Structural assessment of the effects of amino acid substitutions on protein stability and protein protein interaction

A structure-based approach is described for predicting the effects of amino acid substitutions on protein function. Structures were predicted using a homology modelling method. Folding and binding energy differences between wild-type and mutant structures were computed to quantitatively assess the effects of amino acid substitutions on protein stability and protein protein interaction, respectively. We demonstrated that pathogenic mutations at the interaction interface could affect binding energy and destabilise protein complex, whereas mutations at the non-interface might reduce folding energy and destabilise monomer structure. The results suggest that the structure-based analysis can provide useful information for understanding the molecular mechanisms of diseases.     

Influence of age and gender on cold pressor response in Indian population

Cold pressor test (CPT) is a simple and well documented laboratory test to evaluate the propensity for hypertension and sympathetic autonomic functions. Role of sex hormones was tested in the present study for the cold pressor response (CPR) in young adults of both sexes and in elderly population. The subjects comprised of young male (n=55), female (n=32) medical students of 17-25 years and elderly males (n=39) and females (n=25) of 50-70 years of age. The CPT was carried out in young and elderly males and females with one minute immersion of one hand in ice cold water (0-4 degrees C). Both in young males and females the absolute rise in SBP and DBP in response to Cold pressor test (CPT) was highly significant, with diastolic percent rise exceeding systolic. In comparison to young males, the females showed greater percent rise in SBP and DBP. Similarly, in elderly groups of both sexes, CPR was associated with significant absolute rise in SBP and DBP with diastolic percent rise more than systolic in males only. Both in young versus elderly males and young versus elderly females comparison yielded comparable percent rise in SBP and DBP. The SBP and DBP percent rise was again comparable between elderly males and females. The greater responsiveness to CPT in young females could be attributed to increased pain sensitivity to cold, and/or genetic and hereditary factors overwhelming the hormonal protection offered by estrogen and nitric oxide (NO).     

Prostate specific antigen ratio: for diagnosis and assessment of aggressiveness of malignancy of prostate

The search for a perfect tumour marker, which would be able to distinguish benign from malignant enlargement of prostate accurately, is still not complete. Total Prostate Specific Antigen (TPSA), a good test, has it's own inadequacies but Free Prostate Specific Antigen (FPSA) to TPSA ratio is emerging as a better adjuvant to it. This prospective study was done to verify the utility of FPSA to TPSA ratio in diagnosis of malignancy of prostate and its relationship to Gleason grading (indicating the aggressiveness) of adenocarcinoma of prostate. 100 patients with urinary symptoms, who were above fifty years of age and had prostatic enlargement, formed the study group. TPSA and FPSA were assayed by ELISA method and FPSA to TPSA ratio was calculated. Prostatic biopsy of all the cases was obtained and diagnostic histopathology and Gleason grading (in cases where adenocarcinoma was diagnosed) was done. Sensitivity, specificity, predictive value of positive test and predictive value of negative test for TPSA and FPSA to TPSA ratio were calculated. They were found to be 100%, 76.7%, 74.1% and 100% for TPSA and 82%, 100%, 100%, 89% for FPSA/TPSA ratio. Thus making it very obvious that FPSA to TPSA ratio is an excellent adjuvant to TPSA for diagnosis of malignancy of prostate increasing the specificity and predictive value for positive test. An inverse correlation (correlation coefficient = -0.95) was also found between PSA ratio and aggressiveness of prostate cancer, pointing towards its capability to predict the histological (Gleason) grade of the tumour.     

Collaborative learning using Internet2 and remote collections of stereo dissection images

We have investigated collaborative learning of anatomy over Internet2, using an application called remote stereo viewer (RSV). This application offers a unique method of teaching anatomy, using high-resolution stereoscopic images, in a client-server architecture. Rotated sequences of stereo image pairs were produced by volumetric rendering of the Visible female and by dissecting and photographing a cadaveric hand. A client-server application (RSV) was created to provide access to these image sets, using a highly interactive interface. The RSV system was used to provide a "virtual anatomy" session for students in the Stanford Medical School Gross Anatomy course. The RSV application allows both independent and collaborative modes of viewing. The most appealing aspects of the RSV application were the capacity for stereoscopic viewing and the potential to access the content remotely within a flexible temporal framework. The RSV technology, used over Internet2, thus serves as an effective complement to traditional methods of teaching gross anatomy.     

