Search PubMed⌕ Search

Biomedical subjects

M Karp

Publications and source records attributed to M Karp.

At least 55 records · Page 3Linked to original sources
PubMed1997

The development and progression of diabetic retinopathy in type I diabetic patients: a cohort study.

To describe the course and risk factors for development and progression of retinopathy, we studied a cohort of 333 Israeli Jewish patients with Type 1 (insulin-dependent) diabetes mellitus. The median age at diagnosis was 9.5 (range 0.04-26.2) years and the median duration of follow-up was 14 (range 1.6-30) years. Evaluation of both retinae was performed yearly since referral and HbA1 values were tested every 3 months since 1978. During a follow-up of 4070 patient-years, 162 patients developed non-proliferative retinopathy. The median retinopathy-free interval was 14.9 years and after 30 years all patients were affected. Pre-pubertal duration of diabetes was relevant. Independent and significant risk factors for early onset of non-proliferative retinopathy were: poor cumulative glycaemic control (median retinopathy-free interval in the 1st vs 4th quartiles of mean HbA1 values over all years: 18.0 vs 12.5 years, p = 0.0001); onset of diabetes during or after puberty (median retinopathy-free interval in patients with onset of diabetes before, during or after pubescence: 16.3, 13.2 and 14.0 years, respectively, p = 0.0001); and non-Ashkenazi Jewish origin (median retinopathy-free interval 15.8 years in Ashkenazi vs 14.0 in non-Ashkenazi patients, p = 0.0004). Of 162 patients with non-proliferative retinopathy, progression to proliferative retinopathy occurred in 37, during 707 patient-years. The first event of proliferative retinopathy was diagnosed within the 1st year after non-proliferative retinopathy evolved, and at 6.3 years since onset of non-proliferative retinopathy 75% of the patients were still free of proliferative changes. Risk factors significantly and independently associated with an early progression to the proliferative stage were: poor glycaemic control in the last 3 years prior to the development of proliferative retinopathy and non-Ashkenazi Jewish origin. All patients in the 4th quartile of HbA1 values were affected by proliferative retinopathy within 11.6 years after onset of non-proliferative retinopathy.

Adolescent↗
PubMed1997

Epitope mapping of human prostate specific antigen and glandular kallikrein expressed in insect cells.

Two human prostate gland proteases were expressed in insect cells using recombinant baculovirus expression system. Prostate specific antigen (PSA) is an established serum marker of prostate cancer whereas the clinical utility of its close homologue, human glandular kallikrein (hK2) is presently unknown. The production levels using Trichoplusia ni cells were roughly 300 &mgr;g/l and 6 mg/l for hK2 and PSA, respectively. On western-blot we estimated the size for both proteins to be approximately 33 kDa which was consistent with PSA purified from seminal plasma. Nine anti-PSA monoclonal antibodies (Mabs) out of 26 tested, representing five independent epitopes, also reacted with hK2. The results obtained in this study may help in designing more accurate diagnostic assays for detection and monitoring of prostate cancer.

Journal Article↗
PubMed1997

Recombinant luminescent bacteria for measuring bioavailable arsenite and antimonite.

Luminescent bacterial strains for the measurement of bioavailable arsenite and antimony were constructed. The expression of firefly luciferase was controlled by the regulatory unit of the ars operon of Staphylococcus aureus plasmid pI258 in recombinant plasmid pTOO21, with S. aureus RN4220, Bacillus subtilis BR151, and Escherichia coli MC1061 as host strains. Strain RN4220(pTOO21) was found to be the most sensitive for metal detection responding to arsenite, antimonite, and cadmium, the lowest detectable concentrations being 100, 33, and 330 nM, respectively. Strains BR151(pTOO21) and MC1061(pTOO21) responded to arsenite, arsenate, antimonite, and cadmium, the lowest detectable concentrations being 3.3 and 330 microM and 330 and 330 nM with BR151(pTOO21), respectively, and 3.3, 33, 3.3, and 33 microM with MC1061(pTOO21), respectively. In the absence of the mentioned ions, the expression of luciferase was repressed and only a small amount of background light was emitted. Other ions did not notably interfere with the measurement in any of the strains tested. Freeze-drying of the cells did not decrease the sensitivity of the detection of arsenite; however, the induction coefficients were somewhat lower.

Antimony↗
PubMed1996

Isolation of strong promoters from Clavibacter xyli subsp. cynodontis using a promoter probe plasmid.

