The cellulase complex of Neurospora crassa: activity, stability and release

The temperature and pH optima, and the temperature and pH stability, of crude and purified enzymes of the cellulase complex of the cellulolytic ascomycete fungus Neurospora crassa were investigated. The effects of some non-ionic surfactants and fatty acids on the production/release of enzymes of cellulase complex were also examined. For the different enzymes of the complex, activity maxima occurred between pH 4.0 and 7.0, with pH 5.0 being close to optimal for stability of all. Temperature optima for activity ranged between 45 and 65 degrees C, with the stability optimum between 45 and 50 degrees C. The presence of C18 fatty acids and surfactants resulted in increased production of both endoglucanase and exoglucanase in the medium. Oleic acid was the most effective fatty acid tested, and Tween 80 the most effective surfactant. Oleic acid had no detectable effect on production of beta-glucosidase, and Tween 80 actually reduced its production.     

The alteration of PLCζ protein expression in unexplained infertile and asthenoteratozoospermic patients: A potential effect on sperm fertilization ability

Failed oocyte activation has been observed in unexplained infertile (UI) and asthenoteratozoospermic (AT) men. The deficiency of phospholipase C‐zeta (PLCζ) could be a possible reason for such failures and has not been studied yet. We investigated the expression and localization of PLCζ protein in the sperms of patients with UI and AT conditions. The relationships between PLCζ‐related parameters with male age, sperm characteristics, DNA integrity, and cellular maturity were assessed. Semen samples were collected from fertile (n = 40), UI (n = 40), and AT (n = 40) men. Subsequently, semen analysis, DNA fragmentation, hyaluronic acid‐binding ability, and PLCζ level along with its distribution were evaluated using computer‐assisted sperm analyzer, sperm chromatin structure assay (SCSA), hyaluronic acid‐binding assay (HBA), western blot analysis and immunofluorescence microscopy, respectively. Unlike SCSA, the values of HBA, and PLCζ expression were significantly reduced in UI and AT patients compared to fertile men, whereas no significant differences were observed among the experimental groups in terms of PLCζ localization patterns. The regression analysis also showed that HBA is the only variable associated with PLCζ levels. Furthermore, the correlation of male age with PLCζ localization in postacrosomal, equatorial, and acrosomal+postacrosomal+equatorial (A+PA+E) patterns, as well as the relation of normal morphology, with the (A+PA+E) pattern, remained in the regression model. Our findings indicated that reduced PLCζ level along with the increased DNA fragmentation and impaired maturation may be possible etiologies of decreased fertilization in the studied subjects.     

Comparison of PCR-RFLP with 21-plex PCR and rDNA Sequencing for Identification of Clinical Yeast Isolates

Non-albicans Candida species and other rare yeasts have emerged as major opportunistic pathogens in fungal infections. Identification of opportunistic yeasts in developing countries is mainly performed by phenotypic assay, which are time-consuming and prone to errors. The aim of the present study was to evaluate PCR-RFLP as a routinely used identification technique for the most clinically important Candida species in Iran and make a comparison with a novel multiplex PCR, called 21-plex PCR. One hundred and seventy-three yeast isolates from clinical sources were selected and identified with sequence analysis of the D1/D2 domains of rDNA (LSU rDNA) sequencing as the gold standard method. The results were compared with those obtained by PCR-RFLP using MspI restriction enzyme and the 21-plex PCR. PCR-RFLP correctly identified 93.4% of common pathogenic Candida species (C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, and P. kudriavsevii (=?C. krusei)) and was able to identify 45.5% of isolates of the uncommon yeast species compared to the D1/D2 rDNA sequencing. Compared with PCR-RFLP, all common Candida species and 72.7% of uncommon yeast species were correctly identified by the 21-plex PCR. The application of the 21-plex PCR assay as a non-sequence-based molecular method for the identification of common and rare yeasts can reduce turnaround time and costs for the identification of clinically important yeasts and can be applied in resource-limited settings.

