Three low molecular weight cysteine proteinase inhibitors of human seminal fluid: Purification and enzyme kinetic properties

The cystatins form a superfamily of structurally related proteins with highly conserved structural folds. They are all potent, reversible, competitive inhibitors of cysteine proteinases (CPs). Proteins from this group present differences in proteinase inhibition despite their high level of structural similarities. In this study, three cysteine proteinase inhibitors (CPIs) of low molecular weight were isolated from human seminal fluid (HSF) by affinity chromatography on carboxymethyl (CM)-papain–Sepharose column, purified using various chromatographic procedures and checked for purity on sodium-dodecyl PAGE (SDS-PAGE). Matrix-assisted laser desorption-ionization-time-of flight-mass spectrometry (MALDI-TOF-MS) identified these proteins as cystatin 9, cystatin SN, and SAP-1 (an N-terminal truncated form of cystatin S). All three CPIs suppressed the activity of papain potentially and showed remarkable heat stability. Interestingly SAP-1 also inhibits the activity of trypsin, chymotrypsin, pepsin, and PSA (prostate specific antigen) and acts as a cross-class protease inhibitor in in vitro studies. Using Surface Plasmon Resonance, we have also observed that SAP-1 shows a significant binding with all these proteases. These studies suggest that SAP-1 is a cross-class inhibitor that may regulate activity of various classes of proteases within the reproductive systems. To our knowledge, this is the first report about purification of CPIs from HSF; the identification of such proteins could provide better insights into the physiological processes and offer intimation for further research.     

Evaluation of a diospyrin derivative as antileishmanial agent and potential modulator of ornithine decarboxylase of Leishmania donovani

World health organization has called for academic research and development of new chemotherapeutic strategies to overcome the emerging resistance and side effects exhibited by the drugs currently used against leishmaniasis. Diospyrin, a bis-naphthoquinone isolated from Diospyros montana Roxb., and its semi-synthetic derivatives, were reported for inhibitory activity against protozoan parasites including Leishmania. Presently, we have investigated the antileishmanial effect of a di-epoxide derivative of diospyrin (D17), both in vitro and in vivo. Further, the safety profile of D17 was established by testing its toxicity against normal macrophage cells (IC₅₀ ∼ 20.7 μM), and also against normal BALB/c mice in vivo. The compound showed enhanced activity (IC₅₀ ∼ 7.2 μM) as compared to diospyrin (IC₅₀ ∼ 12.6 μM) against Leishmania donovani promastigotes. Again, D17 was tested on L. donovani BHU1216 isolated from a sodium stibogluconate-unresponsive patient, and exhibited selective inhibition of the intracellular amastigotes (IC₅₀ ∼ 0.18 μM). Also, treatment of infected BALB/c mice with D17 at 2 mg/kg/day reduced the hepatic parasite load by about 38%. Subsequently, computational docking studies were undertaken on selected enzymes of trypanothione metabolism, viz. trypanothione reductase (TryR) and ornithine decarboxylase (ODC), followed by the enzyme kinetics, where D17 demonstrated non-competitive inhibition of the L. donovani ODC, but could not inhibit TryR.     

Chemistry,antioxidant and antimicrobial potential of nutmeg (Myristica fragrans Houtt)

Antioxidant and antimicrobial activities of nutmeg (Myristica fragrans Houtt) seed extracts were evaluated. Seeds were extracted with acetone, ethanol, methanol, butanol and water. All the extracts have shown significant antioxidant and antimicrobial activities against the tested microorganisms. Among all extracts, acetone extract has shown the highest antioxidant activity. The acetone extract showed 93.12 ± 1.48 mg gallic acid equivalents (GAE)/100 g dry weight total phenolic content, DPPH scavenging activity of 63.04 ± 1.56%, chelating activity of 64.11 ± 2.21% and 74.36 ± 1.94% inhibition of β-carotene bleaching, at 1 mg/mL extract concentration. Out of all extracts, acetone extract was able to exert antimicrobial activity against all tested bacteria and fungi. Acetone extract has shown the strongest antibacterial and antifungal activity with Staphylococcus aureus (13.8 ± 0.42 mm) and Aspergillus niger (14.4 ± 0.37 mm), respectively. GC–MS analysis of acetone extract has revealed the presence of 32 compounds of extract representing 99.49%. Sabinene (28.61%) has shown the highest occurrence in the extract. β-Pinene (10.26), α-pinene (9.72), myristicin (4.30%), isoeugenol (2.72%), p-cymene (1.81%), carvacrol (1.54%), eugenol (0.89%) and β-caryophellene (0.82%) were reported as possible contributor for antioxidant and antimicrobial activity of nutmeg.     

