Structural and mutational analyses of the molecular interactions between the catalytic domain of factor XIa and the Kunitz protease inhibitor domain of protease nexin 2

Factor XIa (FXIa) is a serine protease important for initiating the intrinsic pathway of blood coagulation. Protease nexin 2 (PN2) is a Kunitz-type protease inhibitor secreted by activated platelets and a physiologically important inhibitor of FXIa. Inhibition of FXIa by PN2 requires interactions between the two proteins that are confined to the catalytic domain of the enzyme and the Kunitz protease inhibitor (KPI) domain of PN2. Recombinant PN2KPI and a mutant form of the FXI catalytic domain (FXIac) were expressed in yeast, purified to homogeneity, co-crystallized, and the structure of the complex was solved at 2.6 angstroms (Protein Data Bank code 1ZJD). In this complex, PN2KPI has a characteristic, disulfide-stabilized double loop structure that fits into the FXIac active site. To determine the contributions of residues within PN2KPI to its inhibitory activity, selected point mutations in PN2KPI loop 1 11TGPCRAMISR20 and loop 2 34FYGGC38 were tested for their ability to inhibit FXIa. The P1 site mutation R15A completely abolished its ability to inhibit FXIa. IC50 values for the wild type protein and the remaining mutants were as follows: PN2KPI WT, 1.28 nM; P13A, 5.92 nM; M17A, 1.62 nM; S19A, 1.86 nM; R20A, 5.67 nM; F34A, 9.85 nM. The IC50 values for the M17A and S19A mutants were not significantly different from those obtained with wild type PN2KPI. These functional studies and activated partial thromboplastin time analysis validate predictions made from the PN2KPI-FXIac co-crystal structure and implicate PN2KPI residues, in descending order of importance, Arg15, Phe34, Pro13, and Arg20 in FXIa inhibition by PN2KPI.     

Postnatal hypoxic-ischemic brain injury alters mechanisms mediating neuronal glucose transport

We examined the effect of hypoxic ischemia and hypoxia vs. normoxia on postnatal murine brain substrate transporter concentrations and function. We detected a transient increase in the neuronal brain glucose transporter isoform (GLUT-3) in response to hypoxic ischemia after 4 h of reoxygenation. This increase was associated with no change in GLUT-1 (blood-brain barrier/glial isoform), monocarboxylate transporter isoforms 1 and 2, synapsin I (neuronal marker), or Bax (proapoptotic protein) but with a modest increase in Bcl-2 (antiapoptotic mitochondrial protein) protein concentrations. At 24 h of reoxygenation, the increase in GLUT-3 disappeared but was associated with a decline in Bcl-2 protein concentrations and the Bcl2:Bax ratio, an increase in caspase-3 enzyme activity (apoptotic effector enzyme), and extensive DNA fragmentation, which persisted later in time (48 h) only in the hippocampus. Hypoxia alone in the absence of ischemia was associated with a transient but modest increase in GLUT-3 and synapsin I protein concentrations, which did not cause significant apoptosis and/or necrosis. Assessment of glucose transporter function by 2-deoxyglucose (2-DG) uptake using two distinct techniques, namely positron emission tomography (PET) and the modified Sokoloff method, revealed a discrepancy due to glucose uptake by extracranial Harderian glands that masked the accurate detection of intracranial brain glucose uptake by PET scanning. The modified Sokoloff method assessing 2-DG uptake revealed that the transient increase in GLUT-3 was critical in protecting against a decline in brain glucose uptake. We conclude that hypoxic-ischemic brain injury is associated with transient compensatory changes targeted at protecting glucose delivery to fuel cellular energy metabolism, which then may delay the processes of apoptosis and cell necrosis.     

Simultaneous determination of amlodipine and metoprolol in their combined dosage form using a synchronous fluorescence spectrofluorimetric method

Highly sensitive, rapid, accurate and precise synchronous fluorescence spectrofluorimetric method has been developed for simultaneous analysis of a mixture of amlodipine (AMD) and metoprolol (MET). The method relies on measuring the relative synchronous fluorescence intensity of both drugs at Δλ of 90 nm in acetate buffer solution at pH 5. The experimental parameters influencing the developed method were investigated and optimized. The method was linear over the ranges 0.2–2 μg/ml and 0.5–10 μg/ml for AMD and MET, respectively. The limits of detection were 50 ng/ml for AMD and 130 ng/ml for MET while the limits of quantitation were 150 ng/ml for AMD and 390 ng/ml for MET. The developed method was applied successfully for the determination of the two drugs in their co‐formulated tablet. The mean percent recoveries were found to be 100.51 and 99.57 for AMD and MET, respectively.     

Molecular docking analysis of bioactive compounds from Mollugo cerviana (L.) SER with DHFR for antifungal activity

Fungal infections have been increasing in recent years due to growing number of high-risk patients particularly immuno compromised hosts. Candida is the third- or fourth-most-common isolate in nosocomial bloodstream infections. The increase of fungal resistance to classical drugs, the treatment costs, and the fact that most available antifungal drugs have only fungistatic activity, justify the search for new strategies. Identification of therapeutic compounds from plants has been the centre of attraction ever since they were discovered. It is of interest to document the molecular docking analysis of bioactive compounds present in Mollugo cerviana (L.) SER with the DHFR protein target for antifungal activity. We show the optimal binding features of several compounds from the extract with in vivo and in vitro activities. Results of this showed that all compounds showed good antimicrobial activity and a very good antifungal activity against the target DHFR protein. So, these compounds may act as potential drug molecules after the experimental validation.     

