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Biomedical subjects

A Cerami

Publications and source records attributed to A Cerami.

At least 19 recordsLinked to original sources
PubMed1992

Exogenous advanced glycosylation end products induce complex vascular dysfunction in normal animals: a model for diabetic and aging complications.

Advanced glycosylation end products (AGEs) have been implicated in many of the complications of diabetes and normal aging. Markedly elevated vascular tissue and circulating AGEs were linked recently to the accelerated vasculopathy of end-stage diabetic renal disease. To determine the pathogenic role of AGEs in vivo, AGE-modified albumin was administered to healthy nondiabetic rats and rabbits alone or in combination with the AGE-crosslink inhibitor aminoguanidine. Within 2-4 weeks of AGE treatment, the AGE content of aortic tissue samples rose to six times the amount found in controls (P < 0.001). Cotreatment with aminoguanidine limited tissue AGE accumulation to levels two times that of control. AGE administration was associated with a significant increase in vascular permeability, as assessed by 125I label tracer methods. This alteration was absent in animals that received aminoguanidine in addition to AGE. Significant mononuclear cell migratory activity was observed in subendothelial and periarteriolar spaces in various tissues from AGE-treated rats compared to normal cellularity noted in tissues from animals treated with aminoguanidine. Blood pressure studies of AGE-treated rats and rabbits revealed markedly defective vasodilatory responses to acetylcholine and nitroglycerin compared to controls (P < 0.001), consistent with marked NO. inactivation; aminoguanidine treatment significantly prevented this defect. These in vivo data demonstrate directly that AGEs, independent of metabolic or genetic factors, can induce complex vascular alterations resembling those seen in diabetes or aging. AGE administration represents an animal model system for the study of diabetic and aging complications as well as for assessing the efficacy of newly emerging therapies aimed at inhibiting advanced glycosylation.

Acetylcholine↗
PubMed1992

Application of DNA amplification to pneumocystosis: presence of serum Pneumocystis carinii DNA during human and experimentally induced Pneumocystis carinii pneumonia.

Pneumocystis carinii pneumonia is a leading cause of morbidity and mortality in patients with the acquired immunodeficiency syndrome (AIDS). Much remains unknown about the basic biology of P. carinii and studies of this infection have been hampered by the lack of cultivation methods. We developed a sensitive and specific assay for P. carinii by utilizing DNA amplification of the P. carinii dihydrofolate reductase (DHFR) gene. By this method, P. carinii DNA was detected in the lungs of rats with experimentally induced P. carinii pneumonia 2 wk before the onset of histopathological changes. DNA amplification analysis of serum demonstrated that by 10 wk of corticosteroid treatment, 12 of 12 (100%) infected rats had circulating DHFR DNA. P. carinii DHFR DNA also was detected in the serum of patients with AIDS and active P. carinii pneumonia (12 of 14 sera collected prospectively). Patients with advanced AIDS but without a history of P. carinii pneumonia were negative by this assay (0 of 6 sera examined). Serum polymerase chain reaction may facilitate investigations into the natural history and epidemiology of P. carinii infection, provide insight into the pathogenesis of parasite dissemination, and offer a useful, noninvasive diagnostic test for the detection of human pneumocystosis.

Acquired Immunodeficiency Syndrome↗
PubMed1992

Hemoglobin-AGE: a circulating marker of advanced glycosylation.

Advanced glycosylation end products (AGEs) form spontaneously from glucose-derived Amadori products and accumulate on long-lived tissue proteins. AGEs have been implicated in the pathogenesis of several of the complications of aging and diabetes, including atherosclerosis and renal disease. With the use of recently developed AGE-specific antibodies, an AGE-modified form of human hemoglobin has been identified. Termed hemoglobin-AGE (Hb-AGE), this modified species accounts for 0.42 percent of circulating hemoglobin in normal individuals but increases to 0.75 percent in patients with diabetes-induced hyperglycemia. In a group of diabetic patients treated with the advanced glycosylation inhibitor aminoguanidine, Hb-AGE levels decreased significantly over a 1-month period. Hemoglobin-AGE measurements may provide an index of long-term tissue modification by AGEs and prove useful in assessing the contribution of advanced glycosylation to a variety of diabetic and age-related complications.

Adult↗
PubMed1992

Myelosuppressive effects in vivo of purified recombinant murine macrophage inflammatory protein-1 alpha.

