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PubMed1997

[Serum protein changes in diabetes mellitus].

AIM: To evaluate the serum protein pattern in a wide sample of diabetic patients using the simple method of electrophoresis, identifying the characteristics for each condition and correlating the various components. EXPERIMENTAL DESIGN: A retrospective study was made using medical records. SETTING: The Institute of Clinical Medicine I at Palermo University Hospital during the years 1990, 1991 and 1992. PATIENTS: 156 patients suffering from diabetes mellitus, of whom 68 were Type 1 (IDDM) and 88 Type 2 (NIDDM). The latter were divided into non-obese (NO-NIDDM), obese (O-NIDDM) and "secondary failures" (SF-NIDDM) to oral hypoglycemic agents (receiving mixed treatment, TM-NIDDM, or insulin alone, I-NIDDM). PARAMETERS: In addition to personal and anthropometric data and clinical data, in particular dysmetabolic data, an electropherogram was performed in each patient. RESULTS: Higher serum albumin concentrations were found in patients with IDDM, NIDDM compared to I-NIDDM, with an overall inverse correlation between the duration of diabetes and albumin serum levels. On the contrary, serum levels of alpha-2 globulins were higher in I-NIDDM patients compared to IDDM, and directly correlated with the age and duration in the overall series. beta-globulin levels were lower in IDDM patients compared to all other groups, and were directly correlated with age and body mass index (BMI) in the overall series. No statistical significance or correlation was found between the two groups in relation to alpha 1 and gamma-globulins. CONCLUSIONS: The study showed a characteristic serum protein pattern for each group of diabetic patients examined, analysing the role of insulin but also the duration and typical pathological events of the natural history of diabetes on protein metabolism, not only with regard to the amino acid metabolism but also on the distribution of circulating proteins.

Adult↗
PubMed1984

Platelet-derived growth factor.

Platelet-derived growth factor (PDGF) is a basic (pI congruent to 10) 30 000 molecular weight protein circulating in normal blood sequestered within the platelet alpha-granules. It binds with high affinity (Kd = 10(-11) M) to a specific cell-surface receptor found on many connective tissue cell types in culture. It is active in stimulating the metabolism and multiplication of connective tissue cells at very low concentrations (ED50 = 10(-11) M). It is likely that PDGF is released from platelets at sites of vascular damage and that it contributes toward the cell proliferation and connective tissue formation seen in healing wounds and in arteriosclerotic lesions. PDGF which does not bind to responsive cells at the wound site is largely inactivated by a plasma binding protein and is rapidly cleared from the circulation.

Adult↗
PubMed1988

Purification of residualizing glycoconjugate labels for protein by reversed-phase high-pressure liquid chromatography.

Residualizing labels are radioactive or fluorescent tracers used for identifying the tissue and cellular sites in which circulating proteins are catabolized in the body. When attached to protein the labels do not affect normal mechanisms of protein catabolism, but remain at the cellular site of protein uptake, after the carrier protein itself is degraded to diffusible catabolites. Until recently these labels consisted of biologically indigestible carbohydrates attached to a radioactive reporter molecule. In this report we describe the synthesis and purification of a new fluorescent residualizing label, N,N-dilactitol-N'-fluoresceinyl-ethylenediamine. The label is prepared by first derivatizing ethylenediamine 1:1 with fluorescein isothiocyanate and then coupling lactose to the remaining primary amino group by reductive amination. A rapid one step purification of this and other glycoconjugate labels by reversed-phase high-pressure liquid chromatography is described.

Affinity Labels↗
PubMed2001

Plasma protein oxidation and antioxidant defense during aging.

BACKGROUND: Oxidative stress is an important process that occurs in vivo during aging and is considered one of the main causes of molecular damage to cellular and tissue structures. These changes can accumulate in biological structures during aging. OBJECTIVE: The aim of this work is to evaluate plasma protein oxidative damage, measured as carbonyl groups content, and the concentration of some antioxidant molecules (vitamins and carotenoids) in 122 healthy volunteers (50 males and 72 females), 25 to 89 years old. RESULTS: Total plasma proteins slightly decreased with age, but the level of carbonyl groups was similar in the adult (< 65 years) and in the old, and was similar in both sexes. Plasma concentration of antioxidant molecules such as alpha-tocopherol, beta-carotene and other carotenoids, increased with age and correlated with the level of lipoproteins; plasma total cholesterol and triglycerides were significantly correlated with age as well. CONCLUSIONS: The surprisingly normal level of plasma protein carbonyl groups in our older subjects suggests two possibilities: a) the older people in our study are healthy and free from pathologies because of better protection against oxidative injury during their lifetimes, i.e., they maintained low-level oxidative damage on plasma proteins; or b) the level of carbonyl groups is normal because of the high turnover in plasma: the oxidized circulating proteins are preferentially and quickly removed; in this case oxidative damage is not discernible in plasma proteins but may proceed silently in other tissues.

