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Biomedical subjects

I Dotan

Publications and source records attributed to I Dotan.

32 records · Page 2Linked to original sources
PubMed1996

Increased leukocyte adhesiveness/aggregation in patients with inflammatory bowel disease during remission. Further evidence for subclinical inflammation.

PURPOSE: We have used a novel leukocyte adhesiveness/aggregation test (LAAT) to show that many patients with inflammatory bowel disease (IBD) in clinical remission have a subclinical low grade inflammation. METHODS: Included in the study are 500 controls, 96 patients with IBD in remission, and 106 patients in relapse. RESULTS: The percent of aggregated white blood cells detected in the peripheral blood was 5.9 +/- 3.9, 9.1 +/- 5.9, and 18.8 +/- 9.4, respectively. The difference between each group and any other was significant at P < 0.0001. Similar results were obtained when other acute phase reactants like the erythrocyte sedimentation rate, white blood cell count, differential count, and C-reactive protein level were examined. However, in a linear regression analysis, LAAT was the only significant (P < 0.0006) variable that could classify correctly each subject to the appropriate category of control and IBD in remission or relapse. CONCLUSIONS: Identification of patients with IBD in clinical remission who have ongoing inflammation may be of clinical-therapeutic relevance. The LAAT is a simple, rapid, and convenient test. The present study indicates that it is also very sensitive.

Adolescent↗
PubMed1996

Inhibition of experimentally induced cirrhosis in rats by hypothyroidism.

The coexistence of hyperkinetic circulation, hypermetabolism, and hyperactivity of the sympathetic nervous system is encountered in both cirrhosis and hyperthyroidism. Several drugs, such as propylthiouracil and propranolol, that are beneficial for treating some patients with chronic liver diseases are also prescribed for the treatment of thyrotoxicosis. We investigated the effects of experimentally induced hypo- and hyperthyroidism on the development of cirrhosis induced in rats by thioacetamide (TAA). We specifically examined whether hypothyroidism could prevent and hyperthyroidism could aggravate liver damage. Hypothyroidism induced by methimazole (MMI, 0.04%), propylthiouracil (PTU 0.05%), and by thyroidectomy was confirmed by a significant elevation of thyroid-stimulating hormone (TSH) levels. Hyperthyroidism (decreased TSH levels) was induced by eltroxin (ELT:50 micrograms/kg). Thirteen groups of 10 rats each were studied: euthyroid controls (3 groups: water, TAA 1.5 months, and TAA 3 months), hypothyroid (6 groups: MMI, PTU, surgical, MMI-TAA, PTU-TAA, surgical-TAA), and hyperthyroid (4 groups:ELT 1.5 months and 3 months, and ELT-TAA for 1.5 months and 3 months). Hepatic fibrosis (scored from 0 to 3) was significantly reduced (P < .0001) in hypothyroid rats as compared with euthyroid controls, and was aggravated in TAA-treated hyperthyroid rats (P < .0001). Quantitative microscopic analysis of liver biopsy specimens from all groups confirmed the semiquantitative histopathological scores (P < .001). Direct intrasplenic pressure measurement revealed a significant portal pressure elevation in the TAA and the ELT-treated rats (from 4.7 +/- 0.1 in the euthyroid group to 8.1 +/- 2.3 and 10.2 +/- 2.1 and 12.5 +/- 1.6 in the TAA, ELT and ELT-TAA groups, respectively). However, in the hypothyroid-TAA groups, the portal pressure was found to be within the euthyroid normal range (4.6 +/- 1.2 and 5.8 +/- 0.6 in the PTU-TAA and surgical-TAA, respectively). After 12 weeks, the mean spleen weight of rats receiving only TAA was significantly higher than the TAA-treated hypothyroid rats (P < .0001), indicating that the hypothyroid TAA-treated rats were less portal hypertensive. These results suggest that induced hypothyroidism can inhibit, whereas hyperthyroidism can aggravate, the development of cirrhosis in a rat model.

Animals↗
PubMed1996

Increased leukocyte adhesiveness/aggregation is the single best biochemical indicator of disease activity in adolescent patients with inflammatory bowel disease.

