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Nascent transcript-binding protein of the pea chloroplast transcriptionally active chromosome.

This study describes the nascent RNA-binding protein of the pea chloroplast transcriptional complex. The protein has been identified by photoaffinity labelling of the transcriptionally active chromosome (TAC) which utilizes the endogenous plastid DNA as template. UV irradiation of lysed chloroplast or the isolated TAC under conditions optimized for transcription photocross-links nascent radiolabelled transcripts (up to 250 nucleotides in length) to a 48 kDa protein. The photoaffinity labelling of the transcript-binding protein is dependent on UV irradiation, is maximal after about 30 min of irradiation, and is completely dependent on transcriptional activity; no cross-linkage has been observed with pre-synthesized RNA. Cross-linkage is influenced by salts and inhibitors in accordance with their effects on transcription. The photoconjugate is composed of protein and RNA moieties, and can be hydrolysed by several proteases. However, the cross-linked transcript is protected from nucleases until the protein is removed. Manganese enhances photoaffinity labelling of the transcript-binding protein, and this is paralleled by an increase in total transcriptional activity of TAC. This protein was isolated by 2-dimensional polyacrylamide gel electrophoresis and the sequence of 15 amino acid residues at the amino terminus was determined. The nascent transcript-binding protein appears to be involved in the transcription of all three classes of chloroplast genes. We also found a polypeptide of identical molecular weight to get cross-linked to nascent transcripts in chloroplasts isolated from other legumes such as Cicer arietenum, Vigna radiata and Phaseolus vulgaris, and monocots like Zea mays, Oryza sativa and Pennisetum americanum.

Affinity Labels↗

Protective characteristics of the antigen common to Pseudomonas and Vibrio and significance of bacterial endotoxins in protection against selected bacterial infections.

The protective effect of different vaccines against the killing activity of Pseudomonas aeruginosa and Vibrio parahaemolyticus for mice was studied. Cross experiments confirmed the high protective effect of the Pseudomonas common antigen (original endotoxin protein) contained in Pseudomonas vaccine, which, together with other soluble components, substantial afforded protection of mice against vibrios and thus confirmed the antigenic relationship between Pseudomonas and Vibrio. Satisfactory results were also obtained by vaccination with selected bacterial endotoxin lipopolysaccharides derived from some members of the Enterobacteriaceae family, which in some cases afforded 70-90% and in others only a 20-50% protection against Pseudomonas and Vibrio infection. The protective effect was reduced by up to 2/3 was obtained with vaccines subjected to alkaline hydrolysis. A single dose of Propionibacterium parvum cells not followed by specific vaccination afforded up to 50% protection against challenge with P. aeruginosa or V. parahaemolyticus. Special characteristics of the vaccines studied and their protective effects in relation to some immunological aspects of nonspecific resistance are discussed.

Animals↗

Mammalian 3-methyladenine DNA glycosylase protects against the toxicity and clastogenicity of certain chemotherapeutic DNA cross-linking agents.

DNA repair status is recognized as an important determinant of the clinical efficacy of cancer chemotherapy. To assess the role that a mammalian DNA glycosylase plays in modulating the toxicity and clastogenicity of the chemotherapeutic DNA cross-linking alkylating agents, we compared the sensitivity of wild-type murine cells to that of isogenic cells bearing homozygous null mutations in the 3-methyladenine DNA glycosylase gene (Aag). We show that Aag protects against the toxic and clastogenic effects of 1,3-bis(2-chloroethyl)-1-nitrosourea and mitomycin C (MMC), as measured by cell killing, sister chromatid exchange, and chromosome aberrations. This protection is accompanied by suppression of apoptosis and a slightly reduced p53 response. Our results identify 3-methyladenine DNA glycosylase-initiated base excision repair as a potentially important determinant of the clinical efficacy and, possibly, the carcinogenicity of these widely used chemotherapeutic agents. However, Aag does not contribute significantly to protection against the toxic and clastogenic effects of several chemotherapeutic nitrogen mustards (namely, mechlorethamine, melphalan, and chlorambucil), at least in the mouse embryonic stem cells used here. We also compare the Aag null phenotype with the Fanconi anemia phenotype, a human disorder characterized by cellular hypersensitivity to DNA cross-linking agents, including MMC. Although Aag null cells are sensitive to MMC-induced growth delay and cell cycle arrest, their sensitivity is modest compared to that of Fanconi anemia cells.

