Efficient synthesis of the benzo[a]pyrene metabolic adducts of 2'-deoxyguanosine and 2'-deoxyadenosine and their direct incorporation into DNA

A new and efficient method is described for the synthesis in gram quantities of the benzo[a]pyrene (B[a]P) metabolic adducts of 2'-deoxyguanosine (dG) and 2'-deoxyadenosine (dA) substituted, respectively, at the N(2)- and N(6)- positions. When the racemic form of the tris(benzoyloxy)amine 5 (related to the notoriously carcinogenic epoxydiol 2) is coupled with the bromoinosine derivative 6 by means of a Buchwald-Hartwig reaction, the expected pair of diastereomers, 7 and 8, is obtained in high (combined) yield. Selective deblocking of this mixture then gave cleanly the pair of diastereomers 9. These were used in the synthesis of a series of DNA oligomers via their 5'-O-DMT-3'-O-phosphoramidites (10) using standard automated methods. Coupling efficiencies were 94-98% at the point of introduction of the xeno-2'-deoxynucleoside, and in all cases the mixtures of the two diastereomeric oligomers (DMT-off stage) were easily separated by HPLC. By a similar sequence of reactions beginning with 5 and the protected 6-bromopurine 2'-deoxynucleoside 11, it was possible with equal efficiency to introduce the N(6)-modified diastereomers (16) of dA into oligomeric DNA. Circular dichroism measurements were used to establish the fundamental configurations at the xeno-2'-deoxynucleoside site for each of the oligomers. Mass spectral data in both the dG and the dA series confirmed the presence of the xeno-2'-deoxynucleoside in the oligomers. This was complemented by enzymatic degradation of one of the oligomers from each of the series. In both of these cases, after HPLC separation, circular dichroism measurements on the reisolated xenonucleoside also confirmed its presence in the oligomer.     

Coil occlusion of the small patent arterial duct without arterial access

BACKGROUND: Arterial access is traditionally considered mandatory during coil occlusion of the patent arterial duct. Arterial access necessitates heparinization and carries the risk of femoral artery occlusion in small children. METHODS AND RESULTS: Between July 1999 and May 2001, we attempted coil occlusion of patent arterial ducts in 104 patients without arterial access. The patients were aged from 3 months to 14 years. The median age was 2 years. They weighed 3-35 kg. The median weight was 9.8 kg. The duct diameter at pulmonary artery insertion was 1.8-3.5 mm. The patients were selected on basis of echocardiographic evaluation of duct diameter at pulmonary artery insertion and morphology of the ampulla. Doppler color flow imaging was used in the catheterization laboratory to confirm duct closure. Arterial access was required in 21 patients. The reasons included accidental puncture in 5 patients, failure to obtain venous access in 1 patient, aortic embolization in 3 patients, poor echo images in 2 patients, requirement for additional coils in 8 patients and, failure to cross the duct from pulmonary artery in 2 patients. The fluoroscopic time ranged from 2.2 to 20 min with a mean of 5.3+/-3.8 min. Immediate closure was achieved in 98 patients and this included 79 of the 83 patients in whom arterial access was avoided. Color Doppler 3-24 h later showed residual flow in 2 patients. Four patients had new-onset left pulmonary artery turbulence with peak gradients below 5 mm of mercury. Coil embolization occurred in 6 patients and all coils were retrieved. Three-month follow up information was available for 78 patients. Small residual ductal leaks were seen in 4 patients, 2 of whom had leaks at 24 h. Two patients had recanalized their ducts. CONCLUSION: It is feasible to occlude small patent arterial ducts with coils using venous access alone in carefully selected patients with excellent immediate and short-term results.     

Quantitative determination of aristolochic acid-derived DNA adducts in rats using 32P-postlabeling/polyacrylamide gel electrophoresis analysis.

Aristolochic acids (AA) are nephrotoxic and carcinogenic nitroaromatic compounds produced by the Aristolochiaceae family of plants. Ingestion of these phytotoxins by humans results in a syndrome known as AA nephropathy, characterized by renal tubulointerstitial fibrosis and upper urothelial cancer. After activation by cellular enzymes, AA I and II react with DNA to form covalent adducts and as such represent potential biomarkers for studies of AA toxicity. Using site-specifically modified oligodeoxynucleotides as standards, we have developed a method for quantifying 7-(deoxyadenosin-N(6)-yl) aristolactam-DNA or 7-(deoxyguanosin-N(2)-yl) aristolactam-DNA adducts in tissues of Wistar rats using an assay in which (32)P-postlabeling techniques are coupled with nondenaturing polyacrylamide gel electrophoresis. The limit of detection with this technique is five adducts in 10(9) nucleotides for a 5-microg DNA sample. In contrast to previous reports, we find that the levels of AA adducts in renal tissues of Wistar rats treated p.o. with AA for 1 week with 5 mg/kg/day of AA I or AA II were much higher than that in the forestomach. Highest adduct levels were observed in rats treated with AA II, suggesting that this compound may be more genotoxic than AA I. Treatment of rats with aristolactam I, an end-product of AA I metabolism, resulted in a much lower level of adduction. This study establishes the feasibility of using AA-DNA adducts as intermediate biomarkers of exposure in studies of AA nephropathy and its associated urothelial cancer.     

