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Intestinal infarction associated with mesenteric vascular thrombotic disease in the horse.

Intestinal infarction associated with cranial mesenteric artery thrombosis, without strangulation obstruction of the intestine, was studied in 18 horses. Findings normally of value in evaluating horses with acute abdominal pain (heart rate, rectal palpation findings, gastric reflux) or classification of the degree of pain were not helpful in diagnosing the problem. Similarly, packed cell volume, plasma total protein, circulating white blood cell count, peritoneal white blood cell count, and peritoneal fluid total protein were not of value in predicting severity of the intestinal damage, location of infarction, or patient survival. In one half of the cases, peritoneal fluid color was of aid in distinguishing intestinal infarction from strangulation. Findings on gross dissection and limited histologic examination did not support the premise that thromboembolism was the major mechanism for production of infarction. In most of the horses, a thrombus or embolus was not found in the peripheral mesenteric vessels.

Animals↗

Circulating immune complexes following food: delayed clearance in idiopathic glomerulonephritis.

Following a meal containing a variety of animal and vegetable proteins circulating immune complexes (CIC) have been found in sera from eight normal subjects. Levels of CIC rose to significantly higher levels in ten patients with idiopathic immune complex glomerulonephritis and return to fasting levels was significantly delayed. The type of CIC detected bore no relation to those in renal biopsy material. The CIC which accumulated in GN were small (MW similar to or approximately 350,000), and plasma exchange did not influence the extent or duration of CIC rise following the meal. An immunological defect manifested by impaired clearance of frequently encountered antigens may exist in subjects who develop GN. The CIC detected in the serum of these patients may be markers of this state and cannot be assumed to be the pathogenic agents in the disease.

Antigen-Antibody Complex↗

Increased glycation of plasma lipoproteins in diabetic cynomolgus monkeys.

Hyperglycemia associated with diabetes mellitus increases glycation of hemoglobin and serum proteins in human and nonhuman primates. It also has been documented that numerous other circulating proteins may be glycated. In this study we found that most of the major subclasses of lipoproteins (low-density, very low-density, and high-density) from diabetic cynomolgus monkeys were significantly more glycated than were lipoproteins from age- and sex-matched controls. Correlations between levels of glycemic control and glycation of lipoproteins also were significant. Because glycation of lipoproteins has been shown to affect their normal metabolism, this animal model may be useful in determining the interaction between lipoproteins, diabetes mellitus, and the increased risk of atherosclerosis.

Animals↗

Evidence of free and bound leptin in human circulation. Studies in lean and obese subjects and during short-term fasting.

Little is known about leptin's interaction with other circulating proteins which could be important for its biological effects. Sephadex G-100 gel filtration elution profiles of 125I-leptin-serum complex demonstrated 125I-leptin eluting in significant proportion associated with macromolecules. The 125I-leptin binding to circulating macromolecules was specific, reversible, and displaceable with unlabeled leptin (ED50: 0.73 +/- 0.09 nM, mean +/- SEM, n = 3). Several putative leptin binding proteins were detected by leptin-affinity chromatography of which either 80- or 100-kD proteins could be the soluble leptin receptor as approximately 10% of the bound 125I-leptin was immunoprecipitable with leptin receptor antibodies. Significantly higher (P < 0.001) proportions of total leptin circulate in the bound form in lean (46.5 +/- 6.6%) compared with obese (21.4 +/- 3.4%) subjects. In lean subjects with 21% or less body fat, 60-98% of the total leptin was in the bound form. Short-term fasting significantly decreased basal leptin levels in three lean (P < 0.0005) and three obese (P < 0.005) subjects while refeeding restored it to basal levels. The effects of fasting on free leptin levels were more pronounced in lean subjects (basal vs. 24-h fasting: 19.6 +/- 1.9 vs. 1.3 +/- 0.4 ng/ml) compared with those in obese subjects (28.3 +/- 9.8 vs. 14.7 +/- 5.3). No significant (P > 0.05) decrease was observed in bound leptin in either group. These studies suggest that in obese individuals the majority of leptin circulates in free form, presumably bioactive protein, and thus obese subjects are resistant to free leptin. In lean subjects with relatively low adipose tissue, the majority of circulating leptin is in the bound form and thus may not be available to brain receptors for its inhibitory effects on food intake both under normal and food deprivation states.