Animals↗

Cross-linking of surface IgM in the Burkitt's lymphoma cell line ST486 provides protection against arsenite- and stress-induced apoptosis that is mediated by ERK and phosphoinositide 3-kinase signaling pathways.

The ST486 cell line, derived from a human Burkitt's lymphoma, is a model for antigen-induced clonal deletion in germinal center B-lymphocytes, with apoptosis induced upon cross-linking of surface IgM. Moreover, this cell line is highly sensitive to the induction of apoptosis by many chemicals, including sodium arsenite, a significant environmental contaminant with immunotoxic activity. In contrast to arsenite and other chemicals, surface IgM cross-linking induces apoptosis in ST486 cells with delayed kinetics. Moreover, the initial signaling events following IgM stimulation are associated with cell survival and proliferation and include activation of the extracellular-signal regulated kinase (ERK) and the phosphoinositide 3-kinase (PI3K) pathways. We examined the question of whether IgM-mediated activation of the ERK and PI3K pathways can influence the apoptotic response of ST486 cells following exposure to arsenite and selected drugs with different molecular targets, including cycloheximide, etoposide, and camptothecin, and a physical stress, hyperthermia. Our findings show that IgM-stimulated cells are significantly protected against arsenite and drug-induced apoptosis during a window of several hours after surface IgM cross-linking, as evidenced by an inhibition of cleavage of poly(ADP-ribose) polymerase and lack of morphological changes indicative of apoptosis. Significantly, surface IgM cross-linking also protects against arsenite-induced mitochondrial depolarization as well as caspase-9 cleavage. Furthermore, we demonstrate that this IgM-mediated protection requires the activation of the ERK and PI3K pathways, because inhibition of either pathway blocks the ability of antigen receptor activation to protect against apoptosis. Our study also provides evidence for p90(S6) ribosomal kinase as a point of convergence between the two signaling pathways resulting in the phosphorylation of the pro-apoptotic Bcl-2 family member Bad at serine 112. This investigation demonstrates, for the first time, that specific signals transduced by activation of the B-cell receptor protect cells at a common point of regulation in the apoptotic pathways for diverse stresses.

Apoptosis↗

Protection of chimpanzees from type B hepatitis by immunization with woodchuck hepatitis virus surface antigen.

Two chimpanzees immunized with woodchuck hepatitis virus (WHV) surface antigen (WHsAg) developed antibodies cross-reactive with hepatitis B virus (HBV) surface antigen (HBsAg). After challenge with HBV, one animal was completely protected and the other experienced a subclinical infection, without evidence of liver disease. Three woodchucks immunized with HBsAg developed antibodies to HBsAg which did not cross-react with WHsAg. After challenge with WHV, all three woodchucks developed typical acute infections with associated hepatic lesions. Serological studies with the cross-reactive antibodies raised in chimpanzees suggested that the protective epitopes of WHsAg were related to the group a specificity of HBsAg. These studies indicated that cross-protective epitopes are shared by HBV and WHV; however, the humoral response to these epitopes can vary among species.

Animals↗

A urokinase receptor mRNA binding protein from rabbit lung fibroblasts and mesothelial cells.

