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Biomedical subjects

M Karp

Publications and source records attributed to M Karp.

At least 19 recordsLinked to original sources
PubMed2001

Expression and purification of polyhistidine-tagged firefly luciferase in insect cells--a potential alternative for process scale-up.

The coleopteran firefly, Photinus pyralis, luciferase was produced in lepidopteran Trichoplusia ni insect cells using a baculovirus expression vector. The recombinant protein was equipped with a polyhistidine affinity tag at the carboxyl terminus and purified by immobilized metal-ion affinity chromatography in combination with an expanded bed adsorption system. This approach enabled an efficient, one-step purification protocol of a genetically modified luciferase with properties similar to those of the authentic counterpart. According to light emission measurements, the final yield of highly purified protein was 23 mg l(-1) of cell culture. In addition, no specific interaction of interfering substances, such as, ATP, adenylate kinase, nucleoside diphosphokinase, as well as, creatine kinase of the final preparation were identified. Together, the results presented here clearly show that the baculovirus expression system in combination with immobilized metal-ion affinity chromatography is a potential strategy for process scale-up of polyhistidine tagged insect luciferase.

Animals↗
PubMed2001

Automated color correction method for Vibrio fischeri toxicity test. Comparison of standard and kinetic assays.

We demonstrate in this study that the toxicity of solid and highly colorful samples can be measured with kinetic bioassay using luminescent bacterium Vibrio fischeri. The Flash assay, named after the test protocol, is performed with a tube luminometer. In this method, each sample acts as a reference for itself, and therefore, the color correction is possible with minimal hands-on-time. The bacteria are dispensed into the sample and the signal is recorded continuously. The maximum signal received after immediately dispensing is compared to the signal after an incubation period. With many chemicals, the toxic effects are obtained after a very short contact time. However, different chemicals have different modes of toxicity. Thus, kinetic data from sample analyses after 15 or 30 min for this bacterium gives an additional dimension for obtaining reliable results. The performance of the test was compared to the standardized photobacteria test protocol with reference chemicals. The repeatability of the test was excellent. The coefficient of variation was normally below 1% with 10 replicates.

Automation↗
PubMed2001

Simultaneous quantification of human glandular kallikrein 2 and prostate-specific antigen mRNAs in peripheral blood from prostate cancer patients.

We present a multiplexed and internally calibrated quantitative reverse transcription-PCR (QRT-PCR) assay to detect human glandular kallikrein 2 (hK2) and prostate-specific antigen (PSA) transcripts in blood samples from healthy subjects and prostate cancer (PC) patients. The assay detected 50 copies of hK2 and PSA mRNA, and 1 PSA- and 10 hK2-expressing LNCaP cells in the presence of 2.5 x 10(6) PSA- and hK2-negative cells. In PC patients, 20 of 25 and 19 of 25 gave detectable PSA and hK2 mRNAs, respectively. Number of hK2 mRNA copies was significantly higher than that of PSA mRNA copies in patients with biochemically progressive (P = 0.02) PC, and with locally advanced and metastasized (P = 0.004) PC. Patients with rapidly progressive and hormone refractory PC gave detectable hK2 mRNA only in 2 of 8 and PSA mRNA in 3 of 8 patients. Neither PSA nor hK2 mRNAs were detected in 16 healthy subjects. PSA and hK2 discriminated PC patients with biochemically progressive and advanced disease from the controls and from the aggressive distant metastatic disease. The assay provides a reliable quantification of the number of hK2 and PSA mRNA copies, allows to discriminate PC cases from healthy subjects, and offers a tool for further studies on molecular staging of PC.

Calibration↗
PubMed2001

Factor analysis of thermal and vibration thresholds in young patients with Type 1 diabetes mellitus.

AIMS: To identify factors that represent relationships among sets of interrelated thermal and vibration threshold variables and to find clinical correlates that are significantly associated with these factors. METHODS: Thermal and vibration perception thresholds were tested in the hands and feet of Type 1 diabetic patients treated in an outpatient clinic for juvenile-onset diabetes. Factor analysis was used to identify factors that represent relationships among sets of thermal and vibration threshold variables. RESULTS: One hundred and forty-eight patients (47.3% males, median current age 22.3 years and median duration of diabetes 11.4 years) were evaluated. Three factors explained 77% of the total variance: 'hand sensation' factor, underlying cold, warmth and vibration perception thresholds in the hand; 'foot sensation' factor, underlying the same sensory thresholds in the foot; and 'heat-related pain' factor, underlying heat pain perception threshold in both limbs. The 'foot sensation' factor was the only factor that significantly correlated with diabetes-related variables (e.g. duration and cumulative glycaemic control of the disease) and concurrent diabetic microangiopathy. Male sex was associated with higher values of the 'heat-related' factor, while the 'hand sensation' factor did not correlate with any of the study variables. CONCLUSIONS: The distribution of the various thermal and vibration threshold variables according to the three factors may point at length-dependent mechanism of axonal degeneration. Cold, warmth and vibration perception thresholds in the foot may be the only valuable psychophysical parameters in the evaluation of early sensory impairment associated with diabetes.