Purified recombinant murine macrophage inflammatory protein-1 alpha (rmuMIP-1 alpha), a cytokine with myelopoietic activity in vitro, was assessed in vivo by injection into C3H/HeJ mice for effects on proliferation (percentage of cells in S phase DNA synthesis of the cell cycle) and absolute numbers of granulocyte-macrophage, erythroid, and multipotential progenitor cells in the femur and spleen, and on nucleated cellularity in the bone marrow, spleen, and blood. rmuMIP-1 alpha rapidly decreased cycling rates (at 2 to 10 micrograms/mouse i.v.) and absolute numbers (at 5 to 10 micrograms/mouse i.v.) of myeloid progenitor cells in the marrow and spleen. These effects were dose- and time-dependent and reversible. Suppressive effects were noted within 3 to 24 h for cell cycling and absolute numbers of progenitor cells in the marrow and spleen, and by 48 h for circulating neutrophils. A study comparing the effects of i.v. injection of rmuMIP-1 alpha versus rmuMIP-1 beta, a biochemically similar molecule but with no myelosuppressive effects in vitro, demonstrated myelosuppression in vivo by rmuMIP-1 alpha, but not by rmuMIP-1 beta. The results suggest that rmuMIP-1 alpha has myelosuppressive activity in vivo and offers the possibility that it may be a useful adjunct to treatments involving cytotoxic drugs because of its reversible suppressive effects on normal progenitor cell cycling.

Animals↗
PubMed1992

Preliminary crystallographic analysis of murine macrophage inflammatory protein 2.

Macrophage inflammatory protein 2 (MIP-2) has been crystallized by vapor diffusion of an 11 mg/ml protein solution in 100 mM-ammonium acetate against 30 to 40% polyethylene glycol (average molecular mass of 3350 Da). The crystals belong to space group P2(1)2(1)2(1) and have unit cell dimensions of a = 42.7 A, b = 59.3 A, and c = 100.3 A. The molecular mass of the protein and volume of the unit cell suggest that there are four monomers in the asymmetric unit. A data set to 2.3 A has been collected, and the self-rotation function identifies the presence of a non-crystallographic 2-fold axis.

Animals↗
PubMed1992

Macrophage inflammatory protein 1 modulates macrophage function.

Macrophage inflammatory protein 1 (MIP 1), initially purified from the conditioned medium of endotoxin-stimulated macrophages, is a low m.w. heparin-binding protein doublet comprising two peptides, MIP 1 alpha and MIP 1 beta. Although native doublet MIP 1 has previously been shown to exert pyrogenic, mitogenic, and proinflammatory effects on other cell types, its actions on its cell of origin, the macrophage, have not been well catalogued. Our study reports several aspects of macrophage function that are modulated by MIP 1. MIP 1 was not directly cytotoxic for WEHI tumor cells, but MIP 1-treated macrophage exhibited enhanced antibody-independent macrophage cytotoxicity for tumor targets. MIP 1 treatment stimulated proliferation of mature tissue macrophages, and this effect was enhanced upon costimulations with either CSF-1 or granulocyte-macrophage-CSF. Thioglycollate-elicited peritoneal exudate macrophages incubated with native doublet MIP 1-secreted bioactive TNF and IL-6, as well as immunoreactive IL-1 alpha, and these effects were enhanced significantly when the cells were costimulated with IFN-gamma. Purified preparations of the recombinantly derived MIP 1 alpha peptide alone stimulated the secretion of TNF, IL-1 alpha, and IL-6 by peritoneal macrophages, but MIP 1 beta did not. In fact, as little as eightfold excess MIP 1 beta blocked TNF-induction by MIP 1 alpha to a significant degree. By contrast to these apparent "macrophage activating" properties of MIP 1, the cytokine failed to trigger the macrophage oxidative burst, or to up-regulate the expression of Ia on the macrophage surface. Taken together, these data reveal that MIP 1 peptides act as autocrine modulators of their cells of origin, and raise the possibility that MIP 1 peptides may play a role in modulating macrophage responses to inflammatory stimuli in vivo.

Animals↗
PubMed1992

Identification of cyclophilin as a proinflammatory secretory product of lipopolysaccharide-activated macrophages.

