Protein Profiling of RGS6, a Pleiotropic Gene Implicated in Numerous Neuropsychiatric Disorders, Reveals Multi-Isoformic Expression and a Novel Brain-Specific Isoform.

A metanalysis identified regulator of G-protein signaling 6 (RGS6) as one of 23 loci with pleiotropic effects on four or more human psychiatric disorders. This finding is significant as it confirms/extends the findings of numerous other studies implicating RGS6 in CNS function and pathology. RGS6 is a highly conserved member of the RGS protein family whose cellular roles are likely affected by mRNA splicing and alternative domain inclusion/exclusion. Indeed, we previously identified multiple RGS6 splice variants predicted to produce 36 distinct protein isoforms containing either long (RGS6L) or short (RGS6S) N-terminal domains, an incomplete or intact GGL domain, and nine alternative C termini. Unfortunately, sequence similarities between the isoforms have made it difficult to confirm their individual existence and/or to determine their unique functions. Here, we developed three RGS6-specific antibodies that recognize all RGS6 protein isoforms (RGS6-fl), the N-terminus of RGS6L isoforms (RGS6-L), and an 18-amino acid alternate C-terminal sequence (RGS6-18). Using these antibodies, we demonstrate that RGS6L(+GGL) isoforms, predominating in both mouse (both sexes) CNS and peripheral tissues, are most highly expressed in the CNS. We further identify three novel RGS6 protein bands that are larger (61, 65, and 69-kDa) than the ubiquitously expressed 53- to 57-kDa RGS6L(+GGL) proteins. Importantly, we show that the 69-kDa protein is a brain-specific dephospho form of the 65-kDa band, the first identified phosphorylated RGS6 isoform. Together, these data begin to define the functional significance behind the complexity of RGS6 gene processing and further clarifies RGS6's physiological roles by resolving tissue-specific RGS6 protein expression.

Dragon fruit (Hylocereus undatus) peel as antioxidant dietary fibre on quality and lipid oxidation of chicken nuggets.

In this study, the efficacy of dragon fruit peel (DFP) powder as antioxidant dietary fibre (ADF), at two different concentrations (T1-1.5% and T2-3.0%), on quality improvement and susceptibility to lipid oxidation of chicken nuggets during 20 days of refrigerated storage was assessed. DFP, rich in dietary fibre (56.91%) with higher insoluble dietary fibre, phenolics (36-39 mgGAE/100 g) content and possessing good radical scavenging activity as well as reducing power, contained 10.36% protein, 4.48% fat and 2.34% ash. HPLC analysis revealed presence of high concentrations of gallic and ferulic acid, among the phenolics. Incorporation of DFP in nuggets although decreased the pH but improved emulsion stability as well as cooking yield and had higher protein, ash and lower fat content. Further, the treated nuggets had significantly (p < 0.05) higher dietary fibre and total phenolics content than control. Incorporation of DFP decreased the hardness, gumminess and chewiness and improved (p < 0.05) the products' redness values. Sensory evaluation of the products revealed significant improvement in the appearance score and non-significant (p > 0.05) increase in the scores of other attributes compared to control samples. DFP significantly decreased lipid peroxidation, odour scores and microbial load in chicken nuggets during 20 days of storage period. From the study, it could be deduced that DFP rich in bioactive components had positive influence on the nutritional quality of chicken nuggets and could also be used as ADF in muscle food without affecting the quality and acceptability of products.

Molecular epidemiology of goat pox viruses.

Goat pox disease outbreaks were observed in different places affecting Black Bengal Goats in West Bengal (WB) and Tellicherry, Vembur and non-descriptive breeds in Tamil Nadu (TN) causing severe lesions and mortality up to 30%. Clinical specimens from all the outbreaks were screened by polymerase chain reaction followed by restriction fragment length polymorphism (PCR-RFLP) and confirmed the diseases as Goat Pox. Virus isolation in Vero cell line was done with randomly selected ten samples, cytopathic effects (CPE) characterized by syncytia and intracytoplasmic inclusion bodies were observed after several blind passages. Nucleotide sequence of complete p32 gene using randomly selected two isolates and three clinical specimens revealed presence of Goat pox virus (GTPV)-specific signature residues in all the sequences. Phylogenetic analysis using the present five sequences along with GenBank data of GTPV complete p32 gene sequences showed all the GTPV sequences cluster together except Pellor strain (NC004003) and FZ Chinese strain (KC951854). The five sequences either from WB or TN cluster more closely with GTPV isolates of Maharashtra state that were responsible for cross species outbreak of pox disease in both sheep (KF468759) and goats (KF468762) in India during the year 2010. All the Indian goat pox viruses, including the Mukteswar strain, isolated in 1946 and sequence reported in 2004 clustered together with the GTPVs causing the recent outbreaks. It was observed that GTPVs caused similar clinical manifestation irrespective of their geographical locations and breed characteristics, no variation observed among the Indian isolates based on p32 gene over the period of seventy years and disease outbreaks could not be observed or reported in vaccinated goats.