To isolate promoters from Clavibacter xyli subsp. cynodontis (C. xyli subsp. cynodontis), we constructed a new promoter probe plasmid and made a C. xyli subsp. cynodontis promoter probe library. Two promoters gave over 2500-times stronger expression than the parental plasmid. The promoters were sequenced and compared to other bacterial promoters. These C. xyli subsp. cynodontis promoter regions are GC-rich and do not resemble E. coli promoters, but do resemble a few individual promoters found in streptomycetes.

Actinomycetales↗
PubMed1996

Epitope mapping of nine monoclonal antibodies against osteocalcin: combinations into two-site assays affect both assay specificity and sample stability.

Nine monoclonal antibodies (Mabs) were raised against human recombinant osteocalcin fusion protein (rGST-hOC) or bovine osteocalcin (bOC) and selected to develop two-site immunoassays for human osteocalcin (hOC). The detection system was based on the time-resolved measurement of the fluorescence of europium chelates conjugated to the tracer Mabs. Based on the ability of the Mabs to recognize different forms of hOC (carboxypeptidase Y-digested, alkylated hOC, thermally decarboxylated hOC, recombinant forms of hOC, and tryptic peptides derived from hOC) and the information obtained from combinations of the Mabs in two-site assays, an epitope map was created. The epitope map was useful in understanding the behavior of the two-site combinations of the Mabs with serum samples. The two-site combinations could be divided into subgroups detecting either full-length hOC or full length+large NH2-terminal fragment as stimulated by the carboxypeptidase Y-digested form of hOC (it lacks four COOH-terminal residues), which with intact specific assays showed cross-reactivities ranging from 7 to 14% when compared with full-length hOC. In addition, differences were observed in the ability of the combinations to detect thermally decarboxylated hOC (lacks gamma-carboxylation at residues 17, 21, and 24) with cross-reactivities ranging from 8 to 85% when compared to gamma-carboxylated hOC. The analysis of human serum samples showed considerable differences in the concentration and stability of serum OC. This was attributed as the varying ability of the Mabs to detect different proteolytic fragments derived from hOC and/or differences in the degree of gamma-carboxylation of hOC. The in vitro generation of the large NH2-terminal fragment during incubation of the serum samples at room temperature (RT) and during prolonged storage at -20 degrees C in an undercooled state was detectable as loss of immunoreactivity (ranging from -42 +/- 17 to -50 +/- 15% in 16 h at RT, n = 3) with Mab combinations detecting only full-length hOC. Two-site combinations detecting full-length+large NH2-terminal fragment showed no loss of immunoreactivity after incubation of the serum samples at RT for 16 h. With all assays, an increase of serum OC ranging from 16 to 38% was found in postmenopausal samples (n = 24) when compared with premenopausal samples (n = 17), but the degree of statistical significance varied from not significant to p < 0.01.

Animals↗
PubMed1996

Purification and characterization of recombinant osteocalcin fusion protein expressed in Escherichia coli.

Human osteocalcin (hOC) is a 49-amino-acid peptide produced mainly by bone osteoblasts. The amount of hOC in the circulation reflects the status of bone metabolism and it is used to monitor various bone-related diseases. The aim of this study was to produce recombinant human osteocalcin (rhOC) in Escherichia coli and use it for designing new osteocalcin fluorescence immunoassays. Recombinant DNA technology was used to fuse synthetic hOC coding sequences to an affinity handle system based on glutathione S-transferase (GST) gene. GST-rhOC fusion protein was produced in a bacterial intracellular expression system mainly in a soluble form. The affinity-purified fusion protein was cleaved with activated protease factor X releasing the rhOC portion. The structure of rhOC was confirmed by mass spectrometry and amino acid sequencing. The fusion protein and its proteolytic cleavage product proved to be immunoreactive as shown by Western blotting analysis and by a new osteocalcin immunoassay based on time-resolved fluorescence. When osteocalcin was tested for its ability to bind to hydroxyapatite, there were no differences between the recombinant forms and native human osteocalcin purified from bone, suggesting that the Gla residues might be important only in oriented high-affinity binding.

Amino Acid Sequence↗
PubMed1996

Identification of biotinylated molecules using a baculovirus-expressed luciferase-streptavidin fusion protein.

