Characterization of Neurospora crassa α-Actinin

α-Actinin, an actin-binding protein of the spectrin superfamily, is present in most eukaryotes except plants. It is composed of three domains: N-terminal CH-domains, C-terminal calcium-binding domain (with EF-hand motifs), and a central rod domain. We have cloned and expressed Neurospora crassa α-actinin as GST and GFP fusion proteins for biochemical characterization and in vivo localization, respectively. The intracellular localization pattern of α-actinin suggests that this protein is intimately associated with actin filaments and plays an important role in the processes of germination, hyphal elongation, septum formation, and conidiation. These functions were confirmed by the experiments on the effect of α-actinin gene deletion in N. crass

Ca²⁺-Dependent Protease I from <i>Allomyces arbuscula</i>

A monomeric Ca²⁺-dependent protease (CDP I) of 39 kDa active at neutral pH has been purified from the aquatic fungusAllomyces arbuscula.The enzyme elutes at NaCl molarity of 0.07 M from the DEAE (DE52)-cellulose columns in contrast to the second Ca²⁺-dependent protease (CDP II) characterized earlier which elutes at 0.18 M NaCl. The enzyme has no basal activity in the absence of Ca²⁺ and requires 1.7 mM Ca²⁺ for half maximum activation of thein vitroenzyme activity. The enzyme prefers substrates with Arg in P₁position but this specificity also depends strongly on the nature of the subsite residues, for example Pro in P₂ position. The enzyme is glycosylated and contains essential cysteine residues in the active site. It appears to be an atypical cysteine protease as it is inactivated to varying degree with some serine protease inhibitors

Purification, Properties and Developmental Regulation of a Ca²⁺-Independent Serine—Cysteine Protease from <i>Allomyces</i> arbuscula

Reproductive differentiation in Allomyces takes place against the background of substrate limitation, a sharp increase in intracellular probeolysine and the induction of at least one specific protease. The aim of this report is to describe the purification, properties and developmental regulation of this enzyme. The enzyme has been partially purified by a combination of ion exchange chromatography, ultrogel filtration and affinity chromatography. The purified enzyme in SDS-PAGE appeared as a doublet of Mr 40–43 kDa. Two bands corresponding to a relative molecular mass of 40–43 kDa were also apparent in activity gels. The protein has an apparent molecular mass in the region of 43 kDa as estimated by gel filtration. The enzyme therefore, seems to be a monomer of 43 kDa. The second band in SDS-PAGE and activity gels is probably the proteolyzed form of the enzyme. The protease recognized alanine and to a lesser extent phenylalanine in the P1 position when assayed with a range of synthetic peptides. The active site of the enzyme detains a reactive serine residue, as shown by its inhibition with PMSF and soya bean trypsin inhibitor. There is probably a reactive cysteine residue as well since the enzyme activity is strongly inhibited by HgCl₂, a thiol group binding reagent. The enzyme is present in zoospores but disappears progressively during germination and hyphal growth. It reappears when actively growing cultures are transferred to dilute salt solution. In conclusion, we have purified a serine-cysteine protease of Mr 43 kDa. This enzyme has a very restricted substrate specificity and appears to be developmentally regulated

A novel Nα-acetyl alanine aminopeptidase from Allomyces arbuscula

An Nα-acetyl alanine aminopeptidase has been purified from the aquatic fungus Allomyces arbuscula. The apparent molecular mass of the enzyme was estimated to be 280 kDa by gel filtration through calibrated Sephacryl S300 column. In SDS-PAGE, the purified enzyme appeared as a single band of Mr 80 kDa. Catalytic activity of the enzyme was inhibited by specific serine protease inhibitors, 3,4-DCI and APMSF, as well as SH reacting compounds, HgCl2 and iodoacetate, indicating that the enzyme is a serine protease with some functional SH group(s) involved in the catalytic reaction. 3H-DFP was used to label the reactive serine of the enzyme. When the labeled protein was analyzed in SDS-PAGE, most of the label appeared in the Mr 80 kDa band, however, a few additional faster migrating minor bands were also seen, probably representing a minor degradation product of the enzyme. The enzyme cleaved mainly Nα-acetlylated alanine, although a small but negligible activity was also obtained with acetylated leucine and phenylalanine. The role of the enzyme in N-end rule proteolysis is discusse

Proteolytic Cleavage of a Spectrin-Related Protein by Calcium-Dependent Protease in Neurospora crassa

To investigate the functional significance of a cytoskeletal spectrin-like protein, we studied its localization pattern in Neurospora crassa and sought the answer to whether it is a substrate for another apically localized protein, the calcium-dependent protease (CDP II). Immunoblots of crude extracts from exponentially growing mycelia, separated by one- and two-dimensional sodium dodecyl sulfate–polyacrylamide gel electrophoresis using antichicken α/β-spectrin antibodies, revealed a single band of approximately relative mass (Mr) 100 kDa with an isoeletric point (pI) in the range of 6.5 to 7.0. Despite rigorous efforts, we could not confirm the presence of an Mr 240- to 220-kDa spectrin-like protein in N. crassa. The immunofluorescence- and immunogold-labeling Mr 100-kDa protein showed its predominance along the plasma membrane of the conidia during the swelling phase of germination. In contrast, in the germ tubes and the growing hyphae, the localization was polarized and concentrated mainly in the apical region. The in vitro proteolysis experiments showed that indeed this protein is a preferred substrate of CDP II which is, as mentioned previously, also localized in the apical regions of the hyphae. These results indicate a putative functional relationship between these two proteins (spectrin-like protein and CDP II) in the dynamics of tip growth

Prevalence and correlates of tobacco use among adolescents in the schools of Kalaiya, Nepal: a cross-sectional questionnaire based study

Introduction Adolescent students are vulnerable group for tobacco addiction. Tobacco use among school children is becoming a serious problem in developing countries. This study was carried out to estimate the prevalence of tobacco use and to determine associated factors among adolescent students of Kalaiya municipality. Material and Methods A cross sectional survey was carried out by self-administered questionnaire adapted from Global Youth Tobacco Survey to assess tobacco use among the representative sample of 1540 adolescent students selected by stratified random sampling from December 2014 to May 2015. Results Overall prevalence of ‘ever users’ of tobacco products was 25.3 %. Prevalence among boys and girls was 31 and 14.4 % respectively. Mean age at initiation of using tobacco was 13.38 ± 1.62 years. The correlates of tobacco use were: sex, ethnicity, family members and friends using tobacco products, and students exposed at home and public place. Conclusions School based interventions and tobacco education are necessary to prevent initiation and cessation of tobacco use. Legislations related to tobacco control should be enforced to decrease availability, accessibility and affordability of tobacco products. Social norms of tobacco use among parents and others at home as well as at public place should be modified to curb the tobacco use among school students

Characterization of Neurospora crassa α-Actinin

α-Actinin, an actin-binding protein of the spectrin superfamily, is present in most eukaryotes except plants. It is composed of three domains: N-terminal CH-domains, C-terminal calcium-binding domain (with EF-hand motifs), and a central rod domain. We have cloned and expressed Neurospora crassa α-actinin as GST and GFP fusion proteins for biochemical characterization and in vivo localization, respectively. The intracellular localization pattern of α-actinin suggests that this protein is intimately associated with actin filaments and plays an important role in the processes of germination, hyphal elongation, septum formation, and conidiation. These functions were confirmed by the experiments on the effect of α-actinin gene deletion in N. crassa