Symmetric vs asymmetric PCR and molecular beacon probe in the detection of a target gene of adenovirus

A DNA fragment (307 bp) from the conserved region of an adenovirus gene (hexon) was amplified by symmetric and by asymmetric polymerase chain reaction (PCR). Two amplifications, one in the absence other in the presence of a molecular beacon probe were conducted by both symmetric and asymmetric PCR. The probe sequence was complementary to an internal segment of the amplified fragment. The product amplified in the absence and presence of the probe was detected by agarose gel and fluorescence analysis, respectively. A symmetric PCR results in exponentially grown double stranded DNA. An asymmetric PCR generates one of the strands by linear ampIlification and a fraction of its total product as double-stranded DNA limited by the concentration ratio of the primers used. Thus asymmetric PCR provided lower intensity signal hence less sensitivity than symmetric PCR by agarose gel analysis as expected. However, signal from a beacon probe based PCR assay is generated only from the probe fraction that hybridizes successfully competing against the strand complementary to the target strand of the product generated by PCR. The symmetric PCR has so far been used for the molecular beacon based fluorescent signal detection. The present study compared the level of fluorescent signal detectable from a symmetric PCR with that from an asymmetric PCR. The fluorescent data analysis demonstrated that a significant higher level of fluorescent signal hence higher sensitivity of detection is obtainable using asymmetric PCR than symmetric PCR performed in presence of the molecular beacon probe.     

Brain-Targeted Nasal Clonazepam Microspheres

Gelatin-chitosan mucoadhesive microspheres of clonazepam were prepared using the emulsion cross linking method. Mirospheres were evaluated using the in vitro and ex vivo drug release patterns. In vivo CNS drug distribution studies were carried out in rats by administering the clonazepam microspheres intra-nasally and clonazepam solution intravenously. From the drug levels in plasma and CSF, drug targeting index and drug targeting efficiency were calculated. Results obtained indicated that intranasally administered clonazepam microspheres resulted in higher brain levels with a drug targeting index of 2.12. Gelatin-chitosan cross linked mucoadhesive microspheres have the potential to be developed as a brain-targeted drug delivery system for clonazepam.     

Calcific pancreatitis-induced gastroduodenal artery pseudoaneurysm: Non-surgical management

Hemorrhage associated with pancreatitis has a high morbidity and mortality in the early phase of the illness. In a small number of patients, bleeding is from major pancreatic or peripancreatic vessels which necessitates emergency intervention. However, most such reports are confined to adults. We report a 6-year-old girl with chronic calcific pancreatitis who presented with hematemesis and melena without any acute exacerbation of her underlying illness. Pseudoaneurysm of the gastroduodenal artery was detected by angiography which was then effectively treated at the same time by embolization with gel foam and a steel coil, thus obviating the need for surgical intervention.     

Dietary variation of protein-carbohydrate: effect on hypothalamic and hippocampal GABA-glutamate in relation to aging

Dietary protein variation has been found to alter brain regional neurochemistry with aging. In the present investigation, we studied the effect of short-term treatment of protein-carbohydrate variable diet to rat on hypothalamic and hippocampal gamma-amino butyric acid (GABA)-glutamate metabolism with increase of age. Exposure of male albino rats with diet containing normal protein (20%)-normal carbohydrate (68%) increased GABA metabolism and decreased glutamate metabolism in both hypothalamus and hippocampus with the increase of age. GABA-glutamate metabolism of rats having low protein (8%)-high carbohydrate (80%) diet for short-term period (STP), was activated in young age (3 months) and decreased in old age (18 months) in both the brain regions. On the contrary, intake of high protein (50%)-low carbohydrate (38%) diet under similar condition decreased GABA-glutamate metabolism in both hypothalamus and hippocampus of young brain and increased only in hypothalamus of aged brain. In hippocampus of aged brain the same diet decreased glutamate metabolism without changing its GABA metabolism. These results suggest that an age-associated change in GABA-glutamate metabolism depends on the amount of dietary protein and carbohydrate and also on the brain region.     

