Identification of the AMP binding sites of rabbit phosphofructo-1-kinase isozymes B and C

Rabbit liver phosphofructo-1-kinase, designated isozyme B, and rabbit brain phosphofructokinase, which contains all three isozymes as heteropolymers, have been modified by [14C]fluorosulfonylbenzoyladenosine (FSBAdo). Several lines of evidence supported modification at the binding site for AMP. The modification proceeded to the extent of 2 to 4 mol of reagent incorporated per mol of tetramer, and AMP protected against the reaction. The kinetic properties of modified isozymes A and B and of modified brain phosphofructokinase were examined and compared to their unmodified forms. It was observed that modification greatly diminished ATP inhibition of all of the isozymes. Furthermore, equilibrium binding studies of modified phosphofructokinase B showed a greatly diminished capacity and affinity for cyclic AMP. Cyclic AMP had little or no influence on the properties of modified A isozyme or brain phosphofructokinase, but was capable of further deinhibiting modified B isozyme, apparently at sites remaining unmodified by FSBAdo. Phosphofructokinase B, modified by radiolabeled FSBAdo, was digested by trypsin, and the digest separated by high-pressure liquid chromatography. The labeled peptide was isolated and sequenced to provide the sequence: Asn-Tyr-Gly-Thr-Lys-Leu-Gly-Val-Lys, with the lysine in the fifth position being the site of modification. To isolate isozyme C, a monoclonal antibody to this isozyme was produced by injecting purified rabbit brain phosphofructokinase into mice, and subsequently selecting for those clones that recognized brain phosphofructokinase but not purified phosphofructokinases A and B. The selected monoclonal was specific for native rabbit isozyme C and would not recognize mouse or rat brain phosphofructokinases. Linking the antibody to an inert phase provided an efficient means of purifying rabbit isozyme C from rabbit brain. The enzyme so recovered retained little of its original activity, but the method provided a simple technique for the preparation of enzyme for protein chemistry studies. The modified C isozyme was isolated on the immuno-affinity column and digested with trypsin. A tryptic peptide bearing the label was isolated and sequenced to provide the structure: Asn-Phe-Gly-Thr-Lys-Ile-Ser-Ala-Arg, with position 5 being the site of modification. The sequences of isozymes B and C are homologous to the site of modification of the A isozyme by FSBAdo.     

Age-associated alterations in human natural killer cells. 2. Increased frequency of selective NK subsets

We have analyzed the peripheral blood lymphocytes from healthy volunteers (20 to 94 years) for the expression of natural killer (NK) cell surface markers, NK activity, and B-cell proliferative response. An increase (2- to 3.5-fold) in relative percentage and absolute number of lymphocytes expressing Leu-7 (HNK-1) or Leu-11a (CD 16) antigen was found in the elderly group (greater than 80 years) as compared to young adults (less than 40 years). A two-color immunofluorescence analysis revealed that the age-associated increment was both progressive and selective; the actual increase occurred in Leu-7+11a+ and Leu-7+11a- populations (subsets with variable and weak NK activity) but not in the Leu-7-11a+ (most active) subset. There is a corresponding decrease in the 7-11a- cells. The ratios of 7+11a+/7-11a+ and 7+11a-/7-11a+ cells doubled with advancing age. Linear regression analysis suggests that the 7-11a+ cells are highly preserved through human senescence and the ratio of 7+11a- cells to the most stable subset, 7-11a+, could expand nearly 100-fold from birth to old age. Further analysis of Leu-7+ cells for the coexpression of Leu-11c (an epitope of Leu-11a) confirmed a similar pattern of changes in 7+11c+ and 7+11c- NK subsets with advancing age. The frequency of Leu-11+ (epitopes 11a+ or 11c+), but not of the subsets of 7+ phenotype (7+11a- or 7+11c-), correlates well with the NK activity (spontaneous killing of K562 tumor cell line). The 7+11c+ cells may directly or indirectly be responsible for the increase in NK activity observed with a majority of aged donors. The inverse relationship observed between the mitogenic response of lymphocytes to pokeweed mitogen (PWM) and the initial frequency of 7+11a-, but not other phenotypes, raises a potential functional significance for the expansion of the 7+11a-(7+11c-) subset. These age-associated NK phenotypic changes provide a cellular basis for our observations on age-associated increase in NK activity and decrease in mitogenic response to PWM.     