Adult↗
PubMed1980

[Sexual organogenesis and circulating androgens in the rabbit fetus. Study after active immunization of mothers against testosterone (author's transl)].

The circulating level of free androgens (testosterone and dihydrotestosterone) in fetal rabbit was reduced by active immunization of mothers against testosterone. The level of testosterone and dihydrotestosterone binding to circulating proteins was about 100% in immunized fetuses and newborns. The testosterone and dihydrotestosterone binding capacities were higher than their circulating concentrations. Macroscopically, all newborns exhibited feminine external genitalia. Histological examinations showed the presence of complete wolffian ducts differentiated in epididymis, deferent ducts and seminal vesicles with normal structure. Prostatic development was strongly inhibited; anterior prostates were present but lateral and posterior prostates were frequently absent. In most animals the masculinization of the genital tubercle was prevented: 60% of males exhibited feminine external genitalia, 40% severe hypospadias. These results suggest: 1 degrees that fetal free circulating androgens (testosterone and/or dihydrotestosterone) induced masculinization of urogenital sinus and genital tubercle; 2 degrees that testicular testosterone was responsible for stabilization and differentiation of the wolffian duct.

Androgens↗
PubMed1995

Effect of high-lipid high-nitrogen intravenous nutrition on total body nitrogen, visceral protein synthesis and nitrogen balance.

The impact of high-lipid intravenous nutrition (IVN) on selected indices of nitrogen retention following major surgical resection was studied. Twenty-two patients, randomly allocated to two equal well matched groups, received either high-lipid IVN (75 per cent non-protein calories supplied as lipid) or isocaloric isonitrogenous glucose IVN (100 per cent non-protein calories supplied as glucose). Total body nitrogen (assessed by in vivo neutron activation analysis), nitrogen balance and levels of circulating proteins were measured. Mean(s.d.) total body nitrogen and fat-free mass decreased (P = 0.04) in patients receiving high-lipid IVN, -109(36) gN and -1.7(0.4) kg respectively, but not in those given glucose-only IVN, 8(43) gN and 0.1(1.0) kg. This small loss of body protein does not appear to be clinically significant because postoperative hospital stay, complication rates and the acute-phase protein response (immunological and visceral) were similar in the two groups.

Body Composition↗
PubMed1975

A study of the influence of various antirheumatic drug regimens on serum acute-phase proteins, plasma tryptophan, and erythrocyte sedimentation rate in rheumatoid arthritis.

In patients with rheumatoid arthritis neither indomethacin nor aspirin influenced the levels of the erythrocyte sedimentation rate (e.s.r) or serum acute-phase proteins fibrinogen, haptoglobin, C-reactive protein and alphaI acid-glycoprotein). Treatment with D-penicillamine, sodium aurothiomalate, or alclofenac produced a significant reduction both in acute-phase protein levels and in e.s.r. Each of the drugs displaced L-tryptophan from plasma proteins in vivo but withdrawal of indomethacin and aspirin was followed immediatley by excessive binding of this amino acid to circulating proteins:this phenomenon was not observed when alclofenac, sodium aurothiomalate or D-penicillamine were withdrawn. It has been demonstrated that disease activity in rheumatoid arthritis is reflected in acute-phase protein concentrations and in the extent to which L-tryptophan is bount to plasma protein. It is suggested that drugs which profoundly affect these parameters provide not only symptomatic relieft but also possible beneficial effects upon the disease process itself.

Adult↗
PubMed1983

Prednisolone disposition and protein binding in oral contraceptive users.

Combined estrogen-progestogen oral contraceptives (OC) have been shown to alter the metabolism of certain drugs, including corticosteroids, as well as affect circulating protein concentrations. To assess these effects with regard to prednisolone, the pharmacokinetics and protein binding of this steroid were evaluated in eight female OC users and compared with results from eight male and five female non-OC users. All volunteers received 40 mg prednisolone, iv, and steroid concentrations were measured by high pressure liquid chromatography. Plasma clearance of total prednisolone in females on OC was 96 +/- 9 (SD) ml/min X 1.73 m2, significantly (P less than 0.001) lower than those in both male and female controls (205 +/- 46 and 187 +/- 22 ml/min X 1.73 m2). The prednisolone half-life and mean residence time were longer, while the steady state volume of distribution was smaller for OC users. Unbound prednisolone was measured by equilibrium dialysis, and pharmacokinetic and protein binding parameters were calculated from free prednisolone concentrations. A significantly higher (2-fold) concentration of transcortin was found in OC users. Evaluation of free prednisolone parameters showed a significantly lower clearance and decreased volume of distribution, without alteration of the mean residence time for the OC users. Dual OC effects on binding and elimination of prednisolone occur with the net result of a 2-fold increase in the area under the free concentration-time curve, indicative of a marked reduction in the biotransformation rate of the steroid.

