Glycosylphosphatidylinositol-anchored proteins play an important role in the biogenesis of the Alzheimer's amyloid beta-protein.

The Alzheimer's amyloid protein (Abeta) is released from the larger amyloid beta-protein precursor (APP) by unidentified enzymes referred to as beta- and gamma-secretase. beta-Secretase cleaves APP on the amino side of Abeta producing a large secreted derivative (sAPPbeta) and an Abeta-bearing C-terminal derivative that is subsequently cleaved by gamma-secretase to release Abeta. Alternative cleavage of the APP by alpha-secretase at Abeta16/17 releases the secreted derivative sAPPalpha. In yeast, alpha-secretase activity has been attributed to glycosylphosphatidylinositol (GPI)-anchored aspartyl proteases. To examine the role of GPI-anchored proteins, we specifically removed these proteins from the surface of mammalian cells using phosphatidylinositol-specific phospholipase C (PI-PLC). PI-PLC treatment of fetal guinea pig brain cultures substantially reduced the amount of Abeta40 and Abeta42 in the medium but had no effect on sAPPalpha. A mutant CHO cell line (gpi85), which lacks GPI-anchored proteins, secreted lower levels of Abeta40, Abeta42, and sAPPbeta than its parental line (GPI+). When this parental line was treated with PI-PLC, Abeta40, Abeta42, and sAPPbeta decreased to levels similar to those observed in the mutant line, and the mutant line was resistant to these effects of PI-PLC. These findings provide strong evidence that one or more GPI-anchored proteins play an important role in beta-secretase activity and Abeta secretion in mammalian cells. The cell-surface GPI-anchored protein(s) involved in Abeta biogenesis may be excellent therapeutic target(s) in Alzheimer's disease.     

Glutathione <Emphasis Type="BoldItalic">S</Emphasis>-transferase P1 gene polymorphisms and susceptibility to coronary artery disease in a subgroup of north Indian population

The present study aimed to investigate the association of \(\hbox {g}.313\hbox {A}{>}\hbox {G}\) and \(\hbox {g}.341\hbox {C}{>}\hbox {T}\) polymorphisms of GSTP1 with coronary artery disease (CAD) in a subgroup of north Indian population. In the present case–control study, CAD patients (\(n = 200\)) and age-matched, sex-matched and ethnicity-matched healthy controls (\(n = 200\)) were genotyped for polymorphisms in GSTP1 using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Genotype distribution of \(\hbox {g}.313\hbox {A}{>}\hbox {G}\) and \(\hbox {g}.341\hbox {C}{>}\hbox {T}\) polymorphisms of GSTP1 gene was significantly different between cases and controls (\(P = 0.005\) and 0.024, respectively). Binary logistic regression analysis showed significant association of A/G (odds ratio (OR): 1.6, 95% CI: 1.08–2.49, \(P = 0.020\)) and G/G (OR: 3.1, 95% CI: 1.41–6.71, P \(=\) 0.005) genotypes of GSTP1 \(\hbox {g}.313\hbox {A}{\!>\!}\hbox {G}\), and C/T (OR: 5.8, 95% CI: 1.26–26.34, \(P = 0.024\)) genotype of GSTP1 \(\hbox {g}.341\hbox {C}{>}\hbox {T}\) with CAD. The A/G and G/G genotypes of \(\hbox {g}.313\hbox {A}{>}\hbox {G}\) and C/T genotype of \(\hbox {g}.341\hbox {C}{>}\hbox {T}\) conferred 6.5-fold increased risk for CAD (OR: 6.5, 95% CI: 1.37–31.27, \(P = 0.018\)). Moreover, the recessive model of GSTP1 \(\hbox {g}.313\hbox {A}{>}\hbox {G}\) is the best fit inheritance model to predict the susceptible gene effect (OR: 2.3, 95% CI: 1.11–4.92, \(P = 0.020\)). In conclusion, statistically significant associations of GSTP1 \(\hbox {g}.313\hbox {A}{>}\hbox {G}\) (A/G, G/G) and \(\hbox {g}.341\hbox {C}{>}\hbox {T}\) (C/T) genotypes with CAD were observed.     

Heterogeneous nanomechanical properties of type I collagen in longitudinal direction

Biomechanics and Modeling in Mechanobiology - Collagen is an abundant structural biopolymer in mammal vertebrates, providing structural support as well as mechanical integrity for connective...     