We determined the relative utility of inflammatory markers in evaluating disease activity in adolescent patients with inflammatory bowel disease (IBD). Sixty-one adolescent patients and 50 age and sex matched volunteers who served as controls were evaluated prospectively. The erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), white blood cell count (WBCC), and the leukocyte adhesiveness/aggregation (LAA) test were determined in each patient. There were 28 subjects in remission and 33 in relapse. Their percentage of aggregated white blood cells in the peripheral blood was 7 +/- 5 and 16 +/- 8, respectively, when compared with the control group (5 +/- 3) (p < 0.0001). Moreover, the LAA test was the only one which could effectively classify patients with IBD into their correct diagnostic categories of remission, mild-moderate, or moderate-severe disease activity. The LAA test is superior to other acute phase reactants used in daily practice to detect the presence of inflammation as well as for the assessment of its severity in adolescent patients with IBD. In addition, our findings may have biological relevance to the disease process and its potential manipulation by anti-adhesive agents.

Adolescent↗
PubMed1995

Relation between uroporphyrin excretion, acute attacks of hereditary coproporphyria and successful treatment with haem arginate.

1. The increased urinary excretion of porphyrins as well as of their precursors was studied in a patient with hereditary coproporphyria during two acute attacks in which symptoms differed markedly in character and severity. 2. The increase in urinary coproporphyrin was similar in the 'mild' and in the 'severe' attack, indicating a lack of correlation between coproporphyrin level and clinical symptoms. 3. Aminolaevulinic acid, porphobilinogen and uroporphyrin exhibited significantly higher values during the 'severe' attack than during the 'mild' attack. During the severe attack these three compounds were increased 18-, 14- and 46-fold, respectively, compared with increases of 3-, 3- and 8-fold, respectively, during the mild attack. 4. The striking rise in the formation of uroporphyrin was reflected in the plasma porphyrin profile, which revealed predominance of uroporphyrin. In accordance with this finding, an increase in erythrocyte porphobilinogen deaminase of 130% was recorded. 5. The fluorescence emission spectra of saline-diluted plasma (excitation of 405 nm) showed a distinct peak at 618 nm during the 'severe' episode and a small peak during the 'mild' attack, pointing to the possibility of diagnosing an attack simply by following the fluorometric screen of plasma. 6. The 'severe' attack of coproporphyria was treated with daily infusions of haem arginate, 3 mg/kg, every day for 4 days, at the end of which period a dramatic clinical response was observed. The relief of symptoms was found to be clearly related to the moderate decrease in uroporphyrin excretion observed rather than to the steep decline in the precursors.

Acute Disease↗
PubMed1995

Characterization of recombinant murine leukemia virus integrase.

Retroviral integration involves two DNA substrates that play different roles. The viral DNA substrate is recognized by virtue of specific nucleotide sequences near the end of a double-stranded DNA molecule. The target DNA substrate is recognized at internal sites with little sequence preference; nucleosomal DNA appears to be preferred for this role. Despite this apparent asymmetry in the sequence, structure, and roles of the DNA substrates in the integration reaction, the existence of distinct binding sites for viral and target DNA substrates has been controversial. In this report, we describe the expression in Escherichia coli and purification of Moloney murine leukemia virus integrase as a fusion protein with glutathione S-transferase, characterization of its activity by using several model DNA substrates, and the initial kinetic characterization of its interactions with a model viral DNA substrate. We provide evidence for functionally and kinetically distinct binding sites for viral and target DNA substrates and describe a cross-linking assay for DNA binding at a site whose specificity is consistent with the target DNA binding site.

Base Sequence↗
PubMed1995

Increased leucocyte adhesiveness/aggregation is a most useful indicator of disease activity in patients with inflammatory bowel disease.