Carrier Proteins↗

Regulation of blood coagulation by the protein C anticoagulant pathway: novel insights into structure-function relationships and molecular recognition.

The protein C system provides important control of blood coagulation by regulating the activities of factor VIIIa (FVIIIa) and factor Va (FVa), cofactors in the activation of factor X and prothrombin, respectively. The system comprises membrane-bound and circulating proteins that assemble into multi-molecular complexes on cell surfaces. Vitamin K-dependent protein C, the key component of the system, circulates in blood as zymogen to an anticoagulant serine protease. It is efficiently activated on the surface of endothelial cells by thrombin bound to the membrane protein thrombomodulin. The endothelial protein C receptor (EPCR) further stimulates the protein C activation. Activated protein C (APC) together with its cofactor protein S inhibits coagulation by degrading FVIIIa and FVa on the surface of negatively charged phospholipid membranes. Efficient FVIIIa degradation by APC requires not only protein S but also intact FV, which like thrombin is a Janus-faced protein with both procoagulant and anticoagulant potential. In addition to its anticoagulant properties, APC has antiinflammatory and antiapoptotic functions, which are exerted when APC binds to EPCR and proteolytic cleaves protease-activated receptor 1 (PAR-1). The protein C system is physiologically important, and genetic defects affecting the system are the most common risk factors of venous thrombosis. The proteins of the protein C system are composed of multiple domains and the 3-dimensional structures of several of the proteins are known. The molecular recognition of the protein C system is progressively being unraveled, giving us new insights into this fascinating and intricate molecular scenario at the atomic level.

Blood Coagulation↗

Admixture-mapping analysis reveals genetic determinants of the human plasma proteome.

Protein profiling and genetic findings can be integrated to define the genetic architecture of the circulating proteome in chronic diseases. Most self-identified African American (AA) individuals have both African and European genetic ancestry. Admixture mapping can detect genomic association regions in which causal variants exist with substantial differences in allele frequency or effect sizes between genetic ancestries. We performed admixture mapping of the circulating proteome in 1,989 participants from the Jackson Heart Study (JHS), investigating the relation of local African ancestry within genomic regions with levels of circulating proteins. We conditioned protein-local ancestry association models on variants previously found to be associated with those proteins in genome-wide association studies (GWASs). We replicated findings in 196 AA participants from the Multi-Ethnic Study of Atherosclerosis (MESA). 62 proteins were associated with local African ancestry. 21 of 62 remained statistically significant after conditioning on protein-associated variants observed in previous GWASs. 48 of 54 available protein-local ancestry associations were replicated in the MESA. Proteins associated with local African ancestry included chemokines, factors associated with vascular biology and inflammation, and other biologically interesting proteins. Admixture associations unexplained by previously reported protein-associated variants in conditional analysis suggest the existence of causal variants missed by standard GWAS techniques.

Aged↗

Differential expression of insulin receptor tyrosine kinase inhibitor (fetuin) gene in a model of diet-induced obesity.

The Differential Display technique has been used to identify differences in mRNA expression in adipose tissue after the introduction of a high fat diet to two strains of rat (OM and S5B/PI) that differ in their susceptibility to develop obesity on this diet. The insulin receptor tyrosine kinase inhibitor protein (fetuin) was shown to be differentially expressed in OM but not S5B/PI rats. This circulating protein may play a role in the development of peripheral insulin resistance associated with high fat diets.

Animals↗

Cloning and characterization of two cDNAs coding for human von Willebrand factor.

