A Bethlem myopathy Gly to Glu mutation in the von Willebrand factor A domain N2 of the collagen alpha3(VI) chain interferes with protein folding.

A single G1679E mutation in the amino-terminal globular domain N2 of the alpha3 chain of type VI collagen was found in a large family affected with Bethlem myopathy. Recombinant production of N2 ( approximately 200 residues) in transfected mammalian cells has now been used to examine the possibility that the mutation interfered with protein folding. The wild-type form and a G1679A mutant were produced at high levels and shown to fold into a stable globular structure. Only a small amount of secretion was observed for mutants G1679E and G1679Q, which apparently were efficiently degraded within the cells. Homology modeling onto the related von Willebrand factor A1 structure indicated that substitution of G1679 by the bulky E or Q cannot be accommodated without considerable changes in the folding pattern. This suggests protein misfolding as a molecular basis for this particular mutation in Bethlem myopathy, in agreement with radioimmunoassay data showing reduced levels of domain N2 in cultured fibroblasts from two patients.     

N-Terminal sequencing by mass spectrometry through specific fluorescamine labeling of α-amino groups before tryptic digestion

We present a single-step procedure for the specific mass labeling of unblocked protein N termini. We show that the dye fluorescamine, which is commonly assumed to require mildly alkaline conditions for undergoing a nonspecific reaction with α- and ε-amino groups associated with amino acids, in fact shows a specific reaction only with α-amino groups present at protein N termini when mildly acidic conditions are used. We use this finding to label, identify, and sequence the trypsinolysis-derived N-terminal peptide of lysozyme, using only mass spectrometry, to illustrate how this method could be used with other proteins.     

Genes involved in phosphatidylcholine biosynthesis correlate with nuclear factor-κB in biliary tract cancer patients: Evidence from 1H NMR and computational analyses

Gallbladder cancer (GBC) is an aggressive malignancy of gastrointestinal tract. Due to uncontrolled growth, GBC cells rapidly synthesize biomolecules including lipids. The lipids are integral component of cell membrane with a wide range of cellular functions. In this study, we measured the clinicopathological features in 40 cases of histologically confirmed GBC and 16 cases of chronic cholecystitis (CC). The female to male ratio in the GBC and CC groups were 3.44:1 and 2.2:1, respectively. The GBC patients exhibited well to poorly differentiated tumor. In the CC group, all patients showed cholecystitis with no evidence of dysplasia or malignancy. The majority of GBC and CC patients reported pain. Using ¹H NMR spectroscopy, we observed 4-folds increase in the level of choline containing phospholipids (CCPLs) in the gallbladder of GBC patients as compared to CC patients. Other lipid metabolites such as cholesterol ester, C18-cholesterol and saturated fatty acids were insignificantly changed between GBC and CC patients. Moreover, the level of CCPLs in the GBC patients with BMI <25 kg/m² was significantly higher as compared to CC patients. Further, a significant increase in the CCPLs level was observed in GBC female patients in comparison to CC patients. From the computational analyses, we observed that the genes involved in the biosynthesis of phosphatidylcholine (PtdCho) indirectly interact with the RELA, which encodes the NF-κB p65 subunit. The genes involved in the PtdCho biosynthesis were also correlated with the overall and disease-free survival of cholangiocarcinoma patients. The study opens new window for exploring the diagnostic and therapeutic potential of CCPLs in GBC patients.     