A genetic fusion between streptavidin of Streptomyces avidinii and luciferase of Pyrophorus plagiophthalamus was constructed. The fusion protein was produced in the Sf9 insect cell line using the baculovirus expression vector system (BEVS). Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the proteins from cells infected with the recombinant virus, VL1393-LucGR-StreptAv, revealed that the fusion protein migrated with an apparent molecular weight of 75 kDa. Light emission measurements showed that the infected cells produced about 255 mg of the chimeric protein per liter of cell culture (127.5 micrograms/1 x 10(6) cells). Precipitation of the LucGR-StreptAv fusion protein with biotinylated acrylic beads as well as immunoblot analyses using biotinylated immunoglobulins indicated that both fusion moieties of the chimeric protein product were functional with respect to their physical and enzymatic activities.

Animals↗
PubMed1996

Sodium-lithium countertransport: a predictor of diabetic nephropathy in Jewish insulin-dependent diabetes mellitus patients of different ethnic origin?

Diabetic nephropathy in Jewish insulin-dependent diabetes mellitus (IDDM) patients has been found to correlate to their ethnic origin. It has also been found that increased sodium-lithium countertransport (SLC) in erythrocytes, as a genetic marker for essential hypertension, may identify those patients at risk for diabetic nephropathy. The purpose of this study was to investigate a possible correlation between this genetic marker and the ethnic origin of Jewish IDDM patients and their parents and the risk for developing diabetic nephropathy. Although SLC was slightly increased in IDDM patients with microalbuminuria, SLC was not correlated with the existence of diabetic nephropathy nor with the ethnic origin and blood pressure of these Jewish IDDM patients. Thus, other genetic factors may play a role in the different prevalence of diabetic nephropathy in Jewish IDDM patients of different ethnic origin.

Adult↗
PubMed1996

Immunofluorometric assay for sensitive and specific measurement of human prostatic glandular kallikrein (hK2) in serum.

Prostate-specific antigen (PSA) and human prostatic glandular kallikrein (hK2) have 79% identity with the primary structure. When we used recombinant hK2 protein, only 7 of 23 monoclonal anti-PSA IgGs (monoclonal antibodies, MAbs) cross-reacted with hK2, which enabled us to design a novel immunofluorometric MAb-MAb assay for the specific detection of hK2. In the first incubation, an excess of MAb 2H11, which does not cross-react with hK2, is added to prevent both free and complexed PSA from reacting in subsequent immunoreactions. In the second incubation, biotinylated MAb H50, which cross-reacts with hK2 by an epitope overlapping with MAb 2H11, served to bind only hK2 to the microtitration wells coated with streptavidin. In the third step, Eu-labeled MAb H117, which cross-reacts with hK2, detected the immobilized hK2. The hK2 assay was calibrated with recombinant hK2. The detection limit of the assay was 0.1 microgram/L, and the cross-reactivity with recombinant PSA was < or = 0.7%. The concentration of hK2 was measured in serum samples from 334 males with total PSA concentrations ranging from 1 to 3400 microgram/L. Most of the samples (57%) had hK2 concentrations below the detection limit. The proportions of hK2 relative to total PSA were 0-2% in 79%, 2-5% in 14%, 5-10% in 4%, and >10% in 3% of the samples. Gel filtration of 10 serum samples with increased hK2 concentrations showed a single peak of hK2 immunoreactivity with an apparent molecular size of approximately 30 kDa, corresponding to that of recombinant hK2 and free PSA.

Antibodies, Monoclonal↗
PubMed1995

Production of recombinant PSA and HK2 and analysis of their immunologic cross-reactivity.

Measurements of prostate-specific antigen (PSA) in serum are widely used to monitor patients with prostate cancer, but the attenuation of the assay response by PSA complexed to protease inhibitors has been shown to affect the results in certain assay designs. Moreover, the human glandular kallikrein-2 (hK2), a kallikrein-like serine protease that is 80% similar to PSA, might interfere with the specific detection of PSA by immunological cross-reactivity. We have expressed hK2 and PSA in eucaryotic cells using the Semliki Forest Virus expression system and studied the reactivity of 18 monoclonal anti-PSA IgGs. Five of them cross-reacted with identical affinities to recombinant hK2 whereas 13 recognized PSA alone. The antibodies that recognized both PSA and hK2 bind to a region of the protein that is exposed when PSA is complexed to alpha-1-antichymotrypsin.

Antibodies, Monoclonal↗
PubMed1995

Two human alpha 2-adrenoceptor subtypes alpha 2A-C10 and alpha 2B-C2 expressed in Sf9 cells couple to transduction pathway resulting in opposite effects on cAMP production.