A cDNA library was prepared in lambda gt11 bacteriophage from poly(A)+ RNA isolated from primary cultures of endothelial cells from human umbilical vein. Approximately 2.5 million independent recombinants were screened and 2 of those were found to synthesize a fusion protein with beta-galactosidase that reacted with rabbit antibody against human von Willebrand factor. Comparison of the amino acid sequence translated from the cDNA insert of the two clones with the amino acid sequence determined by Edman degradation of the protein established that both phage isolates code for von Willebrand factor. The first clone (lambda HvWF1) contained an insert of 404 nucleotides that corresponded to amino acid residues 1-110 in the mature protein circulating in blood, in addition to a portion (24 amino acids) of a prepro leader sequence. The second cDNA clone (lambda HvWF3) contained an insert of 4.9 kilobases that coded for the carboxyl-terminal 1525 amino acids of von Willebrand factor, a stop codon of TGA, 134 nucleotides of 3' noncoding sequence, and a poly(A) tail of 150 nucleotides. The two clones together code for greater than 80% of the molecule circulating in blood. The same carboxyl-terminal lysine residue was identified in the mature protein as well as in the cDNA, indicating that all of the proteolytic processing that occurs during the biosynthesis and assembly of von Willebrand factor is associated with the amino-terminal portion of the precursor protein. The amino acid sequence of von Willebrand factor indicates the presence of two different internal gene duplications and one triplication. These repetitive amino acid sequences account for about one-half of the amino acids present in the mature protein. The tetrapeptide sequence of Arg-Gly-Asp-Ser, which mediates the cell attachment and platelet binding activity of fibronectin, was also identified in the carboxyl-terminal portion of von Willebrand factor.

Amino Acid Sequence↗

The nasal epithelium as a factory for systemic protein delivery.

We have previously shown that recombinant Sendai virus (SeV) produces efficient in vivo airway epithelial gene transfer. The ability to produce therapeutic levels of circulating proteins following noninvasive gene transfer would have widespread clinical application. Here, we compared nose, lung, and skeletal muscle for the ability to produce circulating levels of the secreted mouse antiinflammatory cytokine interleukin-10 (IL10) following SeV-mediated gene transfer. High levels of serum IL10 were obtained from each site with a potency order of lung > nose > muscle for a given viral titer. Serum levels from each site were within the likely required range for anti-inflammatory effects. The combination of a high-efficiency gene transfer agent (SeV) and sites that can be assessed noninvasively (nose or lung) may circumvent several current challenges to gene therapy.

Animals↗

Characterization of "plasma proteins" secreted by cultured rat macroglial cells.

The brain is isolated behind a blood-tissue barrier that restricts the access of circulating proteins to neural cells. There is evidence that some of these proteins are synthesized within the central nervous system. The present study examines the synthesis and secretion of such proteins by cultured macroglial cells. Primary glial cultures were derived from cortical and subcortical regions of neonatal rat brains, and subsequent secondary cultures were enriched in type-1 astrocytes, type-2 astrocytes, or oligodendrocytes. Newly synthesized proteins were immunoprecipitated from the culture media using antisera directed against whole rat serum. All three types of glial cells secreted a range of plasma proteins. In general, type-1 astrocytes secreted more of these proteins than did type-2 astrocytes or oligodendrocytes, although the one-dimensional polyacrylamide gel electrophoresis (PAGE) profiles were specific for each cell type. Anti-sera directed against specific plasma proteins identified three of the most abundant proteins secreted by type-1 astrocytes as transferrin, alpha-2-macroglobulin, and ceruloplasmin. Northern blot analysis of cellular RNA confirmed that type-1 astrocytes contained transferrin mRNA, and that it was more abundant in cultures derived from subcortical regions than from cortical regions. In situ hybridization studies revealed that virtually all type-1 and type-2 astrocytes contained transferrin mRNA. Since the proteins identified in this study have been proposed to have a variety of neurotrophic roles in the central nervous system, these data further extend the range of possible functions that glial cells may serve in the CNS.

Animals↗

Blockade of protein C activation reduces microvascular surgical blood loss.

Protein C is a natural anticoagulant glycoprotein which prevents intravascular clot formation. Protein C functions as an anticoagulant when converted to an active serine protease (activated protein C). Activated protein C is formed at the site of the endothelial injury in response to blood clotting and helps limit the size of blood clots. We tested the hypothesis that by temporarily blocking the activation of intrinsic protein C, we could reduce subsequent surgical blood loss from a microvascular surgical wound. The formation of activated protein C was blocked systemically by intravenous administration of a monoclonal antibody (HPC4) which binds to circulating protein C and prevents its conversion to activated protein C. Domestic pigs were blindly pretreated with intravenous HPC4 or saline then underwent partial-thickness skin graft harvesting to create a reproducible microvascular wound. Blood loss was measured from each wound and the hemostatic effect of protein C blockade was compared to intravenous saline alone as well as to topical thrombin or thromboplastin. We found that blocking the activation of protein C significantly (P = 0.005) reduces surgical blood loss in this model by 27% compared to saline control animals. Intravenous HPC4 performed equally as well as topical thrombin or tissue thromboplastin. In addition, topical thrombin acted synergistically with HPC4 to reduce blood loss an additional 44% (P = 0.01) as compared to intravenous HPC4 or topical thromboplastin alone. Autopsies performed 1 week after HPC4 treatment showed no evidence of systemic thrombosis resulting from the protein C blockade.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Renal selectivity properties towards endogenous albumin in minimal change nephropathy.

