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Interaction of serum proteins with lung endothelial glycocalyx: its effect on endothelial permeability.

To examine directly the interaction of circulating proteins (CP) with the glycocalyx of pulmonary endothelium and its effect on endothelial permeability, two types of experiments were carried out. In the first, rats were exchange transfused with graded amounts of FC-43 fluorocarbon emulsion (FCE) resulting in CP concentrations of 25, 10, and 4 mg/ml, respectively. In the second, rats were exchange transfused with FCE to remove 99.9% of CP. The rats were then exchange transfused with 1 ml FCE containing 60 mg/ml of rat serum protein and killed 3.5, 7.5, and 15 min after the administration of protein. In all animals the distribution of albumin and immunoglobulin G (IgG) was visualized by immunocytochemistry, and endothelial permeability to native ferritin was measured by morphometry. In the depletion experiments increased endothelial permeability to ferritin coincided with loss of adsorbed albumin and IgG from the glycocalyx. Conversely, the presence of administered serum proteins in the glycocalyx and in a few luminal vesicles was associated with an endothelial permeability to ferritin indistinguishable from that of controls. These observations suggest that the adsorption of CP to the endothelial glycocalyx renders the underlying endothelium less permeable to ferritin.

Animals↗

Biotransformations of pituitary luteinizing hormone in serum and urine. I. Association with serum components.

In the effort to elucidate the nature of luteinizing hormone (LH) in the circulation, studies in adult male rats have been conducted using a highly purified and well-characterized tritiated and methylated ovine pituitary LH, a derivative which retains full biological activity. Following an intravenous injection of the radioactive hormone, serum chromatograms (molecular exclusion chromatography) are characterized by three radioactive components. The first one, a rapidly formed high molecular weight heterogeneous fraction, involves the non-covalent interaction of the hormone with one or more circulating proteins. These high molecular weight complexes are cleared rather slowly from the circulation, relative to the non-associated hormone. A second component co-chromatographs with control hormone and is rapidly cleared from the circulation. These two fractions retain full biological activity judged by their ability to stimulate testosterone production in Leydig cell suspensions. A third component is observed in the circulation about 15 minutes after injection and was shown to represent tritiated amino acids. The lag period for the appearance of the labeled amino acids in the circulation correlates with the kinetics of hepatic and renal uptake and subsequent lysosomal catabolism of [3H]methylated-LH. The high molecular weight fraction may represent a physiologically important circulatory storage form for LH by conferring a relatively long circulatory half-life. This appears to be brought about mainly by reduced urinary excretion, presumably due to its high molecular weight.

Animals↗

Adipose tissue adiponectin production and adiponectin serum concentration in human obesity and insulin resistance.

The role of adiponectin production for the circulating protein concentration in human obesity and insulin resistance is unclear. We measured serum concentration and sc adipose tissue secretion rate of adiponectin in 77 obese and 23 nonobese women with a varying degree of insulin sensitivity. The serum adiponectin concentration was similar in both groups. In obesity, adiponectin adipose tissue secretion rate per weight unit was reduced by 30% (P = 0.01), whereas total body fat secretion rate was increased by 100% (P < 0.0001). In the group being most insulin resistant (1/3), serum concentration (P < 0.001) and adipose tissue secretion rate per tissue weight (P < 0.05) were reduced, whereas total body fat secretion rate was increased (P < 0.01), by about 30%. The adipose tissue secretion rate of adiponectin was related to the serum concentration (P = 0.005) but explained only about 10% of the interindividual variation in circulating adiponectin and insulin sensitivity. The plasma adiponectin half life was long, 2.5 h. In conclusion, the role of protein secretion for the circulating concentration of adiponectin and insulin sensitivity under these conditions is minor because adiponectin turnover rate is slow. Although increased in obesity and insulin resistance, total body production of adiponectin is insufficient to raise the circulating concentration, may be due to reduced secretion rate per tissue unit.

Adiponectin↗

Protein transport to the epididymal lumen.