Adult↗
PubMed1996

Humoral response after administration of E1-deleted adenoviruses: immune privilege of the subretinal space.

An important limitation of E1-deleted recombinant adenoviruses in gene therapy is the immune response that they engender and that rapidly destroys transduced cells. Transduced cells of the outer retina appear to be an exception. To determine whether differences in immune sequestration of the outer retina contribute to the increased stability of transgene expression in this tissue, we examined the systemic humoral response to an E1-deleted adenovirus injected into the subretinal space. Subretinal injection of Ad.CMVlacZ in mature immunocompetent mice resulted in minimal circulating antibody production. In contrast, subcutaneous administration of equivalent doses of Ad.CMVlacZ resulted in high-titer antibody production against adenoviral proteins. Circulating antibodies in systemically immunized animals had minimal effect on retinal transgene expression patterns measured 2 weeks after subretinal injection of Ad.CMVlacZ. Histologic examination showed minimal retinal toxicity attributable to subretinal adenovirus in naive or immunized mice, although 3/18 (14%) of eyes from the latter set contained a localized granulomatous infiltrate at the ocular injection site. The data demonstrate that the subretinal space is an immune-privileged site regarding humoral immunity. Further, short-term transduction efficiency is not affected by the presence of anti-adenoviral antibodies. The retina may be a favorable environment for replication-defective virus-mediated genetic therapy.

Adenovirus E1 Proteins↗
PubMed1992

An evaluation of different enzymatic cleavage methods for recombinant fusion proteins, applied on des(1-3)insulin-like growth factor I.

Different enzymatic methods for cleavage of recombinant fusion proteins were compared. To find an efficient cleavage method, five different fusion proteins were produced. The fusion proteins differed only in the linker region between the fusion partner and the desired product, human des(1-3)insulin-like growth factor I. A cleavage study was performed with enterokinase, plasmin, thrombin, urokinase, and recombinant H64A subtilisin. Significant cleavage was obtained using thrombin, H64A subtilisin, and enterokinase. Thrombin cleavage was studied on a larger scale and des(1-3)IGF-I was recovered at a final yield of 3 mg/L growth medium. Thrombin and enterokinase were also studied as immobilized proteases and they cleaved the fusion proteins with retained activity. To further improve thrombin cleavage, a continuous reactor was constructed, consisting of a closed system with a thrombin column and an ion exchange column in series. Here, the fusion protein circulated while free des(1-3)IGF-I was bound to the ion exchange column after release from the fusion protein. In the reactor, thrombin was as efficient as the free enzyme but gave a diminished rate of product degradation.

Amino Acid Sequence↗
PubMed1996

Serum protein and zinc levels in patients with thoracic empyema.

The element Zn is the metal component or activator of many important enzymes. The tissue concentrations and activities of Zn metalloenzymes direct the rate of protein and nucleic acid syntheses, thereby influencing tissue growth and reperative processes. Most of the serum Zn is normally bound to circulating proteins. Low serum Zn concentrations might result from depletion of Zn-binding proteins. Serum protein and Zn concentrations have been reported to be depressed in patients with acute and chronic diseases. We compare the serum protein and Zn values of patients with thoracic empyema (n = 20) with those of a control group (n = 20). The values obtained in the empyema group were significantly lower than those in the control group before the study. Test group administered 220 mg zinc sulfate (ZnSO4.7H2O) over 20 d and there was a significant increase in the values for serum protein and Zn after the oral administration of the zinc sulfate.

Administration, Oral↗
PubMed1981

O-(4-Diazo-3,5-di[125I]iodobenzoyl)sucrose, a novel radioactive label for determining organ sites of catabolism of plasma proteins.

