Silver nitrate-induced in vitro shoot multiplication and precocious flowering in <Emphasis Type="Italic">Catharanthus roseus</Emphasis> (L.) G. Don,a rich source of terpenoid indole alkaloids

Catharanthus roseus (L.) G. Don is an economically and medicinally important plant since its leaves and flowers contain terpenoid indole alkaloids. The present study, for the first time, encompasses the influence of silver nitrate (AgNO₃), in consort with cytokinins like N ⁶-benzyladenine (BA) and 6-furfurylaminopurine (kinetin), to regenerate multiple shoots from nodal segments explants and to induce high-frequency precocious flowering of C. roseus under in vitro condition. Synergistic effect of equal concentrations of BA and kinetin was enhanced following the amalgamation of AgNO₃. As high as 98% explants responded to multiple shoot initiation and proliferation in Murashige and Skoog medium supplemented with 3 µM BA, 3 µM kinetin and 0.1 µM AgNO₃. As many as 7 shoots were developed per explant following 12 days of inoculation. Continuous culture in the same medium for 21 days induced precocious flowering from 75% shoots, wherein a maximum of ~?6 (5.67?±?0.88) flowers was observed per in vitro shoot. On the other hand, in the combinations of BA and kinetin excluding AgNO₃, a maximum of 6.67% explants responded and initiated merely 3.33 shoots per explant. Nevertheless, no induction of flower was observed in the media devoid of AgNO₃. Our results on the induction and proliferation of multiple shoots with simultaneous flowering would help the global pharmaceutical industry to produce in vitro shoots and flowers in bulk, as an alternative source of alkaloids.     

Elucidation of molecular mechanism of stability of the heme-regulated eIF2α kinase upon binding of its ligand,hemin in its catalytic kinase domain

The eIF2α kinase activity of the heme-regulated inhibitor (HRI) is regulated by heme which makes it a unique member of the family of eIF2α kinases. Since heme concentrations create an equilibrium for the kinase to be active/inactive, it becomes important to study the heme binding effects upon the kinase and understanding its mechanism of functionality. In the present study, we report the thermostability achieved by the catalytic kinase domain of HRI (HRI.CKD) upon ligand (heme) binding. Our CD data demonstrates that the HRI.CKD retains its secondary structure at higher temperatures when it is in ligand bound state. HRI.CKD when incubated with hemin loses its monomeric state and attains a higher order oligomeric form resulting in its stability. The HRI.CKD fails to refold into its native conformation upon mutation of H377A/H381A, thereby confirming the necessity of these His residues for correct folding, stability, and activity of the kinase. Though our in silico study demonstrated these His being the ligand binding sites in the kinase insert region, the spectra-based study did not show significant difference in heme affinity for the wild type and His mutant HRI.CKD.     

Distal arthrogryposis syndrome

A 5-month-old male infant presented with weak cry, decreased body movements, tightness of whole body since birth, and one episode of generalized seizure on day 4 of life. He was born at term by elective caesarian section performed for breech presentation. The child had failure to thrive, contractures at elbow and knee joints, hypertonia, microcephaly, small mouth, retrognathia, and camptodactyly. There was global developmental delay. Abdominal examination revealed umbilical and bilateral inguinal hernia. Visual evoked response and brainstem evoked response audiometry were abnormal. Nerve conduction velocity was normal. Magnetic resonance imaging of brain revealed paucity of white matter in bilateral cerebral hemispheres with cerebellar and brain stem atrophy. The differential diagnoses considered in the index patient were distal arthrogryposis (DA) syndrome, cerebroculofacioskeletal syndrome, and Pena Shokier syndrome. The index patient most likely represents a variant of DA: Sheldon Hall syndrome.     