RGS6 suppresses Ras-induced cellular transformation by facilitating Tip60-mediated Dnmt1 degradation and promoting apoptosis.

The RAS protooncogene has a central role in regulation of cell proliferation, and point mutations leading to oncogenic activation of Ras occur in a large number of human cancers. Silencing of tumor-suppressor genes by DNA methyltransferase 1 (Dnmt1) is essential for oncogenic cellular transformation by Ras, and Dnmt1 is overexpressed in numerous human cancers. Here we provide new evidence that the pleiotropic regulator of G protein signaling (RGS) family member RGS6 suppresses Ras-induced cellular transformation by facilitating Tip60-mediated degradation of Dmnt1 and promoting apoptosis. Employing mouse embryonic fibroblasts from wild-type and RGS6(-/-) mice, we found that oncogenic Ras induced upregulation of RGS6, which in turn blocked Ras-induced cellular transformation. RGS6 functions to suppress cellular transformation in response to oncogenic Ras by downregulating Dnmt1 protein expression leading to inhibition of Dnmt1-mediated anti-apoptotic activity. Further experiments showed that RGS6 functions as a scaffolding protein for both Dnmt1 and Tip60 and is required for Tip60-mediated acetylation of Dnmt1 and subsequent Dnmt1 ubiquitylation and degradation. The RGS domain of RGS6, known only for its GTPase-activating protein activity toward Gα subunits, was sufficient to mediate Tip60 association with RGS6. This work demonstrates a novel signaling action for RGS6 in negative regulation of oncogene-induced transformation and provides new insights into our understanding of the mechanisms underlying Ras-induced oncogenic transformation and regulation of Dnmt1 expression. Importantly, these findings identify RGS6 as an essential cellular defender against oncogenic stress and a potential therapeutic target for developing new cancer treatments.

Effect of soybean meal substitution by different concentrations of sunflower meal on egg quality traits of white and coloured dwarf dam lines.

1. The effect of soybean meal substitution by different concentrations of sunflower meal on egg quality traits of white and coloured dwarf dam lines was investigated. 2. A total of 144 dwarf hens (38 weeks of age) from the same hatch were randomly divided into 12 groups of 12 birds (4 dietary treatments x 3 replicates). 3. A 2 x 4 factorial design was used to study the effect of 2 lines (Factor A) and substitution of soybean meal (SBM) with 4 concentrations (0, 10, 15 and 20%) of sunflower meal (SFM) [Factor B] on egg quality traits of dwarf dam line hens. 4. All the diets were designed to be isocaloric (113 MJ ME/kg) and isonitrogenous (180 g/kg crude protein) The duration of the experiment was 12 weeks. 5. Analysis of variance indicated a highly significant line effect. There were non-significant effects of substitution of soybean meal with different concentrations of sunflower meal on egg quality traits except for Haugh unit. 6. White plumaged dwarf broiler breeder dam line produced significantly fewer, but larger, eggs than coloured dwarf dam line hens.

Chromium III exposure inhibits brain Na+K+ATPase activity of Clarias batrachus L. involving lipid peroxidation and deficient mitochondrial electron transport chain activity.

The present study elucidated the role of lipid peroxidation and diminished mitochondrial electron transport chain activity in partial dysfunction of brain Na+K+ATPase of Clarias batrachus exposed to chromium III ions. The fish were exposed to 10% and 20% of the derived 96 h LC50 value, 5.69 mg/L and 11.38 mg/L, respectively, and sampled on 20, 40 and 60 days. Exposure to chromium III on fish brain demonstrated an increased lipid peroxidation, production of protein carbonyl and reactive oxygen species and loss of protein thiol groups in synaptosomal fraction with decreased activity of Na+K+ATPase, partial inactivation of mitochondrial electron transport chain activity and energy depletion.

Regulation of the nitric oxide synthesis pathway and cytokine balance contributes to the healing action of Myristica malabarica against indomethacin-induced gastric ulceration in mice.

