In vitro effects of some drugs on human red blood cell glucose-6-phosphate dehydrogenase enzyme activity

In vitro effects of omeprazole, morphine sulphate, remifentanyl, ketamine and vankomycin were investigated on human red blood cell glucose-6 phosphate dehydrogenase (G-6PD) (E.C. 1.1.1.49) enzyme activity purified from human red blood cell by 2', 5'ADP-Sepharose 4B affinity gel. The obtained I50 values of omeprazole, morphine, remifentanil, ketamine and vankomycin were 3.24, 43.58, 97.6, 64.16 and 0.903 mM, respectively and the Ki constants for omeprazole, morphine and vankomycin were 8.22 +/- 2.055, 25.93 +/- 6.482, and 2.71 +/- 0.677 mM, respectively and they were non competitive inhibitors.     

The Interactions of Nitric Oxide and Acetylcholine on Penicillin-Induced Epilepsy in Rats

The aim of this study was to investigate the interaction between nitric oxide (NO) and acetylcholine (ACh) in penicillin-induced experimental epilepsy. Adult male Wistar rats weighing 220 ± 35 g were used in the experiments. The epileptiform activity was induced by microinjection of penicillin (200 IU/1 μl) into the left sensorymotor cortex. Electrocorticogram was recorded by using Ag/AgCl ball electrodes. Sodium nitroprusside (SNP), a NO donor, given intracortically 30 min after penicillin significantly reduced the spike frequency whereas ACh increased the epileptiform activity for 5 min. Atropine, an antagonist for muscarinic receptors, was given intracortically 30 min after penicillin and did not significantly affect epileptiform activity for 30 min. SNP given after atropine significantly suppressed the epileptiform activity. ACh given 10 min after Nω-nitro-L: -arginine methyl ester (L-NAME), a nonspecific nitric oxide synthase inhibitor, did not have a significant effect on spike frequency. When ACh and SNP were administered together, penicillin induced epileptiform activity and spike frequency were significantly suppressed from the 10th minute onwards. It can be concluded that ACh increases the penicillin-induced epileptiform activity while co-administration of ACh and SNP produces a potent anticonvulsant effect as compared to SNP alone.     

Interactions of the antifungal mycosubtilin with ergosterol-containing interfacial monolayers

Mycosubtilin, an antimicrobial lipopeptide produced by Bacillus subtilis, is characterized by strong antifungal activities. The molecular mechanisms of its biological activities on the membranes of the sensitive yeasts or fungi have not yet been clearly elucidated. Our purpose was to mimic the mycosubtilin interactions with these membranes using various Langmuir monolayers. Since the major sterol of yeasts or fungi is ergosterol, the interactions of mycosubtilin with monolayers constituted by ergosterol, DPPC/ergosterol or DPPC/sphingomyelin/ergosterol were examined at different initial surface pressures (Πi). Plotting the mycosubtilin-induced surface pressure increases versus Πi allowed to determine that the exclusion pressures of mycosubtilin from these different monolayers is higher than the surface prevailing within the biological membranes. However, this behavior was lost when mycosubtilin was interacting with ergosteryl acetate-containing monolayers. This suggests the involvement of the sterol alcohol group in the mycosubtilin interactions within membranes. Furthermore, the behavior of mycosubtilin with stigmasterol, similar to that observed with ergosterol, differs from that previously observed with cholesterol, suggesting a role of the alkyl side chain of the sterols. The adsorption of mycosubtilin to ergosterol monolayers induced changes in the lipopeptide orientation at the air-water interface as revealed by polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS). Moreover, imaging the air-water interface by Brewster angle microscopy (BAM) indicates that mycosubtilin induced changes in the organization and morphology of monolayers containing pure ergosterol with the appearance of small condensed dots, suggesting again that the target of mycosubtilin might be the ergosterol present in the membranes of the sensitive yeasts or fungi.     

Antibody-functionalized fluid-permeable surfaces for rolling cell capture at high flow rates

Adhesion-based cell capture on surfaces in microfluidic devices forms the basis of numerous biomedical diagnostics and in vitro assays. However, the performance of these platforms is partly limited by interfacial phenomena that occur at low Reynolds numbers. In contrast, cell homing to porous vasculature is highly effective in vivo during inflammation, stem cell trafficking, and cancer metastasis. Here, we show that a porous, fluid-permeable surface functionalized with cell-specific antibodies promotes efficient and selective cell capture in vitro. This architecture is advantageous due to enhanced transport as streamlines are diverted toward the surface. Moreover, specific cell-surface interactions are promoted due to reduced shear, allowing gentle cell rolling and arrest. Together, these synergistic effects enable highly effective cell capture at flow rates more than an order of magnitude larger than those provided by existing devices with solid surfaces.     

