CUSP: an algorithm to distinguish structurally conserved and unconserved regions in protein domain alignments and its application in the study of large length variations

Background  

Distantly related proteins adopt and retain similar structural scaffolds despite length variations that could be as much as two-fold in some protein superfamilies. In this paper, we describe an analysis of indel regions that accommodate length variations amongst related proteins. We have developed an algorithm CUSP, to examine multi-membered PASS2 superfamily alignments to identify indel regions in an automated manner. Further, we have used the method to characterize the length, structural type and biochemical features of indels in related protein domains.     

HNHDb: A database on pattern based classification of HNH domains reveals functional relevance of sequence patterns and domain associations.

The HNH Database is a collection and sequence-based classification of HNH domain proteins. The database contains about 1913 HNH domain containing proteins, and is classified into 10 subsets based on the sequence pattern. Each of these subsets has unique signature sequences. We have shown a correlation between the subset combination and their domain association and function. Functional divergence of this domain may be due to the combination of these conserved patterns and the large variations in the non-conserved regions. HNHDb is freely available at http://bicmku.in:8081/hnh.     

Image-based modeling of hemodynamics in coronary artery aneurysms caused by Kawasaki disease

Kawasaki Disease (KD) is the leading cause of acquired pediatric heart disease. A subset of KD patients develops aneurysms in the coronary arteries, leading to increased risk of thrombosis and myocardial infarction. Currently, there are limited clinical data to guide the management of these patients, and the hemodynamic effects of these aneurysms are unknown. We applied patient-specific modeling to systematically quantify hemodynamics and wall shear stress in coronary arteries with aneurysms caused by KD. We modeled the hemodynamics in the aneurysms using anatomic data obtained by multi-detector computed tomography (CT) in a 10-year-old male subject who suffered KD at age 3?years. The altered hemodynamics were compared to that of a reconstructed normal coronary anatomy using our subject as the model. Computer simulations using a robust finite element framework were used to quantify time-varying shear stresses and particle trajectories in the coronary arteries. We accounted for the cardiac contractility and the microcirculation using physiologic downstream boundary conditions. The presence of aneurysms in the proximal coronary artery leads to flow recirculation, reduced wall shear stress within the aneurysm, and high wall shear stress gradients at the neck of the aneurysm. The wall shear stress in the KD subject (2.95-3.81 dynes/sq cm) was an order of magnitude lower than the normal control model (17.10-27.15 dynes/sq cm). Particle residence times were significantly higher, taking 5 cardiac cycles to fully clear from the aneurysmal regions in the KD subject compared to only 1.3 cardiac cycles from the corresponding regions of the normal model. In this novel quantitative study of hemodynamics in coronary aneurysms caused by KD, we documented markedly abnormal flow patterns that are associated with increased risk of thrombosis. This methodology has the potential to provide further insights into the effects of aneurysms in KD and to help risk stratify patients for appropriate medical and surgical interventions.     

Tyrosine hydroxylase in secretory granules from bovine adrenal medulla. Evidence for an integral membrane form

Intact secretory granules isolated from bovine adrenal medulla express tyrosine hydroxylase (TH) activity. Granule-associated TH sediments on continuous sucrose gradients with dopamine beta-hydroxylase, a marker for granule membranes, indicating that TH is associated with chromaffin granules. Membranes prepared from lysed granules retain TH, whereas granule contents are free of the enzyme. TH immunoreactivity was detected in granule membranes by immunoblot analysis using a polyclonal antiserum against TH. TH immunoreactivity cannot be removed from membranes by washes in high ionic strength buffers and is only partially removed from membranes by treatment with either urea or Na2CO3. TH can be removed from granule membranes by the detergents Nonidet P-40, Triton X-100, and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Treatment of membranes with a phosphatidylinositol-specific phospholipase C did not remove TH, ruling out the possibility of a glycosyl phosphatidyl anchor. Fractionation of granule membranes by temperature-induced phase separation in Triton X-114 revealed that TH is recovered in phases in which integral (detergent phase) and hydrophobic (phospholipid phase) membrane proteins are typically found. By contrast, TH from adrenal cytosol fractionated exclusively into the aqueous phase along with other soluble proteins. Digestion of granules with various protease enzymes revealed that TH is resistant to degradation, suggesting that the enzyme is embedded within membranes. TH becomes phosphorylated when intact granules are exposed to the catalytic subunit of the cAMP-dependent protein kinase, indicating that at least the N-terminal region of TH is exposed on the cytoplasmic surface of granules. These results establish that a fraction of TH is an integral component of bovine granule membranes. The association of TH with granule membranes may play a role in coordinating TH activity and catecholamine release.     