Lectin- and ionophore-stimulated Ca2+ influx in murine lymphocytes: inhibition by disialoganglioside

Gangliosides suppress lymphocyte mitogenesis when added exogenously to the cells. On the premise that the mechanism of ganglioside action may be an interference with primary induction events, mitogen-induced 45Ca2+ influx in murine lymphocytes was studied. Disialoganglioside (GD1a) at physiopathological concentrations inhibits concanavalin A-induced 45Ca2+ uptake as well as blast transformation. The suppressive action of GD1a is both concentration dependent (50% suppression at 13 microM) and very rapid (within 1 min). GD1a is not cytotoxic nor does it significantly alter the rate of Ca2+ efflux. The uptake studies were extended to A23187, a compound with mitogenic and specific divalent cation ionophore activities. Ca2+ uptake by lymphoid cells from AKR/J, Swiss, and CBA mice is stimulated by A23187; and GD1a, in a dose-dependent manner, inhibits the ionophore-induced 45Ca2+ influx. Pretreatment of thymocytes with GD1a renders the cells greatly insensitive to the subsequent ionophore activity of A23187. The results suggest that exogenous gangliosides may function as an inhibitor of some of the mitogen-triggered early events, including Ca2+ metabolism, and thus influence the immunological behavior of intact lymphoid cells.     

Molecular docking analysis of stachydrine and sakuranetin with IL-6 and TNF-α in the context of inflammation

Inflammation is a process triggered by pro-inflammatory cytokines and anti-inflammatory molecules. Therefore, it is of interest to document the anti-inflammatory activity of Stachydrine and Sakuranetin against the inflammatory target proteins IL-6 and TNF-α by using molecular docking analysis. Both compounds showed good binding features with the selected target proteins. Compared to Sakuranetin, the Stachydrine have low binding energy and good hydrogen bond interactions. Hence, data show that Stachydrine possessed high and specific inhibitory activity on tumor necrosis factor-α and interleukin-6.     

Cation–π Interactions in β-Lactamases: The Role in Structural Stability

β-lactam group of antibiotics is the most widely used therapeutic molecules for treating bacterial infections. The main mode of bacterial resistance to β-lactams is by β-lactamases. In the present study, we report our results on the role of cation–π interactions in β-lactamases and their environmental preferences. The number of interactions formed by arginine is higher than lysine in the cationic group, while tyrosine is comparatively higher than phenylalanine and tryptophan in the π group. Our results indicate that cation–π interactions might play an important role in the global conformational stability of β-lactamases.     

Optimization of a Method for Chromatin Immunoprecipitation Assays in the Marine Invertebrate Chordate Ciona

Chromatin immunoprecipitation (ChIP) assays allow the efficient characterization of the in vivo occupancy of genomic regions by DNA-binding proteins and thus facilitate the prediction of cis-regulatory sequences in silico and guide their validation in vivo. For these reasons, these assays and their permutations (e.g., ChIP-on-chip and ChIP-sequencing) are currently being extended to several non-mainstream model organisms, as the availability of specific antibodies increases. Here, we describe the development of a polyclonal antibody against the Brachyury protein of the marine invertebrate chordate Ciona intestinalis and provide a detailed ChIP protocol that should be easily adaptable to other marine organisms.     

Azetidines and spiro azetidines as novel P2 units in hepatitis C virus NS3 protease inhibitors

Herein, we report the synthesis and structure–activity relationship studies of new analogs of boceprevir 1 and telaprevir 2. Introduction of azetidine and spiroazetidines as a P2 substituent that replaced the pyrrolidine moiety of 1 and 2 led to the discovery of a potent hepatitis C protease inhibitor 37c (EC₅₀ = 0.8 μM).     

Nano-transfersomal formulations for transdermal delivery of asenapine maleate: in vitro and in vivo performance evaluations

Context: Asenapine maleate (ASPM) is an antipsychotic drug for the treatment of schizophrenia and bipolar disorder. Extensive metabolism makes the oral route inconvenient for ASPM.

Objective: The objective of this study is to increase ASPM bioavailability via transdermal route by improving the skin permeation using combined strategy of chemical and nano-carrier (transfersomal) based approaches.