The role of the ariginine-metabolism in the healing action of the methanol extract of Myristica malabarica (rampatri) (RM) and omeprazole (Omez) against indomethacin-induced stomach ulceration in mouse was investigated. Indomethacin (18 mg/kg) was found to induce maximum stomach ulceration in Swiss albino mice on the 3rd day of its administration, which was associated with reduced arginase activity (38.5%, p < 0.05), eNOS expression, along with increased iNOS expression, total NOS activity (5.37 fold, p < 0.001), NO generation (55.1%, p < 0.01), and ratio of pro-/anti-inflammatory cytokines. Besides providing comparable healing as Omez (3 mg/kg × 3 d), RM (40 mg/kg × 3 d, p.o.) shifted the iNOS/NO axis to the arginase/polyamine axis as revealed from the increased arginase activity (59.5%, p < 0.01), eNOS expression, and reduced iNOS expression, total NOS activity (73%, p < 0.001), and NO level (49.8%, p < 0.01). These could be attributed to a favourable anti/pro inflammatory cytokines ratio, generated by RM. The healing by Omez was however, not significantly associated with those parameters.

Molecular pathogenesis of Wilson disease: haplotype analysis, detection of prevalent mutations and genotype-phenotype correlation in Indian patients.

Wilson disease (WD) is an autosomal recessive disorder caused by defects in the copper-transporting P-type ATPase gene (ATP7B) resulting in the accumulation of copper in the liver and the brain. We identified prevalent mutations in the ATP7B of Indian WD patients and attempted to correlate those with the disease phenotype. Patients from 62 unrelated families and their first-degree relatives comprising 200 individuals were enrolled in this study. Three dinucleotide repeat markers flanking WD locus and a few intragenic SNPs were used to determine the genotypes and construct haplotypes of the patients. Seven recurring haplotypes accounting for 58% of the total mutant chromosomes were identified, and four underlying defects in the ATP7B representing 37% of WD chromosomes were detected. In addition, five other rare mutations were characterized. Thus a total of nine mutations including five novel changes were identified in the ATP7B of WD patients. Interestingly, homozygotes for different mutations that would be expected to produce similar defective proteins showed significant disparity in terms of organ involvement and severity of the disease. We also observed WD patients with neurological symptoms with little or no manifestation of hepatic pathogenesis. In one WD family, the proband and a sib had remarkably different phenotypes despite sharing the same pair of mutant chromosomes. These findings suggest a potential role for yet unidentified modifying loci for the observed phenotypic heterogeneity among the WD patients.

Genetic polymorphism revealed by 13 tetrameric and 2 pentameric STR loci in four Mongoloid tribal population.

The short tandem repeat allelic profiles at to 15 autosomal polymorphic loci were analyzed in four tribal populations of Mizoram (India). The analysis was performed on 354 unrelated healthy individuals belonging to Mongoloid races. All the samples were subjected to sex test (Amelogenin marker) besides the STR typing and in all instances; it has shown no deviation from expectation. The allele frequencies for all the analyzed loci in the studied populations are within expected range in comparison to the populations from same racial background. No significant deviation from the Hardy-Weinberg Equilibrium was observed for all the populations. In no cases the observed heterozygosity is less than that of expected values and it varied from 0.978 (Penta E) to as low as 0.425 (THO1). The discriminatory power and exclusion probability values for all the analyzed markers are significantly high and thus reveal high forensic significance. There is no evidence for association of alleles among the 15 studied loci. This allele frequency data will be useful for human identity testing in Mizo population.

Genetic polymorphism at nine microsatellite loci in four high altitude Himalayan desert human populations.

Allele frequencies for the nine STR loci of Profiler Plus STR markers were analyzed in 307 healthy unrelated individuals belonging to four predominant highly important endogamous population groups of Ladakh. The studied loci are found highly informative in human identification and understanding history of peopling of India. This is the first report on DNA Markers on any high altitude desert human population group.

Analysis of CAG and CCG repeats in Huntingtin gene among HD patients and normal populations of India.

We have analysed the distribution of CAG and adjacent polymorphic CCG repeats in the Huntingtin gene in 28 clinically diagnosed unrelated Huntington's disease (HD) patients and in normal individuals belonging to different ethnic groups of India. The range of expanded CAG repeats in HD patients varied from 41 to 56 repeats, whereas in normal individuals this number varied between 11 and 31 repeats. We identified six CCG alleles from a total of 380 normal chromosomes that were pooled across different ethnic populations of India. There were two predominant alleles: (CCG)7 (72.6%) and (CCG)10 (20%). We report here for the first time one four-repeat CCG allele which has not been found in any population so far. We found 30 haplotypes (two loci CAG-CCG) for 380 normal chromosomes. In the present study, no statistically significant preponderance of expanded HD alleles was found on either (CCG)7 or (CCG)10 backgrounds. Our studies suggest that the overall prevalence of HD in Indian populations may not be as high as in Western populations. Further studies are necessary to identify the origin of HD mutation in these populations.

Antibacterial activity of Litsea glutinosa bark.

The methanol extract of the bark of Litsea glutinosa showed antibacterial activity, comparable to chloramphenicol, against all 16 tested microorganisms.

Clinical profile of multiple sclerosis in Bengal.