Childhood systemic lupus erythematosus in South India

Out of 330 adult Systemic Lupus Erythematosus (SLE) cases who attended the Rheumatic Care Centre, Government General Hospital, 59 children were analysed. There was no case with onset before the age of 5 years. There were 49 females and 110 males (M:F =1∶4.9). The initial manifestations were fever (67%), arthritis (61%), skin rash (59%) and lymphadenopathy (27.1%). There was no case of Raynaud's phenomenon. Only 10.1% of patients presented with thrombocytopenic purpura. In the cumulative clinical features, arthritis in 86.6%, fever in 79.8%, skin rash in 69.4%, lymphadenopathy in 61% and hepatosplenomegaly in 39.9% were observed. Renal involvement was seen in 49.1%, neuropsychiatric manifestations in 27.1%, pleuropulmonary in 22% and cardiac manifestations in 10.2%. Anaemia was seen in 50.8%, leukopenia in 18.4%, thrombocytopenia in 11.8%, ANA in 100%, anti-dsDNA in 92.3%, anti-Sm in 34.7%, anti-SSA in 38.5%, anti-SSB in 15.4%, ACL in 30.8%, low C3 in 50% and false positive VDRL in 3.3%. Death occurred in 8 children, 3 due to infection, 2 due to renal causes, I due to cardiac and 2 due to central nervous system involvement.     

ENPP1/PC-1 K121Q polymorphism and genetic susceptibility to type 2 diabetes

Genetic susceptibility modulates the impact of obesity on risk for type 2 diabetes. The present study evaluates the role of ENPP1 K121Q polymorphism in prediction of type 2 diabetes in three populations that differ in susceptibility to diabetes and environmental exposure. The three cohorts included 679 nonmigrant South Asians living in Chennai, India (223 with type 2 diabetes); 1,083 migrant South Asians living in Dallas, Texas (121 with type 2 diabetes); and 858 nonmigrant Caucasians living in Dallas, Texas (141 with type 2 diabetes). Patients with type 2 diabetes were included in these cohorts if they had diabetes onset before the age of 60 years. The prevalence of subjects carrying the polymorphic ENPP1 121Q allele was 25% in the nondiabetic group and 34% in the diabetic group of South Asians living in Chennai (P = 0.01). The prevalence in the nondiabetic and diabetic groups were 33 and 45% (P = 0.01) for the South Asians living in Dallas and 26 and 39% (P = 0.003) for the Caucasians. Although further replication studies are necessary to test the validity of the described genotype-phenotype relationship, our study supports the hypothesis that ENPP1 121Q predicts genetic susceptibility to type 2 diabetes in both South Asians and Caucasians.     

Sclerosing stromal tumour of the ovary--a clinicopathologic spectrum

Majority of the Sclerosing stromal tumours of the ovary documented in the literature are single case reports. We report a series of 4 cases. Among the 4 cases encountered the mean age at presentation was 22.2 years. The clinical presentation varied from asymptomatic mass per abdomen (2 cases), menorrhagia (1 case) and amenorrhoea (1 case). The tumour was unilateral in all the cases with an average size of 10 cms. Grossly the appearances varied from a solid, partly cystic, edematous tumour (2 cases) to solid,firm tumour with yellow flecks (1 case) to unilocular cystic tumour (1 case). Microscopically, the tumour was characterized by cellular pseudolobules composed of a disorderly admixture of collagen-producing fibroblasts and lipid rich lutein cells with shrunken nuclei. In one case the lutein cells had a robust appearance with abundant cytoplasm and vesicular nuclei. The pseudolobules were very vascular and separated by hypocellular dense to oedematous fibrous tissue. Frozen section demonstrated fat in luteinized cells in 3 cases.     