The urokinase receptor (uPAR) influences several biological functions relevant to lung injury and repair, including proteolysis, cell migration, and adhesion. In malignant mesothelioma cells, we recently found that a posttranscriptional mechanism involving a cis-trans interaction between a uPAR mRNA sequence and a cytoplasmic uPAR mRNA binding protein (mRNABP) regulates uPAR gene expression (S. Shetty, A. Kumar, and S. Idell. Mol. Cell Biol. 17: 1075-1083, 1997). In this study, we sought to determine if uPAR expression in lung and pleural cells involves a similar posttranscriptional pathway. We first identified and characterized the uPAR mRNABP in rabbit tissues using gel mobility shift, ultraviolet (UV) cross-linking, and RNase protection assays and detected it in liver, heart, brain, spleen, colon, and lung. Phorbol 12-myristate 13-acetate, lipopolysaccharide, transforming growth factor-beta, tumor necrosis factor-alpha, or cycloheximide induced uPAR and uPAR mRNA expression in cultured rabbit pleural mesothelial cells and lung fibroblasts and concurrently reduced the uPAR mRNA-uPAR mRNABP interaction. Using conventional and affinity chromatography, we purified a 50-kDa uPAR mRNABP that selectively binds to a 51-nucleotide fragment of the uPAR coding region. This protein migrates as a monomer when analyzed by SDS-PAGE and UV cross-linking and does not possess intrinsic RNase activity in vitro. A uPAR mRNABP physicochemically and functionally similar to that of human malignant mesothelioma is constitutively expressed in the rabbit lung and other nonneoplastic tissues. In rabbit lung fibroblasts and mesothelial cells, expression of uPAR involves posttranscriptional regulation whereby the uPAR mRNABP appears to interact with a specific coding region cis-element to decrease the stability of uPAR mRNA.

Animals↗

Indications and operative techniques for combined aortoesophageal resection.

Combined aortoesophageal resection was performed in 8 patients, including 7 with esophageal carcinoma and 1 with aortoesophageal fistula. Aortic resection procedures included segmental resection with permanent aorto-aortic bypass (1 case), segmental resection with graft interposition (1 case), semicircumferential resection with patch aortoplasty (3 cases), wedge resection with lateral aortorrhaphy (1 case), and resection of adventitia (2 cases). Protective methods during aortic cross-clamping included one aorto-aortic permanent bypass, one subclavian-aortic bypass, and three axillo-femoral bypass. Postoperative complications include mediastinal abscess, paresis, arrythmia, and pneumonia. Five patients with esophageal carcinoma died within 6 postoperative months. In 4 of these 5 nonsurvivors, metastasis to distant organs including the liver, bone and peritoneal cavity were found at the time of death or autopsy. Those early recurrence cases were characterized by skip lesions and extensive lymph node metastasis with extranodal invasion. The clinical benefit of aortoesophageal resection will be attained by careful preoperative evaluation for case selection and a sufficient protective method for aortic cross-clamping.

Aged↗

Production and characterization of neutralizing monoclonal antibodies against human metapneumovirus F protein.

Human metapneumovirus (hMPV) F protein promotes fusion of viral and cell membranes, and is thought to be a major antigenic determinant that mediates effective neutralization and protection against hMPV infection. In this paper, the development of two mouse monoclonal antibodies (MAbs) by immunization with hMPVinfected cells is described. Immunofluorescence assay (IFA) using hMPV F protein-expressing cells indicated that two MAbs, designated 1G3 and 9B10, recognized hMPV F protein. Both MAbs were found to be reactive to two groups of hMPV by an IFA using two groups of hMPV-infected cells. The 9B10 mAb had strong neutralizing activity against both groups of hMPV, while the 1G3 MAb had only weak neutralization activity. These results indicate that the hMPV F protein is a major antigenic determinant that mediates extensive cross-lineage neutralization and protection. Production of MAbs to the hMPV F protein is critical for development of diagnostic techniques, vaccine research, and studies on viral pathogenesis.

Animals↗

Vaccines prepared from chimeras of foot-and-mouth disease virus (FMDV) induce neutralizing antibodies and protective immunity to multiple serotypes of FMDV.

The G-H loop of VP1 (residues 132 to 159) of foot-and-mouth disease virus (FMDV) is a prominent feature on the virion surface and has an important role in vaccine efficacy, generation of antigenic variants, and cell binding. Using an infectious cDNA of FMDV, we have constructed serotype A viruses in which the G-H loop has been substituted with the homologous sequences from serotype O or C. These chimeric viruses replicated to high titer and displayed plaque morphologies similar to those of wild-type viruses, demonstrating that the functions provided by the loop can be readily exchanged between serotypes. Monoclonal antibody analyses showed that epitopes contained within the loop were transferred to the chimeras and that epitopes encoded by the type A backbone were maintained. Chemically inactivated vaccines prepared from chimeric viruses induced antibodies in guinea pigs that neutralized both type A and either type O or type C viruses. Swine inoculated with the A/C chimera vaccine also produced cross-reactive antibodies, were protected from challenge with the type A virus, and partially protected against challenge with type C. These studies emphasize the importance of epitopes outside of the G-H loop in protective immunity in swine, which is a natural host of FMDV.

