Neomycin binding preserves extracellular matrix in bioprosthetic heart valves during in vitro cyclic fatigue and storage

Bioprosthetic heart valve (BHV) cusps have a complex architecture consisting of an anisotropic arrangement of collagen, glycosaminoglycans (GAGs) and elastin. Glutaraldehyde (GLUT) is used as a fixative for all clinical BHV implants; however, it only stabilizes the collagen component of the tissue, and other components such as GAGs and elastin are lost from the tissue during processing, storage or after implantation. We have shown previously that the effectiveness of the chemical crosslinking can be increased by incorporating neomycin trisulfate, a hyaluronidase inhibitor, to prevent the enzyme-mediated GAG degradation. In the present study, we optimized carbodiimide-based GAG-targeted chemistry to incorporate neomycin into BHV cusps prior to conventional GLUT crosslinking. This crosslinking leads to enhanced preservation of GAGs during in vitro cyclic fatigue and storage. The neomycin group showed greater GAG retention after both 10 and 50 million accelerated fatigue cycles and after 1 year of storage in GLUT solution. Thus, additional binding of neomycin to the cusps prior to standard GLUT crosslinking could enhance tissue stability and thus heart valve durability.     

Intracellular glutathione and cytotoxicity of platinum complexes

Although there have been a number of reports correlating cellular GSH levels with cytotoxicity of platinum agents, none has examined the relationship between GSH concentrations and cytotoxicity. In this study, using a highly specific HPLC method for measuring GSH and expressing GSH as concentration and also per cell number, we evaluated the correlation between GSH levels and the cytotoxicity to five agents in ten human tumor cell lines. The five platinum agents included the platinum(II) complexes cisplatin, carboplatin and oxaliplatin and platinum(IV) complexes iproplatin and tetraplatin. The correlation between intracellular GSH concentration and cytotoxicity was highly significant only for iproplatin (P=0.002) followed by tetraplatin, which demonstrated a trend toward statistical significance (P=0.06). Cytotoxicity of the other platinum complexes showed no relation to GSH concentration, cisplatin itself showing aP-value of 0.09. In contrast, the GSH levels normalized to cell number showed a statistically significant correlation with the cytotoxicity of four of the five platinum agents, the exception being carboplatin; the strongest correlation observed was that for iproplatin and tetraplatin. Glutathione-S-transferase (GST) activity in these cell lines showed no correlation with cytotoxicity of any of the platinum complexes. Our results, from the analyses of both GSH concentration as well as GSH per cell number, suggest a significantly higher interaction between GSH and iproplatin compared with the other platinum agents. Moreover, our data suggest that relationships between cytotoxicity and GSH levels on a per-cell basis may not persist when differences in cell volume are taken into account.     

Biochemical, ultrastructural and histochemical studies of cat placentae deficient in activity of lysosomal alpha-mannosidase

Lysosomal alpha-mannosidase activity, oligosaccharide profiles, light and electron microscopy and lectin histochemistry studies were performed on full-term placentae obtained from five litters of cats. They resulted from breeding related cats who are obligate heterozygotes for lysosomal alpha-mannosidase deficiency. alpha-Mannosidase activity in placentae from affected kittens was less than 10 per cent of control, while in placentae from presumptive heterozygotes the activity was less than 50 per cent of control. High-pressure liquid chromatographic analysis of oligosaccharides revealed massive accumulation of undegraded oligosaccharides in placentae of affected kittens. A small elevation was found in placentae from presumptive heterozygous kittens, and none was detected in placentae of normal kittens. Light and electron microscopic examinations revealed vacuolization of fetal endothelial and mesenchymal cells only in placentae of affected kittens. Succinylated wheat germ agglutinin and concanavalin A stained the fetal fibroblasts only in placentae of affected kittens.     

Role of ascorbic acid in the modulation of inhibition of platelet aggregation by polymorphonuclear leukocytes

OBJECTIVES: We investigated the modulatory effect of ascorbate on the inhibition of platelet aggregation response by polymorphonuclear leukocytes (PMNs) and characterized the mechanism of the inhibitory response. BACKGROUND: PMNs have been reported to play a significant role in vascular homeostasis by releasing various factors including short-lived reactive oxygen species (ROS) and nitric oxide (NO). NO prevents the activation of circulating platelets and plays a significant role in hemostasis. In addition, PMNs also have the capacity to store very high concentrations of ascorbate. The physiological implications of storing such high concentrations of an antioxidant by a cell-releasing free radicals is unknown, viz. a viz. hemostatic regulation. METHODS: ADP-induced aggregation in human, monkey and rat platelet-rich plasma (PRP) was monitored in the presence of PMNs treated with varying concentrations of ascorbate/dehydroascorbate. NO generation from rat and human PMNs treated with ascorbate was monitored on a FACS Calibur flow cytometer and intraplatelet cyclic guanosine 3',5'-monophosphate (cGMP) levels was also measured. RESULTS: PMNs induced a cell number and time-dependent inhibition of ADP-induced aggregation. The PMNs dependent inhibition was enhanced significantly at 30 min by ascorbate (300 microM). Ascorbate seemed to exert its effects through its oxidized product, dehydroascorbate, as the effects was prevented in the presence of D-glucose (10 mM). Dehydroascorbate elicited significant potentiation of the PMNs induced inhibitory responses and these effects were mediated by the release of NO and subsequent activation of platelet guanylyl cyclase. Flow cytometry experiments with human and rat PMNs confirmed the release of NO and the elevated platelet cGMP levels confirmed NO-mediated activation of guanylyl cyclase. CONCLUSIONS: Ascorbate in circulation seems to prevent the activation of platelets by enhancing the release of antiaggregatory NO, from neighbouring or cohabitant PMNs. The ascorbate effect is mediated through its conversion to dehydroascorbate, subsequently, gets taken up by the cell and converted back to ascorbate. Intracellular ascorbate potentiates the release of NO from the PMNs and subsequently activates guanylyl cyclase in the platelets.     