cDNA Cloning and Molecular Modeling of Procerain B,a Novel Cysteine Endopeptidase Isolated from Calotropis procera

Procerain B, a novel cysteine protease (endopeptidase) isolated from Calotropis procera belongs to Asclepiadaceae family. Purification of the enzyme, biochemical characterization and potential applications are already published by our group. Here, we report cDNA cloning, complete amino acid sequencing and molecular modeling of procerain B. The derived amino acid sequence showed high sequence homology with other papain like plant cysteine proteases of peptidase C1A superfamily. The three dimensional structure of active procerain B was modeled by homology modeling using X-ray crystal structure of actinidin (PDB ID: 3P5U), a cysteine protease from the fruits of Actinidia arguta. The structural aspect of the enzyme is also discussed.     

Prostasin, a membrane-anchored serine peptidase, regulates sodium currents in JME/CF15 cells, a cystic fibrosis airway epithelial cell line

Prostasin is a tryptic peptidase expressed in prostate, kidney, lung, and airway. Mammalian prostasins are related to Xenopus channel-activating protease, which stimulates epithelial Na+ channel (ENaC) activity in frogs. In human epithelia, prostasin is one of several membrane peptidases proposed to regulate ENaC. This study tests the hypothesis that prostasin can regulate ENaC in cystic fibrosis epithelia in which excessive Na+ uptake contributes to salt and water imbalance. We show that prostasin mRNA and protein are strongly expressed by human airway epithelial cell lines, including immortalized JME/CF15 nasal epithelial cells homozygous for the DeltaF508 cystic fibrosis mutation. Epithelial cells transfected with vectors encoding recombinant soluble prostasin secrete active, tryptic peptidase that is highly sensitive to inactivation by aprotinin. When studied as monolayers in Ussing chambers, JME/CF15 cells exhibit amiloride-sensitive, transepithelial Na+ currents that are markedly diminished by aprotinin, suggesting regulation by serine-class peptidases. Overproduction of membrane-anchored prostasin in transfected JME/CF15 cells does not augment Na+ currents, and trypsin-induced increases are small, suggesting that baseline serine peptidase-dependent ENaC activation is maximal in these cells. To probe prostasin's involvement in basal ENaC activity, we silenced expression of prostasin using short interfering RNA targeting of prostasin mRNA's 3'-untranslated region. This drops ENaC currents to 26 +/- 9% of baseline. These data predict that prostasin is a major regulator of ENaC-mediated Na+ current in DeltaF508 cystic fibrosis epithelia and suggest that airway prostasin is a target for therapeutic inhibition to normalize ion current in cystic fibrosis airway.     

Enhancing the efficacy and persistency of Spodoptera litura (Fab.) nuclear polyhedrosis virus using UV irradiation protectants

To enhance the field persistency of S. litura nuclear polyhedrosis virus (SLNPV), three chemicals viz. cupric ammonium nitrate, tinopal and cupric sulphate were tried as protectants (0.01 mg/ml) against natural sunlight (UV) irradiation. On exposure for 8 hr and subsequent bioassaying (diet surface treatment), it was found that cupric sulphate protected the polyhedrosis inclusion bodies (PIBs), recording 95.56% mortality which was statistically at par with unexposed PIBs recording 97.78% mortality.     