Amino Acid Sequence↗

Development of immunity to malaria may not be an entirely active process.

It has never been explained why it takes so long for humans to develop immunity to malaria, although factors such as antigenic variation, antigenic polymorphism, and poor immunological responses to critical antigens are thought to be important. Models of malaria, particularly in rodents, have not been helpful. The course of malaria infection differs considerably between humans and rodents. Mice rapidly develop immunity whereas for most humans it takes several years of exposure for this to occur. Mice typically exhibit high parasitaemias whereas humans typically do not. A significant difference in the immune response of humans and mice to malaria parasites might, in part, explain these differences. Most humans have a preexisting population of activated malaria parasite-specific T cells (cross-reactive T cells) which we have referred to as 'natural' T cells, but such cells have not been observed in mice. These cells, many of which secrete interferon-gamma, might control parasitaemia early in the infection, but a by-product of their further activation by malaria parasites might be disease symptoms. Development of immunity has been thought of as an active process--acquisition of specific antibody and effector T cell responses. However, it might in part reflect induction of tolerance of this preexisting population of disease-inducing T cells as a result of chronic parasitaemia. The initial presence of these Th1-like cells may also impede the development of a Th2-like response necessary for the production of protective antibodies. Persistent cross-reactive stimulation may significantly impede this process.

Animals↗

Interaction of melatonin with model membranes and possible implications in its photoprotective activity.

It is well known that administration of antioxidants represents a successful strategy for preventing the occurrence and for reducing the severity of UV-mediated oxidative damage. Melatonin was recently shown to be an efficacious photoprotective agent. The aim of the present study was to better investigate the interaction of melatonin with model membranes and the possible implications in its photoprotective activity. The antioxidant activity of melatonin was tested in two 'in vitro' experimental models: (a) UV radiation-induced peroxidation in phosphatidylcholine multilamellar vesicles (MLVs); (b) scavenging activity against nitric oxide (NO). Furthermore, we investigated the melatonin/biomembrane interaction by differential scanning calorimetry (DSC) on dimyristoylphosphatidylcholine (DMPC) MLVs and unilamellar vesicles (LUVs). The findings of in vitro antioxidant tests suggest that the photoprotective effect of melatonin should be due, partially at least, to the drug scavenging activity against aqueous and lipophilic free radicals, including NO; besides, melatonin might provide its protective effect against UV radiation-induced damage also by acting as a UV-absorbing screen. The results of DSC experiments have evidenced a good capability of melatonin to interact with DMPC bilayers, causing a significant fluidifying effect; however, the transfer of melatonin in the LUVs is faster than that observed for MLVs, even if both values tend to the maximum values reachable. Our present data allow us to emphasize two points: (1) the fluidifying effect induced by melatonin on lipidic bilayers might act as a cooperative mechanism in its protective effect against peroxidative membrane damage; (2) melatonin appears able to cross biomembranes, so that it could protect intracellular components against peroxidative insult.

Antioxidants↗

Synthesis and gelation of DOPA-modified poly(ethylene glycol) hydrogels.

3,4-Dihydroxyphenylalanine (DOPA) residues are known for their ability to impart adhesive and curing properties to mussel adhesive proteins. In this paper, we report the preparation of linear and branched DOPA-modified poly(ethylene glycol)s (PEG-DOPAs) containing one to four DOPA endgroups. Gel permeation chromatography-multiple-angle laser light scattering analysis of methoxy-PEG-DOPA in the presence of oxidizing reagents (sodium periodate, horseradish peroxidase, and mushroom tyrosinase) revealed the formation of oligomers of methoxy-PEG-DOPA, presumably resulting from oxidative polymerization of DOPA endgroups. In the case of PEG-DOPAs containing two or more DOPA endgroups, oxidative polymerization resulted in polymer network formation and rapid gelation. The amount of time required for gelation of aqueous PEG-DOPA solutions was found to be as little as 1 min and was dependent on the polymer architecture as well as the type and concentration of oxidizing reagent used. Analysis of reaction mixtures by UV-vis spectroscopy allowed the identification of reaction intermediates and the elucidation of reaction pathways. On the basis of the observed reaction intermediates, oxidation of the catechol side chain of DOPA resulted in the formation of highly reactive DOPA-quinone, which further reacted to form cross-linked products via one of several pathways, depending on the presence or absence of N-terminal protecting groups on the PEG-DOPA. N-Boc protected PEG-DOPA cross-linked via phenol coupling and quinone methide tanning pathways, whereas PEG-DOPA containing a free amino group cross-linked via a pathway that resembled melanogenesis. Similar differences were observed for the rate of gel formation as well as the molecular weight between cross-links ((-)M(c)), calculated using equilibrium swelling and the Flory-Rehner equation.