The aim of the study was to determine the comparative usefulness of inflammatory markers, in evaluating disease activity in patients with inflammatory bowel disease. Disease activity was assessed by the Mayo Clinic score for ulcerative colitis, and Harvey-Bradshaw score for Crohn's disease. Five hundred normal blood donors who had no underlying inflammatory condition served as controls. The erythrocyte sedimentation rate, platelet and white blood cell count, C reactive protein, and the leucocyte adhesiveness/aggregation test (LAAT) were determined in each patient. One hundred and twenty four patients with inflammatory bowel disease were tested while in remission and 128 in relapse. Their mean (SD) per cent of aggregated white blood cells in the peripheral blood was 8 (5) and 17 (10) respectively compared with controls 6 (4) (p < 0.0001). Moreover, the LAAT could effectively discriminate between various grades of disease activity, the values in patients with active disease being 13 (6)% in mild, 17 (10)% in moderate, and 26 (10)% in severe disease (p < 0.0001). Other acute phase reactants including the erythrocyte sedimentation rate, the white blood cell count, the platelet count, neutrophil count, as well as, the C reactive protein concentration did not differentiate as well between the various groups. Using logistic regression analysis to differentiate between inflammatory bowel disease patients in remission or relapse, the LAAT was the single best indicator. The addition of any other test did not contribute to the discrimination. Among the different laboratory variables tested only the LAAT significantly discriminated between the five different subgroups of controls, remission and mild, moderate or severe disease activity.

Adolescent↗
PubMed1994

[Neuroleptic malignant syndrome in elderly hospital patients].

Neuroleptic malignant syndrome (NMS) has gained increasing attention over the past decade. Diagnostic features include exposure to major tranquilizers, acute onset of high fever, extrapyramidal symptoms and markedly elevated CPK. Other common signs include changes in mental status, tachycardia and leukocytosis. It usually runs its course within 10 days. Greater awareness and improved treatment have resulted in markedly decreased mortality. However, failure to diagnose and treat properly results in a significant risk of death. Most cases of NMS reported have been in those with psychiatric illness. Few cases have been reported in elderly patients without a psychiatric history. We describe an 85-year-old man, without previous psychiatric illness, who developed NMS while hospitalized on a general medical ward.

Aged↗
PubMed1989

Activation of the superoxide forming NADPH oxidase in a cell-free system by sodium dodecyl sulfate. Absolute lipid dependence of the solubilized enzyme.

The superoxide (O2-)-forming NADPH oxidase of resting macrophages can be activated in a cell-free system by certain anionic amphiphiles, such as sodium dodecyl sulfate (SDS). O2- production requires the cooperation of membrane-associated and cytosolic components. The membrane component can be solubilized by octyl glucoside yielding a highly active oxidase preparation. High performance gel filtration of the solubilized oxidase on Superose 12 in the presence of 40 mM octyl glucoside leads to the total loss of enzymatic activity. This can be restored in previously inactive eluate fractions by "reconstitution" with N-ethylmaleimide or heat (60 degrees C)-inactivated total solubilized membrane. Oxidase activity, that becomes evident upon reconstitution, is eluted from Superose 12 with molecules in the Mr range of 300,000-71,000. The material with reconstitutive capacity is completely dissociated from the oxidase, eluting with molecules in the Mr range of 71,000-11,000. The Superose 12 elution profile of the material responsible for reconstitution coincides with that of membrane-derived phospholipid. Also, the reconstitutive capacity of total solubilized membrane and that of the Mr 71,000-11,000 region of the Superose eluate are recovered in a chloroform extract prepared by the method of Bligh and Dyer. It is concluded that loss of oxidase activity by gel filtration at a high octyl glucoside concentration is the consequence of delipidation. NADPH oxidase activity, revealed by reconstitution of Superose 12 fractions with exogenous phospholipid, correlates closely with the elution profile of cytochrome b559. Reconstitution of activity of delipidated oxidase can also be achieved with natural non-macrophage phospholipids and with synthetic phospholipids. Reconstitution of NADPH oxidase activity by lipids is governed by the following rules: (a) phospholipids are effective; lysophospholipids and neutral lipids are not; (b) phospholipids with polar heads represented by choline, ethanolamine, and serine, as well as cardiolipin, are effective; phosphatidylinositol is much less active; (c) phospholipids with unsaturated fatty acid residues are capable of reconstitution while saturated acyl residues do not confer activity; this specificity appears not to be related to the transition temperature of the phospholipids.