We have isolated an 18-kDa peptide (designated sp18, for 18-kDa secreted protein) from the conditioned medium of lipopolysaccharide-stimulated RAW 264.7 murine macrophages. Purified sp18 had in vivo inflammatory activity and in vitro chemotactic activity for human peripheral blood polymorphonuclear leukocytes and monocytes. Surprisingly, N-terminal sequencing and tryptic mapping studies revealed that sp18 and cyclophilin, an intracellular protein that binds the immunosuppressive drug cyclosporin A, are highly homologous. The in vitro chemotactic activity of sp18 on monocytes was blocked by cyclosporin A but not by cyclosporin H, a structural analog of cyclosporin A that does not bind cyclophilin. Like purified porcine cyclophilin, mouse sp18 exhibited peptidyl-prolyl cis-trans isomerase activity. Medium conditioned by lipopolysaccharide-stimulated resident peritoneal exudate macrophages isolated from C57BL/6 mice contained substantially higher levels of sp18/cyclophilin than medium conditioned by nonstimulated macrophages. The observation that sp18/cyclophilin exhibits proinflammatory activity and is secreted by macrophages in response to endotoxin suggests that this protein may function as a cytokine, and invites the hypothesis that the immunosuppressive action of cyclosporin A results in part from interaction with an extracellular form of cyclophilin released as a mediator of immune and inflammatory functions.

Amino Acid Isomerases↗
PubMed1992

Immunochemical detection of advanced glycosylation end products in vivo.

Reducing sugars react with protein amino groups to form a diverse group of protein-bound moieties with fluorescent and cross-linking properties. These compounds, called advanced glycosylation end products (AGEs), have been implicated in the structural and functional alterations of proteins that occur during aging and long-term diabetes. Although several AGEs have been identified on the basis of de novo synthesis and tissue isolation procedures, the measurement of AGE compounds in vivo has remained difficult. As an approach to the study of AGE formation in vivo, we prepared polyclonal antiserum to an AGE epitope(s) which forms in vitro after incubation of glucose with ribonuclease (RNase). This antiserum proved suitable for the detection of AGEs which form in vivo. Both diabetic tissue and serum known to contain elevated levels of AGEs readily competed for antibody binding. Cross-reactivity studies revealed the presence of a common AGE epitope(s) which forms after the incubation of diverse proteins with glucose. Cross-reactive epitopes also formed with glucose 6-phosphate or fructose. These data suggest that tissue AGEs which form in vivo appear to contain a common immunological epitope which cross-reacts with AGEs prepared in vitro, supporting the concept that immunologically similar AGE structures form from the incubation of sugars with different proteins (Horiuchi, S., Araki, N., and Morino, Y. (1991) J. Biol. Chem. 266, 7329-7332). None of the known AGEs, such as 4-furanyl-2-furoyl-1H-imidazole, 1-alkyl-2-formyl-3,4-diglycosylpyrrole, pyrraline, carboxymethyllysine, or pentosidine, were found to compete for binding to anti-AGE antibody. These data further suggest that the dominant AGE epitope which forms from the reaction of glucose with proteins under native conditions is immunologically distinct from the structurally defined AGEs described to date.

Adolescent↗
PubMed1992

Inhibition by chloroquine of a novel haem polymerase enzyme activity in malaria trophozoites.

The incidence of human malaria has increased during the past 20 years; 270 million people are now estimated to be infected with the parasite. An important contribution to this increase has been the appearance of malaria organisms resistant to quinoline-containing antimalarials such as chloroquine and quinine. These drugs accumulate in the acid food vacuoles of the intraerythrocytic-stage malaria parasite, although the mechanism of their specific toxicity in this organelle is uncertain. The primary function of the food vacuole is the proteolysis of ingested red cell haemoglobin to provide the growing parasite with essential amino acids. Haemoglobin breakdown in the food vacuole releases haem, which if soluble can damage biological membranes and inhibit a variety of enzymes. Rather than degrading or excreting the haem, the parasite has evolved a novel pathway for its detoxification by incorporating it into an insoluble crystalline material called haemozoin or malaria pigment. These crystals form in the food vacuole of the parasite concomitant with haemoglobin degradation, where they remain until the infected red cell bursts. The structure of haemozoin comprises a polymer of haems linked between the central ferric ion of one haem and a carboxylate side-group oxygen of another. This structure does not form spontaneously from either free haem or haemoglobin under physiological conditions, and the biochemistry of its formation is unclear. Here we report the identification and characterization of a haem polymerase enzyme activity from extracts of Plasmodium falciparum trophozoites, and show that this enzyme is inhibited by quinoline-containing drugs such as chloroquine and quinine. This provides a possible explanation for the highly stage-specific antimalarial properties of these drugs.

Animals↗
PubMed1992

Inflammatory cytokines.