Experiments were performed to clarify the debate over the entry of circulating proteins into the epididymal lumen by use of the marker horseradish peroxidase (HRP). Epididymal tubules from the caput epididymidis of the rat were immersed in medium TC 199 containing HRP (3.5 mg/ml) for 5 min to 3 h at 33 degrees C. Sections were examined for the presence of tracer within the epithelial cells by electron microscopy. From 5 min to 3 h, vesicles containing peroxidase reaction products were found throughout the cytoplasm of the principal cells. Vesicles occurred close to both the basal and apical membranes, and many were found opening into the interstitial space and lumen, depending on the length of incubation. By 5 min labelled vesicles were infrequently found in the apical part of the cells. Reaction product was observed in the epididymal lumen adhering to the microvilli from 30 min of incubation onwards. At all periods of incubation peroxidase was present at the base of the epithelium and between the cells, but it was never found within the tight junctional complexes, and no reaction deposits were found within epithelial cells of tubules incubated in the absence of peroxidase. It is concluded that large molecules leaving the capillaries may enter the epididymal lumen in the caput by means of fluid-phase endocytosis.

Animals↗

The role of mass spectrometry in the study of non-enzymatic protein glycation in diabetes.

Mass spectrometry has been applied successfully to the study of non-enzymatic protein glycation, which is a topic of wide interest in the diabetes field. Low- and high-resolution mass spectra, GC/MS, and collisional activation spectroscopy allow the structural identification and quantitative evaluation of advanced glycation end-products, which represent important molecules for monitoring diabetes. More recently available techniques, such as ESI and MALDI/MS, have had a significant impact on analytical problems in diabetes. In particular, MALDI has been applied to the study of protein glycation in in vitro and in vivo conditions, because the number of glucose molecules that condense onto the protein can be easily determined by this approach. In the former case, glycation kinetics have been studied in various sugars and sugar concentrations, proteins, and buffer concentrations; in the latter, comparisons of MALDI spectra of circulating proteins from healthy and diabetic subjects determine the exposure of patients to high glucose levels. The method has been applied to an evaluation of the glycation level of immunoglobulins, and indicates that glycation takes place preferentially on the Fab fragment of the protein; data are relevant in relating immunological impairment with glycation-induced changes in the functionality of immunoglobulins.

Animals↗

Scavenger receptors on sinusoidal liver endothelial cells are involved in the uptake of aldehyde-modified proteins.

Scavenger receptors on sinusoidal liver endothelial cells (SECs) eliminate potentially harmful modified proteins circulating through the liver. It was shown recently that aldehyde-modified proteins bind to scavenger receptors and are associated with the development/progression of alcoholic liver diseases. For these studies, rat livers were perfused in situ with 125I-formaldehyde-bovine serum albumin (f-Alb) or 125I-malondialdehyde-acetaldehyde-bovine serum albumin (MAA-Alb) in the presence of known scavenger receptor ligands as inhibitors. Reverse transcription-polymerase chain reaction (RT-PCR) analysis and scavenger receptor Type A (SRA) knock-out mice were used to assess the role of these receptors in mediating immune responses. The degradation of 125I-f-Alb or 125I-MAA-Alb in whole livers and isolated SECs can be inhibited by known scavenger receptor ligands, including f-Alb, maleylated bovine albumin, and fucoidan. 125I-f-Alb could not be completely inhibited by MAA-Alb. In contrast, 125I-MAA-Alb was only partially inhibited with advanced glycosylated endproduct albumin. RT-PCR data show the presence of a number of scavenger receptors on SECs that may be responsible for the binding of MAA-modified proteins. SRA seems to be one of these receptors involved in the effects mediated by MAA-modified proteins. In a study using SRA knockout mice, it was shown that a decreased antibody response to MAA-Alb resulted. By RT-PCR, CD36, LOX-1, and SR-AI are the scavenger receptors most likely involved in the degradation of MAA-Alb.

Aldehydes↗

Identification of acute-phase proteins (APP) in circulating immune complexes (CIC) in esophageal cancer patients' sera.

The occurrence of increased circulating immune complexes (CIC) in sera of patients with esophageal cancer and their usefulness for diagnosis and prognosis have not been demonstrated. Circulating acute-phase proteins (APP) related to esophageal cancer have been described but without any association with CIC. This is a study to measure CIC, C-reactive protein (CRP), and alpha 1-acidic glycoprotein (AAG) in pretreatment esophageal cancer sera and to analyze the presence of both APP associated with these CIC. Increased CIC levels were found in 57% of sera from esophageal cancer patients; elevated CRP was detected in 87% and AAG in 47%. Western blot analysis showed the presence of CRP and AAG in CIC-derived fractions. We conclude that: (1) CIC, CRP, and AAG are elevated in esophageal cancer sera; (2) they may be considered possible useful clinical parameters in pretreatment esophageal cancer patients; (3) these APPs appear in CIC precipitates and may possibly be involved in their composition.

Acute-Phase Proteins↗

Limited generation of activated protein C during infusion of the protein C activator thrombin analog W215A/E217A in primates.

