Characterization of an Evolutionarily Conserved Metallophosphoesterase That Is Expressed in the Fetal Brain and Associated with the WAGR Syndrome

Among the human diseases that result from chromosomal aberrations, a de novo deletion in chromosome 11p13 is clinically associated with a syndrome characterized by Wilms' tumor, aniridia, genitourinary anomalies, and mental retardation (WAGR). Not all genes in the deleted region have been characterized biochemically or functionally. We have recently identified the first Class III cyclic nucleotide phosphodiesterase, Rv0805, from Mycobacterium tuberculosis, which biochemically and structurally belongs to the superfamily of metallophosphoesterases. We performed a large scale bioinformatic analysis to identify orthologs of the Rv0805 protein and identified many eukaryotic genes that included the human 239FB gene present in the region deleted in the WAGR syndrome. We report here the first detailed biochemical characterization of the rat 239FB protein and show that it possesses metallophosphodiesterase activity. Extensive mutational analysis identified residues that are involved in metal interaction at the binuclear metal center. Generation of a rat 239FB protein with a mutation corresponding to a single nucleotide polymorphism seen in human 239FB led to complete inactivation of the protein. A close ortholog of 239FB is found in adult tissues, and biochemical characterization of the 239AB protein demonstrated significant hydrolytic activity against 2',3'-cAMP, thus representing the first evidence for a Class III cyclic nucleotide phosphodiesterase in mammals. Highly conserved orthologs of the 239FB protein are found in Caenorhabditis elegans and Drosophila and, coupled with available evidence suggesting that 239FB is a tumor suppressor, indicate the important role this protein must play in diverse cellular events.

Twin roller quenching technique and preparation of glasses of high melting ionic solids

A rapid quenching technique with a quenching rate of roughly 106°C/sec has been developed to prepare glassy samples of ABO3 type materials. Glasses of potassium lithium niobate have been prepared by this technique. These glasses have been characterized by x-ray diffraction, electron diffraction and differential scanning calorimetry techniques to assess the quality of the obtained glasses.

Triplet repeat polymorphism & fragile X syndrome in the Indian context

Mental retardation due to fragile X syndrome is one of the genetic disorders caused by tripler repeat expansion, CGG repeat involved in this disease is known to exhibit polymorphism even among normal individuals. Here we describe the development of suitable probes for detection of polymorphism in CGG repeat at FMR1 locus as well as the diagnosis of fragile X syndrome. Using these methods polymorphism at the FMR1 locus has been examined in 161 individuals. Ninety eight patients with unclassified mental retardation were examined, of whom 7 were found to have the expanded (CGG) allele at the FMR1 locus, The hybridization pattern for two patients has been presented as representative data.

Mycobacterial Stress Regulation: The Dps ``Twin Sister'' Defense Mechanism and Structure-Function Relationship

In this work, we have tried to emphasize the connection between mycobacterial growth and regulation of gene expression. Utilization of multiple carbon sources and diauxic growth helps bacteria to regulate gene expression at an optimum level so that the inhospitable conditions encountered during nutrient depletion can be circumvented. These aspects will be discussed with respect to mycobacterial growth in subsequent sections. Identification and characterization of genes induced under such conditions is helpful to understand the physiology of the bacterium. Although it is necessary to compare the total expression profile of proteins as they transit from vegetative growth to stationary phase, at times a lot of insights can be deciphered from the expression pattern of one or two proteins. We have compared the protein expression and sigma factor selectivity of two such proteins in M. smegmatis to understand the differential regulation of genes playing diverse function in the same species. Some newer insights on the structure and function of one of the Dps proteins are also explained.

Angelman Syndrome Protein UBE3A Interacts with Primary Microcephaly Protein ASPM, Localizes to Centrosomes and Regulates Chromosome Segregation