Studies onGesonula punctifrons Stål (Orthoptera: Acrididae: Cyrtacanthacridinae) attacking water-hyacinth in India

Résumé L'AcridienGesonula punctifrons St?l attaque la Jacinthe d'eau (Eichhornia crassipes) dans plusieurs parties de l'Inde et est adapté à un habitat aquatique ou semi-aquatique. Les œufs, pondus dans les pétioles des feuilles, éclosent en 3 à 4 semaines. Les larves se développent en 4 à 5 semaines et demi, avec 5 mues. Les adultes vivent au maximum 4 à 5 mois en s'alimentant seulement de Jacinthe d'eau. En essais de laboratoire cet Acridien se nourrit activement deCanna orientalis dont les tiges sont percées de trous pour la ponte par les femelles, mais aucun œuf n'est déposé. Quarante trois autres plantes d'intérêt économique ont été expérimentées: plusieurs d'entre elles ont été l'objet d'attaques légères à modérées. L'analyse de la littérature concernant les plantes-h?tes du genreGesonula et la distribution deG. punctifrons montrent que cette espèce s'est adaptée secondairement àE. crassipes. Bien que cet Acridien s'observe en grand nombre dans quelques régions, l'ensemble de ses dégats à la mauvaise herbe n'est pas suffisant pour assurer le contr?le de celle-ci.

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This research has been financed in part by a grant made by the United States Department of Agriculture under P. L. 480.     

A unique and essential role for insulin in the phenotypic expression of rat mammary epithelial cells unrelated to its function in cell maintenance

Mammary explants from pregnant rats can be induced in regard to casein synthesis and alpha-lactalbumin activity when cultured in the presence of hydrocortisone, prolactin and levels of insulin approaching physiological concentrations. No detectable induction occurs in the absence of insulin. Although epidermal growth factor and multiplication stimulating activity, in the presence of hydrocortisone, can maintain the initial level of NADH-cytochrome c reductase as well as insulin, neither can substitute effectively for insulin in the induction of the milk proteins. Proinsulin, nerve growth factor, platelet-derived growth factor and fibroblast growth factor are also ineffective substitutes for insulin in this regard. Whereas prolonged tissue exposure to multiplication stimulating activity, hydrocortisone and prolactin does not result in induction of alpha-lactalbumin activity, subsequent addition of insulin leads to prompt response. The results suggest that the ability of insulin to function as a unique, essential factor in the induction of rat milk proteins is independent of its cell-maintenance activity. Thus, in addition to its well established functions in metabolic processes, insulin appears to play a vital role in certain developmental processes.     

Recent trends in remote homology detection: an Indian Medley

The development of remote homology detection methods is a challenging area in Bioinformatics. Sequence analysis-based approaches that address this problem have employed the use of profiles, templates and Hidden Markov Models (HMMs). These methods often face limitations due to poor sequence similarities and non-uniform sequence dispersion in protein sequence space. Search procedures are often asymmetrical due to over or under-representation of some protein families and outliers often remain undetected. Intermediate sequences that share high similarities with more than one protein can help overcome such problems. Methods such as MulPSSM and Cascade PSI-BLAST that employ intermediate sequences achieve better coverage of members in searches. Others employ peptide modules or conserved patterns of motifs or residues and are effective in overcoming dependencies on high sequence similarity to establish homology by using conserved patterns in searches. We review some of these recent methods developed in India in the recent past.     