Detection and identification of phytoplasma associated with witches’ broom and little leaf disease in Arundo donax: first report from India

During the survey of two successive years 2012–2013, in nearby places of Gorakhpur districts, Uttar Pradesh, India, Arundo donax plants were found to be exhibiting witches’ broom, excessive branching accompanied with little leaf symptoms with considerable disease incidence. Nested PCR carried out with universal primers pair R16F2n/R16R2 employing the PCR (P1/P7) product as a template DNA (1:20) resulted in expected size positive amplification ~1.2 kb in all symptom-bearing plants suggested the association of phytoplasma with witches’ broom disease of Narkat plants. BLASTn analysis of the 16S rRNA gene sequence showed the highest (99%) sequence identity with Candidatus phytoplasma asteris (16SrI group). In phylogenetic analysis, the sequence data showed close relationships with the members of 16SrI phytoplasma and clustered within a single clade of 16SrI group and closed to B subgroup representatives. This is a first report of 16Sr I-B group phytoplasma associated with witches’ broom accompanied with little leaf disease of Narkat in India.     

Apparent Strength Conceals Instability in a Model for the Collapse of Historical States

An explanation for the political processes leading to the sudden collapse of empires and states would be useful for understanding both historical and contemporary political events. We examine political disintegration across eras, cultures and geographical scale to form a simple hypothesis that can be expressed verbally yet formulated mathematically. Factions within a state make choices described by game-theory about whether to accept the political status quo, or to attempt to better their circumstances through costly rebellion. In lieu of precise data we verify our model using sensitivity analysis. We find that a small amount of dissatisfaction is typically harmless to the state, but can trigger sudden collapse when there is a sufficient buildup of political inequality. Contrary to intuition, a state is predicted to be least stable when its leadership is at the height of its political power and thus most able to exert its influence through external warfare, lavish expense or autocratic decree.     

Transcriptome generation and analysis from spleen of Indian catfish, Clarias batrachus (Linnaeus, 1758) through normalized cDNA library

Catfishes are commercially important fish for both the fisheries and aquaculture industry. Clarias batrachus, an Indian catfish species is economically important owing to its high demand. A normalized cDNA library was constructed from spleen of the Indian catfish to identify genes associated with immune function. One thousand nine hundred thirty seven ESTs were submitted to the GenBank with an average read length of approximately 700 bp. Clustering analysis of ESTs yielded 1,698 unique sequences, including 184 contigs and 1,514 singletons. Significant homology to known genes was found by homology searches against data in GenBank in 576 (34 %) ESTs, including similarity to functionally annotated unigenes for 158 ESTs. Additionally, 433 ESTs revealed similarity to unigenes and ESTs in the dbEST but the remaining 658 EST sequences (39 %) did not match any sequence in GenBank. Of a total of 1,698 ESTs generated, 65 ESTs were found to be associated with immune functions. Gene Ontology and KEGG pathway analyses of C. batrachus ESTs collectively revealed a preponderance of immune relevant pathways apart from the presence of pathways involved in protein processing, localization, folding and protein degradation. This study constitutes first EST analysis of lymphoid organ in aquaculturally important Indian catfish species and could pave the way for further research of immune-related genes and functional genomics in this catfish.     

Establishment and characterization of an epithelial cell line from thymus of Catla catla (Hamilton, 1822)

A cell line, CTE, derived from catla (Catla catla) thymus has been established by explant method and subcultured for more than 70 passages over a period of 400 days. The cell line has been maintained in L-15 (Leibovitz) medium supplemented with 10% fetal bovine serum. CTE cell line consists of homogeneous population of epithelial-like cells and grows optimally at 28 °C. Karyotype analysis revealed that the modal chromosome number of CTE cells was 50. Partial amplification, sequencing and alignment of fragments of two mitochondrial genes 16S rRNA and COI confirmed that CTE cell line originated from catla. Significant green fluorescent signals were observed when the cell line was transfected with phrGFP II-N mammalian expression vector, indicating its potential utility for transgenic and genetic manipulation studies. The CTE cells showed strong positivity for cytokeratin, indicating that cell line was epithelial in nature. The flow cytometric analysis of cell line revealed a higher number of cells in S-phase at 48 h, suggesting a high growth rate. The extracellular products of Vibrio cholerae MTCC 3904 were toxic to the CTE cells. This cell line was not susceptible to fish betanodavirus, the causative agent of viral nervous necrosis in a large variety of marine fish.