Molecular diversity of Glanzmann thrombasthenia in southern India: new insights into mRNA splicing and structure-function correlations of alphaIIbbeta3 integrin (ITGA2B, ITGB3)

The molecular basis of Glanzmann thrombasthenia (GT) was studied in 40 families from southern India. Of 23 identified mutations (13 in the alphaIIb (ITGA2B) gene and 10 in the beta3 (ITGB3) gene), 20 were novel and three were described previously. Three mutations in the beta3 gene-p.Leu143Trp (Leu117Trp), p.Tyr307Stop (Tyr281Stop), and p.Arg119Gln (Arg93Gln)-were detected in 12, three, and two families, respectively, with definite founder effects observed for the first two mutations. Alternative splicing was predicted in silico for the normal variant and a missense variant of the beta3 gene, and for 10/11 frameshift or nonsense mutations in alphaIIb or beta3. The prediction was confirmed experimentally for a c.2898_2902dupCCCCT mutation in exon 28 of the alphaIIb gene that induced exon skipping. Seven out of nine missense mutations substituted highly conserved amino acids buried in the proteins' cores, predicting structural abnormalities. Among these, a beta3 substitution, p.Cys39Gly (Cys13Gly) was found to cause intracellular degradation of the beta3 subunit, in contrast to previous findings that mutations at Cys435, the partner of Cys13 in a disulfide bond, cause constitutive activation of alphaIIbbeta3. The two patients with a beta3 Arg93Gln mutation had normal clot retraction, consistent with a recent finding that this substitution is associated with normal surface expression of alphaIIbbeta3. In conclusion, this study demonstrates that a variety of mutations account for GT in southern Indian patients, provides new insights into mRNA splicing, and highlights the role of specific amino acids in structure-function correlations of alphaIIbbeta3.     

Transplanted embryonic stem cells successfully survive, proliferate, and migrate to damaged regions of the mouse brain

An understanding of feasibility of implanting embryonic stem cells (ESCs), their behavior of migration in response to lesions induced in brain tissues, and the mechanism of their in vivo differentiation into neighboring neural cells is essential for developing and refining ESC transplantation strategies for repairing damages in the nervous system, as well as for understanding the molecular mechanism underlying neurogenesis. We hypothesized that damaged neural tissues offer a niche to which injected ESCs can migrate and differentiate into the neural cells. We inflicted damage in the murine (C57BL/6) brain by injecting phosphate-buffered saline into the left frontal and right caudal regions and confirmed neural damage by histochemistry. Enhanced yellow fluorescent protein-expressing ESCs were injected into the nondamaged left caudal portion of the brain. Using immunohistochemistry and fluorescent microscopy, we observed migration of ESCs from the injection site (left caudal) to the damaged site (right caudal and left frontal). Survival of the injected ESCs was confirmed by the real-time polymerase chain reaction analysis of stemness genes such as Oct4, Sox2, and FGF4. The portions of the damaged neural tissues containing ESCs demonstrated a fourfold increase in expression of these genes after 1 week of injection in comparison with the noninjected ESC murine brain, suggesting proliferation. An increased level of platelet-derived growth factor receptor demonstrated that ESCs responded to damaged neural tissues, migrated to the damaged site of the brain, and proliferated. These results demonstrate that undifferentiated ESCs migrate to the damaged regions of brain tissue, engraft, and proliferate. Thus, damaged brain tissue provides a niche that attracts ESCs to migrate and proliferate.     

Skeletal stability after correction of maxillary hypoplasia by the Glasgow extra-oral distraction (GED) device

Maxillary distraction osteogenesis delivers excellent results, particularly in patients with clefts. In the past, devices such as the conventional facemask and the rigid external distraction device have been used to correct maxillary hypoplasia after a Le Fort I osteotomy. We describe a new device, the Glasgow extra-oral distraction device. The extent of skeletal and dental stability of corrections achieved in 10 patients with maxillary hypoplasia associated with clefts was satisfactory. This device costs little, can be produced in developing countries, and provides effective treatment for severe secondary deformity associated with clefts.