Adolescent↗
PubMed2001

Measurement of effects of antibiotics in bioluminescent Staphylococcus aureus RN4220.

The spread of antibiotic resistance among pathogenic bacteria is a serious threat to humans and animals. Therefore, unnecessary use should be minimized, and new antimicrobial agents with novel mechanisms of action are needed. We have developed an efficient method for measuring the action of antibiotics which is applied to a gram-positive strain, Staphylococcus aureus RN4220. The method utilizes the firefly luciferase reporter gene coupled to the metal-inducible cadA promoter in a plasmid, pTOO24. Correctly timed induction by micromolar concentrations of antimonite rapidly triggers the luciferase gene transcription and translation. This sensitizes the detection system to the action of antibiotics, and especially for transcriptional and translational inhibitors. We show the results for 11 model antibiotics with the present approach and compare them to an analytical setup with a strain where luciferase expression is under the regulation of a constitutive promoter giving only a report of metabolic inhibition. The measurement of light emission from intact living cells is shown to correlate extremely well (r = 0.99) with the conventional overnight growth inhibition measurement. Four of the antibiotics were within a 20% concentration range and four were within a 60% concentration range of the drugs tested. This approach shortens the assay time needed, and it can be performed in 1 to 4 h, depending on the sensitivity needed. Furthermore, the assay can be automatized for high-throughput screening by the pharmaceutical industry.

Anti-Bacterial Agents↗
PubMed2001

Psychological impact of islet cell antibody screening.

The purpose of this study was to evaluate the psychological impact of autoantibody screening and its results on at-risk individuals and family members. Individuals who were antibody positive (AP) were identified through a large-scale screening program conducted at our institute. The sample consisted of nine families in whom 10 AP youngsters (7 M, 3 F) were identified, ranging in age from 6-18 years (mean 11.8, median 10 yr). Seventeen parents and eight diabetic youngsters (mean age 15.2, median 16 yr) participated in the study. Reaction to autoantibody positivity was assessed with the Impact of Event scale (IES). The IES was answered twice: within a week from the disclosure of the AP status, and 3 months later. Parents scored higher than their diabetic children and AP children on both measures of the IES, Intrusion and Avoidance. Three months later both scores were significantly reduced in both the parents and the AP children; however, parents still scored significantly higher on both scores than the AP children. The results suggest that learning one's AP status induces significant anxiety, especially in parents of AP youngsters. Although this initial anxiety dissipates over time it still remains quite high after 3 months. The results highlight the importance of psychosocial counseling for all members of diabetes mellitus screening and prevention trials.

Adolescent↗
PubMed2001

Dual-label detection of amplified products in quantitative RT-PCR assay using lanthanide-labeled probes.

Quantitative RT-PCR (QRT-PCR) enables the sensitive and specific detection of mRNA with a small copy number. We used the QRT-PCR method and dual-label analysis of amplification products for the detection of prostate-specific antigen (PSA) mRNA. The QRT-PCR assay employed a PSA-like internal standard (IS) mRNA, which was used to quantify the PSA mRNA copies and to control the variations during the whole assay procedure from the RNA extraction to the detection of QRT-PCR amplification products by hybridization assay. After co-amplification, the PSA and IS products were detected in a microplate using Eu3+ chelate-labeled PSA and Tb3+ chelate-labeled IS hybridization probes. The detection probes allowed the simultaneous and dual-label detection of PSA and IS products in the same microtiter well. Compared to the single-label assay, the dual-label detection improved the within- and between-assay CV% from 21.7 to 7.5 and from 36.0 to 30.3, respectively. The between- and within-assay variation of the dual-label assay was further studied using PSA-producing LNCaP cells. The cells were found to express 980 +/- 170 (mean +/- SD) copies of PSA-mRNA with the within-assay CV% of 17.7 and 890 +/- 220 (mean +/- SD) copies of PSA-mRNA with the between-assay CV% of 25.0. The methodology developed may help in future studies to obtain reliable quantification of PSA mRNA generated by circulating prostate cancer cells.

