Crystal Structure of Full-length Mycobacterium tuberculosis H37Rv Glycogen Branching Enzyme: INSIGHTS OF N-TERMINAL β-SANDWICH IN SUBSTRATE SPECIFICITY AND ENZYMATIC ACTIVITY*

The open reading frame Rv1326c of Mycobacterium tuberculosis (Mtb) H37Rv encodes for an α-1,4-glucan branching enzyme (MtbGlgB, EC 2.4.1.18, Uniprot entry {"type":"entrez-protein","attrs":{"text":"Q10625","term_id":"1707934","term_text":"Q10625"}}Q10625). This enzyme belongs to glycoside hydrolase (GH) family 13 and catalyzes the branching of a linear glucose chain during glycogenesis by cleaving a 1→4 bond and making a new 1→6 bond. Here, we show the crystal structure of full-length MtbGlgB (MtbGlgBWT) at 2.33-Å resolution. MtbGlgBWT contains four domains: N1 β-sandwich, N2 β-sandwich, a central (β/α)₈ domain that houses the catalytic site, and a C-terminal β-sandwich. We have assayed the amylase activity with amylose and starch as substrates and the glycogen branching activity using amylose as a substrate for MtbGlgBWT and the N1 domain-deleted (the first 108 residues deleted) MtbΔ108GlgB protein. The N1 β-sandwich, which is formed by the first 105 amino acids and superimposes well with the N2 β-sandwich, is shown to have an influence in substrate binding in the amylase assay. Also, we have checked and shown that several GH13 family inhibitors are ineffective against MtbGlgBWT and MtbΔ108GlgB. We propose a two-step reaction mechanism, for the amylase activity (1→4 bond breakage) and isomerization (1→6 bond formation), which occurs in the same catalytic pocket. The structural and functional properties of MtbGlgB and MtbΔ108GlgB are compared with those of the N-terminal 112-amino acid-deleted Escherichia coli GlgB (ECΔ112GlgB).     

Introductory gestures before songbird vocal displays are shaped by learning and biological predispositions

Numerous animal displays begin with introductory gestures. For example, lizards start their head-bobbing displays with introductory push-ups, and many songbirds begin their vocal displays by repeating introductory notes (INs) before producing their learned song. Among songbirds, the acoustic structure and the number of INs produced before song vary considerably between individuals in a species. While similar variation in songs between individuals is a result of learning, whether variations in INs are also due to learning remains poorly understood. Here, using natural and experimental tutoring with male zebra finches, we show that mean IN number and IN acoustic structure are learned from a tutor. Interestingly, IN properties and how well INs were learned, were not correlated with the accuracy of song imitation and only weakly correlated with some features of songs that followed. Finally, birds artificially tutored with songs lacking INs still repeated vocalizations that resembled INs, before their songs, suggesting biological predispositions in IN production. These results demonstrate that INs, just like song elements, are shaped both by learning and biological predispositions. More generally, our results suggest mechanisms for generating variation in introductory gestures between individuals while still maintaining the species-specific structure of complex displays like birdsong.     

Binding mechanism of caffeic acid and simvastatin to the integrin linked kinase for therapeutic implications: a comparative docking and MD simulation studies

Abstract

Integrin linked kinase (ILK) is a Ser/Thr kinase, which regulates various integrin mediated signaling pathways, and is involved in cell adhesion, migration and differentiation. Alteration in the ILK is responsible for abnormal functioning of the cell system, which may lead to the cancer progression and metastasis. Caffeic acid (CA) and simvastatin are used as antioxidant and possess anticancer properties. Thus, inhibiting the kinase activity of ILK by CA and simvastatin may be implicated in the cancer therapy. In this study, we have performed molecular docking followed by 100?ns MD simulations to understand the interaction mechanism of ILK protein with the CA and simvastatin. Average potential energy was found to be highest in case of ILK–CA complex (?770,949?kJ/mol). Binding free energy was found to be higher in case of simvastatin than CA. Our results indicate that simvastatin binds more effectively to the active pocket of ILK. We further performed MTT assay to understand its anticancer potential. Simvastatin shows the IC₅₀ values for HepG2 and MCF-7 as 19.18?±?0.12 and 13.84?±?0.22?µM, respectively. However, the IC₅₀ value of CA on HepG2 and MCF-7 was reported as 175.50?±?1.44 and 144.90?±?1.53?µM, respectively. Our study provides a deeper insight into the binding mechanism of simvastatin and CA to ILK, which further opens a promising channel for their implications in cancer therapy.     

