The mitochondrial genomes of two Pre-historic Hunter Gatherers in Sri Lanka.

Sri Lanka is an island in the Indian Ocean connected by the sea routes of the Western and Eastern worlds. Although settlements of anatomically modern humans date back to 48,000 years, to date there is no genetic information on pre-historic individuals in Sri Lanka. We report here the first complete mitochondrial sequences for Mesolithic hunter-gatherers from two cave sites. The mitochondrial haplogroups of pre-historic individuals were M18a and M35a. Pre-historic mitochondrial lineage M18a was found at a low prevalence among Sinhalese, Sri Lankan Tamils, and Sri Lankan Indian Tamil in the Sri Lankan population, whereas M35a lineage was observed across all Sri Lankan populations with a comparatively higher frequency among the Sinhalese. Both haplogroups are Indian derived and observed in the South Asian region and rarely outside the region.

Oleanolic acid from antifilarial triterpene saponins of Dipterocarpus zeylanicus induces oxidative stress and apoptosis in filarial parasite Setaria digitata in vitro.

Absence of a drug that kills adult filarial parasites remains the major challenge in eliminating human lymphatic filariasis (LF); the second leading cause of long-term and permanent disability. Thus, the discovery of novel antifilarial natural products with potent adulticidal activity is an urgent need. In the present study, methanol extracts of leaves, bark and winged seeds of Dipterocarpus zeylanicus (Dipterocarpaceae) were investigated for macro and microfilaricidal activity. Two antifilarial triterpene saponins were isolated from winged seed extracts by bioactivity guided chromatographic separation and identified using Nuclear Magnetic Resonance and mass spectroscopic analysis as oleanolic acid 3-O-ß-D- glucopyranoside (1) (IC50 = 20.54 µM for adult worms, 19.71 µM for microfilariae ) and oleanolic acid 3-O-α-L-arabinopyranoside (2) (IC50 = 29.02 µM for adult worms, 25.99 µM for microfilariae). Acid hydrolysis of both compounds yielded oleanolic acid (3) which was non or least toxic to human peripheral blood mono nuclear cells (Selectivity index = >10) while retaining similar macrofilaricidal (IC50 = 38.4 µM) and microfilaricidal (IC50 = 35.6 µM) activities. In adult female worms treated with 50 and 100 µM doses of oleanolic acid, condensation of nuclear DNA, apoptotic body formation and tissue damage was observed by using Hoechst 33342 staining, TUNEL assay and Hematoxylin and Eosin staining respectively. A dose dependent increase in caspase 3/CED3 activity and decrease in total protein content were also observed in these parasites. A dose dependant DNA fragmentation was observed in adult parasites and microfilariae. Decreased levels of reduced glutathione (GSH) and elevated levels of glutathione S transferase (GST), superoxide dismutase (SOD) and reactive oxygen species (ROS) were also observed in parasites treated with oleanolic acid indicating an oxidative stress mediated apoptotic event. Compound 3/oleanolic acid was thus identified as a potent and safe antifilarial compound in vitro.

Rhizome extracts of Curcuma zedoaria Rosc induce caspase dependant apoptosis via generation of reactive oxygen species in filarial parasite Setaria digitata in vitro.

