Molecular Characterization of β-Thalassemia Mutations Via the Amplification Refractory Mutation System-Polymerase Chain Reaction Method at the North Waziristan Agency,Pakistan

β-Thalassemia (β-thal) is a monogenic disease characterized by mutations on the HBB gene, affecting the production of globin that results in hypochromic and microcytic anemia. The aim of this study was to determine the prevalence of six common β-thal mutations, and their frequency and inheritance pattern in affected populations of North Waziristan Agency, Pakistan. In this study, 130 blood samples from 37 unrelated β-thalassemic families having a minimum of one transfusion-dependent child with β-thal major (β-TM), were retrieved either from the Thalassaemia Centre for Women and Children Hospital Bannu or their home towns situated in Noth Waziristan Agency. All samples were analyzed by the amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) using six allele-specific primers for the presence of the six β-thal mutations common in the Pakistani population. Of the six common mutations, our study demonstrated five HBB mutations comprising HBB: c.27_28insG, HBB: c.92+5G>C, HBB: c.126_129delCTTT, HBB: c.92+1G>T and HBB: c.17_18delCT from the families studied, while mutation HBB: c.47G>A [codon 15 (G>A)] was not detected in any of the studied families. Furthermore, the HBB: c.27_28insG and HBB: c.92+5G>C were noted to be the most common with frequencies of 42.85 and 31.42%, respectively. The findings of the present study may be useful in launching carrier screening and prenatal diagnosis (PND) programs by screening analyzed and other unanalyzed affected families for the possible presence of common mutations through the ARMS-PCR technique that will help to control the disease.     

Left ventricular rotational deformation changes by speckle tracking imaging before and 24 hours after transcatheter closure of large secundum atrial septal defects in children

Background: Large atrial septal defects (ASDs) in children cause increased volume overload of the right side of the heart which in turn lead to impairment of left ventricular (LV) performance. Aim: The aim of this study was to evaluate immediate LV rotational deformation changes in children with large ASDs post-device closure and removal of right ventricle (RV) volume overload. Patients and Methods: Twenty children who underwent transcatheter closure (TCC) of large secundum ASDs were included in the study. LV rotational deformation was assessed pre- and 24 hours post-device closure using speckle tracking imaging (STI). Results: 55% were females with mean age 6.1 ± 3.5 years. LV peak basal clockwise rotation improved significantly (−6.9 ± 2.6° before vs −10.3 ± 4.1° after TCC, P = .005), and time to peak clockwise rotation (345.1 ± 124.7 milliseconds (ms) before vs 282.2 ± 82.9 ms after closure, P = .02). There was no significant difference in apical rotational parameters including peak counterclockwise rotation (P > .05 for both). LV twist (11.3 ± 3.8° before vs 17.5 ± 7.1° after closure, P = .001) and torsion (2.1 ± 0.7°/cm before vs 3.1 ± 1.2°/cm after closure, P = .01) were significantly improved, mainly as the result of improvement of LV basal rotation. LV revealed a significant increase in LV end-diastolic volumes (P = .02) 24 hour after TCC with no significant change (P > .05) in end-systolic volumes after closure. Conclusion: Increased peak LV twisting and torsion were attributed to the improved peak systolic clockwise basal rotation after TCC of large ASDs in children.     

Changes in beta 2-adrenoceptor and other signaling proteins produced by chronic administration of 'beta-blockers' in a murine asthma model

BACKGROUND: We have previously reported that chronic treatment with certain 'beta-blockers' reduces airway hyperresponsiveness (AHR) to methacholine in a murine model of asthma. METHODS: Airway resistance was measured using the forced oscillation technique in ovalbulmin-sensitized and ovalbulmin-challenged mice treated with several beta-adrenoceptor (beta-AR) ligands. We used the selective beta 2-AR ligand ICI 118,551 and the preferential beta 1-AR ligand metoprolol to investigate the receptor subtype mediating the beneficial effect. Expression of beta-ARs was evaluated using immunofluorescence. We evaluated several signaling proteins by western blot using lung homogenates, and measured the relaxation of the isolated trachea produced by EP2 and IP receptor agonists. RESULTS: Four findings were associated with the decreased AHR after chronic beta-blocker treatment: (1) the highly selective beta 2-AR antagonist/inverse agonist, ICI 118,551 produced the bronchoprotective effect; (2) beta 2-AR up-regulation resulted from chronic 'beta-blocker' treatment; (3) reduced expression of certain proteins involved in regulating bronchial tone, namely, Gi, phosphodiesterase 4D and phospholipase C-beta 1; and (4) an enhanced bronchodilatory response to prostanoid agonists for the IP and EP2 receptors. CONCLUSIONS: These data suggest that in the murine model of asthma, several compensatory changes associated with either increased bronchodilator signaling or decreased bronchoconstrictive signaling, result from the chronic administration of certain 'beta-blockers'.     