Many proteins associated with the phenotype microcephaly have been localized to the centrosome or linked to it functionally. All the seven autosomal recessive primary microcephaly (MCPH) proteins localize at the centrosome. Microcephalic osteodysplastic primordial dwarfism type II protein PCNT and Seckel syndrome (also characterized by severe microcephaly) protein ATR are also centrosomal proteins. All of the above findings show the importance of centrosomal proteins as the key players in neurogenesis and brain development. However, the exact mechanism as to how the loss-of-function of these proteins leads to microcephaly remains to be elucidated. To gain insight into the function of the most commonly mutated MCPH gene ASPM, we used the yeast two-hybrid technique to screen a human fetal brain cDNA library with an ASPM bait. The analysis identified Angelman syndrome gene product UBE3A as an ASPM interactor. Like ASPM, UBE3A also localizes to the centrosome. The identification of UBE3A as an ASPM interactor is not surprising as more than 80% of Angelman syndrome patients have microcephaly. However, unlike in MCPH, microcephaly is postnatal in Angelman syndrome patients. Our results show that UBE3A is a cell cycle regulated protein and its level peaks in mitosis. The shRNA knockdown of UBE3A in HEK293 cells led to many mitotic abnormalities including chromosome missegregation, abnormal cytokinesis and apoptosis. Thus our study links Angelman syndrome protein UBE3A to ASPM, centrosome and mitosis for the first time. We suggest that a defective chromosome segregation mechanism is responsible for the development of microcephaly in Angelman syndrome.

Odd-even oscillations of SHG efficiencies in twin NLO chromophores

Twin NLO chromophores having two azobenzene units linked by a flexible polymethylene spacer of varying lengths are shown to exhibit odd-even oscillations in their second harmonic generation (SHG) efficiencies, when measured in the powder form. These twin systems were designed to also exhibit liquid cystallinity, and indeed most of them do exhibit a nematic mesophase. The anticipated odd-even oscillations, in both their isotropization transition temperatures (Ti) and isotropization entropies (Delta Si), were also observed. The odd-even oscillation of the SHG efficiencies has been ascribed to a more effective cancellation of mesogenic dipoles in the even twins as compared to their odd counterparts, due to a preferred centrosymmetric packing in the former case. Based on the behaviour of these twin chromophoric molecules, it may be anticipated that such odd-even oscillations will also be observed in the analogous main chain NLO polymers.

Relaxation behavior of twin nonlinear optical chromophores: Effect of the spacer length

A new series of twin nonlinear optical (NLO) molecules, having two 4-nitrophenol chromophores that are linked via a flexible polymethylene spacer of varying length [(CH2)(n), n = 1-12], were synthesized. Powder second harmonic generation measurements of these twin samples indicated a pronounced odd-even oscillation, with the odd twins exhibiting a high SHG value while the even ones gave no measurable SH signal. This behavior reflects the crystal packing preferences in such twin NLO systems that have odd and even numbers of atoms linking them - the even ones appear to prefer a centrosymmetric packing arrangement. The orientational/disordering dynamics of these twin NLO molecules, doped in a polymer (poly(methyl methacrylate)) matrix, has also been studied using SHG in electric field poled samples. Interestingly, the maximum attainable SH signal, chi((2)), in, the poled samples also showed an odd-even oscillation; the odd ones again having a higher value of chi((2)) This unprecedented odd-even oscillation in such molecularly doped systems is rationalized as being due to the intrinsically greater ease of a parallel alignment of the two chromophores in the twins with an odd spacer than in those with an even one. Further, the temporal stability of the SHG intensity at 70 degrees C, after the removal of the applied corona, was also studied. The relaxation of all the twin chromophores followed a biexponential decay; the characteristic relaxation time (tau(2)) for the slow decay component suggests that while the twin with a single methylene unit relaxes relatively slowly, the relaxation is significantly faster in cases where n = 2 and 3. In the twins with even longer spacer segments, the relaxation again becomes slower and reaches a saturation value. The observed minimum appears to reflect the interplay of two competing factors that affect the chromophore alignment in such twin systems, namely, the electrostatic repulsion between neighboring oriented dipoles and the intrinsic flexibility of the spacer.

Surface effects on stacking fault and twin formation in fcc nanofilms: A first-principles study

The free surface effects on stacking fault and twin formation in fcc metals (Al, Cu, and Ni) were examined by first-principles calculations based on density functional theory (DFT). It is found that the generalized planar fault (GPF) energies of Ni are much larger than bulk Ni with respect to Al and Cu. The discrepancy is attributed to the localized relaxation of Ni nanofilm to accommodate the large expansion of the inter-planar separation induced at the fault plane. The localized relaxation can be coupled to the electronic structure of Ni nanofilms. (C) 2011 Elsevier B.V. All rights reserved.

