Ram Doolman

The Ubiquitin-Proteasome Pathway Mediates the Regulated Degradation of Mammalian 3-Hydroxy-3-methylglutaryl-coenzyme A Reductase

3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR), the key regulatory enzyme in the mevalonate (MVA) pathway, is rapidly degraded in mammalian cells supplemented with sterols or MVA. This accelerated turnover was blocked byN-acetyl-leucyl-leucyl-norleucinal (ALLN), MG-132, and lactacystin, and to a lesser extent byN-acetyl-leucyl-leucyl-methional (ALLM), indicating the involvement of the 26 S proteasome. Proteasome inhibition led to enhanced accumulation of high molecular weight polyubiquitin conjugates of HMGR and of HMGal, a chimera between the membrane domain of HMGR and β-galactosidase. Importantly, increased amounts of polyubiquitinated HMGR and HMGal were observed upon treating cells with sterols or MVA. Cycloheximide inhibited the sterol-stimulated degradation of HMGR concomitantly with a marked reduction in polyubiquitination of the enzyme. Inhibition of squalene synthase with zaragozic acid blocked the MVA- but not sterol-stimulated ubiquitination and degradation of HMGR. Thus, similar to yeast, the ubiquitin-proteasome pathway is involved in the metabolically regulated turnover of mammalian HMGR. Yet, the data indicate divergence between yeast and mammals and suggest distinct roles for sterol and nonsterol metabolic signals in the regulated ubiquitination and degradation of mammalian HMGR.

Prospective Study of Serum Homocysteine and Risk of Ischemic Stroke Among Patients With Preexisting Coronary Heart Disease

Background and Purpose— Substantial evidence is accumulating suggesting that hyperhomocysteinemia may be a risk factor for ischemic stroke. Results of prospective studies are, however, conflicting, and the role of hyperhomocysteinemia in patients with preexisting atherosclerotic vascular disease is not clear. Our aim was to assess prospectively the risk of incident ischemic stroke conferred by serum total homocysteine among patients with preexisting stable coronary heart disease (CHD). Methods— We obtained baseline fasting serum samples from patients with chronic CHD enrolled in the Bezafibrate Infarction Prevention (n=3090) secondary prevention study cohort. With a nested case-control design, we measured baseline total homocysteine concentration by a high-performance liquid chromatography–based method in sera (n=160) of matched case-control pairs: patients who developed ischemic stroke during a mean follow-up of 8.2 years (cases) and age- and sex-matched controls without subsequent cardiovascular events. Results— An increase of 1 natural log unit in homocysteine concentration was associated with a >3-fold increase in relative odds of incident ischemic stroke (3.3; 95% CI, 1.2 to 10.2). Homocysteine concentrations at the highest quartile (>17.4 &mgr;mol/L) were associated with significantly higher odds of ischemic stroke compared with the lowest quartile in matched-pair analysis (3.1; 95% CI, 1.1 to 9.8) and after multivariable adjustments (4.6; 95% CI, 1.3 to 18.9). Adding fibrinogen or soluble intercellular adhesion molecule-1 concentrations, markers of inflammation, to the model did not attenuate this association. The linear trends across the quartiles were significant for all models (P <0.05). Conclusions— Serum total homocysteine concentration is a strong predictor for incident ischemic stroke among patients at increased risk because of chronic CHD. The graded association observed is independent of traditional risk factors or inflammatory markers and indicates the importance of serum homocysteine levels in patients with preexisting vascular disease.

Recurrent aphthous stomatitis and thiamine deficiency

Recurrent aphthous stomatitis is a disease of unknown cause. To examine whether thiamine (vitamin B1) deficiency is associated with recurrent aphthous stomatitis, we studied vitamin B1 levels in 70 patients with recurrent aphthous stomatitis and in 50 members of a control group. The vitamin B1 level was determined as thiamine pyrophosphate effect on transketolase activity in red blood cell lysates. Low levels of vitamin B1 were detected in 49 patients but in only two members of the control group (p < 0.0001). These low levels were not associated with patient age, sex, or underlying disease causing recurrent aphthous stomatitis. Our finding suggests an association between thiamine deficiency and recurrent aphthous stomatitis.