Human lymphatic filariasis (LF) is mainly caused by filarial parasite Wuchereria bancrofti and is the second leading cause of long term and permanent disability in tropical countries. To date, incapability to eliminate long lived adult parasites by current drugs remains the major challenge in the elimination of LF. Hence, in the current study, the efficacy of rhizome extracts of Curcuma zedoaria (a plant traditionally used in Sri Lanka in the management of LF) was evaluated as an effective filaricide in vitro. Sequential solvent extracts of C. zedoaria rhizomes were screened for in vitro antifilarial activity at 0.01-1 mg/mL concentrations by motility inhibition assay and 3-(4, 5 dimethylthiazol-2-yl)-2, 5 diphenyl tetrazolium bromide (MTT) reduction assay using cattle parasite Setaria digitata as a model organism. Exposure of parasites to hexane and chloroform extracts of C. zedoaria caused a dose dependant reduction in motility and viability of microfilariae (IC50 = 72.42 µg/mL for hexane extract, 191.14 µg/mL for chloroform extract) and adult parasites (IC50 = 77.07 µg/mL for hexane extract, 259.87 µg/mL for chloroform extract). Both extracts were less toxic to human peripheral blood mononuclear cells when compared to filariae. A dose dependant increase in caspase 3/CED 3 and a decrease in total protein content, cyclooxygenase (COX) and protein tyrosine phosphatase (PTP) activities were observed in adult parasites treated with hexane or chloroform extract. A significant degree of chromatin condensation and apoptotic body formation were also observed in these worms by Hoechst 33342 and terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labeling (TUNEL) staining respectively. Dose dependant chromosomal DNA laddering was observed in treated adult worms but not in microfilariae in response to both extracts. Oxidative stress parameters such as reduction in reduced glutathione (GSH) levels and increase in glutathione s transferase (GST), superoxide dismutase (SOD) and catalase activities, increased reactive oxygen levels (ROS) and lipid peroxidation were also observed indicating that an apoptotic event is induced by reactive oxygen species.

Growth hormone releasing hormone receptor codon 72 mutation in a cohort of Sri Lankan patients with growth hormone deficiency.

INTRODUCTION: Growth hormone releasing hormone receptor (GHRH-R) codon 72 mutation is recognised as a common genetic cause of growth hormone deficiency (GHD) in the Indian subcontinent resulting in a characteristic lean phenotype. Genetic studies have not been previously carried out in Sri Lankans with GHD. METHODS: Patients with GHD presenting to a tertiary care referral centre were studied for GHRH-R codon 72 mutation by PCR amplification and sequencing. The phenotype of the cohort was described as the BMI SDS (Body mass index standard deviation score) based on the anthropometric data at the time of diagnosis. RESULTS: Among 91 patients from 88 families studied, eight (6 boys) carried the codon 72 mutation. The presence of this mutation was low among the Sinhalese ethnicity (3 out of 68) than among Tamil and Moor ethnicities. BMI SDS of <-2 was seen in 71% of mutation positive and 45.8% of mutation negative patients. CONCLUSIONS: Prevalence of GHRH-R codon 72 mutation in this group of GH deficient patients was 8.8%. The lean phenotype observed in 71% of the mutation positive patients was not a significant association when compared to a similar phenotype in 45.8% of the mutation negative patients.

Microcystin-LR-induced cytotoxicity and apoptosis in human embryonic kidney and human kidney adenocarcinoma cell lines.

Microcystin-LR (MC-LR) is a potent hepatotoxin, and increasing evidence suggests that it might also induce kidney injury. The aim of the present work was to evaluate the cytotoxicity and possible apoptotic effects of MC-LR on a human embryonic kidney cell line (HEK-293) and human kidney adenocarcinoma cell line (ACHN). Cells were exposed for 24 h to pure MC-LR (1.0-200 µM) and the cytotoxic effects were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and sulphorhodamine B (SRB) cell viability assays. Cell viability in both cell lines was significantly decreased after treatment with MC-LR at 50 µM for 24 h (P<0.001). Moreover, MC-LR-treated ACHN and HEK-293 cells exhibited a marked dose-dependent loss of confluence as judged by phase-contrast microscopy. Similarly, fluorescence microscopic observations following acridine orange-ethidium bromide (AO/EB) staining confirmed that both cell types were undergoing apoptosis after treatment with MC-LR for 24 h. Expression of three apoptosis-related genes, Bax, Survivin and p53, was analysed by quantitative reverse transcriptase PCR analysis. Both Bax and p53 functioned as promoters of MC-LR-mediated apoptosis in ACHN and HEK-293 cells. The Survivin gene acted as a suppressor of apoptosis at lower MC-LR concentration (1 µM) and the gene was upregulated at higher MC-LR concentration (10 µM) (P<0.001). Significant increases of caspase 3 (P<0.0001) and caspase 9 (P<0.0001) activity were detected in both cell lines after exposure to MC-LR for 24 h, indicating the MC-LR induces cytotoxicity and a marked apoptosis in both ACHN and HEK-293 kidney cell lines.