Gap phenomenon in the bundle of his conduction]

His bundle electrograms recorded during atrial stimulation with the extrastimulus technique permitted the analysis of the so called gaps in bundle branch conduction. Three different types of gaps were identified each with a distinct electrophysiological mechanism. In type I gap a CLBBB pattern disappeared at shorter coupling (A1-A2) intervals because the more premature impulses encountered a greater degree of the delay at the A-V node therefore reaching the left bundle branch after its effective refractory period had ended. During this part of the cycle the H1-H2 were longer and H2-V2 intervals shorter than when CRBBB was present. In type II gap a CRBBB pattern disappeared at shorter coupling intervals because the premature impulses met a greater delay in the proximal portions of the His-Purkinje system. In consequence, they arrived at the right bundle branch when its effective refractory period had expired. When this occurred the H1-H2 interval were shorter and the H2-V2 longer than those at which CRBBB had been present. The patient with type III gap had CLBBB. A-V conduction was possible through the right bundle branch very late in the cycle. However, it could not occur at shorter coupling intervals finally reappearing at even shorter coupling intervals. True supernormal conduction or vagal pulsatile discharges could have been present. Yet, we favor, as the most likely possibility, the existence of 2:1 phase 4 block with latent or abortive escapes not recorded in the surface leads.     

The hypoglycaemic and insulinotropic activity of Tinospora crispa: studies with human and rat islets and HIT-T15 B cells

Summary In Malaysia, Tinospora crispa extract is taken orally by Type 2 (non-insulin-dependent) diabetic patients to treat hyperglycaemia. We have evaluated the claimed hypoglycaemic property by adding aqueous extract to the drinking water of normal and alloxan-diabetic rats. After one week, fasting blood glucose levels were significantly (p<0.01) lower and serum insulin levels were significantly (p<0.01) higher in treated diabetic animals (10.4±1.0 mmol/l and 12.8±1.1 U/ml respectively) compared to untreated diabetic controls (17.4±1.7 mmol/l and 8.0±0.7 U/ml respectively). The insulinotropic action of T. crispa was further investigated in vitro using isolated human or rat islets of Langerhans and HIT-T15 cells. In static incubations with rat islets and HIT-T15 B cells, the extract induced a dosage dependent stimulation and potentiation of basal and glucose-stimulated insulin secretion respectively. This insulinotropic effect was also evident in perifused human and rat islets and HIT-T5 B-cells. The observations that (i) in all three models insulin secretory rates rapidly returned to basal levels on removal of the extract and (ii) in rat islets, a second challenge with T. crispa induced an additional, stimulated response, are all consistent with physiological release of insulin by B cells. Moreover, the rate of HIT-T15 glucose utilisation was not affected by incubation with T. crispa, suggesting that the cells were viable throughout. These are the first studies to provide biochemical evidence which substantiates the traditional claims for an oral hypoglycaemic effect of Tinospora crispa, and which also show that the hypoglycaemic effect is associated with increased insulin secretion.     

Insulinotropic activity of Tinospora crispa extract: effect on ß-cell Ca2+ handling

The mechanism of insulinotropic action of Tinospora crispa was investigated in vitro using an insulin-secreting clonal ß-cell line, HIT-T15. The aqueous extract sensitizes the ß-cell to extracellular Ca²⁺ and promotes intracellular Ca²⁺ accumulation which in turn causes increased insulin release. The increase in cytosolic Ca²⁺ concentration is due to stimulation of Ca²⁺ uptake from the extracellular medium and inhibition of Ca²⁺ efflux from the cytosol. That the mechanism of insulinotropic action of T. crispa is physiological suggests that the insulin secretagogue/s present in the extract could indeed be a potential source of a specific oral hypoglycaemic agent for the treatment of non-insulin-dependent diabetes mellitus. © 1998 John Wiley & Sons, Ltd.