It is well accepted that the molecular charge and conformation of serum proteins are major determinants of their glomerular filtration, but few studies characterizing the molecular features of circulating proteins in renal diseases are currently available. In 11 children affected by minimal change nephropathy (MCN) we determined the electrical charge and the fluorescence quantum yield of Tyrosine (Tyr) and Tryptophan (Trp) (taken as index of conformation) of serum and urinary albumin before and after steroid-induced remission of proteinuria. In all proteinuric children at the onset of the disease, urinary albumin was formed by one band with an isoelectric point (pI) of 4.7 (pI of the native protein), and by numerous other, less anionic bands with pIs between 4.8 and 5.5 accounting for about 50% of the total amount of this protein. The normalization of proteinuria which followed steroid therapy was characterized by the disappearance in urines of the less anionic fraction and by the appearance of numerous isoforms with a pI still more anionic (pI less than 4.7) than normal. At the same time, in the proteinuric phase, the fluorescence quantum yield of Trp of urinary albumin was markedly quenched, returning to near normal levels after steroid-induced remission of proteinuria. These data indicate that in MCN the charge-dependent renal selectivity properties are partially maintained and that the less anionic isoforms of albumin are a main component of urinary albumin. Together with the electrical charge, the conformation of albumin as a major determinant of its urinary excretion in MCN must also be considered.

Adolescent↗

[Changes in the protein and amino acid composition of the blood in patients with peptic ulcer complicated by stenosis].

Hypo- and disproteinemia, a decrease of total circulating proteins, an increased nitrogen excretion in the urine and aminoacid disbalance in blood serum were noted in patients with ulcerous disease complicated with stenosis. These changes were mostly pronounced in decompensated stenosis. Substitutive proteinotherapy in the preoperative period results in normalization of protein metabolism indices in blood only in patients with compensated stenosis.

Adolescent↗

High affinity immunoreactive FGF receptors in the extracellular matrix of vascular endothelial cells--implications for the modulation of FGF-2.

We recently characterized three FGF-binding proteins (FGF-BPs) which are soluble forms of the extracellular domains of the high affinity FGF receptors (Hanneken, A. M., W. Ying, N. Ling, and A. Baird. Proc. Natl. Acad. Sci. USA. 1994. 91:9170-9174). These proteins circulate in blood and have been proposed to modulate the biological activity of the FGF family of proteins. Immunohistochemical studies now demonstrate that these soluble, truncated FGF receptors are also present in the basement membranes of retinal vascular endothelial cells. These immunoreactive proteins can be detected with antibodies raised to the extracellular domain of FGFR-1 but not with antibodies raised to either the juxtamembrane domain or the cytoplasmic domain of FGFR-1. Western blotting of human retinal extracts with the antibody raised to the extracellular domain of FGFR-1 detects specific, low molecular mass proteins at 85 kD and 55 kD, corresponding in size to the FGF-BPs, which are not detected with antibodies against the cytoplasmic domain of the receptor. The interaction of this receptor with the extracellular matrix is not dependent on the presence of FGF-2. Immunoreactive receptors are still detected in vascular basement membranes after the removal of FGF-2 with heparitinase. In addition, the recombinant extracellular domain of FGFR-1 continues to bind to corneal endothelial cell matrix after endogenous FGF-2 has been removed with 2 M NaCl. Acid treatment, which has been shown to disrupt protein interactions with the extracellular matrix, leads to a significant reduction in the presence of the matrix form of the FGF receptor. This loss can be restored with exogenous incubations of the recombinant extracellular domain of FGFR-1. This report is the first demonstration that a truncated form of a high affinity growth factor receptor can be localized to the extracellular matrix. These findings add to the list of binding proteins associated with the extracellular matrix (IGFBP-5) and suggest a potentially new regulatory mechanism for controlling the biological availability of FGF, and other peptide growth factors, in the extracellular matrix.