Biocompatible Materials↗

Purification of a protective antigen from a saline extract of Pasteurella multocida.

It has been shown previously that soluble material extracted from Pasteurella multocida P-1059 by a 2.5% NaCl solution protects turkeys from generalized septicemia at a subsequent challenge exposure to the organism. In the present study, a protective antigen was purified from the crude soluble material by chromatographic methods. Four protein peaks were obtained by gel filtration with Sephadex G-200. The protective antigen was detected only in the first peak fraction, which contained a substantial amount of carbohydrate. The peak 1 fraction was adsorbed onto DEAE-cellulose and eluted by a linear gradient of NaCl. Fractions corresponding to a single protein peak were pooled and passed through an immunoadsorbent column to remove any possible serum component originating from the growth medium. The purified antigen had a carbohydrate/protein ratio of 1.5 and formed a single precipitin line with rabbit antiserum against the crude material in gel diffusion and immunoelectrophoresis analyses. The antigen produced antibodies in rabbits and turkeys which formed a single precipitin line against the crude material. Upon sodium dodecyl sulfate-polyacryl-amide gel electrophoresis, the purified antigen showed three protein bands, corresponding to molecular weights of 44,000, 31,000, and 25,000, and one carbohydrate band. The carbohydrate band did not correspond to any of the three protein bands. Upon isoelectric focusing gel analysis, the purified antigen showed two bands (pI = 3.5 to 4.0 and 4.5 to 5.5), but the two bands were antigenically identical by isoelectric focusing crossed immunoelectrophoresis. The 50% protective dose of the purified antigen was between 10 and 50 mug of protein in trials where two doses were given at 14-day intervals to 10- to 20-week-old turkeys.

Animals↗

Calcium-stressed erythrocyte membrane structure and function for assessing glipizide effects on transglutaminase activation.

A proposed mechanism of action of hypoglycemic sulfonylureas is the prevention of transglutaminase-mediated endocytosis of insulin receptors. When activated by high levels of intracellular calcium, transglutaminase (TG) catalyzes the cross-linking of intracellular proteins to membrane proteins and modifies membrane structure and function. This study examined the effects of the sulfonylurea glipizide on TG activity in an erythrocyte model by assessing various membrane ATPase activities and high molecular weight protein polymer formation using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. To activate TG, red blood cells were exposed to 1 mM intracellular Ca2+ using 10(-5) M Ca2(+)-ionophore A23187. In Ca2(+)-stressed cells, calmodulin stimulation (0.1 micrograms/ml) of (Ca2+ + Mg2+)-ATPase was decreased to 21.2% of control activity. Increasing concentrations of calmodulin (0.1-3.0 micrograms/ml) could not overcome the inhibitory effects of TG on the (Ca2+ + Mg2+)-ATPase in Ca2(+)-stressed cells with or without glipizide. An increased Ca2+ sensitivity of calmodulin-independent (Ca2+ + Mg2+)-ATPase due to Ca2+ stress was seen in all Ca2(+)-stressed cells even in the presence of 1 mM glipizide. Structural changes were observed in the form of high molecular weight polymer formation. Cells exposed to high Ca2+ and glipizide (3 x 10(-5)-10(-3) M) showed no improvement in ATPase activity or protection from protein cross-linking compared with cells without the drug. We conclude that in this model glipizide fails to inhibit TG induced protein cross-linking and does not prevent the decrease in (Ca2+ + Mg2+)-ATPase activation in Ca2(+)-stressed red blood cells. This finding considerably weakens the proposal that sulfonylureas act by inhibiting TG activity.