Hyperhomocysteinemia and vitamin score: correlations with silent brain ischemic lesions and brain atrophy.

Elevated fasting plasma total homocysteine concentration (tHcy) and lower vitamin status are associated with atherosclerotic states. Silent brain ischemic lesions and brain atrophy, prevailing in the elderly, are affected by tHcy and vitamin status. The study was performed on 56 outpatients who had undergone brain computed tomography (CT) before the onset of the study. According to brain CT evaluation, three groups were set: minor brain ischemia, brain atrophy and control. Brain CT, tHcy, plasma pyridoxal phosphate (PLP), vitamin B12, folic acid and cognitive and functional capacities were measured or evaluated in all of the subjects. Plasma vitamin score for three vitamins was calculated. In subjects with minor brain ischemic lesions (n = 21), tHcy was higher by 5.6 µM, whereas vitamin score and cognitive function were lower than in controls (n = 24). In subjects with brain atrophy (n = 11), plasma PLP and cognitive function were lower. Particular attention should be paid to tHcy monitoring, vitamin status assessment and brain impairment evaluation.

Serum Homocysteine and Long-Term Risk of Myocardial Infarction and Sudden Death in Patients with Coronary Heart Disease

We have prospectively evaluated the risk of incident coronary events in association with serum total homocysteine in patients with preexisting chronic coronary heart disease. A nested case-control design was used. Total homocysteine concentration was measured in baseline fasting serum samples from patients with chronic coronary heart disease enrolled in the Bezafibrate Infarction Prevention Study (n = 3,090) who developed coronary events during 6.2 years of follow-up (n = 69). They were matched for age and gender with controls without subsequent cardiovascular events. Elevated homocysteine levels were associated with 2.5 times higher risk of subsequent coronary events and each 5 µmol/l increment was associated with a 25% higher risk.

Effect of Elevated Homocysteine Levels on Clinical Restenosis following Percutaneous Coronary Intervention

Since hyperhomocysteinemia confers a prothrombotic effect and promotes proliferation of smooth muscle cells in response to vascular injury, it might be implicated in the pathogenesis of restenosis after percutaneous coronary intervention (PCI). Our study comprised 55 patients who underwent successful PCI in the acute myocardial infarction (AMI) course. Homocysteine levels were determined within 24 h of admission. During a 1-year follow-up, 16 patients (31%) underwent repeated coronary angiography for recurrent angina or re-infarction, which demonstrated re-narrowing of ≧50% at the qualifying PCI site (clinical restenosis). Irrespective of stent deployment, clinical restenosis was not associated with higher homocysteine levels (12 ± 7 vs. 14 ± 11µmol/l, p = 0.77). There was no correlation between homocysteine levels and time to restenosis (r2 = 0.06, p = 0.35). In conclusion, elevated homocysteine levels do not predict a higher incidence of restenosis after PCI.

Analysis of Cell-Free Human α1 Integrin with a Monoclonal Antibody to the I-Domain: Detection in Ocular Fluid and Function as an Adhesion Substrate

The α1β1 integrin, an inserted (I) domain containing collagen receptor, is expressed in the cell surface membrane of normal and malignant cells, and may play a role in their migration through tissues or in metastatic spread. Here we report that a functional anti-human α1β1 integrin monoclonal antibody (mAb) (1B3.1) directly and specifically binds plastic bound recombinant human α1 I-domain protein containing the collagen binding site. Detection was diminished by acidification of the I-domain protein but was enhanced by increasing concentrations of Mg2+ cation. Furthermore, we detected binding of the mAb to proteins from the ocular fluids of 6 patients, with the highest concentration, corresponding to 22.1 ng/ml of I-domain, found in a sample from the eye of a patient with metastatic lung adenocarcinoma. Interestingly, we found that both SKNSH neuroblastoma cells and virally transformed human T cells adhered specifically to plastic wells coated with either immobilized collagen IV oral I-domain. MAb 1B3.1 inhibited adhesion to collagen IV but not to immobilized I-domain. These results suggest a novel function for cell free α1 I-domain as a substrate for cellular adhesion, which may have relevance in tumor spread in vivo.