A method is described for radiolabelling proteins with O-(4-diazo-3,5-di[125I]iodobenzoyl)sucrose (DD125IBS). When proteins so labelled were degraded within lysosomes, the radioactive fragments were largely retained within the organelle. High specific radioactivities were obtained without changing the properties of the protein. The validity of the method was demonstrated in vivo in rats using the short-lived protein lactate dehydrogenase, isoenzyme M4, and the long-lived protein bovine serum albumin. Derivatization with DD125IBS did not alter the clearance of either protein. Uptake of DD125IBS-labelled lactate dehydrogenase, isoenzyme M4, by liver and spleen of rats was determined. Radioactivity in these tissues increased up to about 2 h after injection (at this time the protein has been almost completely cleared from the blood) and subsequently declined with a half-life of approx. 20 h. After differential fractionation of liver, radioactivity was largely found in the mitochondrial and lysosomal fraction. The results of these studies establish that DD125IBS covalently coupled to plasma proteins should be a useful radioactive tracer for identifying the tissue and cellular sites of catabolism of relatively long-lived circulating proteins.

Animals↗
PubMed1992

Monoclonal antibodies against human T cell adhesion molecules--modulation of immune function in nonhuman primates.

The cytotoxic T cell is thought to be a primary effector of allograft rejection. In vitro studies have demonstrated that the interaction between cytotoxic T cells and target cells involves cell surface adhesion molecules that result in conjugate formation, with subsequent antigen recognition, T cell activation, and target cell lysis. Experiments have also demonstrated the ability of monoclonal antibodies with specificity for two human T cell adhesion molecules, lymphocyte function associated (LFA) antigen-1 (LFA-1, CD11a, alpha-chain/CD18, beta-chain) and LFA-2 (CD2), to inhibit conjugate formation in vitro. Studies in a nonhuman primate model were undertaken to determine whether the in vivo administration of monoclonal antibodies with specificity for the alpha chain of LFA-1 (CD11a) or with specificity for CD2 could modulate in vivo T cell function. Cynomolgus monkeys (Macaca fascicularis) received 10 daily intravenous infusions of either anti-CD11a, anti-CD2 or both anti-CD11a and anti-CD2 monoclonal antibodies. Antibody administration was well tolerated and resulted in high levels of circulating murine monoclonal antibody in the peripheral circulation. Nearly all the animals generated antimurine antibodies that were specific for both idiotypic and nonidiotypic determinants of the infused mouse protein. Circulating lymphocytes and T cells were not depleted by treatment with anti-CD11a or anti-CD2 mAbs; in fact, treatment with the combination of anti-CD11a plus anti-CD2 or anti-CD11a alone led to increased numbers of circulating lymphocytes and T cells. Modulation of the LFA-1 molecule on circulating T cells occurred as a result of treatment with anti-CD11a (or the combination of anti-CD11a plus anti-CD2), whereas treatment with anti-CD2 (or anti-CD11a plus anti-CD2) did not result in modulation of the CD2 antigen despite detectable levels of circulating anti-CD2 mAb. In vivo T cell function was assessed by placement of skin allografts. As compared with treatment with saline or a control mAb, allograft survival was significantly prolonged in animals treated with anti-CD11a or combination treatment but not in animals receiving anti-CD2 alone. We conclude that the in vivo administration of anti-LFA-1 mAb may be useful for the blockade of effector T cell activity during allograft rejection, that saturation of antigen and antigen modulation may be important for efficacy of such antibody effects in vivo, and that monoclonal antibodies with specificity for functionally important T cell surface molecules may alter T cell function in vivo without lymphocyte depletion.

Animals↗
PubMed1985

Endocytic uptake, transport, and catabolism of proteins by epithelial cells.

Adsorptive and/or receptor-mediated endocytosis of proteins is a universal cell property, which is highly expressed in epithelial cells. Some absorbed proteins are transported intact across cells and in this manner subserve specialized functions such as the transference of immunity from mother to child. Mainly, however, absorbed proteins are transported to lysosomes, where they undergo complete hydrolysis to amino acids. This process is essential for the homeostasis of circulating proteins. This brief review considers the intracellular pathways taken by endocytosed proteins and the quantitative aspects and determinants of protein uptake and catabolism by epithelial cells. The topics to be briefly discussed are initial internalization sites, transport organelles (endosomes), and lysosomal and nonlysosomal pathways of transport; intracellular sorting of internalized proteins, membranes, and receptors; kinetics and selectivity of renal cell uptake of low-molecular-weight proteins and proteohormones; receptor-mediated endocytosis of larger proteins (e.g., glycoproteins) by hepatocytes; and lysosomal catabolism of absorbed proteins and its dependence on protein load and endosomal-lysosomal pH and function. The perspectives of the field and some of the outstanding unsolved problems are briefly discussed.

Adsorption↗
PubMed1999

Na-K-ATPase inhibitor dissociated from hypertension-associated plasma protein.