Materials and methods: Transfersomes were prepared by the thin film hydration method using soy-phosphatidylcholine (SPC) and sodium deoxycholate (SDC). Transfersomes were characterized for particle size, polydispersity index (PDI), zeta potential (ZP), entrapment efficiency, surface morphology, and in vitro skin permeation studies. Various chemical enhancers were screened for skin permeation enhancement of ASPM. Optimized transfersomes were incorporated into a gel base containing suitable chemical enhancer for efficient transdermal delivery. In vivo pharmacokinetic study was performed in rats to assess bioavailability by transdermal route against oral administration.

Results and discussion: Optimized transfersomes with drug:SPC:SDC weight ratio of 5:75:10 were spherical with an average size of 126.0?nm, PDI of 0.232, ZP of??43.7?mV, and entrapment efficiency of 54.96%. Ethanol (20% v/v) showed greater skin permeation enhancement. The cumulative amount of ASPM permeated after 24?h (Q₂₄) by individual effect of ethanol and transfersome, and in combination was found to be 160.0, 132.9, and 309.3?μg, respectively, indicating beneficial synergistic effect of combined approach. In vivo pharmacokinetic study revealed significant (p?Conclusion: Dual strategy of permeation enhancement was successful in increasing the transdermal permeation and bioavailability of ASPM.     

Circulating miRNA-33: a potential biomarker in patients with coronary artery disease

Background: Circulating microRNAs (miRNA) are present in body fluids in stable, cell-free form. Likewise, these miRNAs can be identified in various stages of coronary artery disease (CAD) such as inflammation, endothelial dysfunction, proliferation and atherosclerosis among others. miRNA expression levels can be identified.

Aims and objectives: To determine the expression of circulating miRNAs (miR-126, miR-92, miR-33, miR-145 and miR-155) in CAD patients of Indian origin.

Material and methods: miRNA profiling analysis in blood plasma was performed by quantitative real-time-PCR (qRT-PCR) in 60 angiographically verified subjects including 30 CAD patients and 30 age- and gender-matched controls. Association between the expression of all five circulating miRNAs and clinical characteristics of patients with CAD were analysed using Medcalc statistics. The severity of CAD was assessed using SYNTAX score (SS).

Results: Expression of plasma miR-33 increased by 2.9 folds in CAD patients than in control group (p value ≥0.002) also it was found that miR-33 expression levels in mild cases (SS: ≤22) were significantly higher than CAD controls. There was a modest negative correlation between miR-33 and total cholesterol/high density lipoprotein ratio, triglycerides and very low density lipoprotein.

Conclusion: The study reports a significant association between increased levels of plasma miR-33 and CAD. Thus, plasma miR-33 appears to be a promising non-invasive biomarker, but requires further validation in a large cohort.     

Modification of Axial Fiber Contact Residues Impact Sickle Hemoglobin Polymerization by Perturbing a Network of Coupled Interactions

The identity of intermolecular contact residues in sickle hemoglobin (HbS) fiber is largely known. However, our knowledge about combinatorial effects of two or more contact sites or the mechanistic basis of such effects is rather limited. Lys16, His20, and Glu23 of the α-chain occur in intra-double strand axial contacts in the sickle hemoglobin (HbS) fiber. Here we have constructed two novel double mutants, HbS (K16Q/E23Q) and (H20Q/E23Q), with a view to delineate cumulative impact of interactions emanating from the above contact sites. Far-UV and visible region CD spectra of the double mutants were similar to the native HbS indicating the presence of native-like secondary and tertiary structure in the mutants. The quaternary structures in both the mutants were also preserved as judged by the derivative UV spectra of liganded (oxy) and unliganded (deoxy) forms of the double mutants. However, the double mutants displayed interesting polymerization behavior. The polymerization behaviour of the double mutants was found to be non-additive of the individual single mutants. While HbS (H20Q/E23Q) showed inhibitory effect similar to that of HbS (E23Q), the intrinsic inhibitory propensity of the associated single mutants was totally quelled in HbS (K16Q/E23Q) double mutant. Molecular dynamics (MD) simulations studies of the isolated α-chains as well as a module of the fiber containing the double and associated single mutants suggested that these contact sites at the axial interface of the fiber impact HbS polymerization through a coupled interaction network. The overall results demonstrate a subtle role of dynamics and electrostatics in the polymer formation and provide insights about interaction-linkage in HbS fiber assembly.