The baculovirus expression vector system utilizing the strong polyhedrin gene promoter of the Autographa californica nuclear polyhedrosis virus (AcNPV) was used for high level expression of the two alpha 2-adrenoceptor subtypes alpha 2A-C10 and alpha 2B-C2 in Spodoptera frugiperda (Sf-9) insect cells. For rapid screening of recombinant viruses the luciferase gene was expressed under the early ETL-promoter (early transcript large) in the same plasmid. Both receptor subtypes showed the same rank order of binding affinity for four agonists tested: dexmedetomidine > l-medetomidine = clonidine > noradrenaline. For the alpha 2A-C10 subtype, these agonists inhibited forskolin stimulated cAMP production through pertussis toxin sensitive G-proteins. In contrast, for the alpha 2B-C2 subtype the agonists stimulated both basal and forskolin stimulated cAMP production.

Animals↗
PubMed1995

Mathematical formulae for the prediction of the residual beta cell function during the first two years of disease in children and adolescents with insulin-dependent diabetes mellitus.

On the basis of a retrospective study of 71 children followed for 24 months after diagnosis of type I insulin dependent diabetes a fitted mathematical model was constructed for the prediction of the course of beta cell function from the time of diagnosis. Two equations were derived, one for the maximal basal (B-max) and the other for the maximal i.v. glucagon stimulated peak C-peptide (P-max) levels reached during the remission period. The prognostic variables selected for analysis were: peak C-peptide levels at diagnosis (Po), age sex, degree of obesity, pubertal rating, the presence of islet cell antibodies (ICA) and levels of GHb. Multivariate analysis of the data showed that Po (p = 0.0006), puberty (p = 0.041). obesity (p = 0.0021), sex (p = 0.031), ICA (p = 0.0045) and GHb(p = 0.0066) significantly contributed to the prediction formula obtained for B-max whereas the contribution of the above variables for P-max were: Po (p = 0.0019), puberty (p = 0.0187), obesity (p = 0.0058), sex (p = 0.0598), ICA (p = 0.0187) and GHb (p = 0.0027). The residuals of the observed values from the values fitted by the predicted equations served to define two separate groups demonstrating distinct differences in the natural course of beta cell function in type I diabetes. This fitted model may thus be useful in distinguishing between newly diagnosed young patients who will undergo remission, requiring lower insulin doses, and those who have little chance for remission. It might also be helpful in the selection of patients most likely to benefit from immunosuppression or modulation, to maximize the benefit to risk ratio for such patients.

Adolescent↗
PubMed1995

Diabetic nephropathy and pregnancy: the effect of ACE inhibitors prior to pregnancy on fetomaternal outcome.

BACKGROUND: Diabetic nephropathy is associated with an increase in perinatal mortality and morbidity in uncontrolled pregnant patients. Recently angiotensin-converting enzyme inhibitor (ACE-I) was shown to improve the disease status in non-pregnant subjects. The purpose of this study was to examine the effect of prepregnancy treatment of insulin-dependent diabetes mellitus (IDDM) nephrotic women with captopril angiotensin converting enzyme inhibitor (ACE-1), on maternal renal function throughout pregnancy and on the fetomaternal outcome. METHODS: Eight IDDM nephrotic patients planning pregnancy were treated with captopril for a minimum of 6 months prior to conception together with intensive insulin management. Conception was allowed when proteinuria was < 500 mg/day and euglycaemia was achieved. At conception captopril was discontinued. RESULTS: At the beginning of captopril treatment, proteinuria was 1633 +/- 666 mg/day. At conception, proteinuria dropped to 273 +/- 146 mg/day (P = 0.0000) and increased gradually over the three trimesters to 593 +/- 515, 783 +/- 813, and 1000 +/- 1185 mg/day respectively (P = 0.2 between the trimesters); declining to 619 +/- 411 mg/day (P = 0.0002 vs conception) 3 months after delivery. Only in two patients (25%) did proteinuria exceed 1000 mg/day during pregnancy. There was no significant change in any of the other renal function tests: CCT, serum creatinine, uric acid, K+ and blood pressure. However, there were three cases of PET just prior to delivery. Maternal glycaemic control improved significantly prior to conception (P = 0.002) and remained euglycaemic (reflected by daily glucose profile, HbA1C and fructosamine) throughout gestation. Perinatal outcome was excellent. CONCLUSION: Captopril treatment before pregnancy has a prolonged protective effect on maternal renal functions during pregnancy and results in a favourable maternal-fetal outcome.

