An unexpected phosphate binding site in Glyceraldehyde 3-Phosphate Dehydrogenase: Crystal structures of apo, holo and ternary complex of Cryptosporidium parvum enzyme

<p>Abstract</p> <p>Background</p> <p>The structure, function and reaction mechanism of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) have been extensively studied. Based on these studies, three anion binding sites have been identified, one 'Ps' site (for binding the C-3 phosphate of the substrate) and two sites, 'Pi' and 'new Pi', for inorganic phosphate. According to the original flip-flop model, the substrate phosphate group switches from the 'Pi' to the 'Ps' site during the multistep reaction. In light of the discovery of the 'new Pi' site, a modified flip-flop mechanism, in which the C-3 phosphate of the substrate binds to the 'new Pi' site and flips to the 'Ps' site before the hydride transfer, was proposed. An alternative model based on a number of structures of <it>B. stearothermophilus </it>GAPDH ternary complexes (non-covalent and thioacyl intermediate) proposes that in the ternary Michaelis complex the C-3 phosphate binds to the 'Ps' site and flips from the 'Ps' to the 'new Pi' site during or after the redox step.</p> <p>Results</p> <p>We determined the crystal structure of <it>Cryptosporidium parvum </it>GAPDH in the apo and holo (enzyme + NAD) state and the structure of the ternary enzyme-cofactor-substrate complex using an active site mutant enzyme. The <it>C. parvum </it>GAPDH complex was prepared by pre-incubating the enzyme with substrate and cofactor, thereby allowing free movement of the protein structure and substrate molecules during their initial encounter. Sulfate and phosphate ions were excluded from purification and crystallization steps. The quality of the electron density map at 2Å resolution allowed unambiguous positioning of the substrate. In three subunits of the homotetramer the C-3 phosphate group of the non-covalently bound substrate is in the 'new Pi' site. A concomitant movement of the phosphate binding loop is observed in these three subunits. In the fourth subunit the C-3 phosphate occupies an unexpected site not seen before and the phosphate binding loop remains in the substrate-free conformation. Orientation of the substrate with respect to the active site histidine and serine (in the mutant enzyme) also varies in different subunits.</p> <p>Conclusion</p> <p>The structures of the <it>C. parvum </it>GAPDH ternary complex and other GAPDH complexes demonstrate the plasticity of the substrate binding site. We propose that the active site of GAPDH can accommodate the substrate in multiple conformations at multiple locations during the initial encounter. However, the C-3 phosphate group clearly prefers the 'new Pi' site for initial binding in the active site.</p

CURRY LEAVES AS ALTERNATIVE MEDICINE IN HEAVY METAL INDUCED OCCUPATIONAL HEALTH HAZARDS

Workers in different industries are regularly exposed to heavy metals. Those metals enter their body through several routes (inhalation, food contamination etc.,) and accumulate in the tissues and induce generation of reactive oxygen species (ROS) leading to oxidative damages. Chronic, regular exposures result in health hazards. Certain physiological, biochemical and behavioural dysfunctions cumulate to pathological conditions. Many of the symptoms complained by those industry workers are hardly recognized to be related to occupational exposure to heavy metals, often unidentified as occupational health hazards with a story of metal induced oxidative stress beneath their etiology. Most of the synthetic conventional drugs which are extensively prescribed by clinicians for treatments of these diseases have adverse side effects and potent cytotoxicity. Herbal remedy can be a safe substitute. The heavy metals induce generation of ROS and the phyto-components have the potential to scavenge those and boost the body's endogenous antioxidant system. They have no reported cytotoxic or adverse side effects, if not over consumed. Some specific or a perfect blend of potent phyto-constituent(s) from curry leaves can be suggested for or adapted as alternative medicine or integrative medicine for preventing or treating or curing or protecting against heavy metal-induced occupational health hazards

Algorithmic construction of Lyapunov functions for continuous vector fields via convex semi-infinite programs

This article presents a novel numerically tractable technique for synthesizing Lyapunov functions for equilibria of nonlinear vector fields. In broad strokes, corresponding to an isolated equilibrium point of a given vector field, a selection is made of a compact neighborhood of the equilibrium and a dictionary of functions in which a Lyapunov function is expected to lie. Then an algorithmic procedure based on the recent work [DACC22] is deployed on the preceding neighborhood-dictionary pair and charged with the task of finding a function satisfying a compact family of inequalities that defines the behavior of a Lyapunov function on the selected neighborhood. The technique applies to continuous nonlinear vector fields without special algebraic structures and does not even require their analytical expressions to proceed. Several numerical examples are presented to illustrate our results.Comment: 26 pages. Submitte