The immune system produces cytokines and other humoral factors to protect the host when threatened by inflammatory agents, microbial invasion, or injury. In some cases this complex defense network successfully restores normal homeostasis, but at other times the overproduction of immunoregulatory mediators may actually prove deleterious to the host. Some examples of immune system-mediated injury have been extensively investigated including anaphylactic shock, autoimmune disease, and immune complex disorders. More recently it has become clear that the cytokine cachectin/tumor necrosis factor (TNF) occupies a key role in the pathophysiology associated with diverse inflammatory states and other serious illnesses including septic shock and cachexia. For example, when cachectin/TNF is produced by resident macrophages during early microbial infection, it mediates an inflammatory response that may alienate and repel the attacking organisms. If the infection spreads, however, the subsequent release of large quantities of cachectin/TNF into the circulation may be catastrophic and trigger a state of lethal shock. These toxic effects occur by direct action of TNF on host cells and by the interaction with a cascade of other endogenous mediators including interleukin-1 and interferon-gamma. The biology of cachectin/TNF will be reviewed, along with the potential for modulating the effects of this pluripotent molecule in a variety of pathologic states.

Animals↗
PubMed1992

Cloning, sequencing, and demonstration of polymorphism in trypanothione reductase from Crithidia fasciculata.

Trypanothione reductase (TR) is a target for drug design since it is unique to trypanosomatids, substituting for the otherwise ubiquitous enzyme, glutathione reductase. We report the cloning and sequencing of several cDNAs and genes encoding Crithidia fasciculata TR, the structure of which has recently been solved by crystallography. Single base polymorphisms are detected in cDNAs (containing 80% of the coding sequence) and two different genomic clones, including a glutamine to glutamate change in the C-terminal region of the TR coding region; other nucleotide changes are silent. Homology (from genomic clones, both of which contained signals appropriate for expression) to the Trypanosoma congolense gene was 63% at the nucleic acid level, with 68% amino acid identity; the significance of homologies to human and Escherichia coli glutathione reductase sequences is discussed. Polymorphic sites in the genomic clones included sites found in the cDNAs, indicating that differences existing in the genomic sequence are real, and propagated to RNA.

Amino Acid Sequence↗
PubMed1992

Interleukin-1 alpha expression is inducible by cholinergic stimulation in the rat adrenal gland.

Interleukin-1-like immunoreactivity has earlier been demonstrated by immunohistochemistry in the noradrenaline-containing chromaffin cells of the rat adrenal gland [Schultzberg et al. (1989) Neuroscience 30, 805-810; Schultzberg et al. (1987) J. Neurosci. Res. 18, 184-189]. In this study, we examine the regulation, upon cholinergic stimulation, of the expression of the cytokine interleukin-1 alpha in the rat adrenal gland. Interleukin-1 alpha and interleukin-1 alpha mRNA levels in the adrenal gland are affected by systemic administration of the cholinergic agonists nicotine (0.5 mg/kg, i.p.) and carbachol (0.5 mg/kg, i.p.). Both drugs cause an increase in interleukin-1 alpha mRNA levels. In contrast to the increased mRNA levels, nicotine and carbachol reduce the interleukin-1 alpha protein level measured in the rat adrenal gland: nicotine by approximately 30%, 60 min after injection, and carbachol by approximately 55%, 30 min after injection. The interleukin-1 alpha protein level returns to control level 90 min after nicotine injection, and 120 min after carbachol injection. We thus found a large, constitutively expressed and inducible pool of interleukin-1 alpha in the rat adrenal gland, which appears to be sensitive to cholinergic stimulation and which may be responsible for some of the local and systemic effects of interleukin-1 alpha. Experiments with Escherichia coli lipopolysaccharide show that this substance, which induces interleukin-1 expression and secretion in macrophages, is also able to induce the expression of interleukin-1 alpha mRNA and interleukin-1 alpha in the adrenal gland when injected at the dose of 2 mg/kg, i.p.

Adrenal Glands↗
PubMed1992

Effects of trypanothione on the biological activity of irradiated transforming DNA.

Held et al. (1984a,b) demonstrated previously that glutathione (GSH), a negatively charged thiol, is significantly less efficient in the hydrogen atom donation repair reaction with radicals induced by radiation in transforming DNA (t-DNA) than are other thiol compounds. Fahey et al. (1991a,b) postulated that the charge on thiols can influence their ability to radioprotect DNA. GSH, which is excluded from the vicinity of DNA due to its negative charge, is less protective than neutral or positively charged thiols. We have investigated this phenomenon further with trypanothione, the conjugate of glutathione and spermidine, N1,N8-bis (L-gamma-glutamyl-L-hemicystinyl-glycyl)-spermidine. Trypanothione exists in aerobic solution largely as the disulphide (T(S)2) but is maintained in the cell in the reduced form (T(SH)2) by means of an NADPH-dependent flavo-enzyme, trypanothione reductase (TR). Experimental data show that T(S)2 in the presence of TR radioprotects t-DNA in the absence of oxygen much better than GSH or spermidine alone or in combination. Little radioprotection by T(S)2 is seen when TR is not present. The results obtained with reduced trypanothione at low concentrations suggest that radioprotection of t-DNA in hypoxia occurs predominantly by H atom donation and slightly by .OH radical scavenging, and the protection is greater than that by GSH or spermidine because the polyamine moiety in trypanothione allows a greater concentration of GSH near the DNA molecule.

