Expression, Purification and Characterization of Peanut (Arachis hypogaea) Agglutinin (PNA) from Baculovirus Infected Insect Cells

Peanut (Arachis hypogaea) seed lectin, PNA is widely used to identify tumor specific antigen (T-antigen), Gal1-3GalNAc on the eukaryotic cell surface. The functional amino acid coding region of a cDNA clone, pBSH-PN was PCR amplified and cloned downstream of the polyhedrin promoter in the Autographa californica nucleopolyhedrovirus (AcNPV) based transfer vector pVL1393. Co-transfection of Spodoptera frugiperda cells (Sf9) with the transfer vector, pAcPNA and AcRP6 (a recombinant AcNPV having B-gal downstream of the polyhedrin promoter) DNAs produced a recombinant virus, AcPNA which expresses PNA. Infection of suspension culture of Sf9 cells with plaque purified AcPNA produced as much as 9.8 mg PNA per liter (2.0 × 10⁶ cells/ml) of serum-free medium. Intracellularly expressed protein (re-PNA) was purified to apparent homogeneity by affinity chromatography using ECD-Sepharose. Polyclonal antibodies against natural PNA (n-PNA) cross-reacted with re-PNA. The subunit molecular weight (30kDa), hemagglutination activity, and carbohydrate specificity of re-PNA were found to be identical to that of n-PNA, thus confirming the abundant production of a functionally active protein in the baculovirus expression system.     

2'-5' Oligoadenylate synthetase plays a critical role in interferon-gamma inhibition of respiratory syncytial virus infection of human epithelial cells

Respiratory syncytial virus (RSV), associated with bronchiolitis and asthma, is resistant to the antiviral effects of type-I interferons (IFN), but not IFN-gamma. However, the antiviral mechanism of IFN-gamma action against RSV infection is unknown. The molecular mechanism of IFN-gamma-induced antiviral activity was examined in this study using human epithelial cell lines HEp-2 and A549. Exposure of these cells to 100-1000 units/ml of IFN-gamma, either before or after RSV infection, results in a significant decrease in RSV infection. After 1 h of exposure, IFN-gamma induces protein expression of IFN regulatory factor-1 (IRF-1) but not IRF-2, double-stranded RNA-activated protein kinase, and inducible nitric-oxide synthase in these cells. The mRNA for IRF-1, p40, and p69 isoforms of 2'-5' oligoadenylate synthetase (2-5 AS) are detectable, respectively, at 1 and 4 h of IFN-gamma exposure. Studies using cycloheximide and antisense oligonucleotides to IRF-1 indicate a direct role of IRF-1 in activating 2-5 AS. Cells transfected with 2-5 AS antisense oligonucleotides inhibit the antiviral effect of IFN-gamma. A stable cell line of HEp-2 overexpressing RNase L inhibitor, RLI-14, which exhibits an IFN-gamma-induced gene expression pattern similar to that of the parent cell line, shows a significant reduction in RNase L activity and IFN-gamma-mediated antiviral effect, compared with HEp-2 cells. These results provide direct evidence of the involvement of 2-5 AS in IFN-gamma-mediated antiviral activity in these cells.     

High irradiance-induced changes in carotenoid composition and increase in non-photochemical quenching of Chl a fluorescence in primary wheat leaves

The effect of acclimation to high irradiance stress (HIS, 250 Wm-2) in wheat leaves grown under three different irradiances was investigated by HPLC analyses of pigments, chlorophyll a fluorescence parameters and photochemical activities of chloroplasts. Significant loss of beta-carotene was observed compared to the xanthophylls in all three types of seedlings exposed to HIS. However, the effect of HIS on neoxanthin and lutein contents was not significant. The loss of partial electron transport (Asc-DCPIP to MV, PSI activity) was less than the whole chain (H2O to MV) and PS II activity (H2O to DCPIP) suggesting that PS I is less susceptible to HIS compared to PS II. The percent of reductions in Fv/Fm and phi PS II were less in plants grown under high irradiance (HI-1, 30 Wm-2 and HI-2, 45 Wm-2) compared to those grown under moderate irradiance (MI, 15 Wm-2). On the other hand, the percent of NPQ increased more in the leaves of HI plants compared to the leaves of MI when exposed to HIS which suggests a more efficient non-radiative dissipation of excess excitation energy in HI plants compared to MI. These observations suggest that plants grown under relatively high irradiance are better adapted to HIS condition.     