Leader RNA binding ability of chandipura virus P protein is regulated by its phosphorylation status: a possible role in genome transcription-replication switch

AbstractThe molecular events associated with the transcriptive and replicative cycle of negative-stranded RNA viruses are still an enigma. We took Chandipura virus, a member of the Rhabdoviridae family, as our model system to demonstrate that Phosphoprotein P, besides Nucleocapsid protein N, also acts as a leader RNA-binding protein in its unphosphorylated form, whereas CKII-mediated phosphorylation totally abrogates its RNA-binding ability. However, interaction between P protein and leader RNA can be distinguished from N-mediated encapsidation of viral sequences. Furthermore, P protein bound to leader chain can successively recruit N protein on RNA while itself being replaced. We also observed that the accumulation of phosphorylation null mutant of P protein in cells results in enhanced genome RNA replication with concurrent increase in the viral yield. All these results led us to propose a model explaining viral transcription-replication switch where Phosphoprotein P acts as a modulator of genome transcription and replication by its ability to bind to the nascent leader RNA in its unphosphorylated form, promoting read-through of the transcription termination signals and initiating nucleocapsid assembly on the nascent RNA chain

AQUEOUS BARK EXTRACT OF TERMINALIA ARJUNA PROTECTS AGAINST PHENYLHYDRAZINE INDUCED OXIDATIVE DAMAGE IN GOAT RED BLOOD CELL MEMBRANE BOUND AND METABOLIC ENZYMES

Objective: The objective of the present study is to determine the phenylhydrazine (PHZ) induced oxidative stress mediated alteration in the metabolic status and morphology of the red blood cells (RBC) and amelioration of the same by aqueous bark extract of Terminalia arjuna (TA).Methods: Fresh goat blood collected from local Kolkata Corporation approved slaughter house, was used for the present study. Packed cells were prepared from the freshly collected goat blood and were divided into four groups as follows for further studies i. e Group I: Control (CON), Group II: TA bark extract treated, named T5C (5 mg/ml, incubation mixture; positive control), Group III: PHZ treated (1 mM), Group IV: PHZ treated+TA bark extract at a dose of 5 mg/ml, named as P+T5. ROS, superoxide anion radical, and hydroxyl radical scavenging activity were determined. Intracellular iron and intracellular nitrate concentration were estimated. Activities of various membrane-bound enzymes like Na+/K+-ATPase, Mg2+-ATPase and Ca2+-ATPase and Ach-E were determined. Moreover, the activities of some metabolic enzymes like glucose 6-phosphate dehydrogenase (G6PDH), hexokinase, aldolase, lactase dehydrogenase were also studied. In addition, the morphological structure of RBCs was also determined.Results: PHZ treatment caused significant alterations in RBC morphology as well as altered the activities of membrane-bound as well as metabolic enzymes. All these changes following oxidative stress were found to be ameliorated when the RBCs were co-treated with PHZ and aqueous bark extract of TA. However, aqueous bark extract of TA alone did not exhibit any such changes in RBC.Conclusion: The aqueous bark extract of TA ameliorates PHZ-induced oxidative damages in goat RBC possibly by an antioxidant mechanism(s). The aqueous bark extract of TA may have future therapeutic relevance in oxidative stress-induced damages in RBCs.Keywords: Antioxidant enzymes, Aqueous bark extract, Oxidative stress, Phenylhydrazine, Red blood cells, Terminalia arjun

A prospective, randomized, double blinded comparative study between 0.5% bupivacaine and 0.5% bupivacaine with pentazocine as an adjuvant in supraclavicular brachial plexus block for post operative analgesia in upper limb surgery: Pentazocine as adjuvant in brachial plexus block