Animals↗
PubMed2000

A new label technology for the detection of specific polymerase chain reaction products in a closed tube.

A novel signal generation principle suitable for real time and end-point detection of specific PCR products in a closed tube is described. Linear DNA probes were labeled at their 5'-ends with a stable, fluorescent terbium chelate. The fluorescence intensity of this chelate is lower when it is coupled to single-stranded DNA than when the chelate is free in solution. The synthesized probes were used in the real time monitoring of PCR using a prototype instrument that consisted of a fluorometer coupled to a thermal cycler. When the probe anneals to a complementary target amplicon, the 5'-->3' exonucleolytic activity of DNA polymerase detaches the label from the probe. This results in an enhanced terbium fluorescence signal. Since terbium has a long excited state lifetime, its fluorescence can be measured in a time-resolved manner, which results in a low background fluorescence and a 1000-fold signal amplification. The detection method is quantitative over an extremely wide linear range (at least 10-10(7)initial template molecules). The label strategy can easily be combined with existing label technologies, such as TaqMan 5'-exonuclease assays, in order to carry out multiplex assays that do not suffer from overlapping emission peaks of the fluorophores.

Chelating Agents↗
PubMed2000

Effect of Cd-containing wood ash on the microflora of coniferous forest humus.

The use of wood ash in forestry has been questioned because the cadmium (Cd) concentration of ash, which varies between 1 and 20 mg kg(-1) ash, exceeds the level allowed for fertilizers (3 mg kg(-1)) used in agriculture. To investigate the combined and separated effects of Cd and ash on the forest humus microflora, pumice or wood ash, spiked with a water-soluble (CdCl(2)) or -insoluble (CdO) form of Cd at three levels (0, 400 and 1000 mg kg(-1)), were applied at a fertilization level of 5000 kg ha(-1) in a laboratory microcosm study. The trial consisted of 60 microcosms (five replications per treatment), which were incubated in darkness at +20 degrees C and a constant relative air humidity of 60%. After two months the humus in the microcosms was sampled. Analyses of CO(2) evolution to measure the overall microbial activity and of phospholipid fatty acid (PLFA) pattern to measure microbial community structure were performed. The substrate-use patterns of Biolog EcoPlates were analyzed as a measure of bacterial functionality. Finally the bacterial (3)H-thymidine incorporation in the presence of different concentrations of Cd and the number of colony forming units (cfu) of bacteria on nutrient agar in the presence of 0, 5 and 20 mg Cd l(-1) agar were applied to measure Cd tolerance. The use of pumice (pH of humus under the pumice 4.0) did not induce any changes in the above variables compared to two untreated microcosms (humus pH 3.9). Pumice was therefore used to distribute the Cd evenly over the humus surface in order to estimate the possible effect of Cd without ash (pH of humus under the ash 7.0). The application of ash increased the microbial activity, changed the PLFA and substrate-use patterns and increased cfu compared to the humus under pumice. The form and level of Cd in the ash had no further effect on this result. In the humus under pumice the level, but not the form of Cd decreased the microbial activity and changed the PLFA pattern compared to the unspiked pumice. None of the treatments induced bacterial tolerance to Cd. Ash thus protected the humus microflora from the harmful effects of Cd.

Journal Article↗
PubMed2000

A group-specific microbiological test for the detection of tetracycline residues in raw milk.

The potentiality of using a luminescent Escherichia coli strain for the specific detection of tetracycline residues in raw bovine milk was investigated. The sensor cells contain a reporter plasmid carrying the bacterial luciferase operon of Photorhabdus luminescens under the control of the tetracycline responsive control region from transposon Tn10. Incubation of the cells with the sample containing tetracyclines increases the light emission of the sensor cells. The most sensitive tetracycline detection was achieved in 120 min and by using CDTA as a chelating agent in the assay. Heat-treatment of milk before the assay decreased the variations in background luminescence signals and in tetracycline-induced luminescence between different milk samples. The detection limits for tetracycline, oxytetracycline, chlortetracycline, doxycycline, methacycline, demeclocycline, and minocycline were between 2 and 35 ng/mL. Nontetracycline antibiotics did not significantly interfere with the detection of tetracyclines.

Animals↗
PubMed2000

Microbial testing methods for detection of residual cleaning agents and disinfectants-prevention of ATP bioluminescence measurement errors in the food industry.