It has been demonstrated that human plasma contains a low molecular weight sodium-potassium-stimulated adenosine triphosphatase (Na-K-ATPase) inhibitor, which can be dissociated from a circulating protein with a molecular weight of approximately 12,000 daltons. The dissociated factor was found to have a molecular weight <500 daltons, and shared many characteristics with ouabain. Similar to ouabain, this factor was found to be a potent inhibitor of both the Na-K-ATPase and potassium-stimulated para-nitrophenyl phosphatase (K-pNPPase) enzyme systems, and to bind to both high- and low-affinity binding sites on Na-K-ATPase, but unlike ouabain did not cross-react with digoxin antibody. The factor was further separated by HPLC and electrochemical detection into two active compounds (p-NKAI-1 and p-NKAI-2). P-NKAI-1 was demonstrated on mass spectroscopy to have a molecular weight of 408 daltons. In a vasoconstrictor assay employing rabbit femoral artery segments, this compound was a direct vasoconstrictor and potentiated the vasoconstriction produced by norepinephrine. It behaved similarly to ouabain in counteracting the relaxing effect on rabbit femoral artery of increasing potassium concentrations in the tissue bath.

4-Nitrophenylphosphatase↗
PubMed1977

Heterogeneity of the cold-insoluble globulin of human plasma (CIg), a circulating cell surface protein.

The cold-insoluble globulin of human plasma (CIg), a circulating cell surface protein, exists in multiple molecular forms. Most molecules are found as two chain (MR approximately 220 000 per chain) disulfide-bridged dimeric units but several minor components of smaller size have also been identified; based upon their migration rates in dodecyl sulfate gel electrophoretic experiments, the smaller molecules characterized in this study range in molecular size from 235 000 to 146 000. The component of molecular weight 235 000 apparently represents a two chain disulfide-bridged derivative of larger parent molecules (one chain of 220 000 plus a smaller remnant), whereas smaller CIg components appear to be single chain proteins. These observations plus electrophoretic analyses of samples of plasmic digests of CIg indicate that the interchain disulfide bridging in the two chain molecule is located in a segment within approx. 175 residues of the NH2- or COOH-terminus.

Dithiothreitol↗
PubMed1990

Nutritional state of patients with lung cancer undergoing thoracotomy.

Preoperative nutritional assessment was carried out on 39 consecutive patients with bronchial carcinoma who underwent thoracotomy. For 18 patients the body mass index and triceps and subscapular skinfold thickness fell below the 25th centile. In 23 patients the creatinine height index was less than 80% of the predicted value. The mean (SEM) serum albumin concentration was 40.3 (0.57) g/l (reference range 35-50 g/l) and mean (SEM) serum transferrin 1.77 (0.1) g/l (reference range 2.0-3.0 g/l). Although only three patients were hypoalbuminaemic, transferrin concentrations were depressed in 26 patients. There was a significant fall in the serum concentrations of both prealbumin and transferrin in the first postoperative week. Nutritional insufficiency was particularly severe in the four patients who developed an early bronchopleural fistula. It is concluded that protein-energy malnutrition is common in patients with operable bronchial carcinoma and that routine postoperative feeding does not prevent further depletion of circulating proteins. A larger prospective study is needed to examine the relation between preoperative nutritional state and outcome.

Adenocarcinoma↗
PubMed1992

The function of conserved elements in the promoter of the mouse angiotensinogen gene.

The angiotensinogen gene encodes the precursor protein for the potent vasoconstrictor angiotensin II. Although the gene is expressed in several tissues, the liver is the major source of circulating protein. In previous in-vivo studies we have found that a mini-gene containing 750 bp of 5'-flanking sequence is transcribed in a manner which largely parallels the expression of the endogenous gene. In this report, we characterized conserved elements in the promoter region, in order to determine their role in the transcription of the angiotensinogen gene. Constructs fused to the chloramphenicol acetyl transferase (CAT) reporter gene were transfected into hepatocarcinoma Hep G2 cells as well as into nonhepatic cell lines. We found that 5'-deletion mutant constructs, containing sequences from +25 to -90 bp and -321 to -750 bp, were each able to activate transcription. These constructs contain the TATA box and core promoter sequences, including an Sp1-binding site, and two glucocorticoid responsive elements respectively. In the non-hepatic cell lines, HeLa and Jeg-3, we found that the constructs were transcribed at a much lower rate when compared with the expression of a plasmid containing the Rous sarcoma virus long terminal repeat fused to the CAT gene. Constructs which included sequence 5' to -244 were oestrogen inducible. An element which is conserved between rodent and human angiotensinogen promoters is contained within a sequence which is oestrogen responsive, while another binds the liver-enriched transcriptional activator hepatocyte nuclear factor 1. However, the role of this transactivator in the transcription of angiotensinogen remains uncertain.

Angiotensinogen↗