Clinical inquiries: Do glucosamine and chondroitin worsen blood sugar control in diabetes?

Despite theoretical risks based on animal models given high intravenous doses, glucosamine/chondroitin (1500 mg/1200 mg daily) does not adversely affect short-term glycemic control for patients whose diabetes is well-controlled, or for those without diabetes or glucose intolerance (SOR: A, consistent, good-quality patient-oriented evidence). Some preliminary evidence suggests that glucosamine may worsen glucose intolerance for patients with untreated or undiagnosed glucose intolerance or diabetes (SOR: C, extrapolation from disease-oriented evidence). Long-term effects are unknown; however, no compelling theoretical or incidental data suggest that long-term results should be different (SOR: C, expert opinion). Further studies are required to clarify the effects of glucosamine on patients with poorly controlled diabetes or glucose intolerance.     

Brain regional gamma-aminobutyric acid (GABA) and locomotor activity: effect of long-term aldrin exposure

Aldrin-induced stimulation of locomotor activity (LA) under nontolerant conditions was restored to control value after 20 or more consecutive days of aldrin administration. In contrast to the inhibition of GABAergic activity with aldrin under short-term conditions as observed in our previous study, the measurement of steady-state level of GABA, the activities of its metabolizing enzymes, turnover and the specific binding of GABA to its receptor in different regions of the brain of rats treated with aldrin (2 or 5 mg/kg/day, po) under long-term (for 30 consecutive days) conditions showed no change in the GABAergic activity in any regions of the rat brain. Moreover, the studies of the interaction between neurotransmitters (using agonist(s) and antagonist(s) of the respective neurotransmitter receptors) showed that long-term administration of aldrin restored the LA to control value by upregulation of central GABAergic activity through the interaction with dopaminergic and cholinergic systems. Thus, this result suggests that long-term aldrin exposure upregulated the central GABAergic activity inhibition under short-term aldrin treatment which may be a cause of restoration of LA stimulated by the short-term aldrin administration to its control value.     

Ventricular tachycardia in congenital adrenal hyperplasia

Severe hyperkalaemia in patients with congenital adrenal hyperplasia in association with aggravating factors such as acidosis and hypocalcaemia can cause life-threatening ventricular arrhythmias. Treatment of the underlying cause may be the only modality required in such cases. We report a 20-day-old male presenting with ventricular tachycardia due to electrolyte abnormalities in salt-losing congenital adrenal hyperplasia. Sudden cardiac deaths reported earlier in such cases thus gain credence.     

The mcm17 mutation of yeast shows a size-dependent segregational defect of a mini-chromosome

Mini-chromosome-maintenance (mcm) mutants were described earlier as yeast mutants which could not stably maintain mini-chromosomes. Out of these, the ARS-specific class has been more extensively studied and is found to lose chromosomes and mini-chromosomes due to a defect in the initiation of DNA replication at yeast ARSs. In the present study we have identified a number of mcm mutants which show size-dependent loss of mini-chromosomes. When the size of the mini-chromosome was increased, from about 15 kb to about 60 kb, there was a dramatic increase in its mitotic stability in these mutants, but not in the ARS-specific class of mutants. One mutant, mcm17, belonging to the size-dependent class was further characterized. In this mutant, cells carried mini-chromosomes in significantly elevated copy numbers, suggesting a defect in segregation. This defect was largely suppressed in the 60-kb mini-chromosome. A non-centromeric plasmid, the TRP1ARS1 circle, was not affected in its maintenance. This mutant also displayed enhanced chromosome-III loss during mitosis over the wild-type strain, without elevating mitotic recombination. Cloning and sequencing of MCM17 has shown it to be the same as CHL4, a gene required for chromosome stability. This gene is non-essential for growth, as its disruption or deletion from the chromosome did not affect the growth-rate of cells at 23 °C or 37 °C. This work suggests that centromere-directed segregation of a chromosome in yeast is strongly influenced by its length. Received: 20 October 1996 / 2 May 1997