In upper limb surgery supraclavicular brachial plexus (SCBP) block with 0.5% bupivacaine is commonly used for anesthesia. To increase the duration of sensory block opioids are used along with bupivacaine, but data on the effect of pentazocine as adjuvant with bupivacaine in SCBP block is still lacking. The study aimed to compare the duration of postoperative analgesia, sensory and motor block between 0.5% bupivacaine and 0.5% bupivacaine with pentazocine as an adjuvant in SCBP. Methods: The study was conducted on 60 consenting patients, posted for upper limb orthopedic surgery involving the forearm under SCBP block. Patients were randomly divided into two groups. Group B received 19 ml of 0.5 % bupivacaine with 1 ml of normal saline; Group BP received 19 ml of 0.5 % bupivacaine with 1 ml pentazocine (30 mg). Block characteristics, duration of postoperative analgesia, and side effects if any were recorded. Statistical analysis was done using the student t-test and Chi-square test for continuous and categorical variables respectively. Results: The onset of sensory (11.47 ± 1.57 vs. 16.8 ± 2.23 min) and motor (8.17 ± 1.14 vs. 13.9 ± 2.44 min) block was significantly faster in the BP group. Duration of sensory (392.33 ± 9.92 vs. 357.2 ± 8.76 min) and motor (379.27 ± 9.28 vs. 347.27 ± 9.13 min) block was also prolonged in group BP (p < 0.0001). Duration of postoperative analgesia was more (p < 0.0001) in the BP group (407.43 ± 10.46 vs 367.3 ± 8.74min). Conclusion: Pentazocine as an adjuvant with bupivacaine in SCBP block provides faster onset, prolonged duration of the block, and postoperative analgesia

SILYMARIN PROTECTS AGAINST COPPER-ASCORBATE INDUCED INJURY TO GOAT CARDIAC MITOCHONDRIA IN VITRO: INVOLVEMENT OF ANTIOXIDANT MECHANISM(S)

Silymarin, 'one of the component of the Milk thistle seeds Silybum marianum (L.) is used in traditional food and medicine in India. In the present study, we investigated the antioxidant activities of Silymarin against copper-ascorbate induced toxic injury to mitochondria obtained from goat heart, in vitro. Incubation of isolated cardiac mitochondria with copper-ascorbate resulted in elevated levels of lipid peroxidation and protein carbonylation of the mitochondrial membrane, a reduced level of mitochondrial GSH and altered status of antioxidant enzymes as well as decreased activities of pyruvate dehydrogenase and the Kreb's cycle enzymes, altered mitochondrial morphology, mitochondrial swelling and di-tyrosine level. All these changes were found to be ameliorated when the cardiac mitochondria were co-incubated with copper-ascorbate and Silymarin, in vitro. Silymarin, in our in vitro experiments, was found to scavenge hydrogen peroxide, superoxide anion free radicals, hydroxyl radicals and DPPH radical, in a chemically defined system, indicating that this compound may provide protection to cardiac mitochondria against copper-ascorbate induced toxic injury through its antioxidant activities. The results of this study suggest that Silymarin may be considered as a future therapeutic antioxidant and may be used singly or as a co-therapeutic in the treatment of diseases associated with mitochondrial oxidative stress

Hemolytic activity and biofilm-formation among clinical isolates of group B streptococcus causing acute urinary tract infection and asymptomatic bacteriuria

Streptococcus agalactiae, also known as group B Streptococcus, is an aetiological agent of urinary tract infection (UTI) in adults, including cystitis, pyelonephritis and asymptomatic bacteriuria (ABU). Whereas ABU-causing S. agalactiae (ABSA) have been shown to grow and achieve higher culture denstity in human urine compared to uropathogenic S. agalactiae (UPSA) other phenotypic distinctions between S. agalactiae isolated from different forms of UTI are not known. Here, we define the hemolytic activities and biofilm-formation of a collection of clinical isolates of UPSA, ABSA and recurrent S. agalactiae bacteriuria (rSAB) strains to explore these phenotypes in the context of clinical history of isolates. A total of 61 UPSA, 184 ABSA, and 47 rSAB isolates were analyzed for relative hemolytic activity by spot assay on blood agar, which was validated using a erythrocyte lysis suspension assay. Biofilm formation was determined by microtiter plate assay with Lysogeny and Todd-Hewitt broths supplemented with 1% glucose to induce biofilm formation. We also used multiplex PCR to analyze isolates for the presence of genes encoding adhesive pili, which contribute to biofilm formation. Comparing the hemolytic activities of 292 isolates showed, surprisingly, that ABSA strains were significantly more likely to be highly hemolytic compared to other strains. In contrast, there were no differences between the relative abilities of strains from the different clinical history groups to form biofilms. Taken together, these findings demonstrate a propensity of S. agalactiae causing ABU to be highly hemolytic but no link between clinical history of UTI strains and ability to form biofilm