In vitro anti- biofilm and anti-bacterial activity of Sesbania grandiflora extract against Staphylococcus aureus

The main objective of this research is to investigate the anti-biofilm and anti-bacterial activity of Sesbania grandiflora (S. grandiflora) against Staphylococcus aureus. S. grandiflora extract were prepared and analyzed with UV –Vis spectroscopy, Fourier transform infrared spectroscopy, Dynamic light scattering. Biofilm forming pathogens were identified by congo-red assay. Quantification of Extracellular polymeric substance (EPS) particularly protein and carbohydrate were calculated. The efficacy of the herbal extract S. grandiflora and its inhibition against the pathogenic strain of S. aureus was also evaluated. The gradual decrease or disappearance of peaks reveals the reduction of protein and carbohydrate content in the EPS of S. aureus when treated with S. grandiflora. The antibacterial activity of S. grandiflora extract against the bacterial strain S. aureus showed that the extract were more active against the strain. To conclude, anti-biofilm and antibacterial efficacy of S. grandiflora plays a vital role over biofilm producing pathogens and act as a good source for controlling the microbial population.     

Facile synthesis, ex-vivo and in vitro screening of 3-sulfonamide derivative of 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxylic acid piperidin-1-ylamide (SR141716) a potent CB1 receptor antagonist

Facile synthesis of biaryl pyrazole sulfonamide derivative of 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxylic acid piperidin-1-ylamide (SR141716, 1) and an investigation of the effect of replacement of the –CO group in the compound 1 by the –SO₂ group in the aminopiperidine region is reported. Primary ex-vivo pharmacological testing and in vitro screening of sulfonamide derivative 2 showed the loss of CB1 receptor antagonism.     

Identification of the major TG4 cross‐linking sites in the androgen‐dependent SVS I exclusively expressed in mouse seminal vesicle

SVS I was exclusively expressed in seminal vesicle in which the protein was immunolocalized primarily to the luminal epithelium of mucosal folds. The developmental profile of its mRNA expression was shown to be androgen‐dependent, manifesting a positive correlation with the animal's maturation. There are 43 glutamine and 43 lysine residues in one molecule of SVS I, which is one of the seven major monomer proteins tentatively assigned on reducing SDS–PAGE during the resolution of mouse seminal vesicle secretion. Based on the fact that SVS I‐deduced protein sequence consists of 796 amino acid residues, we produced 7 recombinant polypeptide fragments including residues 1–78/F1, residues 79–259/F2, residues 260–405/F3, residues 406–500/F4, residues 501–650/F5, residues 651–715/F6, and residues 716–796/F7, and measured the covalent incorporation of 5‐(biotinamido)pentylamine (BPNH₂) or biotin‐TVQQEL (A25 peptide) to each of F1‐to‐F7 by type 4 transglutaminase (TG₄) from the coagulating gland secretion. F2 was active to a greater extent than the other fragments during the BPNH₂‐glutamine incorporation, and a relatively low extent of A25‐lysine cross link was observed with all of the seven fragments. The MS analysis of BPNH₂‐F2 conjugate identified Q²³² and Q²⁵⁴ as the two major TG₄ cross‐linking sites. This was substantiated by the result that much less BPNH₂ was cross‐linked to any one of the three F2 mutants, including Q232G and Q254G obtained from single‐site mutation, and Q232G/Q254G from double‐site mutation. J. Cell. Biochem. 107: 899–907, 2009. © 2009 Wiley‐Liss, Inc.     

Astrocytes are poised for lactate trafficking and release from activated brain and for supply of glucose to neurons

Brain is a highly-oxidative organ, but during activation, glycolytic flux is preferentially up-regulated even though oxygen supply is adequate. The biochemical and cellular basis of metabolic changes during brain activation and the fate of lactate produced within brain are important, unresolved issues central to understanding brain function, brain images, and spectroscopic data. Because in vivo brain imaging studies reveal rapid efflux of labeled glucose metabolites during activation, lactate trafficking among astrocytes and between astrocytes and neurons was examined after devising specific, real-time, sensitive enzymatic fluorescent assays to measure lactate and glucose levels in single cells in adult rat brain slices. Astrocytes have a 2- to 4-fold faster and higher capacity for lactate uptake from extracellular fluid and for lactate dispersal via the astrocytic syncytium compared to neuronal lactate uptake from extracellular fluid or shuttling of lactate to neurons from neighboring astrocytes. Astrocytes can also supply glucose to neurons as well as glucose can be taken up by neurons from extracellular fluid. Astrocytic networks can provide neuronal fuel and quickly remove lactate from activated glycolytic domains, and the lactate can be dispersed widely throughout the syncytium to endfeet along the vasculature for release to blood or other brain regions via perivascular fluid flow.     