Unique Utilization of a Phosphoprotein Phosphatase Fold by a Mammalian Phosphodiesterase Associated with WAGR Syndrome

Metallophosphoesterase-domain-containing protein 2 (MPPED2) is a highly evolutionarily conserved protein with orthologs found from worms to humans. The human MPPED2 gene is found in a region of chromosome 11 that is deleted in patients with WAGR (Wilms tumor, aniridia, genitourinary anomalies, and mental retardation) syndrome, and MPPED2 may function as a tumor suppressor. However, the precise cellular roles of MPPED2 are unknown, and its low phosphodiesterase activity suggests that substrate hydrolysis may not be its prime function. We present here the structures of MPPED2 and two mutants, which show that the poor activity of MPPED2 is not only a consequence of the substitution of an active-site histidine residue by glycine but also due to binding of AMP or GMP to the active site. This feature, enhanced by structural elements of the protein, allows MPPED2 to utilize the conserved phosphoprotein-phosphatase-like fold in a unique manner, ensuring that its enzymatic activity can be combined with a possible role as a scaffolding or adaptor protein. (C) 2011 Elsevier Ltd. All rights reserved.

Tar characterisation in new generation agro-residue gasifiers - Cyclone and downdraft open top twin air entry systems

This paper deals with the characterisation of tar from two configurations of bioresidue thermochemical conversion reactors designed for producer gas based power generation systems. The pulverised fuel reactor is a cyclone system (R1) and the solid bioresidue reactor (denoted R2) is an open top twin air entry system both at 75-90 kg/h capacity (to generate electricity similar to 100 kVA). The reactor, R2, has undergone rigorous test in a major Indo-Swiss programme for the tar quantity at various conditions. The former is a recent technology development. Tars collected from these systems by a standard tar collection apparatus at the laboratory at Indian Institute of Science have been analysed at the Royal Institute of Technology (KTH), Sweden. The results of these analyses show that these thermochemical conversion reactors behave differently from the earlier reactors reported in literature in so far as tar generation is concerned. The extent of tar in hot gas is about 700-800 ppm for R1 and 70-100 ppm for R2. The amounts of the major compounds - naphthalene and phenol-are much lower that what is generally understood to happen in the gasifiers in Europe. It is suggested that the longer residence times at high temperatures allowed for in these reactors is responsible for this behavior. It is concluded the new generation reactor concepts extensively tried out at lower power levels hold promise for high power atmospheric gasification systems for woody as well as pulverisable bioresidues.

Familial Diarrhea Syndrome Caused by an Activating GUCY2C Mutation

BACKGROUND Familial diarrhea disorders are, in most cases, severe and caused by recessive mutations. We describe the cause of a novel dominant disease in 32 members of a Norwegian family. The affected members have chronic diarrhea that is of early onset, is relatively mild, and is associated with increased susceptibility to inflammatory bowel disease, small-bowel obstruction, and esophagitis. METHODS We used linkage analysis, based on arrays with single-nucleotide polymorphisms, to identify a candidate region on chromosome 12 and then sequenced GUCY2C, encoding guanylate cyclase C (GC-C), an intestinal receptor for bacterial heat-stable enterotoxins. We performed exome sequencing of the entire candidate region from three affected family members, to exclude the possibility that mutations in genes other than GUCY2C could cause or contribute to susceptibility to the disease. We carried out functional studies of mutant GC-C using HEK293T cells. RESULTS We identified a heterozygous missense mutation (c.2519G -> T) in GUCY2C in all affected family members and observed no other rare variants in the exons of genes in the candidate region. Exposure of the mutant receptor to its ligands resulted in markedly increased production of cyclic guanosine monophosphate (cGMP). This may cause hyperactivation of the cystic fibrosis transmembrane regulator (CFTR), leading to increased chloride and water secretion from the enterocytes, and may thus explain the chronic diarrhea in the affected family members. CONCLUSIONS Increased GC-C signaling disturbs normal bowel function and appears to have a proinflammatory effect, either through increased chloride secretion or additional effects of elevated cellular cGMP. Further investigation of the relevance of genetic variants affecting the GC-C-CFTR pathway to conditions such as Crohn's disease is warranted. (Funded by Helse Vest Western Norway Regional Health Authority] and the Department of Science and Technology, Government of India.)