Stability and anatomical parameters of irradiated potato cultivars under drought stress

This study was carried out in Desert Research Center and Faculty of Agriculture, Zagazig University, Egypt, under North Sinai conditions during three growing seasons, i.e., summer 2018, fall 2018/2019 and summer 2019 to assess the effect of radiation mutants on leaf histological features and genetic stability of the productivity of some potato cultivars under drought stress conditions. Results reveal that the genotypes can be statistically classified based on regression coefficient (bi), deviation from regression (S²di) to 4 groups (with low in S²di are considered in all groups) as: (i) Genotype with elevated average, bi = 1, it is considered as stable genotype where Cara cultivar (both generations) was included. (ii) Genotype with elevated average, bi > 1 as genotype with average stability where spunta cultivar was involved. (iii) Genotype with low mean, bi < 1 as genotype with low stability where hermes (both primary (M₁) and secondary (M₂) radiated generations) and Caruso (2nd generation) cultivars were involved. (iv) Genotypes including a few bi values in one generation, as genotype including low stability but are not recommended for use in this generation where Caruso cv in M₁ was included. The results indicated that 20 Gy irradiation exposure revealed that Spunta cultivar produced markedly high mean combined over yield during M₁ (11.771 ton/fed) and M₂ (10.97 ton/fed) generations than other genotypes and ranked first over all environments. It proves that Hermes could be employed as anti-stress genotypes under stress conditions (negative conditions or poor yielding). However, spunta followed by cara cv. represented a good performance in M₂ production yield (10.97 and 8.51 ton/fed, respectively), slight drift from the regression line and coefficient close to 1, therefore, both cultivars were excellent between genotypes in shape of yield stability and is recommended for different conditions. According to anatomical studies, 80 % from field capacity (FC) decreased the thickness of medvein and lamina of potato cv. spunta, also, dimensions of medvein bundle and mean diameter of vessels. In conclusion, plants treated with gamma ray at level 20 and grown under 80 % FC induced prominent increase in all previous characters.     

Energetics underlying the process of long-chain fatty acid transport.

The gram negative bacterium Escherichia coli has evolved a highly specific system for the transport of exogenous long-chain fatty acids (C12-C18) across the cell envelope that requires the outer membrane protein FadL and the inner membrane associated fatty acyl CoA synthetase. The transport of oleate (C18:1) across the cell envelop responds to metabolic energy. In order to define the source of metabolic energy which drives this process, oleate transport was measured in wild-type and ATP synthase-defective (Deltaatp) strains which were (i) subjected to osmotic shock and (ii) starved and energized with glucose or d-lactate in the presence of different metabolic inhibitors. Osmotic shock did not eliminate transport but rather reduced the rate to 33-55% of wild-type levels. These results suggested a periplasmic protein may participate in this process or that osmotic shock disrupts the energized state of the cell which in turn reduces the rate of oleate transport. Transport systems which are osmotically sensitive also require ATP. The process of long-chain fatty acid transport requires ATP generated either by substrate-level or oxidative phosphorylation. Following starvation, the basal rate of transport for wild-type cells was 340.4 pmol/min/mg protein compared to 172.0 pmol/min/mg protein for the Deltaatp cells. When cells are energized with glucose, the rates of transport were increased and comparable (1242.6 and 1293.8 pmol/min/mg protein, respectively). This was in contrast to cells energized with d-lactate in which only the wild-type cells were responsive. The role of ATP is likely due to the ATP requirement of fatty acyl CoA synthetase for catalytic activity. The process of oleate transport is also influenced by the energized state of the inner membrane. In the presence of carbonyl cyanide-m-chlorophenylhydrazone oleate transport is depressed to 30-50% of wild-type levels in wild-type and Deltaatp strains under starvation conditions. These results are mirrored in cells energized with glucose and d-lactate, indicating that an energized membrane is required for optimal levels of oleate transport. These data support the hypothesis that the fatty acid transport system of E. coli responds to both intracellular pools of ATP and an energized membrane for maximal proficiency.     

Decreased Cholinergic Receptor Expression in the Striatum: Motor Function Deficit in Hypoglycemic and Diabetic Rats

Hypoglycemic brain injury is a common and serious complication of insulin therapy associated with diabetes. This study evaluated the effect of insulin-induced hypoglycemia and STZ-induced diabetes on striatal cholinergic receptors and enzyme expression and on motor function. Cholinergic enzymes: AChE and ChAT gene expression, radioreceptor binding assay and immunohistochemistry of muscarinic M1, M3 receptors and α7nAChR were carried out. Motor performance on grid walk test was analysed. AChE and ChAT expression significantly downregulated in hypoglycemic and diabetic rats. Total muscarinic and Muscarinic M3 receptor binding decreased in hypoglycemic rats compared to diabetic rats whereas muscarinic M1 receptor binding increased in hypoglycemic rats compared to diabetic rats. Real-time PCR analysis and confocal imaging of muscarinic M1, M3 receptors confirmed the changes in muscarinic receptor binding in hypoglycemic and diabetic rats. In hypoglycemic rats, α7nAChR expression significantly up regulated compared to diabetic rats. Grid walk test demonstrated the impairment in motor function and coordination in hypoglycemic and hyperglycemic rats. Neurochemical changes along with the behavioral data implicate a role for impaired striatal cholinergic receptor function inducing motor function deficit induced by hypo and hyperglycemia. Hypoglycemia exacerbated the neurobehavioral deficit in diabetes which has clinical significance in the treatment of diabetes.