The role of cysteine proteases in hypoxia-induced rat renal proximal tubular injury.

The role of the lysosomal proteases cathepsins B and L and the calcium-dependent cytosolic protease calpain in hypoxia-induced renal proximal tubular injury was investigated. As compared to normoxic tubules, cathepsin B and L activity, evaluated by the specific fluorescent substrate benzyloxycarbonyl-L-phenylalanyl-L-arginine-7-amido-4-methylcoumarin, was not increased in hypoxic tubules or the medium used for incubation of hypoxic tubules in spite of high lactate dehydrogenase (LDH) release into the medium during hypoxia. These data in rat proximal tubules suggest that cathepsins are not released from lysosomes and do not gain access to the medium during hypoxia. An assay for calpain activity in isolated proximal tubules using the fluorescent substrate N-succinyl-Leu-Tyr-7-amido-4-methylcoumarin was developed. The calcium ionophore ionomycin induced a dose-dependent increase in calpain activity. This increase in calpain activity occurred prior to cell membrane damage as assessed by LDH release. Tubular calpain activity increased significantly by 7.5 min of hypoxia, before there was significant LDH release, and further increased during 20 min of hypoxia. The cysteine protease inhibitor N-benzyloxycarbonyl-Val-Phe methyl ester (CBZ) markedly decreased LDH release after 20 min of hypoxia and completely prevented the increase in calpain activity during hypoxia. The increase in calpain activity during hypoxia and the inhibitor studies with CBZ therefore supported a role for calpain as a mediator of hypoxia-induced proximal tubular injury.

The killing of Leishmania major by human macrophages is mediated by nitric oxide induced after ligation of the Fc epsilon RII/CD23 surface antigen.

Serum IgE concentrations and the expression of the low-affinity receptor for IgE (Fc epsilon RII/CD23) are increased in cutaneous leishmaniasis or after immune challenge with Leishmania antigens. In vitro, the ligation of CD23 by IgE-anti-IgE immune complexes (IgE-IC) or by anti-CD23 monoclonal antibody (mAb) induces nitric oxide (NO) synthase and the generation of various cytokines by human monocytes/macrophages. The present study shows that IgE-IC, via CD23 binding, induce intracellular killing of Leishmania major in human monocyte-derived macrophages through the induction of the L-arginine:NO pathway. This was demonstrated by increased generation of nitrite (NO2-), the stable oxidation product of NO, and by the ability of NG-monomethyl-L-arginine to block both NO generation and parasite killing. A similar NO-dependent effect was observed with interferon gamma-treated cells. Tumor necrosis factor alpha is involved in this process, since both the induction of NO synthase and the killing of parasites caused by anti-CD23 mAb were inhibited by an anti-tumor necrosis factor alpha mAb. Treatment of noninfected CD23+ macrophages with IgE-IC provided protection against subsequent in vitro infection of these cells by Leishmania major promastigotes. Thus, IgE-IC promote killing of L. major by inducing NO synthase in human macrophages.

Identification, purification, and characterization of a zyxin-related protein that binds the focal adhesion and microfilament protein VASP (vasodilator-stimulated phosphoprotein).

VASP (vasodilator-stimulated phosphoprotein), an established substrate of cAMP- and cGMP-dependent protein kinases in vitro and in living cells, is associated with focal adhesions, microfilaments, and membrane regions of high dynamic activity. Here, the identification of an 83-kDa protein (p83) that specifically binds VASP in blot overlays of different cell homogenates is reported. With VASP overlays as a detection tool, p83 was purified from porcine platelets and used to generate monospecific polyclonal antibodies. VASP binding to purified p83 in solid-phase binding assays and the closely matching subcellular localization in double-label immunofluorescence analyses demonstrated that both proteins also directly interact as native proteins in vitro and possibly in living cells. The subcellular distribution, the biochemical properties, as well as microsequencing data revealed that porcine platelet p83 is related to chicken gizzard zyxin and most likely represents the mammalian equivalent of the chicken protein. The VASP-p83 interaction may contribute to the targeting of VASP to focal adhesions, microfilaments, and dynamic membrane regions. Together with our recent identification of VASP as a natural ligand of the profilin poly-(L-proline) binding site, our present results suggest that, by linking profilin to zyxin/p83, VASP may participate in spatially confined profilin-regulated F-actin formation.

Tonic stimulation of renin gene expression by nitric oxide is counteracted by tonic inhibition through angiotensin II.