Anticoagulation with activated protein C (APC) reduces the mortality of severe sepsis. We investigated whether the circulating protein C (PC) pool could be utilized for sustained anticoagulation by endogenous APC. To generate APC without procoagulant effects, we administered the anticoagulant thrombin mutant W215A;E217A (WE) to baboons. In preliminary studies, administration of high dose WE (110 microg kg(-1) i.v. bolus every 120 min; n = 2) depleted PC levels by 50% and elicited transient APC bursts and anticoagulation. The response to WE became smaller with each successive injection. Low dose WE infusion (5 microg kg(-1) loading + 5 microg kg(-1) h(-1) infusion; n = 5) decreased plasma PC activity by 15%, from 105% to 90%, to a new equilibrium within 60 min. APC levels increased from 7.5 ng mL(-1) to 86 ng mL(-1) by 40 min, then declined, but remained elevated at 34 ng mL(-1) at 240 min. A 22-fold higher dose WE (n = 5) decreased PC levels to 60% by 60 min without significant further depletion in 5 h. The APC level was 201 ng mL(-1) at 40 min and decreased to 20 ng mL(-1) within 120 min despite continued activator infusion. Co-infusion of WE and equimolar soluble thrombomodulin (n = 5) rapidly consumed about 80% of the PC pool with significant temporal increase in APC generation. In conclusion, low-grade PC activation by WE produced sustained, clinically relevant levels of circulating APC. Limited PC consumption in WE excess was consistent with the rapid depletion of cofactor activity before depletion of the PC zymogen. Reduced utilization of circulating PC might have similar mechanism in some patients.

Amino Acid Substitution↗

The effects of acute administration of ethanol on jejunal protein synthesis and circulating insulin-like growth factor (IGF)-1 and IGF binding proteins in ad libitum fed and nutritionally restricted rats.

The effects of acute ethanol administration (75 mmol/kg body weight) to male Wistar rats (either ad libitum fed or nutritionally restricted) on fractional rates of protein synthesis in the jejunum was assessed together with the changes in IGF-1 and IGF binding protein concentrations. Acute administration of ethanol resulted in significant decreases in fractional rates of protein synthesis in the whole jejunum and jejunal seromuscular layers of both the ad libitum fed and nutritionally restricted animals. The synthesis rate per unit RNA (k(RNA), mg protein/day/mg RNA) in whole jejunum was reduced by 29% and 24% in the nutritionally restricted and ad libitum fed animals, respectively. Mean IGF-1 levels were lower in the nutritionally restricted group (871 +/- 36.9 microg/l) than the ad libitum animals (960 +/- 27.3 microg/l) although this did not reach significance. In contrast, administration of alcohol to both groups markedly reduced circulating IGF-1 levels (ad libitum: 518 +/- 19.8 microg/l, nutritionally restricted: 417 +/- 33.7 microg/l). Furthermore, ethanol treatment resulted in a three-fold increase in the intensity of a 30 kDa IGF binding protein (IGFBP) in the ad libitum fed animals and a fourfold increase in both 30 and 32 kDa IGFBP bands in the nutritionally restricted group as visualized by Western ligand blotting. Decreases in levels of IGF-1 allied with increased circulating small molecular weight IGFBPs may contribute to the reduction in fractional rates of protein synthesis in the gastrointestinal tract of ethanol-treated rats.

Journal Article↗

Serum amyloid P component controls chromatin degradation and prevents antinuclear autoimmunity.

Serum amyloid P component (SAP), a highly conserved plasma protein named for its universal presence in amyloid deposits, is the single normal circulating protein that shows specific calcium-dependent binding to DNA and chromatin in physiological conditions. The avid binding of SAP displaces H1-type histones and thereby solubilizes native long chromatin, which is otherwise profoundly insoluble at the physiological ionic strength of extracellular fluids. Furthermore, SAP binds in vivo both to apoptotic cells, the surface blebs of which bear chromatin fragments, and to nuclear debris released by necrosis. SAP may therefore participate in handling of chromatin exposed by cell death. Here we show that mice with targeted deletion of the SAP gene spontaneously develop antinuclear autoimmunity and severe glomerulonephritis, a phenotype resembling human systemic lupus erythematosus, a serious autoimmune disease. The SAP-/- mice also have enhanced anti-DNA responses to immunization with extrinsic chromatin, and we demonstrate that degradation of long chromatin is retarded in the presence of SAP both in vitro and in vivo. These findings indicate that SAP has an important physiological role, inhibiting the formation of pathogenic autoantibodies against chromatin and DNA, probably by binding to chromatin and regulating its degradation.