Fabrication of hierarchical hybrid structures using bio-enabled layer-by-layer self-assembly

Development of versatile and flexible assembly systems for fabrication of functional hybrid nanomaterials with well-defined hierarchical and spatial organization is of a significant importance in practical nanobiotechnology applications. Here we demonstrate a bio-enabled self-assembly technique for fabrication of multi-layered protein and nanometallic assemblies utilizing a modular gold-binding (AuBP1) fusion tag. To accomplish the bottom-up assembly we first genetically fused the AuBP1 peptide sequence to the C'-terminus of maltose-binding protein (MBP) using two different linkers to produce MBP-AuBP1 hetero-functional constructs. Using various spectroscopic techniques, surface plasmon resonance (SPR) and localized surface plasmon resonance (LSPR), we verified the exceptional binding and self-assembly characteristics of AuBP1 peptide. The AuBP1 peptide tag can direct the organization of recombinant MBP protein on various gold surfaces through an efficient control of the organic-inorganic interface at the molecular level. Furthermore using a combination of soft-lithography, self-assembly techniques and advanced AuBP1 peptide tag technology, we produced spatially and hierarchically controlled protein multi-layered assemblies on gold nanoparticle arrays with high molecular packing density and pattering efficiency in simple, reproducible steps. This model system offers layer-by-layer assembly capability based on specific AuBP1 peptide tag and constitutes novel biological routes for biofabrication of various protein arrays, plasmon-active nanometallic assemblies and devices with controlled organization, packing density and architecture.     

Trehalose transporter from African chironomid larvae improves desiccation tolerance of Chinese hamster ovary cells

Dry preservation has been explored as an energy-efficient alternative to cryopreservation, but the high sensitivity of mammalian cells to desiccation stress has been one of the major hurdles in storing cells in the desiccated state. An important strategy to reduce desiccation sensitivity involves use of the disaccharide trehalose. Trehalose is known to improve desiccation tolerance in mammalian cells when present on both sides of the cell membrane. Because trehalose is membrane impermeant the development of desiccation strategies involving this promising sugar is hindered. We explored the potential of using a high-capacity trehalose transporter (TRET1) from the African chironomid Polypedilum vanderplanki[21] to introduce trehalose into the cytoplasm of mammalian cells and thereby increase desiccation tolerance. When Chinese hamster ovary cells (CHO) were stably transfected with TRET1 (CHO-TRET1 cells) and incubated with 0.4M trehalose for 4h at 37°C, a sevenfold increase in trehalose uptake was observed compared to the wild-type CHO cells. Following trehalose loading, desiccation tolerance was investigated by evaporative drying of cells at 14% relative humidity. After desiccation to 2.60g of water per gram dry weight, a 170% increase in viability and a 400% increase in growth (after 7days) was observed for CHO-TRET1 relative to control CHO cells. Our results demonstrate the beneficial effect of intracellular trehalose for imparting tolerance to partial desiccation.     

The bioactive peptides salusins and apelin-36 are produced in human arterial and venous tissues and the changes of their levels during cardiopulmonary bypass

This study aimed to examine the effects of CPB on salusin-α, salusin-β and apelin-36 bioactive peptides in people who are planned to undergo coronary artery bypass graft (CABG) operation due to coronary artery disease and to explore whether these peptides are produced in human aortic, saphenous and arterial tissues. The study included age and BMI matched 15 patients who underwent CABG operation by CPB. In order to determine salusin-α, salusin-β and apelin-36 levels, venous blood samples were collected before induction of anesthesia (T1), before CPB (T2), 5min before the removal of cross-clamp (T3), 5min after the removal of cross-clamp (T4), upon arrival in the intensive care (T5), at postoperative 24th hour (T6) and 72nd hour (T7). Salusin and apelin expressions of the tissues were shown by immunohistochemical method. Peptide amounts of sera and tissues were measured using ELISA. Salusins production by vessels occurs in fibroblast cells of the media in the aorta and smooth muscle cells of the media in the LIMA and saphena. Apelin is produced by endothelial cells of the intima and fibroblast cells of the media in the aorta and by smooth muscle cells of the media in the LIMA and saphena. Changes in the levels of salusin-β and apelin-36 were significant during CPB. Salusin-α, salusin-β and apelin-36 are locally synthesized in the arteries and veins. Salusins and apelin-36 might be important markers in the CPB, and also that salusin-β was more specific in comparison to salusin-α.     

Mutations in RIPK4 cause the autosomal-recessive form of popliteal pterygium syndrome

The autosomal-recessive form of popliteal pterygium syndrome, also known as Bartsocas-Papas syndrome, is a rare, but frequently lethal disorder characterized by marked popliteal pterygium associated with multiple congenital malformations. Using Affymetrix 250K SNP array genotyping and homozygosity mapping, we mapped this malformation syndrome to chromosomal region 21q22.3. Direct sequencing of RIPK4 (receptor-interacting serine/threonine kinase protein 4) showed a homozygous transversion (c.362T>A) that causes substitution of a conserved isoleucine with asparagine at amino acid position 121 (p.Ile121Asn) in the serine/threonine kinase domain of the protein. Additional pathogenic mutations-a homozygous transition (c.551C>T) that leads to a missense substitution (p.Thr184Ile) at a conserved position and a homozygous one base-pair insertion mutation (c.777_778insA) predicted to lead to a premature stop codon (p.Arg260ThrfsX14) within the kinase domain-were observed in two families. Molecular modeling of the kinase domain showed that both the Ile121 and Thr184 positions are critical for the protein's stability and kinase activity. Luciferase reporter assays also demonstrated that these mutations are critical for the catalytic activity of RIPK4. RIPK4 mediates activation of the nuclear factor-κB (NF-κB) signaling pathway and is required for keratinocyte differentiation and craniofacial and limb development. The phenotype of Ripk4(-/-) mice is consistent with the human phenotype presented herein. Additionally, the spectrum of malformations observed in the presented families is similar, but less severe than the conserved helix-loop-helix ubiquitous kinase (CHUK)-deficient human fetus phenotype; known as Cocoon syndrome; this similarity indicates that RIPK4 and CHUK might function via closely related pathways to promote keratinocyte differentiation and epithelial growth.