This study was designed to examine the possible involvement of prostaglandins and nitric oxide (NO) in the renin stimulatory effect of angiotensin II (AngII) antagonists. To this end, plasma renin activities (PRAs) and renal renin mRNA levels were assayed in rats that were treated with the Ang-converting enzyme inhibitor ramipril or with the AngII AT1-receptor antagonist losartan. Ramipril and losartan increased PRA values from 7.5 +/- 1.6 to 86 +/- 6 and 78 +/- 22 ng of AngI per h per ml and renin mRNA levels from 112 +/- 9% to 391 +/- 20% and 317 +/- 10%, respectively. Inhibition of prostaglandin formation with indomethacin did not influence basal or ramipril-affected PRA. Basal renin mRNA levels also were unchanged by indomethacin, while increases in renin mRNA levels after ramipril treatment were slightly reduced by indomethacin. Inhibition of NO synthase by nitro-L-arginine methyl ester (L-NAME) reduced PRA values to 3.2 +/- 0.9, 34 +/- 13, and 12.1 +/- 2.7 ng of AngI per h per ml in control, ramipril-treated, and losartan-treated animals, respectively. Renin mRNA levels were reduced to 77 +/- 14% under basal conditions and ramipril- and losartan-induced increases in renin mRNA levels were completely blunted after addition of L-NAME. The AngII antagonists, furthermore, induced an upstream recruitment of renin-expressing cells in the renal afferent arterioles, which was also blunted by L-NAME. These findings suggest that renin mRNA levels are tonically increased by NO and that the action of NO is counteracted by AngII.

Cerebrovascular alterations in mice lacking neuronal nitric oxide synthase gene expression.

Nitric oxide (NO) is known to mediate increases in regional cerebral blood flow elicited by CO2 inhalation. In mice with deletion of the gene for neuronal NO synthase (NOS), CO2 inhalation augments cerebral blood flow to the same extent as in wild-type mice. However, unlike wild-type mice, the increased flow in mutants is not blocked by the NOS inhibition, N omega-nitro-L-arginine, and CO2 exposure fails to increase brain levels of cGMP. Topical acetylcholine elicits vasodilation in the mutants which is blocked by N omega-nitro-L-arginine, indicating normal functioning of endothelial NOS. Moreover, immunohistochemical staining for endothelial NOS is normal in the mutants. Thus, following loss of neuronal NOS, the cerebral circulatory response is maintained by a compensatory system not involving NO.

Incorporation of glutamine repeats makes protein oligomerize: implications for neurodegenerative diseases.

Many transcription factors and some other proteins contain glutamine repeats; their abnormal expansion has been linked to several dominantly inherited neuro-degenerative diseases. Having found that poly(L-glutamine) alone forms beta-strands held together by hydrogen bonds between their amide groups, we surmised that glutamine repeats may form polar zippers, an unusual motif for protein-protein interactions. To test this hypothesis, we have engineered a Gly-Gln10-Gly peptide into the inhibitory loop of truncated chymotrypsin inhibitor 2 (CI2), a small protein from barley seeds, by both insertion and replacement. Gel filtration resolved both mutant inhibitors into at least three fractions, which analytical ultracentrifugation identified as monomers, dimers, and trimers of the recombinant protein; the truncated wild-type CI2 formed only monomers. CD difference spectra of the dimers and trimers versus wild type indicated that their glutamine repeats formed beta-pleated sheets, while those of the monomers versus wild type were more suggestive of type I beta-turns. The CD spectra of all three fractions remained unchanged even after incubation at 70 degrees C; neither the dimers nor the trimers dissociated at this temperature. We argue that the stability of all three fractions is due to the multiplicity of hydrogen bonds between extended strands of glutamine repeats in the oligomers or within a beta-hairpin formed by the single glutamine repeat of each monomer. Pathological effects may arise when expanded glutamine repeats cause proteins to acquire excessively high affinities for each other or for other proteins with glutamine repeats.

Cloning, baculovirus expression, and characterization of a second mouse prolyl 4-hydroxylase alpha-subunit isoform: formation of an alpha 2 beta 2 tetramer with the protein disulfide-isomerase/beta subunit.