Ammonium Sulfate↗
PubMed1989

Use of DNA amplification methods for clinical diagnosis in autoimmune diseases.

Autoimmune disease is generally felt to result from the interaction of genetic and environmental factors. In recent years, significant advances have been made in using recombinant DNA methods to analyze specific genetic factors and infectious agents. However, new techniques are needed that are more rapid, inexpensive, and suitable for small tissue biopsies obtained early in the course of disease. New methods of DNA amplification based on polymerase chain reaction (PCR) and Q beta-replicase (Q beta R) have recently been reported. These methods are briefly reviewed, and their potential applications to patients with autoimmune disease are presented. Several types of applications can be considered, including detection of: a) specific HLA-D alleles in order to predict prognosis and better utilize existing medications; b) bacterial, fungal, and spirochete infections in joint aspirates or synovial biopsies; c) human immunodeficiency virus (HIV) and other viruses (e.g., EBV, CMV) that may be associated with immune dysregulation in certain patients; and d) neoplastic transformation in blood or tissues by determining monoclonal gene rearrangements, karyotypic alterations or oncogene activation. It is likely that routine clinical laboratories will soon begin implementing DNA amplification methods in order to screen blood products for infectious agents (especially HIV and hepatitis B virus). Because these techniques will be readily available, rheumatologists/clinical immunologists should begin developing strategies that will allow them to use these methods in a cost-effective manner for diagnosis and monitoring treatment.

Autoimmune Diseases↗
PubMed1987

Isolation and purification of a rat liver 3-hydroxy-3-methylglutaryl-coenzyme reductase activating protein (RAP).

A protein with an estimated subunit mass of 19 kDa was isolated and purified from perfused rat liver cytosol. This protein activates hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase (NADPH) (EC 1.1.1.34), the rate-limiting enzyme in the cholesterol biosynthetic pathway. The activation process by this HMG-CoA reductase activating protein (RAP) is time-dependent and requires NADPH. Maximal activity of HMG-CoA reductase induced by RAP is comparable to that obtained in the presence of thiols, such as GSH, and can exceed 100-fold the activity obtained when thiols are omitted. Purified RAP lacks ability to reduce 5,5'-dithiobis-(2-nitrobenzoic acid). RAP was purified to homogeneity utilizing DEAE- and phenyl-Sepharose CL-4B column chromatography. The purified RAP migrates as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and shows multiple interconvertible aggregational forms on native polyacrylamide gel electrophoresis. A monospecific antibody against RAP was prepared by immunization of hens and extracted from either their egg yolks or serum. The catalytic activity of RAP might be responsible for the physiological activation of HMG-CoA reductase and regulation of its activity.

Animals↗
PubMed1985

Reduced glutathione in Chinese hamster ovary cells protects against inactivation of 3-hydroxy-3-methylglutaryl coenzyme A reductase by 2-mercaptoethanol disulfide.

When the disulfide of 2-mercaptoethanol (ESSE) is added to the medium of cultured Chinese hamster ovary (CHO) cells, a time and concentration dependent release of 2-mercaptoethanol to the medium is observed. The reduction of ESSE to 2-mercaptoethanol by cells is a saturable process, the rate being approximately 50 nmoles of 2-mercaptoethanol per mg cell protein for an hour upon exposure to 250 microM ESSE. Reduction rate of ESSE by cells attached to a substratum is independent of glucose and insulin for periods up to 4 hours. However, in detached cells, swirled in suspension, addition of glucose and insulin is necessary in order to obtain a linear reduction rate of ESSE. The rate limiting enzyme in the sterol biosynthetic pathway, 3-hydroxy-3-methyl-glutaryl Coenzyme A reductase (E.C. 1.1.1.34), is inhibited by ESSE when isolated from CHO cells but total nonsaponifiable lipids synthesis from [2-14C]-acetate in intact cells is not affected by ESSE at concentrations up to 500 microM. Cytosolic reduced glutathione can spontaneously exchange disulfide bonds with ESSE and thus prevent it from inhibiting the reductase. Cultured cells respond to ESSE administration by elevating their total and acid-soluble glutathione levels. The use of ESSE as a perturbant of the GSH Status in cells is discussed.