Di-nucleotide repeat polymorphisms of the insulin-like growth factor-1 gene and their association with IGF-1, insulin-like growth factor-binding protein-1 and birth size in a Sri-Lankan cohort.

BACKGROUND: Insulin-like growth factor (IGF)-I is implicated in fetal growth. Ethnic variations of IGF-1 have also been suggested. Di-nucleotide repeat polymorphisms of the IGF-1 gene or their association with IGF-1 levels and birth size have not been studied in Sri Lankans. OBJECTIVES: To describe IGF-1 di-nucleotide repeat polymorphisms and their association with IGF-1 and IGF-binding protein (IGFBP)-1 levels and birth size in a cohort of Sri Lankans. METHODS: A cross-sectional study on 200 mother-newborn pairs was carried out. Maternal and cord blood levels of IGF-1 and IGFBP-1 were measured by enzyme-linked immunosorbent assay. Three di-nucleotide repeat polymorphisms of the IGF-1 gene [cytosine-adenine (CA) repeats in the promoter and 3' regions and intron 2 cytosine-thymine (CT) repeat] were studied using PCR amplification and fragment analysis. RESULTS: Cord blood IGFBP-1 levels correlated negatively with birth weight (p < 0.01) and crown-heel length (p < 0.05). Wild-type alleles of the CA repeat polymorphisms differed from those reported in other populations. Newborn and maternal intron 2 CT repeat polymorphism showed a significant effect on birth weight (p < 0.01 and p < 0.001, respectively), crown-heel length (p < 0.01) and head circumference (p < 0.01 and p < 0.05, respectively). Promoter region CA repeat polymorphism and intron 2 CT repeat polymorphism in the newborns were significantly (p < 0.05) associated with cord blood IGF-1 levels. Almost all these effects were limited to primiparous pregnancies. CONCLUSIONS: In Sri Lankans intron 2 CT repeat polymorphism of the IGF-1 gene appears to be a significant contributor to IGF-1 levels and birth size in primiparous pregnancies.

Association of -2548 G/A polymorphism in the leptin gene with preeclampsia/pregnancy-induced hypertension.

OBJECTIVES: To investigate −2548 G/A polymorphism of LEP gene, plasma leptin and soluble leptin receptor in preeclampsia/pregnancy induced hypertension. METHODS: Sixty-two patients and 63 controls (normal pregnancies) studied in the third trimester. Leptin and soluble leptin receptor measured by enzyme immunosorbent assay and −2548 G/A polymorphism by polymerase chain reaction followed by restriction enzyme digestion. RESULTS: Frequency of the AA genotype was significantly (p < 0.001) higher in patients. The A allele conferred a relative risk of 1.67 for the disease (p < 0.0001). Preeclampsia/pregnancy induced hypertension significantly (p < 0.001 to 0.0001) affected leptin and soluble leptin receptor concentrations, free leptin index and leptin normalized to body mass index. Genotype significantly (p < 0.05) influenced only the soluble leptin receptor concentrations. CONCLUSIONS: Preeclampsia/pregnancy induced hypertension appears to be associated with higher circulating leptin and lower SLR levels, and with the AA genotype of −2548 G/A polymorphism of the leptin gene.

Cord blood leptin levels in normal pregnancies, pregnancy induced hypertension and gestational diabetes mellitus.