The ATP luminescence measurement is based on the presence of an enzymatic reaction and may significantly be affected by cleaning agents and disinfectants. In addition, disinfectants can also reduce the activity of the luciferase enzyme and also act as ATP-releasing agents. The agents disrupt the cell walls but preserve ATP in measurable form, and therefore correlation with culture methods can be poor. Therefore, if a rapid method is used to detect ATP, a control must be used for reliable results. The possible effect of disinfectants can be eliminated with a rapid test to minimize sources of error. In the present study a microbiological residue testing method that is nonspecific for residues was developed. The effects of a total of 38 commercial cleaning agents and disinfectants of various types were assessed using two microbiological methods, the Vibrio fischeri photobacteria test and Micrococcus luteus inhibition zone technique. The results show that the V. fischeri photobacteria test is very sensitive. This test can therefore be used for testing cleaning agent residues on surfaces in very small amounts. A small study was also carried out in a food factory to show applicability in processing facilities. The study showed, that a need for this type of method exists in food processing.

Adenosine Triphosphate↗
PubMed2000

Qualitative detection of tetracycline residues in milk with a luminescence-based microbial method: the effect of milk composition and assay performance in relation to an immunoassay and a microbial inhibition assay.

Performance of Tet-Lux, a newly developed microbiological test for the detection of tetracycline residues in raw milk, based on tetracycline-controlled luminescence activation of the test bacteria, was evaluated in bovine milks with variable amounts of somatic cells, bacteria, fat, protein, and natural inhibitory compounds. The sensitivity of Tet-Lux was also compared to a commercially available tetracycline immunoassay (Snap, Idexx Laboratories Inc.) and to a microbial inhibition test (Delvotest SP, Gist-Brogades). There were slight differences in the luminescence signals between different milk samples, but no single factor could be pointed out to be responsible for them. There appeared to be a modest inverse relationship between luminescence and increasing fat and protein content. The amount of somatic cells, bacteria, and the natural inhibitors lysozyme and lactoferrin did not affect the luminescence response. The test fulfilled the sensitivity requirement specified by the European Union (maximum residue limit 100 ng/ml for tetracyclines). The Tet-Lux test was clearly more sensitive to all tetracyclines tested (oxytetracycline, tetracycline, chlortetracycline, doxycycline, demeclocycline, methacycline, minocycline) than Delvotest SP, and for five tetracyclines out of seven more sensitive than Snap. The test provides a fast, simple, and robust microbial method for the qualitative detection of tetracycline residues in milk.

Animals↗
PubMed2000

Development and evaluation of three immunofluorometric assays that measure different forms of osteocalcin in serum.

BACKGROUND: Circulating human osteocalcin (hOC) has been used as a marker of bone formation. Our aim was to validate three immunofluorometric assays (IFMAs), measuring different forms of hOC. METHODS: The two-site IFMAs were based on previously characterized monoclonal antibodies. Assay 2 recognized intact hOC, assays 4 and 9 measured the NH(2)-terminal mid-fragment and the intact hOC. In addition, assay 9 required hOC to be gamma-carboxylated. RESULTS: A 76-79% increase of serum immunoreactive hOC was found in the postmenopausal group compared with the premenopausal group with all IFMAs. With EDTA-plasma samples, the observed increases were lower (49-65%). The hOC concentration in the postmenopausal group receiving hormone replacement therapy was 42-44% lower than that in the postmenopausal control group in both serum and EDTA-plasma samples. The depressed carboxylation in warfarin-treated patients was accompanied by lower results in assay 9. The ratio of assay 9 to assay 4 totally discriminated the warfarin-treated patients from the controls. Assay 9 showed the smallest decreases in measured hOC after storage of serum or plasma for 4 weeks at 4 degrees C, followed by assay 4 and assay 2. Results from the last assay were <17% of their initial values after 4 weeks of storage. No diurnal variation was observed with assay 9 as opposed to the two other IFMAs. CONCLUSION: The three assays with their distinct specificity profiles (intact vs fragmented and carboxylated vs decarboxylated hOC) may provide valuable tools for investigating the significance of different hOC forms in various bone-related diseases.

Aged↗
PubMed1999

A streptavidin-luciferase fusion protein: comparisons and applications.