GBF1, a Guanine Nucleotide Exchange Factor for Arf,Is Crucial for Coxsackievirus B3 RNA Replication

The replication of enteroviruses is sensitive to brefeldin A (BFA), an inhibitor of endoplasmic reticulum-to-Golgi network transport that blocks activation of guanine exchange factors (GEFs) of the Arf GTPases. Mammalian cells contain three BFA-sensitive Arf GEFs: GBF1, BIG1, and BIG2. Here, we show that coxsackievirus B3 (CVB3) RNA replication is insensitive to BFA in MDCK cells, which contain a BFA-resistant GBF1 due to mutation M832L. Further evidence for a critical role of GBF1 stems from the observations that viral RNA replication is inhibited upon knockdown of GBF1 by RNA interference and that replication in the presence of BFA is rescued upon overexpression of active, but not inactive, GBF1. Overexpression of Arf proteins or Rab1B, a GTPase that induces GBF1 recruitment to membranes, failed to rescue RNA replication in the presence of BFA. Additionally, the importance of the interaction between enterovirus protein 3A and GBF1 for viral RNA replication was investigated. For this, the rescue from BFA inhibition of wild-type (wt) replicons and that of mutant replicons of both CVB3 and poliovirus (PV) carrying a 3A protein that is impaired in binding GBF1 were compared. The BFA-resistant GBF1-M832L protein efficiently rescued RNA replication of both wt and mutant CVB3 and PV replicons in the presence of BFA. However, another BFA-resistant GBF1 protein, GBF1-A795E, also efficiently rescued RNA replication of the wt replicons, but not that of mutant replicons, in the presence of BFA. In conclusion, this study identifies a critical role for GBF1 in CVB3 RNA replication, but the importance of the 3A-GBF1 interaction requires further study.Enteroviruses are small, nonenveloped, positive-stranded RNA viruses that include many important pathogens, such as poliovirus (PV), coxsackievirus, echovirus, and human rhinovirus. Following virus entry and uncoating, the 7.5-kb enteroviral RNA genome is directly translated into a large polyprotein. This polyprotein is proteolytically processed by the virus-encoded proteases 2A^pro, 3C^pro, and 3CD^pro into the structural P1 region proteins and the nonstructural P2 and P3 region proteins that are involved in viral RNA replication.All RNA viruses with a positive-stranded genome induce the remodeling of cellular membranes to create a scaffold for genomic RNA replication. The organelle origin and morphology of these membranous replication sites, however, appear to vary for different viruses. Enteroviruses replicate their RNA genomes in nucleoprotein complexes that are associated with small vesicular membrane structures (6). The enteroviral proteins 2B, 2C, and 3A have been implicated in vesicle formation (4, 6, 27), but the mechanism and pathway of membrane reorganization are poorly understood. There are strong indications that these vesicular membranous structures, which are referred to here as “vesicles,” are derived from the early secretory pathway. Vesicles produced in PV-infected cells may form at the endoplasmic reticulum (ER) by the cellular COP-II budding machinery and may therefore share components with the membranous vesicles mediating ER-to-Golgi network transport (26). Further support for the involvement of the secretory pathway stems from the observation that brefeldin A (BFA), a well-known inhibitor of ER-to-Golgi network transport, completely inhibits enteroviral RNA replication (17, 20). In addition, the autophagocytic pathway appears to contribute to the formation of the membrane vesicles, many of which exhibit a double-membrane morphology characteristic of autophagosomes (18, 27). The utilization of individual components or reactions from different membrane metabolic pathways, rather than subversion of an entire pathway in toto, may represent a common strategy for building viral replication machinery.BFA inhibits activation of the small monomeric GTPase ADP ribosylation factor 1 (Arf1), a major regulator of intracellular protein transport (2). Arf1 cycles between an inactive, GDP-bound, cytosolic state and an active, GTP-bound, membrane-associated state, and this cycling is catalyzed by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (13). BFA blocks the activities of the large GEFs GBF1, BIG1, and BIG2 by stabilizing an intermediate, abortive complex with inactive Arf1 (23), thus efficiently preventing activation of Arf1 and eventually formation of transport intermediates.Not only the fact that BFA blocks enteroviral replication suggests a role for Arf1 and/or its large GEFs in this process; recently, it was shown that Arf1 accumulates on membranes during PV infection (3). Arf1 translocation to membranes can be induced independently by enterovirus protein 3A or 3CD in vitro (5), but the underlying mechanisms seem to differ; the 3A protein specifically triggers the recruitment of GBF1 to membranes, most likely through a direct interaction with this GEF (32, 33), whereas 3CD recruits BIG1 and BIG2 to membranes (3). Here, we report the involvement of Arf1 and its large BFA-sensitive GEFs in coxsackievirus B3 (CVB3) replication.     