OBJECTIVE: To compare cord blood leptin concentrations between normal pregnancy, pregnancy induced hypertension (PIH), and gestational diabetes mellitus (GDM). DESIGN: Cross-sectional study. SETTING: Academic institutes and a tertiary care maternal hospital. METHOD: 48 newborns of normal pregnancies (N=18), pregnancy induced hypertension (N=16), and gestational diabetes mellitus (N=14) were studied. Cord blood samples were collected and newborn anthropometric indices recorded at delivery. Leptin concentrations were measured using an enzyme immunoassay. RESULTS: Cord blood leptin levels were significantly different between the 3 groups (Kruskal-Wallis ANOVA; P=0.0064), and the difference resulted mainly from higher levels in GDM than in PIH [geometric mean (95% CI) for GDM: 10.89 (6.30, 18.84) vs PIH: 3.49 (2.14, 5.69) ng/ml (Dunn's multiple comparison: P<0.01). This pattern persisted even when leptin levels were normalized to the ponderal index (Kruskal-Wallis ANOVA P=0.0035; Dunn's multiple comparison: P<0.01). Leptin levels significantly and positively correlated with the ponderal index in normal pregnancy (Spearman r=0.506, p<0.05) and with birth weight in PIH (r=0.5463, p<0.05). CONCLUSION: In GDM cord blood leptin levels are significantly higher, and a source other than fetal adipocytes appears to contribute to this.

Maternal and cord blood levels of insulin-like growth factors--I and--II and insulin-like growth factor binding protein-1: correlation with birth weight and maternal anthropometric indices.

OBJECTIVES: To study the correlation of maternal and cord blood insulin like growth factor (IGF)-I and -II and IGF binding protein (IGFBP)-1 levels with birth weight and maternal anthropometric indices. DESIGN: Longitudinal prospective study. SETTING: Academic Institutions and a Tertiary Care Maternity Hospital. PARTICIPANTS: Women with uncomplicated singleton pregnancy (N = 35) and their newborns. MEASUREMENTS: Maternal weight, height, symphysiofundal height and serum levels of IGF-I, IGF-II, IGFBP-1 were measured thrice during the antenatal period, within 24 h of delivery and at 6 weeks and 6 months postpartum. Newborn anthropometric indices were recorded at birth, and at 6 weeks and 6 months of age. Cord blood levels of IGF-1, IGF-II, IGFBP-1, paternal height and weight, and placental weight measured. RESULTS: Maternal and cord blood IGF-I levels were lower than values reported for Caucasians. All newborns showed adequate growth at birth, and up to 6 months of age. Cord blood IGF-1 positively correlated with chest circumference (r = 0.4532, P = 0.0262), IGFBP-1, negatively with birth weight (r = -0.4024, P = 0.0461) and IGF-II had no effect. Cord blood IGF-I positively correlated with maternal levels at 28 +/- 2 (r = 0.4571, P = 0.0247) and 36 +/- 2 (r = 0.4291, P = 0.0364) weeks of amenorrhoea, whereas IGF-II and IGFBP-1 did not correlate with maternal values. Maternal IGF-I, IGF-II and IGFBP-1 did not correlate with newborn or maternal anthropometric indices. Placental weight correlated significantly with birth weight (r = 0.5299, P = 0.0348) and head circumference (r = 0.5031, P = 0.0470). CONCLUSIONS: Cord blood IGFBP-1 and placental weight appear to be determinants of birth weight variation even among appropriately grown for gestational age newborns.

Insulin-like growth factors-I and -II and insulin-like growth factor binding protein-1 during normal pregnancy: pattern of secretion and correlation with other placental hormones.