Forty five patients of multiple sclerosis diagnosed on the basis of Poser's criteria from West Bengal were studied. The male-female ratio was 1:1.5, mean age of onset 31.83 years in male and 29.11 years in females. The maximum cases were between the 3rd and 4th decade. Definite MS comprised of 60%, while remaining 40% were probable. Visual impairment (53.33%), weakness of limbs (31.11%) and sensory paraesthesia (20%) were the common presenting symptoms whereas pyramidal tract involvement (93.33%) with absent abdominal reflexes (90%) and optic pallor (64.44%) were common signs. Posterior column and spinothalamic sensations were involved in 55% and 51% of cases respectively. Inter-nuclear ophthalmoplegia was present in 6.66% of cases. Pattern of involvement commonly showed three or more sites of lesion. Optico-spinal affection was present in 22.2% of cases. Relapsing and remitting course was found in 48. 91%, relapsing and progressive course in 33.33% and chronic progressive in 17.8%. MRI of brain showed positive results in 16 out of 23 cases. CSF study showed increased positivity in estimation of immunoglobulin level than oligoclonal band. Findings revalidate the disease pattern as being similar to that in other parts of India as well as Asia.

Profile of non-compressive myelopathy in eastern India: a 2-year study.

Eighty-two patients with non-compressive myelopathy have been studied from July 1994 to June 1996 in Bangur Institute of Neurology and S.S.K.M. Hospital, Calcutta, of which 48 patients were men and 34 patients were women. Presentation was acute in 40 patients (48.78%), subacute in 7 (8.53%), chronic in 27 (32.92%) and history of relapse and remission in 8 (9.75%) patients. Preceding as well as simultaneous fever was observed in 16 cases (19.5%); vaccination (anti-rabies) in 1 case (1.21%); drug abuse in 1 case (1.21%); arthralgia-myalgia and rash in 2 cases (2.42%) and history of electrocution in 2 cases (2.42%). Only pyramidal tract involvement was present in 24 cases (29.26%) and remaining 58 cases (70.73%) had pyramidal tract affection with other sites of involvement. CSF study carried out in 60 cases, revealed rise of protein in 31 (37.8%); oligoclonal band had been detected in 6 (7.31%), pleocytosis in 18 cases (21.95%) and increased IgG index greater than 6.66 in 2 cases (2.42%). CT myelogram done in 23 cases revealed no abnormality. MRI study carried out in 59 cases showed myelomalacia in 1 (1.21%); demyelination plaque in 14 cases (17.07%); atrophy of cord in 3 (3.65%); infarction of cord in 1 (1.21%) and in 40 cases (48.78%) no abnormality could be detected. Etiological diagnosis could be established in 59 (71.95%) cases such as transverse myelitis or myelopathy (post infectious) in 24 (29.26%); demyelination in 16 (19.51%); vascular and vasculitis in 3 (3.65%); toxic in 1 (1.21%); physical (electrocution) in 2 cases (2.42%). In the remaining 23 cases (28.04%) no aetiological factors could be found.

Profile of electrocardiographic changes in Duchenne muscular dystrophy.

Cardiac changes often culminating in cardiac failure are at times a dramatic cause of death in patients of Duchenne muscular dystrophy. These changes are probably invariable in such cases though they may escape detection in early stages by clinical examination or radiological investigation. Electrocardiography serves as a sensitive, non-invasive and inexpensive tool to detect these changes. Fifty-four cases of Duchenne muscular dystrophy were studied clinically and were confirmed by biochemical and electroneuromyographic studies. They were then subjected to electrocardiographic studies. A number of electrocardiographic changes were observed, like tachycardia in 77.77%, deep Q in leads I, aVL, V6 in 53.70%, prolonged VAT in 37.03%, deep Q in leads II, III, aVF in 29.62% and prolonged Q-Tc interval in 25.92% cases. Some of these were distinctive enough to warrant attention for cases of Duchenne muscular dystrophy.

Prognostic factors in intracerebral haemorrhage.

In a prospective study, the prognostic value of clinical characteristics and CT scan findings in 50 patients of intracerebral haemorrhage (ICH) has been examined. Follow up has been done over 6 months period. Each patient has been individually followed up for 8 weeks. At the end of the follow up period 34% of the patients died, 36% were dependent on outside help for daily living while 30% were capable of independent existence. Age of more than 60 yrs, Glasgow Coma Scale (GCS) Score of 6 or less (in a modified Scale of 10) at the time of admission, ICH volume greater than 30 ml., midline shift in CT scan of more than 3 mm and presence of intraventricular haemorrhage (IVH) and hydrocephalus had an adverse impact on outcome. Young age, GCS score of more than 8, ICH volume of less than 20 ml, presence of lobar haemorrhage and absence of IVH/hydrocephalus were associated with relatively favourable outcome.