Prolyl 4-hydroxylase (EC 1.14.11.2) catalyzes the posttranslational formation of 4-hydroxyproline in collagens. The vertebrate enzyme is an alpha 2 beta 2 tetramer, the beta subunit of which is a highly unusual multifunctional polypeptide, being identical to protein disulfide-isomerase (EC 5.3.4.1). We report here the cloning of a second mouse alpha subunit isoform, termed the alpha (II) subunit. This polypeptide consists of 518 aa and a signal peptide of 19 aa. The processed polypeptide is one residue longer than the mouse alpha (I) subunit (the previously known type), the cloning of which is also reported here. The overall amino acid sequence identity between the mouse alpha (II) and alpha (I) subunits is 63%. The mRNA for the alpha (II) subunit was found to be expressed in a variety of mouse tissues. When the alpha (II) subunit was expressed together with the human protein disulfide-isomerase/beta subunit in insect cells by baculovirus vectors, an active prolyl 4-hydroxylase was formed, and this protein appeared to be an alpha (II) 2 beta 2 tetramer. The activity of this enzyme was very similar to that of the human alpha (I) 2 beta 2 tetramer, and most of its catalytic properties were also highly similar, but it differed distinctly from the latter in that it was inhibited by poly(L-proline) only at very high concentrations. This property may explain why the type II enzyme was not recognized earlier, as an early step in the standard purification procedure for prolyl 4-hydroxylase is affinity chromatography on a poly(L-proline) column.

Metallothionein protects against the cytotoxic and DNA-damaging effects of nitric oxide.

In inflammatory states, nitric oxide (.NO) may be synthesized from precursor L-arginine via inducible .NO synthase (iNOS) in large amounts for prolonged periods of time. When .NO acts as an effector molecule under these conditions, it may be toxic to cells by inhibition of iron-containing enzymes or initiation of DNA single-strand breaks. In contrast to molecular targets of .NO, considerably less is known regarding mechanisms by which cells become resistant to .NO. Metallothionein (MT), the major protein thiol induced in cells exposed to cytokines and bacterial products, is capable of forming iron-dinitrosyl thiolates in vitro. Therefore, we tested the hypothesis that overexpression of MT reduces the sensitivity of NIH 3T3 cells to the .NO donor, S-nitrosoacetylpenicillamine (SNAP), and to .NO released from cells (NIH 3T3-DFG-iNOS) after infection with a retroviral vector expressing human iNOS gene. There was a 4-fold increase in MT in cells transfected with the mouse MT-1 gene (NIH 3T3/MT) compared to cells transfected with the promoter-free inverted gene (NIH 3T3/TM). NIH 3T3/MT cells were more resistant than NIH 3T3/TM cells to the cytotoxic effects of SNAP (0.1-1.0 mM) or .NO released from NIH 3T3-DFG-iNOS cells. A brief (1 h) exposure to 10 mM SNAP caused DNA single-strand breaks that were 9-fold greater in NIH 3T3/TM compared to NIH 3T3/MT cells. Electron paramagnetic resonance spectroscopy of NIH 3T3 cells revealed a greater peak at g = 2.04 (e.g., iron-dinitrosyl complex) in NIH 3T3/MT than NIH 3T3/TM cells. These data are consistent with a role for cytoplasmic MT in interacting with .NO and reducing .NO-induced cyto- and nuclear toxicity.

Nitric oxide mediates the increase in local cerebral blood flow during focal seizures.

The role of nitric oxide (NO) in the increase in local cerebral blood flow (LCBF) elicited by focal cortical epileptic seizures was investigated in anesthetized adult rats. Seizures were induced by topical bicuculline methiodide applied through two cranial windows drilled over homotopic sites of the frontal cortex, and LCBF was measured by quantitative autoradiography by using 4-iodo[N-methyl-14C]antipyrine. Superfusion of an inhibitor of NO synthase, N omega-nitro-L-arginine (NA; 1 mM), for 45 min abolished the increase of LCBF induced by topical bicuculline methiodide (10 mM) [164 +/- 18 ml/100 g per min in the artificial cerebrospinal fluid (aCSF)-superfused side and 104 +/- 12 ml/100 g per ml in the NA-superfused side; P < 0.005]. This effect was reversed by coapplication of an excess of L-arginine substrate (10 mM) (218 +/- 22 ml/100 g per min in the aCSF-superfused side and 183 +/- 31 ml/100 g per min in the NA + L-Arg-superfused side) but not by 10 mM D-arginine, a stereoisomer with poor affinity for NO synthase (193 +/- 17 ml/100 g per min in the aCSF-superfused side and 139 +/- 21 ml/100 g per min in the NA + D-Arg-superfused side; P < 0.005). Superfusion of the guanylyl cyclase inhibitor methylene blue attenuated the LCBF increase elicited by topical bicuculline methiodide by 25% +/- 16% (P < 0.05). The present findings suggest that NO is the mediator of the vasodilation in response to focal epileptic seizures.

Nitric oxide inhibits hypothalamic luteinizing hormone-releasing hormone release by releasing gamma-aminobutyric acid.