Immunological responses to recombinant cysteine synthase A of Brucella abortus in BALB/c mice

Brucellosis is a worldwide zoonotic disease. No Brucella vaccine is available for use in humans, and existing animal vaccines have limitations. There is a need to develop a safe and effective vaccine against human and animal brucellosis. In the present study, we generated recombinant cysteine synthase A (rCysK) of Brucella abortus in Escherichia coli and purified it up to homogeneity by metal affinity chromatography. The immunogenicity and protective efficacy of purified rCysK were evaluated in BALB/c mice with Freund’s adjuvant, aluminium hydroxide gel or without any adjuvant. High titres of anti-rCysK IgG antibody predominated by IgG1 were observed in all immunized mice. After stimulation with rCysK, the spleen lymphocytes of mice immunized with CysK formulated with aluminium hydroxide gel produced significant levels of IFN-γ. Protection against challenge with virulent B. abortus strain 544 was determined in BALB/c mice, and significant protection was observed in all CysK immunized groups when compared with PBS control. Among all the CysK vaccine groups, comparatively better protection was observed in mice immunized with aluminium hydroxide gel (1.064 units of protection). Overall, the results of the study suggest that rCysK induces primarily Th2 type of immune response and provides partial protection against B. abortus challenge.     

Callus culture and in vitro biosynthesis of cardiac glycosides from Calotropis gigantea (L.) Ait

Calotropis gigantea (L.) Ait., belonging to the family Asclepiadaceae, is a source of many cardiac glycosides (CGs) and their steroidal moieties (genins). These CGs have been reported to have anti-proliferative activity on tumor cell lines and are potential targets for cancer chemotherapy. However, the abundance of CGs in wild plants is particularly restricted and it is difficult to isolate the desired compound in required quantities. This study is the first attempt to standardize the induction and proliferation of callus from various explants of C. gigantea specifically for the production of CGs. Callus growth was accompanied by CG measurement using high-performance liquid chromatography-tandem mass spectrometry. Murashige and Skoog (MS) and modified Murashige and Skoog (MMS) media were optimized with various combinations and concentrations of auxin and cytokinin for induction and growth of calli from a range of explant sources. While leaves and stem explants resulted in greatest callus induction, MMS medium was found to be optimal. However, no CG was produced from callus grown on this medium. In contrast, the induction and proliferation of callus on MS medium were optimum at primary stages, but growth slowed during the third subculture. Therefore, calli were transferred to MMS medium to promote callus proliferation and production of CGs. As a result, three CGs and two genins were biosynthesized. Furthermore, the callus induction data in MS medium indicated that among different auxins, 2,4-dichlorophenoxyacetic acid was the best for callus induction compared to 1-naphthylacetic acid and indole-3-acetic acid. The data also revealed that the cytokinin/auxin ratio was critical rather than their independent presence for the induction of callus. Thus, the in vitro biosynthesis of targeted CGs may offer an alternative pathway for new source of anti-proliferative agents in required quantities.     

Respiration drives phase synchronization between blood pressure and RR interval following loss of cardiovagal baroreflex during vasovagal syncope

Loss of the cardiovagal baroreflex (CVB), thoracic hypovolemia, and hyperpnea contribute to the nonlinear time-dependent hemodynamic instability of vasovagal syncope. We used a nonlinear phase synchronization index (PhSI) to describe the extent of coupling between cardiorespiratory parameters, systolic blood pressure (SBP) or arterial pressure (AP), RR interval (RR), and ventilation, and a directional index (DI) measuring the direction of coupling. We also examined phase differences directly. We hypothesized that AP-RR interval PhSI would be normal during early upright tilt, indicating intact CVB, but would progressively decrease as faint approached and CVB failed. Continuous measurements of AP, RR interval, respiratory plethysomography, and end-tidal CO2 were recorded supine and during 70-degree head-up tilt in 15 control subjects and 15 fainters. Data were evaluated during five distinct times: baseline, early tilt, late tilt, faint, and recovery. During late tilt to faint, fainters exhibited a biphasic change in SBP-RR interval PhSI. Initially in fainters during late tilt, SBP-RR interval PhSI decreased (fainters, from 0.65±0.04 to 0.24±0.03 vs. control subjects, from 0.51±0.03 to 0.48±0.03; P<0.01) but then increased at the time of faint (fainters=0.80±0.03 vs. control subjects=0.42±0.04; P<0.001) coinciding with a change in phase difference from positive to negative. Starting in late tilt and continuing through faint, fainters exhibited increasing phase coupling between respiration and AP PhSI (fainters=0.54±0.06 vs. control subjects=0.27±0.03; P<0.001) and between respiration and RR interval (fainters=0.54±0.05 vs. control subjects=0.37±0.04; P<0.01). DI indicated respiratory driven AP (fainters=0.84±0.04 vs. control subjects=0.39±0.09; P<0.01) and RR interval (fainters=0.73±0.10 vs. control subjects=0.23±0.11; P<0.001) in fainters. The initial drop in the SBP-RR interval PhSI and directional change of phase difference at late tilt indicates loss of cardiovagal baroreflex. The subsequent increase in SBP-RR interval PhSI is due to a respiratory synchronization and drive on both AP and RR interval. Cardiovagal baroreflex is lost before syncope and supplanted by respiratory reflexes, producing hypotension and bradycardia.