OBJECTIVES: To describe pattern of secretion of insulin-like growth factor (IGF)-I, IGF-II, IGF binding protein (IGFBP)-1 and their correlation with each other and major placental hormones during normal pregnancy. DESIGN: Longitudinal study. SETTING: Academic Institutions and a Tertiary Care Maternity Hospital. PARTICIPANTS: Healthy women with singleton uncomplicated pregnancies (N = 35). MEASUREMENTS: Serum levels of IGF-I, IGF-II, IGFBP-1, chorionic gonadotrophin (HCG), placental lactogen (HPL), prolactin, oestradiol and progesterone were studied thrice during the antenatal period and within 24 h of delivery. RESULTS: IGF-I, IGFBP-1, HPL, prolactin, oestradiol and progesterone increased and HCG decreased significantly with advancing gestation (Repeated measures ANOVA: P < 0.01 to 0.0001). IGF-II levels were not significantly affected by period of gestation. Significant negative correlations (multiple regression analysis) were seen between IGFBP-1 and prolactin at 28 +/- 2 (P = 0.0226) and 36 +/- 2 (P = 0.0417) weeks of amenorrhoea (WOA) and between oestradiol and IGF-II at 36 +/- 2 WOA (P = 0.037). Prolactin and IGF-I at 14 +/- 2 WOA (P = 0.0225) and progesterone and IGFBP-1 at 28 +/- 2 WOA (P = 0.0216) correlated positively. CONCLUSIONS: Maternal IGF-I and IGFBP-1 but not IGF-II significantly increase as pregnancy advances. Components of the IGF system regulate or are affected by some of the placental hormones and the effects vary with the period of gestation.

Maternal prolactin concentrations and lactational behaviour in the early postpartum period in women with lactational amenorrhoea.

OBJECTIVE: To compare early postpartum differences in maternal prolactin concentrations and lactational behaviour between women who have short and long periods of lactational amenorrhoea. METHODS: Healthy lactating women resuming regular menstruation before 24 weeks [short amenorrhoea (SA) group; n = 15], and at or after 24 weeks [long amenorrhoea (LA) group; n = 15] postpartum were studied from 4 to 12 weeks postpartum. Infant feeding pattern and maternal plasma prolactin concentrations were compared between the two groups using repeated measures ANOVA. RESULTS: Breastfeeds/24 h were significantly higher and other feeds/24 h were significantly lower (p < 0.01) at 12 weeks postpartum in the LA group when expressed as a percentage of all feeds. Number of breastfeeds (total/24 h and night) significantly decreased and the number of other feeds significantly increased (p < 0.05 to 0 < 0.001) with time in both groups. Prolactin concentrations (basal, suckling stimulated and increment) were higher in the LA group, but consistent significant differences were observed mainly in the increment (p < 0.05 to p < 0.001). Prolactin concentrations significantly (p < 0.01 to < 0.001) decreased with time postpartum in the SA group, but not in the LA group. CONCLUSIONS: Women with longer periods of lactational amenorrhoea maintain higher prolactin concentrations than those with shorter periods of lactational amenorrhoea during the early postpartum period, even when the breastfeeding frequency is reduced. This could be explained by a greater sensitivity of the pituitary lactotrophe to the suckling stimulus in women who have longer periods of lactational amenorrhoea.

Lactational amenorrhea/anovulation and some of their determinants: a comparison of well-nourished and undernourished women.

OBJECTIVE: To study the effect of maternal nutritional status and some other possible determinants on lactational amenorrhea/anovulation. DESIGN: Prospective matched-pairs study. SETTING: Postpartum wards and community and academic settings. PATIENT(S): Thirty matched pairs of otherwise healthy, well-nourished (body mass index > or = 26.00 kg/m2) and undernourished (body mass index < or = 19.00 kg/m2) postpartum women were selected. INTERVENTION(S): Infant feeding pattern was recorded weekly, and infant weights, maternal body mass index, and maternal PRL levels were estimated every 4 weeks until resumption of menstruation. Ovulatory activity was determined using urinary estrone and pregnanediol glucuronide concentrations. MAIN OUTCOME MEASURE(S): Time of resumption of menstruation, regular and ovulatory. RESULT(S): Well-nourished women resumed regular menstruation significantly earlier than undernourished women but resumed ovulatory menstruation at almost the same time. Undernourished women had fewer anovulatory cycles preceding first postpartum ovulation and a higher prevalence of formula feeding. Effect of body mass index on lactational amenorrhea became nonsignificant when nonintroduction of formula feeds, maternal age, and socioeconomic status were controlled for. CONCLUSION(S): Improved maternal nutritional status has no significant effect on fertility: ovulation is not advanced despite early resumption of regular menstruation.