Nitric oxide synthase (NOS)-containing neurons, termed NOergic neurons, occur in various regions of the hypothalamus, including the median eminence-arcuate region, which plays an important role in controlling the release of luteinzing hormone-releasing hormone (LHRH). We examined the effect of NO on release of gamma-aminobutyric acid (GABA) from medial basal hypothalamic (MBH) explants incubated in vitro. Sodium nitroprusside (NP) (300 microM), a spontaneous releaser of NO, doubled the release of GABA. This release was significantly reduced by incubation of the tissue with hemoglobin, a scavenger of NO, whereas hemoglobin alone had no effect on the basal release of GABA. Elevation of the potassium concentration (40 mM) in the medium increased GABA release 15-fold; this release was further augmented by NP. Hemoglobin blocked the increase in GABA release induced by NP but had no effect on potassium-induced release, suggesting that the latter is not related to NO. As in the case of hemoglobin, NG-monomethyl-L-arginine (NMMA), a competitive inhibitor of NOS, had no effect on basal release of GABA, which indicates again that NO is not significant to basal GABA release. However, NMMA markedly inhibited the release of GABA induced by high potassium, which indicates that NO plays a role in potassium-induced release of GABA. In conditions in which the release of GABA was substantially augmented, there was a reduction in GABA tissue stores as well, suggesting that synthesis of GABA in these conditions did not keep up with release of the amine. Although NO released GABA, there was no effect of the released GABA on NO production, for incubation of MBH explants with GABA had no effect on NO release as measured by [14C]citrulline production. To determine whether GABA had any effect on the release of LHRH from these MBH explants, GABA was incubated with the tissue and the effect on LHRH release was determined. GABA (10(-5) or 10(-6) M) induced a 70% decrease in the release of LHRH, indicating that in the male rat GABA inhibits the release of this hypothalamic peptide. This inhibition in LHRH release induced by GABA was blocked by NMMA (300 microM), which indicates that GABA converts the stimulatory effect of NO on LHRH release into an inhibitory one, presumably via GABA receptors, which activate chloride channels that hyperpolarize the cell. Previous results have indicated that norepinephrine stimulates release of NO from the NOergic neurons, which then stimulates the release of LHRH. The current results indicate that the NO released also induces release of GABA, which then inhibits further LHRH release. Thus, in vivo the norepinephrinergic-driven pulses of LHRH release may be terminated by GABA released from GABAergic neurons via NO.

The activating role of phospho-(Tyr)-calmodulin on the epidermal growth factor receptor

The activity of calmodulin (CaM) is modulated not only by oscillations in the cytosolic concentration of free Ca2+, but also by its phosphorylation status. In the present study, the role of tyrosine-phosphorylated CaM [P-(Tyr)-CaM] on the regulation of the epidermal growth factor receptor (EGFR) has been examined using in vitro assay systems. We show that phosphorylation of CaM by rat liver solubilized EGFR leads to a dramatic increase in the subsequent phosphorylation of poly-L-(Glu:Tyr) (PGT) by the receptor in the presence of ligand, both in the absence and in the presence of Ca2+. This occurred in contrast with assays where P-(Tyr)-CaM accumulation was prevented by the presence of Ca2+, absence of a basic cofactor required for CaM phosphorylation and/or absence of CaM itself. Moreover, an antibody against CaM, which inhibits its phosphorylation, prevented the extra ligand-dependent EGFR activation. Addition of purified P-(Tyr)-CaM, phosphorylated by recombinant c-Src (cellular sarcoma kinase) and free of non-phosphorylated CaM, obtained by affinity-chromatography using an immobilized anti-phospho-(Tyr)-antibody, also increased the ligand-dependent tyrosine kinase activity of the isolated EGFR toward PGT. Also a CaM(Y99D/Y138D) mutant mimicked the effect of P-(Tyr)-CaM on ligand-dependent EGFR activation. Finally, we demonstrate that P-(Tyr)-CaM binds to the same site (645R-R-R-H-I-V-R-K-R-T-L-R-R-L-L-Q660) as non-phosphorylated CaM, located at the cytosolic juxtamembrane region of the EGFR. These results show that P-(Tyr)-CaM is an activator of the EGFR and suggest that it could contribute to the CaM-mediated ligand-dependent activation of the receptor that we previously reported in living cells.