Auxin-binding proteins without KDEL sequence in the moss Funaria hygrometrica

Whereas the important plant growth regulator auxin has multiple effects in flowering plants, it induces a specific cell differentiation step in the filamentous moss protonema. Here, we analyse the presence of classical auxin-binding protein (ABP1) homologues in the moss Funaria hygrometrica. Microsomal membranes isolated from protonemata of F. hygrometrica have specific indole acetic acid-binding sites, estimated to be about 3–5 pmol/mg protein with an apparent dissociation constant (K _d) between 3 and 5 μM. Western analyses with anti-ABP1 antiserum detected the canonical endoplasmic reticulum (ER)-localised 22–24 kDa ABP1 in Zea mays, but not in F. hygrometrica. Instead, polypeptides of 31–33 and 46 kDa were labelled in the moss as well as in maize. In F. hygrometrica these proteins were found exclusively in microsomal membrane fractions and were confirmed as ABPs by photo-affinity labelling with 5-azido-[7-³H]-indole-3-acetic acid. Unlike the classical corn ABP1, these moss ABPs did not contain the KDEL ER retention sequence. Consistently, the fully sequenced genome of the moss Physcomitrella patens, a close relative of F. hygrometrica, encodes an ABP1-homologue without KDEL sequence. Our study suggests the presence of putative ABPs in F. hygrometrica that share immunological epitopes with ABP1 and bind auxin but are different from the classical corn ABP1.     

Genetic Characterization of Hepatitis B Virus in Peripheral Blood Leukocytes: Evidence for Selection and Compartmentalization of Viral Variants with the Immune Escape G145R Mutation

The compartmentalization of viral variants in distinct host tissues is a frequent event in many viral infections. Although hepatitis B virus (HBV) classically is considered hepatotropic, it has strong lymphotropic properties as well. However, unlike other viruses, molecular evolutionary studies to characterize HBV variants in compartments other than hepatocytes or sera have not been performed. The present work attempted to characterize HBV sequences from the peripheral blood leukocytes (PBL) of a large set of subjects, using advanced molecular biology and computational methods. The results of this study revealed the exclusive compartmentalization of HBV subgenotype Ae/A2-specific sequences with a potent immune escape G145R mutation in the PBL of the majority of the subjects. Interestingly, entirely different HBV genotypes/subgenotypes (C, D, or Aa/A1) were found to predominate in the sera of the same study populations. These results suggest that subgenotype Ae/A2 is selectively archived in the PBL, and the high prevalence of G145R indicates high immune pressure and high evolutionary rates of HBV DNA in the PBL. The results are analogous to available literature on the compartmentalization of other viruses. The present work thus provides evidence in favor of the compartment-specific abundance, evolution, and emergence of the potent immune escape mutant. These findings have important implications in the field of HBV molecular epidemiology, transmission, transfusion medicine, organ transplantation, and vaccination strategies.Hepatitis B virus (HBV) is the prototype member of the Hepadnaviridae family and classically has been described to be hepatotropic, causing a wide range of clinical and subclinical manifestations of liver disease (57). Nevertheless, studies of HBV-infected human subjects and woodchucks infected with Woodchuck hepatitis virus (WHV; an animal model of hepadnaviral infection) have reported different molecular forms of replicative intermediates in the lymphatic cells and have established that hepadnaviruses are strongly lymphotropic in nature (29). Moreover, the results of studies of human subjects as well as with animal models have revealed that the life-long occult persistence of replication- and transmission-competent viruses in lymphatic cells is a strict consequence of hepadnaviral infections (29).More interestingly, in animal models, lymphatic system-restricted occult hepadnaviral infection has been found to be transmissible vertically as an asymptomatic, serologically occult infection exclusively confined to the lymphatic system (29). Earlier we provided evidence that occult HBV persisting in the lymphatic cells are transmissible, specifically to the PBL through horizontal intrafamilial modes (9). These observations clearly indicate important immunological, pathogenic, and epidemiological implications of lymphatic system-restricted hepadnaviral infections. Although the involvement of specific viral variants has been suggested to explain this lymphatic system-restricted hepadnaviral infection and transmission (29), the classical belief that hepatocytes are the primary target and only reservoir of HBV has precluded the genetic characterization of hepadnaviruses from extrahepatic sites.Fascinatingly, despite being classically considered a hepatotropic virus, hepatitis C virus (HCV), belonging to the family Flaviviridae, also shows occult persistence and lymphotropism very similar to that of hepadnaviruses (37). Similarly to WHV, HBV, and HCV, other viruses, including HIV (human immunodeficiency virus), small ruminant lentivirus, and Epstein-Barr virus, also have been shown to infect and persist in different anatomical compartments of the body in addition to their classical target cells (38, 40, 43, 45, 50). Furthermore, recent molecular evolutionary analyses based on envelope sequences of these viruses (e.g., HIV, HCV, small ruminant lentivirus, Epstein-Barr virus, etc.) have established clearly that these viruses undergo selection and independent evolution in diverse tissues, leading to the tissue-specific compartmentalization of viral populations (38, 40, 43, 45, 50). In contrast to other viruses, to the best of our knowledge, methodical molecular evolutionary studies to characterize HBV sequences isolated from extrahepatic sites of HBV-infected subjects have not been reported in the literature.We hypothesized that similar to other viruses, HBV also undergo independent evolution in different compartments of the body under the influence of differential immune pressure. To examine our hypothesis, we used the most easily available lymphatic cells, the peripheral blood leukocytes (PBL), determined the HBV envelope sequences from HBV DNA isolated from these cells, and performed advanced genetic, phylogenetic, and mutational analysis. The results of this work demonstrate a highly compartment-specific preponderance of HBV genetic variants in serum and PBL of the same study population, providing evidence in favor of the compartmentalization of HBV genetic variants. The results and important implications of these findings are discussed in this work.     