Animals↗
PubMed1983

Properties of latent and thiol-activated rat hepatic 3-hydroxy-3-methylglutaryl-coenzyme A reductase and regulation of enzyme activity.

The effect of the thiols glutathione (GSH), dithiothreitol (DTT), and dithioerythritol (DTE) on the conversion of an inactive, latent form (El) of rat liver 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase, EC 1.1.1.34) to a catalyticaly active form (Ea) is examined. Latent hepatic microsomal HMG-CoA reductase is activated to a similar degree of activation by DTT and DTE and to a lower extent by GSH. All three thiols affect both Km and Vmax values of the enzyme toward HMG-CoA and NADPH. Studies of the effect of DTT on the affinity binding of HMG-CoA reductase to agarose-hexane-HMG-CoA (AG-HMG-CoA) resin shows that thiols are necessary for the binding of the enzyme to the resin. Removal of DTT from AG-HMG-CoA-bound soluble Ea (active enzyme) does not cause dissociation of the enzyme from the resin at low salt concentrations. Substitution of DTT by NADPH does not promote binding of soluble El (latent enzyme) to AG-HMG-CoA. The enzymatic activity of Ea in the presence of DTT and GSH indicates that these thiols compete for the same binding site on the enzyme. Diethylene glycol disulfide (ESSE) and glutathione disulfide (GSSG) inhibit the activity of Ea. ESSE is more effective for the inhibition of Ea than GSSG, causing a higher degree of maximal inhibition and affecting the enzymatic activity at lower concentrations. A method is described for the rapid conversion of soluble purified Ea to El using gel-filtration chromatography on Bio-Gel P-4 columns. These combined results point to the importance of the thiol/disulfide ratio for the modulation of hepatic HMG-CoA reductase activity.

Animals↗
PubMed1982

Thiol-disulfide-dependent interconversion of active and latent forms of rat hepatic 3-hydroxy-3-methylglutaryl-coenzyme A reductase.

The activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (hydroxymethylglutaryl-CoA reductase, EC 1.1.1.34) in preparations of thiol-deficient rat liver microsomes and microsomes containing thiols have been compared. Unlike microsomes containing thiols, which possess an active hydroxymethylglutaryl-CoA reductase (Ea), thiol-deficient microsomes contain an inactive, latent enzyme (E1) which can be activated by addition of thiols. Ea can be converted to E1 by dialysis. The maximal degree of activation of E1 depends on the activating thiol with the order of effectiveness: dithioerythritol = dithiothreitol greater than glutathione (GSH) greater than cysteine. Ea is inhibited by oxidized glutathione (GSSG). The degree of the inhibition of Ea by GSSG is proportional to the ratio GSSG/thiol in the reaction. E1 was solubilized from microsomes and purified. Its molecular weight is estimated to be 104 000 by gel filtration chromatography on Sepharose 6B. The reducing agents NaBH4, dithionite and ascorbate failed to activate E1. NaBH4 did not inhibit Ea whereas only partial inhibition was caused by ascorbate and dithionite. Soluble Ea binds to both blue dextran/Sepharose 4B and agarose/hexane-3-hydroxy-3-methylglutaryl Coenzyme A affinity resins at low-salt concentrations. By contrast, soluble E1 did not bind to agarose/hexane-hydroxymethylglutaryl-CoA whereas quantitative binding of E1 to blue dextran/Sepharose 4B was still observed at low salt concentrations. These results indicate that thiols are necessary cofactors for hydroxymethylglutaryl-CoA reductase reaction. Their effect on the activation of E1 is not caused by change in the state of aggregation of the enzyme. Rather, the reversible change of the enzyme from E1 to Ea is affected by increasing the affinity of the enzyme to the substrate hydroxymethylglutaryl-CoA.

Animals↗