A synthetic biology-based device prevents liver injury in mice

BACKGROUND & AIMS The liver performs a panoply of complex activities coordinating metabolic, immunologic and detoxification processes. Despite the liver's robustness and unique self-regeneration capacity, viral infection, autoimmune disorders, fatty liver disease, alcohol abuse and drug-induced hepatotoxicity contribute to the increasing prevalence of liver failure. Liver injuries impair the clearance of bile acids from the hepatic portal vein which leads to their spill over into the peripheral circulation where they activate the G-protein-coupled bile acid receptor TGR5 to initiate a variety of hepatoprotective processes. METHODS By functionally linking activation of ectopically expressed TGR5 to an artificial promoter controlling transcription of the hepatocyte growth factor (HGF), we created a closed-loop synthetic signalling network that coordinated liver injury-associated serum bile acid levels to expression of HGF in a self-sufficient, reversible and dose-dependent manner. RESULTS After implantation of genetically engineered human cells inside auto-vascularizing, immunoprotective and clinically validated alginate-poly-(L-lysine)-alginate beads into mice, the liver-protection device detected pathologic serum bile acid levels and produced therapeutic HGF levels that protected the animals from acute drug-induced liver failure. CONCLUSIONS Genetically engineered cells containing theranostic gene circuits that dynamically interface with host metabolism may provide novel opportunities for preventive, acute and chronic healthcare. LAY SUMMARY Liver diseases leading to organ failure may go unnoticed as they do not trigger any symptoms or significant discomfort. We have designed a synthetic gene circuit that senses excessive bile acid levels associated with liver injuries and automatically produces a therapeutic protein in response. When integrated into mammalian cells and implanted into mice, the circuit detects the onset of liver injuries and coordinates the production of a protein pharmaceutical which prevents liver damage.

The influence of coating materials on some properties of alginate beads and survivability of microencapsulated probiotic bacteria

The probiotics, Lactobacillus acidophilus 547, Bifidobacterium bifidum ATCC 1994, and Lactobacillus casei 01, were encapsulated into uncoated calcium alginate beads and the same beads were coated with three types of material, chitosan, sodium alginate, and poly-L-lysine in combination with alginate. The thickness of the alginate beads increased with the addition of coating materials. No differences were detectable in the bead strength by texture analysis or in the thickness of the beads with different types of coating materials by transmission electron microscopy. The survivability of three probiotics in uncoated beads, coated beads, and as free cells (unencapsulated) was conducted in 0.6% bile salt solution and simulated gastric juice (pH 1.55) followed by incubation in simulated intestinal juice with and without 0.6% bile salt. Chitosan-coated alginate beads provided the best protection for L. acidophilus and L. casei in all treatments. However, B. bifidum did not survive the acidic conditions of gastric juice even when encapsulated in coated heads. (C) 2004 Elsevier Ltd. All rights reserved.

Nitric oxide synthase inhibition in sepsis? Lessons learned from large-animal studies

Nitric Oxide (NO) plays a controversial role in the pathophysiology of sepsis and septic shock. Its vasodilatory effects are well known, but it also has pro- and antiinflammatory properties, assumes crucial importance in antimicrobial host defense, may act as an oxidant as well as an antioxidant, and is said to be a vital poison for the immune and inflammatory network. Large amounts of NO and peroxynitrite are responsible for hypotension, vasoplegia, cellular suffocation, apoptosis, lactic acidosis, and ultimately multiorgan failure. Therefore, NO synthase (NOS) inhibitors were developed to reverse the deleterious effects of NO. Studies using these compounds have not met with uniform success however, and a trial using the nonselective NOS inhibitor N-G-methyl-L-arginine hydrochloride was terminated prematurely because of increased mortality in the treatment arm despite improved shock resolution. Thus, the issue of NOS inhibition in sepsis remains a matter of debate. Several publications have emphasized the differences concerning clinical applicability of data obtained from unresuscitated, hypodynamic rodent models using a pretreatment approach versus resuscitated, hyperdynamic models in high-order species using posttreatment approaches. Therefore, the present review focuses on clinically relevant large-animal studies of endotoxin or living bacteria-induced, hyperdynamic models of sepsis that integrate standard day-today care resuscitative measures.

Emulsion strategies in the microencapsulation of cells: Pathways to thin coherent membranes

Microencapsulation of cell spheroids in an immunoselective, highly biocompatible, biomembrane offers a way to create viable implantation options in the treatment of insulin-dependent diabetes mellitus (IDDM). Traditionally the encapsulation process has been achieved through the injection/extrusion of alginate/cell mixtures into a calcium chloride solution to produce calcium alginate capsules around the cells. A novel alternative is explored here through a procedure using an emulsion process to produce thin adherent calcium alginate membranes around cell spheroids. In this study, a thorough investigation has been used to establish the emulsion process parameters that are critical to the formation of a coherent alginate coat both on a model spheroid system and subsequently on cell spheroids. Optical and fluorescence microscopy are used to assess the morphology and coherence of the calcium alginate/ poly-L-ornithine/alginate (APA) capsules produced. (c) 2005 Wiley Periodicals, Inc.