Calpain-1 cleaves Rad21 to promote sister chromatid separation

Defining the mechanisms of chromosomal cohesion and dissolution of the cohesin complex from chromatids is important for understanding the chromosomal missegregation seen in many tumor cells. Here we report the identification of a novel cohesin-resolving protease and describe its role in chromosomal segregation. Sister chromatids are held together by cohesin, a multiprotein ring-like complex comprised of Rad21, Smc1, Smc3, and SA2 (or SA1). Cohesin is known to be removed from vertebrate chromosomes by two distinct mechanisms, namely, the prophase and anaphase pathways. First, PLK1-mediated phosphorylation of SA2 in prophase leads to release of cohesin from chromosome arms, leaving behind centromeric cohesins that continue to hold the sisters together. Then, at the onset of anaphase, activated separase cleaves the centromeric cohesin Rad21, thereby opening the cohesin ring and allowing the sister chromatids to separate. We report here that the calcium-dependent cysteine endopeptidase calpain-1 is a Rad21 peptidase and normally localizes to the interphase nuclei and chromatin. Calpain-1 cleaves Rad21 at L192, in a calcium-dependent manner. We further show that Rad21 cleavage by calpain-1 promotes separation of chromosome arms, which coincides with a calcium-induced partial loss of cohesin at several chromosomal loci. Engineered cleavage of Rad21 at the calpain-cleavable site without activation of calpain-1 can lead to a loss of sister chromatid cohesion. Collectively, our work reveals a novel function of calpain-1 and describes an additional pathway for sister chromatid separation in humans.     

Molecular characterization of distinct YMV (Yellow mosaic virus) isolates affecting pulses in India with the aid of coat protein gene as a marker for identification

The present study was carried out to find out the variations present in different isolates of yellow mosaic virus (YMV) causing yellow mosaic disease of pulses in southern parts of India. The coat protein gene of YMV was amplified using gene specific and deng universal primers with DNA isolated from YMV infected samples. Further, cloning and DNA sequencing of CP gene was carried out. CP gene decrypt sequences revealed that YMV infected samples of Black gram, Cowpea and Green gram were similar to the MYMV-Tamil Nadu isolates. Whereas the YMV infected sample of Horse gram was found to be similar with HYMV. Hence, in the present study, two distinct YMV infecting pulses in Tamil Nadu (MYMV and HYMV species) were identified and it was observed that there exists considerable genetic variation among these species. In addition, Cowpea crop which was earlier supposed not to be susceptible for YMV infection also showed the presence of this virus similar to the MYMV. Overall, the findings of the present study indicate that the CP region is efficient enough to provide a simple, rapid, and reliable method for early detection of YMV infections in pulses, which would help to develop proper management strategies to control these viruses.     

Brevibacillus as a biological tool: a short review

The significance of Brevibacillus has been documented scientifically in the published literature and commercially in heterologous recombinant protein catalogs. Brevibacillus is one of the most widespread genera of Gram-positive bacteria, recorded from the diverse environmental habitats. The high growth rate, better transformation efficiency by electroporation, availability of shuttle vectors, production of negligible amount of extracellular protease, and the constitutive expression of heterologous proteins make some strains of this genus excellent laboratory models. Regarding biotechnological applications, this genus continues to be a source of various enzymes of great biotechnological interest due to their ability to biodegrade low density polyethylene, ability to act as a candidate bio-control agent, and more recently acknowledged as a tool for the overexpression. This article reviews the properties of Brevibacillus spp. as better biological tools with varied applications.