Effect of skim milk supplementation of the maternal diet on lactational amenorrhea, maternal prolactin, and lactational behavior.

Effect of skim milk supplementation of the maternal diet on lactational amenorrhea was studied in 30 pairs of healthy lactating women matched for parity, body mass index, and previous experience of lactational amenorrhea. Supplementation of the maternal diet had no significant effect on the time of resumption of regular menstruation or ovulation, maternal prolactin concentrations, breast-feeding pattern, maternal body mass index, or infant weight. However, the supplemented group breast-fed nearly exclusively (supplemental feeds were introduced but did not exceed 20% of total feeds) for a significantly longer duration (P < 0.05) than did the control group. Previous experience of lactational amenorrhea was significantly positively correlated with the time of resumption of menstruation in the supplemented (P < 0.01) and control (P < 0.05) groups when frequency of breast-feeding, maternal body mass index, and supplementary feeds to the infant were controlled for. Thus, maternal nutritional supplementation does not appear to affect the contraceptive benefit of lactation when the frequency of breast-feeding is not compromised but apparently lengthens the duration of nearly full breast-feeding.

Effect of Momordica charantia on key hepatic enzymes.

The effect of Momordica charantia on certain key hepatic enzymes was investigated using male Sprague-Dawley rats as the animal model. Fruit juice and seed extract of Momordica charantia were administered orally at a daily dose of 1 ml/100 g body weight for 30 days under light ether anaesthesia while the control group received equivalent amounts of distilled water under identical conditions (n = 10 in each case). Serum gamma-glutamyl transferase (P < 0.001) and alkaline phosphatase (P < 0.01-0.001) concentrations were found to be significantly elevated following oral administration of both the fruit juice and the seed extract. Consistent significant histopathological changes in the liver were not observed in either treatment group although the prevalence of dilatation and/or congestion of the central vein sinusoidal system appeared to be twice as high following fruit juice treatment than in the other 2 groups. Thus, Momordica charantia may either contain hepatotoxins capable of causing cellular damage at the molecular level without causing significant histopathological changes or the plant may have an enzyme inducing effect.

Prolactin response to suckling in a group of fully breast feeding women during the early postpartum period.

Prolactin response to suckling was studied in a group of fully breast feeding women (N = 58) between 4-6 weeks postpartum. Basal, suckling stimulated and the increment of prolactin showed wide individual variations. Basal prolactin concentrations varied from 140 to 4,600 mIU/l, suckling stimulated prolactin from 400 to 5,600 mIU/l and the increment of prolactin from 40 to 4,160 mIU/l. Basal (p = 0.0395) and suckling stimulated (p = 0.0423) prolactin concentrations significantly increased as the number of night breast feeds increased and the suckling stimulated (p = 0.0218) prolactin concentrations significantly increased as the number of breast feeds/24 h increased. However, the magnitude of the rise in prolactin in response to suckling was not dependent on basal prolactin concentration. Basal, suckling stimulated or the increment of prolactin were not significantly different between subjects having different breast feeding frequencies, when the subjects were grouped according to the number of breast feeds. These differences may be due to the large individual variation in prolactin concentrations seen in women having similar breast feeding frequencies which may arise from individual variations in hypothalamic--pituitary response to suckling.

Effect of age on serum prolactin concentration in presumably fertile men in Sri Lanka.

OBJECTIVE: To examine possible age related variation in serum prolactin in men. DESIGN: A cross sectional study. SUBJECTS: Healthy married men aged 21 to 85 years with one or more children. MEASUREMENTS: Serum prolactin concentrations measured by immunoradiometric assay. RESULTS: Prolactin concentrations were significantly higher (p < 0.001) from 61 years of age onwards when compared with 31 to 60 years. Prolactin concentrations between 21 to 30 years were significantly higher than between 31 to 50 years (p < 0.05), but lower than between 61 to 70 (p < 0.05) and 75 to 85 (p < 0.01) years. CONCLUSIONS: Serum prolactin concentrations show age related variations in presumably fertile men.