Electrical signals propagate unbiased in cortex

The greater spatial coherence of local field potentials (LFPs) compared with that of spiking activity has been attributed to frequency-dependent propagation of signals through the cortical medium. However, in this issue of Neuron, Logothetis and colleagues show that signal propagation within cortex is largely unbiased across different frequencies, thus suggesting a more functional and interpretable basis of LFP coherence.     

Intracellular adaptor molecules and AR signalling in the tumour microenvironment

Androgen deprivation therapy is the mainstay for treating advanced prostate cancer. A better understanding in the complexity of the androgen receptor (AR) signalling pathway has highlighted that this form of treatment is not sufficient. Since Huggins and Hodges made their crucial observations on the benefits of castration for prostate cancer, significant progress has been achieved in understanding the importance of the cross-talk between the hormone signalling pathway and the kinase signalling network. We now know that preventing androgen production or ligand binding to the AR does not necessarily mark the end of the road for prostate tumour growth. Emerging evidence suggests that there exists a complex set of compensatory mechanisms which allows growth factors to push the transformed cells into a ‘survival adaptation mode’ within the tumour microenvironment. An increase in autocrine and paracrine cascades of growth factor are the most commonly reported events to correlate with progression of androgen-dependent disease to a disseminated androgen independent state. The mechanism of how growth factors can sustain AR activation when cells are deprived of androgens is unknown. This is due to the lack of information about the critical factors linking the intracellular signalling molecules associated with the downstream AR signalling events triggered by growth factors. The aim of this mini review is to highlight a potentially new insight into how intracellular adaptor molecules activated by growth factors may influence and act as a molecular switch to allow the continuation of AR activity in the presence of therapeutic anti-androgens following chemical or surgical castration.     

Structural and docking studies of <Emphasis Type="Italic">Leucaena leucocephala</Emphasis> Cinnamoyl CoA reductase

Lignin, a major constituent of plant call wall, is a phenolic heteropolymer. It plays a major role in the development of plants and their defense mechanism against pathogens. Therefore Lignin biosynthesis is one of the critical metabolic pathways. In lignin biosynthesis, the Cinnamoyl CoA reductase is a key enzyme which catalyzes the first step in the pathway. Cinnamoyl CoA reductase provides the substrates which represent the main transitional molecules of lignin biosynthesis pathway, exhibits a high in vitro kinetic preference for feruloyl CoA. In present study, the three-dimensional model of cinnamoyl CoA reductase was constructed based on the crystal structure of Grape Dihydroflavonol 4-Reductase. Furthermore, the docking studies were performed to understand the substrate interactions to the active site of CCR. It showed that residues ARG51, ASN52, ASP54 and ASN58 were involved in substrate binding. We also suggest that residue ARG51 in CCR is the determinant residue in competitive inhibition of other substrates. This structural and docking information have prospective implications to understand the mechanism of CCR enzymatic reaction with feruloyl CoA, however the approach will be applicable in prediction of substrates and engineering 3D structures of other enzymes as well.     

In silico characterization of thermostable lipases

Thermostable lipases are of high priority for industrial applications as they are endowed with the capability of carrying out diversified reactions at elevated temperatures. Extremophiles are their potential source. Sequence and structure annotation of thermostable lipases can elucidate evolution of lipases from their mesophilic counterparts with enhanced thermostability hence better industrial potential. Sequence analysis highlighted the conserved residues in bacterial and fungal thermostable lipases. Higher frequency of AXXXA motif and poly Ala residues in lid domain of thermostable Bacillus lipases were distinguishing characteristics. Comparison of amino acid composition among thermostable and mesostable lipases brought into light the role of neutral, charged and aromatic amino acid residues in enhancement of thermostability. Structural annotation of thermostable lipases with that of mesostable lipases revealed some striking features which are increment of gamma turns in thermostable lipases; being first time reported in our paper, longer beta strands, lesser beta-branched residues in helices, increase in charged-neutral hydrogen bonding pair, hydrophobic-hydrophobic contact and differences in the N-cap and C-cap residues of the α helices. Conclusively, it can be stated that subtle changes in the arrangement of amino acid residues in the tertiary structure of lipases contributes to enhanced thermostability.