known adult correlations. Role of X-linked factors were indicated for facial and nasal measurements. Maternal factors were influencing almost all the traits. Stepwise multiple regression analysis, involving a total of fifteen independent variables, also revealed similar role of genetic factors.

Dermatoglyphic studies in congenital heart disease in India.

Finger, palm and toe prints of 91 patients (53 male and 38 female) with congenital heart disease (CHD) were compared with those of 100 control subjects (50 male and 50 female). The CHD group included tetralogy of Fallot (TF), patent ductus arteriosus (PDA), pulmonic stenosis (PS), atrial septal defect (ASD) and ventricular septal defect (VSD). The dermatoglyphic traits studied were finger tip patterns, palmar patterns, toe patterns, presence of accessory triradii, absence of c triradius, total finger ridge count, palmar ridge counts (a-b, b-c, c-d, a-d and t-d), main line index and pattern intensity index. When CHD was considered as a whole and when individual classifications of CHD were considered separately, significant differences from the controls were observed in some of the parameters. But, in general, the parameters showing significant differences were not the same from one category to next. However, one parameter stood out for its consistency. There was a considerable decrease in the t-d ridge count in all the categories of CHD studied, showing a distal displacement of the axial triradius to the t' position.

Dermatoglyphic studies in rheumatic heart disease.

62 patients suffering from rheumatic heart disease were taken from Institute of Child Health, Niloufer Hospital, Hyderabad, for the present dermatoglyphic investigation. The finger, palm and toe prints were analysed to see if there was an association between rheumatic heart disease and any of the dermatoglyphic traits. The parameters which were significantly different from the controls are: (1) reduced frequency of arches on the finger tips in males and increased frequency of whorls in females; (2) increased frequency of patterns in the III interdigital area in males; (3) decreased d-t ridge count in females, and (4) higher incidence of multiple axial triradii in females.