Animals↗

[Molecular basis of the variability of epidemic strains of human influenza viruses].

The data from literature and authors own studies are reviewed on variability of human influenza viral strains, isolated during the same epidemic season in different periods of pandemic cycle. The data obtained indicate that variability of epidemic strains of human influenza virus deals with the genes coding for outer membrane proteins (hemagglutinin and neuraminidase) as well as nonglycosylated proteins. Circulation of a number of viral variants of the same serotype, differing in antigenic specificity of outer membrane proteins or in the genes coding for nonglycosylated proteins was registered during one and the same season of one epidemic. During circulation of viral variants of the same serotype recombination may take place. Heterogeneity of viral strains circulating during different epidemic seasons of the same pandemic cycle is different. The possible mechanisms of development of the new epidemic variants of human influenza virus are discussed.

Disease Outbreaks↗

Relation of the hepatic and splenic microcirculations to the development of lesions in experimental amyloidosis.

By the use of a perfusion technique for identifying blood vessels, it was found that amyloid lesions in mice have a close relation to the underlying microcirculation. The earliest lesions develop about arteriolar capillaries. With the onset of the lesions, circulation of plasma not only continues through the affected vessels but also extends into the entire volume of the surrounding lesions. Progression of the lesions follows the underlying microcirculation, and there is a continuing presence of circulating proteins. Other observations, from tracer studies using labeled plasma proteins and studies of unfixed, frozen, tissue sections following saline extraction, indicate that much of the early amyloid lesions in these animals is circulating plasma. It would appear that the onset and site of formation of the lesions is determined by arteriolar capillary injury rather than by polymorphism of SAA proteins. Structural amyloid fibril proteins may gain access to the lesions by either entry from the circulation or local formation.

Amyloidosis↗

The cerebrospinal-fluid soluble form of Alzheimer's amyloid beta is complexed to SP-40,40 (apolipoprotein J), an inhibitor of the complement membrane-attack complex.

The amyloid fibrils deposited in Alzheimer's neuritic plaque cores and cerebral blood vessels are mainly composed of aggregated forms of a unique peptide, 39-42 amino acids long, named amyloid beta (A beta). A similar, although soluble, A beta ('sA beta') has been identified in cerebrospinal fluid, plasma and cell supernatants, indicating that it is normally produced by proteolytic processing of its precursor protein, amyloid precursor protein (APP). Using direct binding experiments we have isolated and characterized an 80 kDa circulating protein that specifically interacts with a synthetic peptide identical with A beta. The protein was unmistakably identified as SP-40,40 or ApoJ, a cytolytic inhibitor and lipid carrier, by means of amino acid sequence and immunoreactivity with specific antibodies. Immunoprecipitation with anti-SP-40,40 retrieved soluble A beta from cerebrospinal fluid, indicating that the interaction occurs in vivo.

Amino Acid Sequence↗

The short and long term effect of gastric partitioning surgery on serum protein levels.

The morbidly obese who undergo elective gastric partitioning surgery serve as models of surgical stress and subsequent severe protein calorie restriction. The short and long term effects of gastric partitioning surgery on circulating proteins and the specific micronutrients carried by the proteins were studied in 22 such patients (ages 23-56 years). Serum micronutrient values paralleled the levels of their carrier proteins. Mean concentrations of both short and long turnover proteins decreased significantly in the early postoperative period, whereas the acute phase reactant ceruloplasmin reached preoperative levels by the seventh postoperative day. Transthyretin and retinol binding protein remained depressed with long term reductions in protein and calorie intake, whereas ceruloplasmin and transferrin were somewhat less sensitive to prolonged protein-calorie restriction. Mean serum albumin, after an initial postoperative fall, rose to the baseline level by 1 month after surgery. Serum albumin levels remained within the normal range despite low protein and calorie intakes.

Adult↗

The magnitude of the acute phase protein response is attenuated by protein deficiency in rats.

