An Uniquely Purified HCV NS3 Protease and NS4A21–34 Peptide Form a Highly Active Serine Protease Complex in Peptide Hydrolysis

The N-terminal domain of the hepatitis C virus (HCV) polyprotein containing the NS3 protease (residues 1027 to 1206) was expressed in Escherichia coli as a soluble protein under the control of the T7 promoter. The enzyme has been purified to homogeneity with cation exchange (SP-Sepharose HR) and heparin affinity chromatography in the absence of any detergent. The purified enzyme preparation was soluble and remained stable in solution for several weeks at 4°C. The proteolytic activity of the purified enzyme was examined, also in the absence of detergents, using a peptide mimicking the NS4A/4B cleavage site of the HCV polyprotein. Hydrolysis of this substrate at the expected Cys–Ala scissile bond was catalyzed by the recombinant protease with a pseudo second-order rate constant (k_cat/K_M) of 205 and 196,000 M⁻¹ s⁻¹, respectively, in the absence and presence of a central hydrophobic region (sequence represented by residues 21 to 34) of the NS4A protein. The rate constant in the presence of NS4A peptide cofactor was two orders of magnitude greater than reported previously for the NS3 protease domain. A significantly higher activity of the NS3 protease–NS4A cofactor complex was also observed with a substrate mimicking the NS4B/5A site (k_cat/K_M of 5180 ± 670 M⁻¹ s⁻¹). Finally, the optimal formation of a complex between the NS3 protease domain and the cofactor NS4A was critical for the high proteolytic activity observed.     

Immune response to leishmania: paradox rather than paradigm

The leishmaniases are a group of diseases caused by protozoan parasites of the genus Leishmania. Various Leishmania species can cause human infection, producing a spectrum of clinical manifestations. It is estimated that 350 million people are at risk, with a global yearly incidence of 1-1.5 million for cutaneous and 500,000 for visceral leishmaniasis (VL). VL is a major cause of morbidity and mortality in East Africa and the Indian subcontinent. Coinfection with HIV enhances the risk of the disease. The only control measure currently available in India is case detection and treatment with antimonial drugs, which are expensive, not always available and cannot be self-administered. Newer drugs like oral miltefosine have not become widely available. Vector and reservoir control is difficult due to the elusive nature of the vector and the diversity of the animal reservoir. A detailed knowledge of immune response to the parasite would help in designing prophylactic and therapeutic strategies against this infection.     

The N Termini of a-Subunit Isoforms Are Involved in Signaling between Vacuolar H+-ATPase (V-ATPase) and Cytohesin-2

Previously, we reported an acidification-dependent interaction of the endosomal vacuolar H⁺-ATPase (V-ATPase) with cytohesin-2, a GDP/GTP exchange factor (GEF), suggesting that it functions as a pH-sensing receptor. Here, we have studied the molecular mechanism of signaling between the V-ATPase, cytohesin-2, and Arf GTP-binding proteins. We found that part of the N-terminal cytosolic tail of the V-ATPase a2-subunit (a2N), corresponding to its first 17 amino acids (a2N(1–17)), potently modulates the enzymatic GDP/GTP exchange activity of cytohesin-2. Moreover, this peptide strongly inhibits GEF activity via direct interaction with the Sec7 domain of cytohesin-2. The structure of a2N(1–17) and its amino acids Phe⁵, Met¹⁰, and Gln¹⁴ involved in interaction with Sec7 domain were determined by NMR spectroscopy analysis. In silico docking experiments revealed that part of the V-ATPase formed by its a2N(1–17) epitope competes with the switch 2 region of Arf1 and Arf6 for binding to the Sec7 domain of cytohesin-2. The amino acid sequence alignment and GEF activity studies also uncovered the conserved character of signaling between all four (a1–a4) a-subunit isoforms of mammalian V-ATPase and cytohesin-2. Moreover, the conserved character of this phenomenon was also confirmed in experiments showing binding of mammalian cytohesin-2 to the intact yeast V-ATPase holo-complex. Thus, here we have uncovered an evolutionarily conserved function of the V-ATPase as a novel cytohesin-signaling receptor.     

Post-Operative Hypertension after Total Knee Arthroplasty and the Effects on Transfusion Rates

Transfusions are a cause of significant patient morbidity as well as expense. Anesthesia literature has examined controlled intraoperative hypotension as a means for reducing blood loss and transfusions. Our hypothesis is that inversely increased blood pressure post-operatively would then lead to increased blood loss and transfusions.We examined 105 consecutive patients who underwent TKA. We found a significant odds ratio of 1.123 for pre-operative hematocrit. For post-operative blood pressure, we calculated an insignificant odds ratio of 1.007, proving no relationship between post-operative blood pressure and transfusions.This is the first study to examine increased post-operative blood pressure''s contribution to transfusion rates. Although we confirmed that low pre-operative hematocrit contributes to increased transfusions, we did not find a relationship between post-operative blood pressure and transfusions.     

Time-Lapse Microscopy of Streptomyces coelicolor Growth and Sporulation

Bacteria from the genus Streptomyces are among the most complex of all prokaryotes; not only do they grow as a complex mycelium, they also differentiate to form aerial hyphae before developing further to form spore chains. This developmental heterogeneity of streptomycete microcolonies makes studying the dynamic processes that contribute to growth and development a challenging procedure. As a result, in order to study the mechanisms that underpin streptomycete growth, we have developed a system for studying hyphal extension, protein trafficking, and sporulation by time-lapse microscopy. Through the use of time-lapse microscopy we have demonstrated that Streptomyces coelicolor germ tubes undergo a temporary arrest in their growth when in close proximity to sibling extension sites. Following germination, in this system, hyphae extended at a rate of ~20 μm h⁻¹, which was not significantly different from the rate at which the apical ring of the cytokinetic protein FtsZ progressed along extending hyphae through a spiraling movement. Although we were able to generate movies for streptomycete sporulation, we were unable to do so for either the erection of aerial hyphae or the early stages of sporulation. Despite this, it was possible to demonstrate an arrest of aerial hyphal development that we suggest is through the depolymerization of FtsZ-enhanced green fluorescent protein (GFP). Consequently, the imaging system reported here provides a system that allows the dynamic movement of GFP-tagged proteins involved in growth and development of S. coelicolor to be tracked and their role in cytokinesis to be characterized during the streptomycete life cycle.     

In vitro culture in combination with aeroponics is an efficient means of mass propagation of Sarcostemma acidum: a rare medicinal plant of Indian arid zone

In Vitro Cellular & Developmental Biology - Plant - In vitro culture in combination with aeroponics is observed to be an efficient means for mass propagation of Sarcostemma acidum in the...