Genetic analysis of an Indian family with members affected with Waardenburg syndrome and Duchenne muscular dystrophy

Purpose: Waardenburg syndrome (WS) is characterized by sensorineural hearing loss and pigmentation defects of the eye, skin, and hair. It is caused by mutations in one of the following genes: PAX3 (paired box 3), MITF (microphthalmia-associated transcription factor), EDNRB (endothelin receptor type B), EDN3 (endothelin 3), SNAI2 (snail homolog 2, Drosophila) and SOX10 (SRY-box containing gene 10). Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder caused by mutations in the DMD gene. The purpose of this study was to identify the genetic causes of WS and DMD in an Indian family with two patients: one affected with WS and DMD, and another one affected with only WS. Methods: Blood samples were collected from individuals for genomic DNA isolation. To determine the linkage of this family to the eight known WS loci, microsatellite markers were selected from the candidate regions and used to genotype the family. Exon-specific intronic primers for EDN3 were used to amplify and sequence DNA samples from affected individuals to detect mutations. A mutation in DMD was identified by multiplex PCR and multiplex ligation-dependent probe amplification method using exon-specific probes. Results: Pedigree analysis suggested segregation of WS as an autosomal recessive trait in the family. Haplotype analysis suggested linkage of the family to the WS4B (EDN3) locus. DNA sequencing identified a novel missense mutation p.T98M in EDN3. A deletion mutation was identified in DMD. Conclusions: This study reports a novel missense mutation in EDN3 and a deletion mutation in DMD in the same Indian family. The present study will be helpful in genetic diagnosis of this family and increases the mutation spectrum of EDN3.

Lacrimal Proline Rich 4 (LPRR4) Protein in the Tear Fluid Is a Potential Biomarker of Dry Eye Syndrome

Dry eye syndrome (DES) is a complex, multifactorial, immune-associated disorder of the tear and ocular surface. DES with a high prevalence world over needs identification of potential biomarkers so as to understand not only the disease mechanism but also to identify drug targets. In this study we looked for differentially expressed proteins in tear samples of DES to arrive at characteristic biomarkers. As part of a prospective case-control study, tear specimen were collected using Schirmer strips from 129 dry eye cases and 73 age matched controls. 2D electrophoresis (2DE) and Differential gel electrophoresis (DIGE) was done to identify differentially expressed proteins. One of the differentially expressed protein in DES is lacrimal proline rich 4 protein (LPRR4). LPRR4 protein expression was quantified by enzyme immune sorbent assay (ELISA). LPRR4 was down regulated significantly in all types of dry eye cases, correlating with the disease severity as measured by clinical investigations. Further characterization of the protein is required to assess its therapeutic potential in DES.

Clinical phenotype and the lack of mutations in the CHRNG, CHRND, and CHRNA1 genes in two Indian families with Escobar syndrome

The objective of this study was to report the clinical phenotype and genetic analysis of two Indian families with Escobar syndrome (ES). The diagnosis of ES in both families was made on the basis of published clinical features. Blood samples were collected from members of both families and used in genomic DNA isolation. The entire coding regions and intron-exon junctions of the ES gene CHRNG (cholinergic receptor, nicotinic, gamma), and two other related genes, CHRND and CHRNA1, were amplified and sequenced to search for mutations in both families. Both families show a typical form of ES. Sequencing of the entire coding regions including the intron-exon junctions of the three genes did not yield any mutations in these families. In conclusion, it is possible that the mutations in these genes are located in the promoter or deep intronic regions that we failed to identify or the ES in these families is caused by mutations in a different gene. The lack of mutations in CHRNG has also been reported in several families, suggesting the possibility of at least one more gene for this syndrome. Clin Dysmorphol 22:54-58 (C) 2013 Wolters Kluwer Health vertical bar Lippincott Williams & Wilkins.

Effect of resonator length and working fluid on the performance of twin thermoacoustic heat engine - experimental and simulation studies

Success in the advancement of thermoacoustic field led the researchers to develop the thermoacoustic engines which found its applications in various fields such as refrigeration, gas mixture separation, natural gas liquefaction, and cryogenics. The objective of this study is to design and fabricate the twin thermoacoustic heat engine (TAHE) producing the acoustic waves with high resonance frequencies which is used to drive a thermoacoustic refrigerator efficiently by the influence of geometrical parameters and working fluids. Twin TAHE has gained significant attention due to the production of high intensity acoustic waves than single TAHE. In order to drive an efficient thermoacoustic refrigerator, a twin thermoacoustic heat engine is built up and its performance are analysed by varying the resonator length and working fluid. The performance is measured in terms of onset temperature difference, resonance frequency and pressure amplitude of the oscillations generated from twin TAHE. The simulation is performed using free software DeltaEC, from LANL, USA. The simulated DeltaEC results are compared with experimental results and the deviations are found within +10%.