Acetylcholinesterase↗

Shell cross-linked Au nanoparticles.

The organic layer of thiol-protected Au nanoparticles (ca.3 nm in diameter) was cross-linked using ring-opening metathesis polymerization or Michael addition of polyfunctional amines. The shell cross-linked nanoparticles showed increased stability toward thermal treatment and oxidative etching. The Au core of cross-linked nanoparticles was removed in an attempt to prepare hollow capsules. However, Au etching resulted in insoluble materials.

Journal Article↗

Ischemic preconditioning in myocardial revascularization with intermittent aortic cross-clamping.

BACKGROUND: This study tests the hypothesis that initial brief periods of ischemia can increase the protection obtained by intermittent aortic cross-clamping. METHODS: In the control group (n = 18), the procedure was performed under intermittent aortic cross-clamping at 32 degrees C. Patients in the preconditioned gorup (n = 17) received a stimulus of two 3-minute periods of cross-clamping followed by 2 minutes of reperfusion prior to standard operation. CKMB, troponin 1, adenosine, and lactate were obtained from the great cardiac vein at the onset of cardiopulmonary bypass (CPB), at the end of the first anastomosis, and at the end of CPB. RESULTS: CKMB and troponin I were slightly higher at the end of CPB in the control group, while there was no difference between adenosine and lactate levels. [table: see text]. CONCLUSION: There was no difference between groups in terms of myocardial protection.

Adenosine↗

Structural requirements for stabilization of vascular elastin by polyphenolic tannins.

Elastin-associated degeneration and calcification are potential causes of long-term failure of glutaraldehyde (Glut) fixed tissue bioprostheses used in cardiovascular surgery. This vulnerability may be attributed to the inability of Glut to cross-link and adequately protect vascular elastin from enzymatic attack. Tannic acid (TA), a poly galloyl glucose (Glc), is compatible with Glut fixation, binds to vascular elastin, improves resistance to degradation and reduces in vivo calcification. While these results provided evidence of a beneficial interaction between elastin and TA, the nature and mechanisms of these interactions are unclear; moreover, TA-elastin binding exhibits a partial instability after long-term interaction with vascular elastin which could contribute to issues of implant toxicity. In present studies, we used resistance to elastase, mechanical properties, and cell viability assays to evaluate the elastin-stabilizing potential and cytotoxicity of TA derivatives and individual TA components such as acetylated TA (AcTA), pentagalloylglucose (PGG), free gallic acid (Gall) and Glc. Our comparative study demonstrates that polyphenolic hydroxyl groups are the main structural groups essential to the interaction between TA and elastin. Furthermore, we show that PGG, the core structure of TA, possesses the same unique elastin-stabilizing qualities of TA, yet it is much less cytotoxic than TA and thus could be potentially useful as an elastin-stabilizing agent for cardiovascular bioprostheses.

Animals↗

Atrial fibrillation after coronary artery bypass grafting: a comparison of cardioplegia versus intermittent aortic cross-clamping.

Supraventricular tachyarrhythmias following coronary artery bypass grafting are a common cause of postoperative morbidity, with a reported incidence of 10-40%. Two techniques of myocardial protection were assessed to determine their influence on the occurrence of postoperative supraventricular tachyarrhythmias. Group I (n = 82) received cold potassium cardioplegia combined with topical hypothermia and systemic cooling to 28 degrees C. Group II (n = 88) were protected by intermittent aortic cross-clamping with a systemic temperature of 32 degrees C. The overall incidence of atrial fibrillation/flutter was 22.3%. No significant difference was detected in the incidence of clinically important atrial fibrillation/flutter between the two groups [21/82 (25.6%) in group I versus 17/88 (19.3%) in group II, P > 0.25]. There was a positive association with age: in patients over 60 years the incidence of arrhythmias (31.8%) was significantly greater than in those less than 60 years (12.9%), P < 0.01. Sex, cardiopulmonary bypass times, aortic cross-clamp times, number of coronary grafts, end-operative creatine kinase myocardial band isoenzyme, right coronary endarterectomy and perioperative myocardial infarction had no association with the occurrence of postoperative atrial tachyarrhythmias.

Atherectomy, Coronary↗