Molecular interplay of autophagy and endocytosis in human health and diseases

Autophagy, an evolutionarily conserved process for maintaining the physio‐metabolic equilibrium of cells, shares many common effector proteins with endocytosis. For example, tethering proteins involved in fusion like Ras‐like GTPases (Rabs), soluble N‐ethylmaleimide sensitive factor attachment protein receptors (SNAREs), lysosomal‐associated membrane protein (LAMP), and endosomal sorting complex required for transport (ESCRT) have a dual role in endocytosis and autophagy, and the trafficking routes of these processes converge at lysosomes. These common effectors indicate an association between budding and fusion of membrane‐bound vesicles that may have a substantial role in autophagic lysosome reformation, by sensing cellular stress levels. Therefore, autophagy–endocytosis crosstalk may be significant and implicates a novel endocytic regulatory pathway of autophagy. Moreover, endocytosis has a pivotal role in the intake of signalling molecules, which in turn activates cascades that can result in pathophysiological conditions. This review discusses the basic mechanisms of this crosstalk and its implications in order to identify potential novel therapeutic targets for various human diseases.     

Batch ethanol production from cassava (<Emphasis Type="Italic">Manihot esculenta</Emphasis> Crantz.) flour using <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> cells immobilized in calcium alginate

The objective of this research was to saccharify cassava flour by acid-acid and acid-enzyme hydrolysis and further conversion of the resulting sugar into ethanol by fermenting with the immobilized (in Ca-alginate) cells of Saccharomyces cerevisiae. The saccharification resulted in higher total sugar recovery by acid-enzyme hydrolysis (72.88 %) than by enzyme-enzyme hydrolysis (58.1 %). Further study on ethanol production was carried out using the hydrolysate obtained from acid-enzyme hydrolysis. The growth of the yeast started in the log phage and maximum ethanol (189?±?3.1 g ethanol/kg flour) production was achieved with 94.74?±?2.187 % sugar conversion during the stationary phase.     

A rationally engineered small antimicrobial peptide with potent antibacterial activity

Antimicrobial resistance (AMR) is a silent pandemic declared by the WHO that requires urgent attention in the post-COVID world. AMR is a critical public health concern worldwide, potentially affecting people at different stages of life, including the veterinary and agriculture industries. Notably, very few new-age antimicrobial agents are in the current developmental pipeline. Thus, the design, discovery, and development of new antimicrobial agents are required to address the menace of AMR. Antimicrobial peptides (AMPs) are an important class of antimicrobial agents for combating AMR due to their broad-spectrum activity and ability to evade AMR through a multimodal mechanism of action. However, molecular size, aggregability, proteolytic degradation, cytotoxicity, and hemolysis activity significantly limit the clinical application of natural AMPs. The de novo design and engineering of a short synthetic amphipathic AMP (≤16 aa, Mol. Wt. ≤ 2 kDa) with an unusual architecture comprised of coded and noncoded amino acids (NCAAs) is presented here, which demonstrates potent antibacterial activity against a few selected bacterial strains mentioned in the WHO priority list. The designer AMP is conformationally ordered in solution and effectively permeabilizes the outer and inner membranes, leading to bacterial growth inhibition and death. Additionally, the peptide is resistant to proteolysis and has negligible cytotoxicity and hemolysis activity up to 150 μM toward cultured human cell lines and erythrocytes. The designer AMP is unique and appears to be a potent therapeutic candidate, which can be subsequently subjected to preclinical studies to explicitly understand and address the menace of AMR.     

Sustainable hydrogen production in the Cyanobacterium Nostoc sp. ARM 411 grown in fructose- and magnesium sulphate-enriched culture

We show that high levels of both carbon sources (sugars) and cations (notably magnesium) in the growth medium enhance the frequency of heterocyst formation by three-fold over the control in Nostoc sp. Such an alteration in the frequency and in enzyme activity (hydrogenase and nitrogenase), induced a proportional increase in hydrogen evolution capacity under a variety of conditions. Nitrogenase and hydrogenase activity also showed an increase in the enriched cultures. Our results therefore suggest that enriching the Nostoc culture with some selected carbon source or a low cost inorganic source like magnesium would result in a high yield of heterocysts in heterocystous cyanobacteria which would be helpful in the production of sustainable bio-fuels.