Circadian variations in the monoamine levels and monoamine oxidase activity in different regions of the rat brain as a function of age

Time-related changes in the levels of norepinephrine (NE), dopamine (DA), 5-hydroxytryptamine (5-HT) and monoamine oxidase (EC 1.4.3.4, MAO) activity have been studied in the cerebral cortex, cerebellum, medulla oblongata, hypothalamus, striatum and midbrain of 21 day, 3, 6, 12 and 24 month old rats maintained at 12 hours of light and 12 hours of dark condition. Maximum NE level was seen during the dark phase in all the regions of 3, 6, 12 and 24 month old rats, whereas in 21 day old, the maximum NE level occurred during the light phase. In the cerebral cortex and in the cerebellum of 21 day old rat DA was absent at all times. In all the other age groups, the maximum DA level was seen during the dark phase, while for 5-HT higher level was during the light phase in all the age groups. MAO activity of 3, 6, 12 and 24 month old rats showed the peak activity at the beginning of the light phase (06:00 hours), whereas cerebral cortex, cerebellum and medulla oblongata of 21 day old rat had its peak MAO activity at 14:00 hours and at 22:00 hours in other regions.     

Bradykinin antagonists have no analgesic effect on incisional pain

Bradykinin, an endogenous nonapeptide and an important mediator of inflammation, is also implicated in the initiation and maintenance of pain. Both des-Arg(8), Leu(8)-bradykinin (dALBK) and HOE-140, the prototypic bradykinin B1 and B2 receptor antagonists, respectively, have been shown to reduce pain behaviors and inflammation in animal models of persistent nociception. We studied them for activity against incision-induced pain behaviors in a rat model for postoperative pain. A 1-cm plantar incision was made in the hind paw of halothane-anesthetized rats and closed with 5-0 nylon. Withdrawal responses to punctate and nonpunctate mechanical stimuli were tested with von Frey filaments and a plastic disk attached to a von Frey filament, respectively. Withdrawal latency to radiant heat was also tested. Rats were tested 1 day before the incision, 1 h after the incision, and 0.5, 1, 1.5, and 2.5 h after the injection of the drug. They were then retested at the same times before and after the injection of the drug on each of the first 2 postoperative days. The rats received the saline vehicle dALBK (0.1, 0.3, 1.0, or 3.0 mg/kg) or HOE-140 (0.1, 0.3, 1.0, or 3.0 mg/kg) IV. Another group of rats had the drug injected 1 h before incision and tested as above. Statistical significance (P < 0.05) was determined with Kruskal-Wallis test and a two-way analysis of variance. None of the doses of either dALBK or HOE-140 affected the responses to punctate or blunt mechanical stimulation or heat, either as a pretreatment or as a posttreatment. These data support the unique mechanisms for incision-induced pain relative to inflammation-related pain. Although inflammation may represent a component of incisional pain, the etiology of inflammation and its role seem different than in other models.     

Physical status of newborns and neonatal outcome

OBJECTIVE: To determine the extent to which physical status at birth is associated with neonatal mortality and the causes of mortality vis-a-vis size at birth and gestational age. METHOD: 11,223 consecutive live births completing 26 weeks of gestation and weighing > or = 500 gm were included in the study. Birth weight and chest circumference were recorded as per WHO guidelines. Gestational age was calculated on the basis of L.M.P. and the new Ballard's score. Deaths occurring in the hospital within 28 days were recorded. Percentile values of gestational age specific birth weights were calculated separately for singletons and multiple births. Percentage of SGA was calculated with reference to WHO recommended values. Birth weight-gestational age-specific mortality rates were calculated at 2 wk and 500 gm intervals. RESULT: Low-birth-weight babies constituted 39.8% of the total, much in excess of WHO recommended figure of 15%. 76% deaths occurred among LBW babies and 56.2% among preterms. Mortality showed remarkable decline as the birth weight increased to 2,000 gm. The lowest mortality was among singletons weighing 2,500-3,000 gm and of 38-40 weeks gestation. Prevalence of SGA at 40 and 42 weeks were 73.7% and 83.6% respectively. But, if SGA babies not categorised as LBW were excluded, the values came down to 32% and 36% respectively. 36% of all deaths occurred during the first 24 hrs of birth; asphyxia and related causes contributing to 50% of it. CONCLUSION: Cut-off value of 2,000 gm instead of 2,500 gm for birth weight may be preferable in countries where most LBW babies are SGAs. Simultaneously, deaths in non-LBW babies due to perinatal causes contribute sgnificantly to total neonatal mortality and need due attention through sensitising obstetricians in essential newbom care and timely Intervention.     

Massive primary chylopericardium in an infant

We report a case of massive spontaneous primary chylopericardium in a 2-month-old infant who was successfully treated with thoracic duct ligation and creation of a posterior pericardial window.