Detection of soluble α1 integrin in human serum

Abstract An enzyme-linked immunosorbent assay (ELISA) for the detection and quantitation of soluble α1β1 integrins (sα1) in human serum samples was developed. Solid phase–bound anti-α1 integrin monoclonal antibody (mAb) TS2/7 was used to capture sα1, and mAb 1B3.1 was used to detect the immobilized integrin. An extract of human placenta (PE) containing 340 ng/mL of VLA-1 molecules served as a positive control, and serum samples from normal donors and patients were assayed. Optimal binding of anti-α1 integrin mAb 1B3.1, expressed as specific optical density (OD), was obtained when a 5 μg/mL solution of anti-α1 integrin “capture” mAb TS2/7 was immobilized to the wells and the PE was added. Solutions of albumin or collagen, in contrast, did not result in binding, confirming the specificity of the assay for sα1. Furthermore, the specific OD of the wells correlated directly with the concentration of PE. A concentration of sα1 above that of a 1:100 dilution of PE—that is, >3.4 ng/mL of integrin, in which the intra-assay correlation of variance was

Microalbuminuria Immunoassay Based on Antibodies Covalently Conjugated to Eupergit C-Coated Beads

OBJECTIVE To develop a reliable, simple, and sensitive assay for microalbuminuria, based on covalent attachment of anti-HSA to oxirane-bearing polymethylmethacrylate beads (Eupergit CB6200). RESEARCH DESIGN AND METHODS Anti-HSA antibodies were coupled to CB6200 beads by reaction of their amino groups with the oxirane groups of the matrix. The capability of the beads to bind HSA from standard solutions or urine was evaluated and compared with the state of the art ELISA test. RESULTS The new bead immunoassay is sensitive and linear in the range of 1–25 mg/L, which is considered the low microalbuminuria range. When HSA levels in urine were tested, the intra- and interassay CV values ranged between 2.7 and 3.9% and between 5.6 and 6.6%, respectively. The long-term storage stability of the antibodies covalently bound on the beads was higher than of the same antibodies adsorbed on ELISA plates. After 16 wk of storage, the CV was about 7.3% with the bead assay, compared with 14% obtained for the ELISA test under the same experimental conditions. CONCLUSIONS A new procedure for microalbuminuria assay was developed, with Eupergit CB6200 beads as a solid support for covalent binding of the first antibody. Accuracy, sensitivity, reproducibility, and precision of the bead immunoassay were similar to those of commonly used immunoassays, as exemplified by the analysis of HSA in 53 clinical urine samples. The bead assay retains a low degree of variability over long storage periods, and the beads may be reapplied after a simple acid-washing procedure.

Klotho response to treatment with growth hormone and the role of IGF-I as a mediator

CONTEXT Klotho is an aging-modulating protein expressed mainly in the kidneys, which can be cleaved and shed to act as a circulating hormone. Several lines of evidence suggest a tight interaction between klotho and the GH-IGF-I axis. We showed previously that klotho levels are decreased in pediatric patients with growth hormone deficiency (GHD). Our aim now is to investigate the effect of GH therapy on klotho levels in these patients and to elucidate the role of IGF-1 in mediating secretion of klotho. BASIC PROCEDURES Klotho levels were measured in 29 GHD pediatric patients (males=15, aged 12.2±3.3years), treated with GH for 2.5±2.8years; nineteen patients had samples obtained both before and during treatment. The effect of IGF-I and its downstream effectors on secretion of klotho to media was studied in COS-7 cells overexpressing klotho. MAIN FINDINGS Klotho levels increased under GH treatment (from 1321±691pg/ml to 3380±2120pg/ml, p<0.001), and were higher compared to controls (1645±778pg/ml, p<0.001), resulting in supraphysiological levels. Fold-increase in klotho correlated with fold-increase in IGF-I (r=0.63, p=0.004). Studies in COS-7 cells overexpressing klotho revealed mTOR-dependent induction of klotho shedding by IGF-I. PRINCIPAL CONCLUSIONS Klotho levels increased during GH treatment of pediatric GHD patients. This increase was associated with an increase in IGF-I levels. Furthermore, we showed, for the first time, a direct role of IGF-I in the regulation of klothos shedding which depends on activation of the AKT-mTOR pathway. Our findings add further support for the close association between klotho and the GH/IGF-I axis.