Amino Acid Sequence↗

Quantitative assessment of a circulating depolarizing factor in shock.

Sustained depolarization of cell membranes and cellular edema are known to accompany various forms of circulatory shock and probably contribute to hypovolemia and cellular dysfunction. It has been proposed that a circulating protein is responsible for these effects. In the present study we have confirmed the existence of a circulating depolarizing factor (CDF) in hemorrhagic shock, burn shock, sepsis, and cardiopulmonary bypass. Plasma samples from pigs or sheep in shock were quantitatively assayed for depolarizing activity using a microelectrode method on rat diaphragm in vitro. The depolarizing effect of CDF in vitro was similar in magnitude to that of shock in situ. We conclude that CDF can entirely account for membrane depolarization during shock. The depolarizing effect of CDF was dose-dependent and saturable; it could be reversed by rinsing the diaphragm with Ringer's or control plasma. CDF activity was detectable in plasma within 5 min after a severe scald and gradually increased over the next 25 min. Resuscitation of hemorrhaged pigs, but not burned sheep, eliminated plasma CDF activity.

Animals↗

Advanced glycation end products/peptides: an in vivo investigation.

Advanced glycation end products/peptides (AGE/peptides) originate by in vivo enzymatic digestion of nonenzymatically glycated proteins, which are produced by reaction of glucose with primary amino groups present in the protein chain following the Maillard pattern. AGE/peptides are highly reactive species and can interact with tissue and circulating proteins, leading to tissue modification and impaired protein functionality. Serum levels of AGE/peptides are reported to be particularly high in diabetes (in terms of higher production) or in end-stage renal disease (in terms of accumulation). For these reasons, their structural identification is of high interest, giving information on their relationship with the pathological state and allowing the design of possible therapeutic interventions. We report here some preliminary results obtained by liquid chromatography/electrospray ionization/mass spectrometry (LC/ESI/MS) and matrix-assisted laser desorption ionization MS (MALDI-MS) investigations carried out on the low-molecular-weight serum peptide fraction from 10 healthy subjects, 10 patients with poorly controlled diabetes, and 10 patients with end-stage nephropathy.

Amines↗

The prionoses and other conformational disorders.

The basic pathogenesis of numerous neurodegenerative disorders is now thought to be related to abnormal protein conformation. The common theme in all these diseases is the conversion of a normal cellular and/or circulating protein into an insoluble, aggregated, beta-sheet rich form which is deposited in the brain, sometimes in the form of amyloid. These deposits are toxic and produce neuronal dysfunction and death. The most common of these illnesses is Alzheimer's disease (AD), in which a central event is the conversion of the normal soluble amyloid beta (sA beta) peptide to amyloid beta (A beta) within neuritic plaques and cerebral vessels. A unique category of the conformational conditions are prion related diseases (or prionoses), where the etiology is thought to be related to conversion of the normal prion protein, PrPC, into an infectious and pathogenic form, PrPSc. In the case of AD and the prionoses, the conformational change can be influenced by the presence of mutations in various gene products, as well as by chaperone proteins. Apolipoprotein E is thought to act as such a chaperone protein in AD; however, among the prionoses such a protein has been hypothesized to exist only by indirect evidence and is called "protein X". Our growing understanding of the mechanisms involved in this category of diseases, raises the possibility of therapeutic approaches based directly on the prevention and reversal of pathologic protein conformation.

Alzheimer Disease↗

Diagnosing protein S deficiency: analytical considerations.

The anticoagulant protein S functions as a cofactor to activated protein C (APC) in the degradation of procoagulant factors Va and VIIIa. In plasma, approximately 70% of circulating protein S is bound to the complement regulatory C4b-binding protein. Deficiency of protein S is an established risk factor for thrombosis, but diagnosing such a condition is not a trivial task. Most protein S deficiencies are quantitative and only few qualitative deficiencies have been characterized. This review is focused on various aspects of protein S: the gene, the protein molecule, and measurement of the protein in plasma. Development of new methods and strict protocols for standardization and sample handling will considerably improve the ability to predict protein S deficiency.

Chromosomes, Human, Pair 3↗