Regulation of carbohydrate metabolism by indole-3-carbinol and its metabolite 3,3′-diindolylmethane in high-fat diet-induced C57BL/6J mice

Indole glucosinolates, present in cruciferous vegetables have been investigated for their putative pharmacological properties. The current study was designed to analyse whether the treatment of the indole glucosinolates—indole-3-carbinol (I3C) and its metabolite 3,3′-diindolylmethane (DIM) could alter the carbohydrate metabolism in high-fat diet (HFD)-induced C57BL/6J mice. The plasma glucose, insulin, haemoglobin (Hb), glycosylated haemoglobin (HbA1c), glycogen and the activities of glycolytic enzyme (hexokinase), hepatic shunt enzyme (glucose-6-phosphate dehydrogenase), gluconeogenic enzymes (glucose-6-phosphatase and fructose-1,6-bisphosphatase) were analysed in liver and kidney of the treated and HFD mice. Histopathological examination of liver and pancreases were also carried out. The HFD mice show increased glucose, insulin and HbA1c and decreased Hb and glycogen levels. The elevated activity of glucose-6-phosphatase and fructose-1,6-bisphosphatase and subsequent decline in the activity of glucokinase and glucose-6-phosphate dehydrogenase were seen in HFD mice. Among treatment groups, the mice administered with I3C and DIM, DIM shows decreased glucose, insulin and HbA1c and increased Hb and glycogen content in liver when compared to I3C, which was comparable with the standard drug metformin. The similar result was also obtained in case of carbohydrate metabolism enzymes; treatment with DIM positively regulates carbohydrate metabolic enzymes by inducing the activity of glucokinase and glucose-6-phosphate dehydrogenase and suppressing the activity of glucose-6-phosphatase and fructose-1,6-bisphosphatase when compared to I3C, which were also supported by our histopathological observations.     

Structure and rearrangements in the carboxy-terminal region of SpIH channels

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) ion channels regulate the spontaneous firing activity and electrical excitability of many cardiac and neuronal cells. The modulation of HCN channel opening by the direct binding of cAMP underlies many physiological processes such as the autonomic regulation of the heart rate. Here we use a combination of X-ray crystallography and electrophysiology to study the allosteric mechanism for cAMP modulation of HCN channels. SpIH is an invertebrate HCN channel that is activated fully by cAMP, but only partially by cGMP. We exploited the partial agonist action of cGMP on SpIH to reveal the molecular mechanism for cGMP specificity of many cyclic nucleotide-regulated enzymes. Our results also elaborate a mechanism for the allosteric conformational change in the cyclic nucleotide-binding domain and a mechanism for partial agonist action. These mechanisms will likely extend to other cyclic nucleotide-regulated channels and enzymes as well.     

Somatic embryogenesis and Agrobacterium-mediated transformation system for scented geraniums (Pelargonium sp. `Frensham')

Plant regeneration via somatic embryogenesis was achieved from leaf petioles of Pelargonium sp. `Frensham' cultured on Murashige and Skoog medium containing 15 μM N⁶-benzyladenine, and 5 μM α-naphthaleneacetic acid (NAA). More than 80% of these somatic embryos converted into plants when isolated and cultured on Murashige and Skoog medium supplemented with 15 μM NAA. Stable transgenic plants were obtained by co-cultivation of the petioles (prior to culture) with Agrobacterium tumefaciens strains LBA4404 (harbouring a binary vector pBI121 carrying the nptII and gus genes) and LBG66 (harbouring a binary plasmid pJQ418 carrying the gus/int:nptII fusion gene). Transformants were selected using kanamycin and transformation was verified by β-glucuronidase histochemical assay and polymerase chain reaction. Southern analysis further confirmed the integration of these genes into the genome of transgenic plants. We report here for the first time, an Agrobacterium-mediated model transformation system coupled with regeneration via somatic embryogenesis for production of transgenics in Pelargonium sp. Received: 20 September 1996 / Accepted: 13 November 1996