We assessed the growth rate and changes in plasma albumin, total protein and alpha 2-macroglobulin concentrations (a major acute phase protein in rats) before and after a subcutaneous injection of turpentine (0.5 mg/kg body wt) in groups of rats receiving one of a series of protein-deficient diets (protein concentrations of 0.5, 1.5, 3.0, 4.5 or 6.0 g/100 g) or a diet containing an adequate level of protein (20 g/100 g) for maximal growth. Increasing protein deficiency in the different groups of animals reduced the basal albumin and total protein concentrations and attenuated the total protein and alpha 2-macroglobulin responses to turpentine. Increasing protein deficiency delayed the time taken for alpha 2-macroglobulin to reach peak concentrations post-injection and its return to basal concentrations. The turpentine-induced hypoalbuminemia was similar in all groups of animals (approximately 10 g/L depression) but restoration to values that were present before turpentine injection was increasingly delayed with increasing protein deficiency. The magnitude of the acute phase response (peak alpha 2-macroglobulin concentration) was found to be directly related to growth rate (r = 0.70, P less than 0.001). We concluded that protein deficiency can alter the pattern and magnitude of the acute phase responses in circulating protein concentrations to an extent that is dependent on the severity of protein deficiency.

Acute-Phase Proteins↗

Chronic effects of hyperproteinemia on blood volume and lymph protein concentration.

The long-term effects of hyperproteinemia on blood volume and lymph protein concentration were studied in six conscious dogs over a 17-day period. Plasma protein concentration (PPC) was increased by daily intravenous infusion of approximately 300 ml of previously collected autologous plasma. By day 17 PPC had increased 2.4 g/dl, and plasma colloid osmotic pressure had increased 51%; however, blood volume was not changed. Also, at this time sulfate space, an index of extracellular fluid volume, had increased 12%, prenodal lymph protein concentration had increased from 1.6 to 5.1 g/dl, mean arterial pressure was unchanged, circulating protein mass was increased, and plasma sodium concentration was decreased slightly. In conclusion, the increase in lymph protein concentration during hyperproteinemia may indicate that interstitial fluid protein concentration also increased. This, in turn, would help to prevent any increase in the transcapillary colloid osmotic pressure gradient and thus attenuate any changes in blood volume.

Animals↗

The gene encoding vitamin K-dependent anticoagulant protein C is expressed in human male reproductive tissues.

Protein C is a vitamin K-dependent protein circulating in plasma as a zymogen to an anticoagulant serine protease. After its activation, protein C cleaves and inactivates coagulation factors Va and VIIIa. Human protein C is synthesized in liver and undergoes extensive post-translational modification during its synthesis. Recently, the protein C inhibitor was demonstrated to be synthesized in several organs of the human male reproductive tract. Moreover, vitamin K-dependent protein S, which functions as a co-factor to activated protein C, was found to be synthesized in the Leydig cells of human testis. The aim of this study was to elucidate whether the protein C gene is also expressed in the male reproductive system. Specific immunostaining of protein C was found in Leydig cells of human testis, in the excretory epithelium of epididymis, and in some epithelial glands of the prostate, whereas no immunostaining was detected in seminal vesicles. Northern blotting and non-radioactive in situ hybridization demonstrated protein C mRNA in Leydig cells, in the excretory epithelium of epididymis, and in some of the epithelial glands of the prostate. The mRNA was distributed perinuclearly and the localization was in accordance with the specific immunostaining for protein C. The epithelium of epididymis was also found to contain both protein S mRNA and immunoreactivity. The demonstration of both protein C and protein S immunoreactivities, as well as their mRNAs, in male reproductive tissues suggests as yet unknown local functions for these proteins.

Aged↗

Processing-independent analysis (PIA)--a new diagnostic tool.

Posttranslational processing is an important phase of the expression of most eucaryotic genes in terms of functional proteins. Among these, secretory proteins and peptides are of particular interest for clinical chemists, since diagnostic measurements of circulating proteins and peptides constitute a major discipline in clinical chemistry. The posttranslational covalent maturation of secretory proteins and peptides involves multiple enzymatic modifications of the corresponding proproteins along the intracellular secretory pathway. During the eighties, an increasing amount of evidence has indicated that sick secretory cells fail to process their secretory products normally. The diseased cells therefore fail to process their secretory products normally. The diseased cells therefore release also incompletely processed precursors and processing-intermediates. In order to measure the degree of disease, assays that measure proteins and peptides independent of the degree of processing are therefore desirable. We have now designed a new analytical principle, according to which secretory proteins, peptides and their precursors can be accurately quantitated irrespective of the degree of processing. This principle, named processing-independent analysis (PIA), is generally applicable to all cellular synthesized substances. The principle has been applied to and developed first for a well-defined secretory peptide system, progastrin and its products. Using this model, the results obtained so far confirm the diagnostic superiority of processing-independent analysis in comparison with conventional assays for bioactive peptides.

Amino Acid Sequence↗