     

Improvement in efficiency of microspore culture to produce doubled haploid lines of Ethiopian mustard

Factors affecting microspore embryogenesis of Ethiopian mustard (Brassica carinata A. Braun) were evaluated, including flower bud length, pollen developmental stage, and microspore density. An embryogenic frequency of 300 embryos per Petri plate was observed with NLN (Nitsch-Lichter-Nitsch) medium supplemented with 13% sucrose, 3.0–3.4-mm-long buds, and a plating density of 65,000 microspores/ml. About 65% of the microspores from buds 3.0–3.4-mm long were at the late uninucleate stage. Microspore-derived embryos were successfully transferred to solid medium for germination. After 4 wk, the resulting plantlets were transplanted to a soilless potting mixture and grew well under greenhouse conditions.     

Spermine as a possible endogenous allosteric activator of carboxypeptidase A: multispectroscopic and molecular simulation studies

Abstract

Carboxypeptidase A (EC.3.4.17.1) is a zinc-containing proteolytic enzyme that removes the C-terminal amino acid from a peptide chain with the free carboxylate-terminal. In this study, the effect of spermine interaction on the structure and thermal stability of Carboxypeptidase A was investigated by ultraviolet???visible spectroscopy, fluorescence spectroscopy, circular dichroism, Kinetic measurement, molecular docking and simulation studies have also been followed at the pH of 7.5. The transition temperature of Carboxypeptidase A, as a criterion of protein thermal stability, in the presence of spermine was enhanced by increasing the concentration of spermine. The results of fluorescence intensity changes, at two temperatures of 308 and 318?K, also suggested that spermine had a great ability to quench the fluorescence of Carboxypeptidase A through the static quenching procedure. The thermodynamic parameters changes, including standard Gibbs free-energy, entropy and enthalpy, showed that the binding of spermine to Carboxypeptidase A was spontaneous and the hydrogen bonding and van der Waals interactions played a major role in stabilizing the Carboxypeptidase A–spermine complex. The changes in the content of the α-helix and the β-sheet of the Carboxypeptidase A with binding to spermine were shown by the CD spectra method. Further, kinetic studies revealed that by increasing concentration of spermine, the activity of Carboxypeptidase A was enhanced. Also, the docking study revealed that the hydrogen bonding and van der Waals interactions played a major role in stabilizing the Carboxypeptidase A–spermine complex. As a result, spermine could be considered as an activator and a stabilizer for Carboxypeptidase A.

Communicated by Ramaswamy H. Sarma     

LncRNAs as potential diagnostic and prognostic biomarkers in gastric cancer: A novel approach to personalized medicine

Gastric cancer is the third leading cause of cancer death with 5-year survival rate of about 30–35%. Since early detection is associated with decreased mortality, identification of novel biomarkers for early diagnosis and proper management of patients with the best response to therapy is urgently needed. Long noncoding RNAs (lncRNAs) due to their high specificity, easy accessibility in a noninvasive manner, as well as their aberrant expression under different pathological and physiological conditions, have received a great attention as potential diagnostic, prognostic, or predictive biomarkers. They may also serve as targets for treating gastric cancer. In this review, we highlighted the role of lncRNAs as tumor suppressors or oncogenes that make them potential biomarkers for the diagnosis and prognosis of gastric cancer. Relatively, lncRNAs such as H19, HOTAIR, UCA1, PVT1, tissue differentiation-inducing nonprotein coding, and LINC00152 could be potential diagnostic and prognostic markers in patients with gastric cancer. Also, the impact of lncRNAs such as ecCEBPA, MLK7-AS1, TUG1, HOXA11-AS, GAPLINC, LEIGC, multidrug resistance-related and upregulated lncRNA, PVT1 on gastric cancer epigenetic and drug resistance as well as their potential as therapeutic targets for personalized medicine was discussed.     

Microsporum fulvum, an Ignored Pathogenic Dermatophyte: A New Clinical Isolation from Iran

Misidentifying with Microsporum gypseum has for a long time been accounted for less prevalence of the geophilic species, Microsporum fulvum in human dermatophytosis. We describe a new case of infection with the species in an Iranian young man. Direct examination of skin scrapings revealed a tinea corporis, and morphological study of the recovered isolate from the culture resulted in the identification of M. gypseum. However, PCR amplification of ITS1-5.8S rDNA-ITS2 region and subsequent ITS-RFLP and sequencing were indicative of M. fulvum as the true causative agent. To recognize M. fulvum in human infections and to validate the morphologically distinguished isolates of M. gypseum, the genetic-based identification is strongly recommended.