Adult↗
PubMed1995

Use of controlled luciferase expression to monitor chemicals affecting protein synthesis.

In this article, we present a new bioluminescent test system for the screening of chemical compounds with an inhibitory effect on protein synthesis. The test is based on the measurement of real-time in vivo light production by Escherichia coli strains expressing different luciferase genes. The eukaryotic lucGR gene from Pyrophorus plagiophthalamus was found to be the best of three types of luciferase genes tested. Chemicals with known inhibitory effects on protein synthesis were used as test chemicals together with some general toxicants. The incubation of a test chemical with cells was performed either prior to or after the induction of protein synthesis, and the difference in the results of the two methods distinguishes the possible influence on protein synthesis from direct metabolic inhibition. Using lyophilized bacteria, the test is performed in less than an hour without any bacterial cultivation, which makes the test suitable for rapid and sensitive screening of chemicals or environmental samples. Compared with the standardized 50% inhibitory concentration calculation method of the bioluminescent cytotoxicity test, the more direct approach of calculation developed in this study proved to be more convenient than and as reliable as the standard method.

Journal Article↗
PubMed1995

An integer programming model to limit hospital selection in studies with repeated sampling.

OBJECTIVE: We describe an integer programming model that, for studies requiring repeated sampling from hospitals, can aid in selecting a limited set of hospitals from which medical records are reviewed. STUDY SETTING: The model is illustrated in the context of two studies: (1) an analysis of the relationship between variations in hospital admission rates across geographic areas and rates of inappropriate admissions; and (2) a validation of computerized algorithms that screen for complications of hospital care. STUDY DESIGN: Common characteristics of the two studies: (1) hospitals are classified into categories, e.g., high, medium, and low; (2) the classification process is repeated several times, e.g., for different medical conditions; (3) medical records are selected separately for each iteration of the classification; and (4) for budgetary and logistical reasons, reviews must be concentrated in a relatively small subset of hospitals. DATA COLLECTION/EXTRACTION METHODS. In each study, hospitals are ranked based on analysis of hospital discharge abstract data. CONCLUSIONS: The model is useful for identifying a subset of hospitals at which more intensive reviews will be conducted.

Bias↗
PubMed1994

Expression of prostate specific antigen on the surface of a filamentous phage.

We have constructed two phagemid vectors containing the gene coding for human Prostate Specific Antigen (PSA) translationally fused to a pelB signal sequence and minor coat protein III of phage fd leading to phage particles that carry PSA on their tips. Phages were characterized by Western blotting and by panning, using biotinylated monoclonal anti-PSA antibodies immobilized onto streptavidin-coated microtitration wells. Binding of PSA-phages was 10(2)- to 5 x 10(3)-fold more effective than that of wild-type phages. Trypsin treatment of the phage abolished the specific binding. Competitive panning with different concentrations of added purified PSA demonstrated that binding was PSA specific. Finally, Western-blot analysis confirmed that full-length and shorter degradation products of the fusion between PSA and protein III were visible as probed with anti-PSA antibody.

Antibodies, Monoclonal↗
PubMed1994

Flow cytometric analysis of bioluminescence emitted by recombinant baculovirus-infected insect cells.

Five recombinant baculoviruses, each containing a different insect luciferase gene encoding a protein with characteristic light emission properties, namely, luc GR (546 nm), luc FF (556 nm), luc YG (560 nm), luc YE (578 nm), and luc OR (593 nm) were constructed. All genes were inserted under the transcriptional control of the polyhedrin gene promoter of the Autographa californica nuclear polyhedrosis virus (AcNPV) and expressed in Spodoptera frugiperda insect cells during viral infection. The biological activity of the different luciferases was characterized by using intact recombinant baculovirus infected cells. Addition of the substrate, D-luciferin, immediately prior to the analysis allowed monitoring of light emission by flow cytometry. Also, the kinetics of the light emission of lucGR was analyzed with the flow cytometer. The emission peaks of the infected cells were clearly separated by wavelength scanning. Especially, the firefly luciferase (lucFF) had a broad peak and transient luminescence. The highest maximal intensity values in vivo were recorded for luc GR and luc YG. SDS-PAGE analysis showed that the major protein expressed had a molecular weight similar to authentic luciferase. Flow cytometry and insect luciferases with clearly separated emission spectra appear to be of value for sensitive in vivo analysis of gene promoter activity.

Animals↗