Stabilization of the E* Form Turns Thrombin into an Anticoagulant

Previous studies have shown that deletion of nine residues in the autolysis loop of thrombin produces a mutant with an anticoagulant propensity of potential clinical relevance, but the molecular origin of the effect has remained unresolved. The x-ray crystal structure of this mutant solved in the free form at 1.55 Å resolution reveals an inactive conformation that is practically identical (root mean square deviation of 0.154 Å) to the recently identified E* form. The side chain of Trp²¹⁵ collapses into the active site by shifting >10 Å from its position in the active E form, and the oxyanion hole is disrupted by a flip of the Glu¹⁹²–Gly¹⁹³ peptide bond. This finding confirms the existence of the inactive form E* in essentially the same incarnation as first identified in the structure of the thrombin mutant D102N. In addition, it demonstrates that the anticoagulant profile often caused by a mutation of the thrombin scaffold finds its likely molecular origin in the stabilization of the inactive E* form that is selectively shifted to the active E form upon thrombomodulin and protein C binding.Serine proteases of the trypsin family are responsible for digestion, blood coagulation, fibrinolysis, development, fertilization, apoptosis, and immunity (1). Activation of the protease requires the transition from a zymogen form (2) and formation of an ion pair between the newly formed amino terminus of the catalytic chain and the side chain of the highly conserved residue Asp¹⁹⁴ (chymotrypsinogen numbering) next to the catalytic Ser¹⁹⁵. This ensures substrate access to the active site and proper formation of the oxyanion hole contributed by the backbone N atoms of Ser¹⁹⁵ and Gly¹⁹³ (3). The zymogen → protease conversion is classically associated with the onset of catalytic activity (3, 4) and provides a useful paradigm for understanding key features of protease function and regulation.Recent kinetic (5) and structural (6, 7) studies of thrombin, the key protease in the blood coagulation cascade (8), have drawn attention to a significant plasticity of the trypsin fold that impacts the function of the enzyme in a decisive manner. The active form of the protease, E, coexists with an inactive form, E*, that is distinct from the zymogen conformation (9). The E* form features a collapse of the 215–217 β-strand into the active site and a flip of the peptide bond between residues Glu¹⁹² and Gly¹⁹³ that disrupts the oxyanion hole. Importantly, the ion pair between Ile¹⁶ and Asp¹⁹⁴ remains intact, suggesting that E* is not equivalent to the zymogen form of the protease and that the E*-E equilibrium is established after the conversion from the zymogen form has taken place. Indeed, existing structures of the zymogen forms of trypsin (10), chymotrypsin (11), and chymase (12) feature a broken Ile¹⁶–Asp¹⁹⁴ ion pair but no collapse of the 215–217 β-strand. Stopped-flow experiments show that the E*-E conversion takes place on a time scale of <10 ms (5), as opposed to the much longer (100–1000 ms) time scale required for the zymogen-protease conversion (13, 14).The E* form is not a peculiarity of thrombin. The collapse of the 215–217 β-strand into the active site is observed in the inactive form of αI-tryptase (15), the high temperature requirement-like protease (16), complement factor D (17), granzyme K (18), hepatocyte growth factor activator (19), prostate kallikrein (20), and prostasin (21). A disrupted oxyanion hole is observed in complement factor B (22) and the arterivirus protease Nsp4 (23). The most likely explanation for the widespread occurrence of inactive conformations of trypsin-like proteases is that the E*-E equilibrium is a basic property of the trypsin fold that fine tunes activity and specificity once the zymogen → protease conversion has taken place (9).The new paradigm established by the E*-E equilibrium has obvious physiological relevance. In the case of complement factors, kallikreins, tryptase, and some coagulation factors must be kept to a minimum until binding of a trigger factor ensues. Stabilization of E* may afford a resting state of the protease waiting for action, as seen for other systems (24–28). For example, factor B is mostly inactive until binding of complement factor C3 unleashes catalytic activity at the site where amplification of C3 activation is most needed prior to formation of the membrane attack complex (29). Indeed, the crystal structure of factor B reveals a conformation with the oxyanion hole disrupted by a flip of the 192–193 peptide bond (22), as observed in the E* form of thrombin (6, 7).The allosteric equilibrium as shown in Scheme 1, involves the rates for the E* → E transition, k₁, and backward, k₋₁, that define the equilibrium constant r = k₋₁/k₁ = [E*]/[E] (5). The value of k_cat/K_m for an enzyme undergoing the E*-E equilibrium is as shown in Equation 1 (30), where s_E is the value of s for the E form, and obviously s_E* = 0. Likewise, the binding of an inhibitor to the enzyme undergoing the E*-E equilibrium is shown in Equation 2, where K_E is the value of the equilibrium association constant K for the E form, and K_E* = 0. As the value of r increases upon stabilization of E*, the values of s and K in Equations 1 and 2 decrease without limits. Hence, stabilization of E* has the potential to completely abrogate substrate hydrolysis (s → 0) or inhibitor binding (K → 0). However, binding of a suitable cofactor could restore activity by triggering the E* → E transition. This suggests a simple explanation for the anticoagulant profile observed in a number of thrombin mutants that have poor activity toward all physiological substrates but retain activity toward the anticoagulant protein C in the presence of the cofactor thrombomodulin (31–34). Here we report evidence that stabilization of E* provides a molecular mechanism to turn thrombin into an anticoagulant.