Luciferases are unique enzymes in being capable of emitting visible light as one of the end-products of their catalysis. Both procaryotic and eucaryotic organisms exist that emit light, and the luciferases from these organisms differ considerably in size as well as chemistry of catalysis. Two main, i.e. most studied groups, are the bacterial luciferases of e.g. Vibrio fisheri, Vibrio harveyi, and Photorhabdus luminescens, responding to FMNH2, long-chain aldehyde and molecular oxygen and the insect luciferases of the fireflies Photinus pyralis and Luciola minengrelica or click beetle Pyrophorus plagiophthalamus, responding to ATP, luciferin and molecular oxygen. An emerging amount of 'new' luciferases from shrimps, fish, jelly fish and overall from marine origin, are finding their way to biotechnological applications. The common feature of these is their ability to produce light within the visible region of the spectrum, i.e. between 450 nm (blue) and 630 nm (red). In this short review, we discuss some of the recent advances on fusion proteins of eucaryotic luciferases and their applications. Special emphasis is placed on a streptavidin-luciferase fusion protein produced by insect cells using the baculovirus expression system.

Animals↗
PubMed1999

Measurement of firefly luciferase reporter gene activity from cells and lysates using Escherichia coli arsenite and mercury sensors.

The structural gene encoding firefly luciferase from Photinus pyralis is a widely used reporter both in traditional monitoring of gene expression and in bacterial sensors. Its activity can be detected from living cells (in vivo) without disruption or from cell-free lysate (in vitro). We compared the two measurement methods by using an overall toxicity detecting strain Escherichia coli MC1061(pCSS810), a mercury-sensing strain E. coli MC1061(pTOO11), and two new arsenic sensor strains MC1061(pTOO31) and AW3110(pTOO31) which were constructed for this study. Plasmid pTOO31 was constructed by inserting the ars promoter and the arsR gene from plasmid R773 to control firefly luciferase gene expression. Both in vivo and in vitro methods correlated well with the strains tested [correlation coefficients R = 0.99484 and 0.99834] and gave highly comparable results with standard solutions of arsenite or mercury ions and from six environmental water samples spiked with the ions. Use of the in vivo method resulted in lower variation between replicates of the same sample (CVs ranging from 3.9 to 7.2%) and also between different samples (from 8.6 to 25.9%) compared to the in vitro method (CVs ranging from 8.6 to 17.8% for replicates and from 13.1 to 36.3% for different samples).

Animals↗
PubMed1999

The effect of alpha-lactalbumin and beta-lactoglobulin hydrolysates on the metabolic activity of Escherichia coli JM103.

Bovine milk proteins alpha-lactalbumin (alpha-la) and beta-lactoglobulin (beta-lg) were hydrolysed with seven different proteolytic enzymes, and the effect of various hydrolysates on a genetically modified luminous Escherichia coli JM103 was tested in vitro with a bioluminescence assay for bacterial growth and metabolism. Undigested proteins did not inhibit the activity of tested E. coli JM103 at a concentration as high as 0.1 g ml-1. At the same concentrations, alpha-la hydrolysed with pepsin or trypsin and beta-lg hydrolysed with alcalase, pepsin or trypsin, showed a lower metabolic activity during the first 8 h of growth. The activity of E. coli JM103 in the presence of 25 mg ml-1 alpha-la or beta-lg hydrolysed with pepsin and trypsin was only 21% of the control after incubation for 6 h. The preliminary results indicated that ultrafiltration through 10 kDa and 1 kDa molecular mass cut-off membranes may be used to enrich bacteriostatic properties.

Escherichia coli↗
PubMed1999

Production and activation of recombinant hK2 with propeptide mutations resulting in high expression levels.

Human glandular kallikrein 2 (hK2) is a serine protease expressed mainly by the prostate gland with 80% identity in primary structure to prostate specific antigen (PSA). hK2 has proven to be a useful marker of prostate cancer which can be used in combination with PSA to better discriminate between prostate cancer and benign prostate hyperplasia. The studies on hK2 have been hampered by its very low phyciological levels (6 microgram.mL-1), its close similarity to PSA, and the low expression levels obtained using recombinant procedures to produce hK2 (0.7 mg.L-1). We have now generated propeptide mutations of hK2 which can be used to isolate stable, inactive prohK2 mutants. Compared with wild-type hK2, expression of the propeptide hK2 mutants increases the expression levels up to 15-40-fold giving 10-30 mg hK2.L-1. These results indicate that the low expression levels of wild-type hK2 are related to the activation or autoactivation of the wild-type enzyme and the instability of the active protease in cell culture and possibly also in tissue. The purified mutant hK2 may be activated by either enterokinase or factor Xa to generate an enzyme for use in functional studies with the characteristics of the original wild-type protein. Further, the stable inactive mutant hK2 protein may be used for immunizations to generate novel monoclonal antibodies, used as standard material for clinical assays or in crystallization studies where large quantities of protein are required.

Amino Acid Sequence↗