Acute infection and macrophage subversion by Mycobacterium tuberculosis require a specialized secretion system

Although many bacterial pathogens use specialized secretion systems for virulence, no such systems have been described for Mycobacterium tuberculosis, a major pathogen of humans that proliferates in host macrophages. In a screen to identify genes required for virulence of M. tuberculosis, we have discovered three components and two substrates of the first Sec-independent secretion pathway described in M. tuberculosis, which we designate the Snm pathway. Here we demonstrate that the proteins Snm1, -2, and -4 are required for the secretion of ESAT-6 and CFP-10, small proteins previously identified as major T cell antigens. Snm2, a member of the AAA ATPase family, interacts with substrates and with Snm1, another AAA ATPase. We show that M. tuberculosis mutants lacking either the Snm system or these substrates exhibit defects in bacterial growth during the acute phase of a mouse infection and are attenuated for virulence. Strikingly, snm mutants fail to replicate in cultured macrophages and to inhibit macrophage inflammatory responses, two well established activities of wild-type M. tuberculosis bacilli. Thus, the Snm secretion pathway works to subvert normal macrophage responses and is a major determinant of M. tuberculosis virulence.     

Inhibition of platelet aggregation by rat globin

This study was undertaken to identify and characterize the anti-aggregatory protein factor present in rat polymorphonuclear leukocytes (PMNs) supernatant. Since the purified protein exhibited sequence homology to beta globin, globin was also isolated from rat blood by acid-acetone precipitation and was purified on Superdex-75 column in FPLC. Elution of rat globin on the gel filtration column yielded two peaks of approximately 60 and 30 kDa as observed in the PMNs supernatant. Purity of globin and eluted fractions was further evaluated by SDS-PAGE. Platelet aggregation induced by agonists viz. adenosine-5'-diphosphate (ADP; 2-5 microM), arachidonic acid (AA; 10 microM), A23187 (2.50 microg/ml) was inhibited by globin and the purified fractions. ADP-induced rise in intracellular calcium levels and expression of CD62 on the platelets were reduced by both globin and active fraction of PMNs supernatant. Results obtained suggest that globin or globin-related protein present in the PMNs supernatant inhibits platelet aggregation response.     

Impact of Opiate–HIV-1 Interactions on Neurotoxic Signaling

Opiate drug abuse exacerbates the pathogenesis of human immunodeficiency virus-1 (HIV-1) in the central nervous system through direct actions on glia and neurons. Opiate abuse causes widespread disruption of astroglial and microglial function, and significant increases in astroglial-derived proinflammatory cytokines and chemokines, which likely contributes to neuronal dysfunction, death, and HIV encephalitis. Neurons are also directly affected by opiate–HIV-1 interactions. HIV-1 and the viral proteins gp120 and Tat activate multiple caspase-dependent and caspase-independent proapoptotic pathways in neurons involving phosphatidylinositol 3-kinase (PI3 kinase)/Akt, as well as p38, c-Jun N-terminal kinase (JNK) and/or other mitogen-activated protein kinases (MAPKs). Opiates appear to decrease the threshold for HIV-1-mediated neurotoxicity by sending convergent signals that exacerbate proapoptotic events induced by viral and cellular toxic products. The synergistic proinflammatory and neurotoxic effects of opiate drugs on glia and neurons are largely mediated through μ opioid receptors, which are expressed by subpopulations of astroglia, microglia, and neurons. Opiate abuse intrinsically modifies the host response to HIV-1. Identification of how this occurs is providing considerable insight toward understanding the mechanisms underlying HIV-1-associated dementia.     

Membrane penetration enhancement of ibuprofen using supersaturation

Permeation enhancement of ibuprofen from supersaturated solutions formed using the cosolvent technique was investigated using silicone as a model membrane. Hydroxpropyl methyl cellulose and hydroxpropyl-beta-cyclodextrin were used to stabilise the supersaturated states. Physical stability studies showed best results for low drug concentrations in a 40:60 propylene glycol/water cosolvent system. Variations in flux across model silicone membranes from saturated solutions were observed as the PG content was increased. The flux of IBU increased with the degree of saturation for solutions prepared in a 40:60 PG/water cosolvent mixture. HPMC and CD were found to be effective in enhancing the stability of supersaturated solutions of IBU. The mechanisms of action are different for the two additives and are discussed.