Bacillus subtilis↗
PubMed1992

Correlation between tumor necrosis factor-alpha (TNF-alpha)-induced cytoskeletal changes and human collagenase gene induction.

Tumor necrosis factor-alpha (TNF-alpha) has been shown not only to induce the biosynthesis and secretion of collagenase but also to change the organization of cytoskeletal components. In the present study we explore the correlation between the biosynthesis of collagenase (by mRNA hybridization, indirect immunofluorescence and collagenolytic activity), and cytoskeletal reorganization (by rhodamine-phalloidin staining of F-actin) induced in fibroblasts by recombinant TNF (rTNF). In the concentration range of 1-100 ng/ml, rTNF increased extracellular collagenase activity 8-fold and collagenase mRNA 4-fold. In addition, whereas the collagenase mRNA was detected as early as 24 h posttreatment, the appearance of extracellular collagenase activity required 48 h. Using phalloidin to follow the organization of the cytoskeleton we observed that rTNF disrupted the parallel array of stress fibers normally observed in the perinuclear region. In contrast to the time required to affect collagenase synthesis, the effect of rTNF on stress fiber organization occurred as early as 6 h post-treatment. Finally, while the number of cells exhibiting this change increased with increasing concentrations of rTNF, a maximum of about 30% of the cells showed this effect. Interestingly, double staining studies demonstrated that both stress fiber changes and procollagenase production occurred in the same cells. This finding, together with the observation that the cytoskeletal disorganization preceded collagenase gene induction by at least 18 h is consistent with the conclusion that the organizational status of the microfilaments may have a role as a regulator of procollagenase gene expression.

Actin Cytoskeleton↗
PubMed1992

Metabolism and functions of trypanothione in the Kinetoplastida.

Trypanosomatids differ from all other organisms in their ability to conjugate the sulfur-containing tripeptide, glutathione, and the polyamine, spermidine, to form trypanothione [N1,N8-bis(glutathionyl)spermidine]. Together with the NADPH-dependent flavoprotein, trypanothione reductase, the dithiol form of trypanothione provides an intracellular reducing environment in these parasites, substituting for glutathione and glutathione reductase found in the mammalian host. Trypanothione and its related enzymes are involved in defense against damage by oxidants, certain heavy metals, and possibly xenobiotics. Trypanothione and its metabolic precursor, glutathionylspermidine, are also implicated in the modulation of spermidine levels during growth. Several existing trypanocidal drugs interact with the trypanothione system, suggesting that trypanothione metabolism may be a good target for the development of new drugs. The purification and properties of three key enzymes (glutathionylspermidine synthetase, trypanothione synthetase, and trypanothione reductase) are discussed, and the catalytic mechanism, substrate-specificity, and the three-dimensional structure of trypanothione reductase are compared to that of glutathione reductase.

Animals↗
PubMed1992

Advanced glycosylation endproducts block the antiproliferative effect of nitric oxide. Role in the vascular and renal complications of diabetes mellitus.

Advanced glycosylation endproducts (AGEs) accumulate on long-lived tissue proteins such as basement membrane collagen and have been implicated in many of the long-term complications of diabetes mellitus. These products originate from glucose-derived Schiff base and Amadori products but undergo a series of complex rearrangement reactions to form ultimately protein-bound, fluorescent heterocycles. AGEs can react with and chemically inactivate nitric oxide (NO), a potent endothelial cell-derived vasodilator and antiproliferative factor. Since mesenchymal cell proliferation is an early and characteristic lesion of diabetic vasculopathy and glomerulopathy, we investigated the possibility that collagen-bound AGEs functionally inactivate the antiproliferative effect of NO. In model cell culture systems, AGEs were found to block the cytostatic effect of NO on aortic smooth muscle and renal mesangial cells. The inactivation of endothelial cell-derived NO by basement membrane AGEs may represent a common pathway in the development of the accelerated vascular and renal disease that accompany long-term diabetes mellitus.

Animals↗