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PubMed2004

Development of a fluorescent microplate assay for determining cyanovirin-N levels in plasma.

A sensitive immunosorbent competition assay was developed for quantitation of the anti-HIV protein cyanovirin-N (CV-N) in plasma using a 96-well plate format and a fluorescent endpoint. The assay is based on the binding of CV-N in plasma to plate-bound anti-CV-N antibodies, followed by removal of the plasma and addition of europium-labeled CV-N (Eu3+ -CV-N) to compete for the remaining antibody sites. Detection by addition of a dissociative fluorescence enhancement solution and time-resolved fluorescence measurements allowed correlation to the concentration of the native CV-N in plasma. A linear detection range of 1-100 nM (r2>0.99) was obtained for CV-N in mouse plasma. This assay was then utilized for analysis of plasma levels of CV-N samples following subcutaneous injection of CV-N into mice. The results of these studies confirmed the reliability and sensitivity of this assay and the feasibility of its use for pharmacokinetic studies in a variety of species.

Animals↗
PubMed2006

Multiple recombinant ELISA for the detection of bovine viral diarrhoea virus antibodies in cattle sera.

The most immunogenic proteins (E0, E2 and NS3) of bovine viral diarrhoea virus (BVDV) (NADL strain) were expressed in the baculovirus/insect cells system. Recombinant antigens were applied to the design of enzyme immunoabsorbent assays (ELISAs) for the detection of specific antibodies in cattle sera. The assays developed were shown to be highly sensitive and specific in comparison with the viral neutralization test, which is the reference test for the serological diagnosis of BVDV. The present results demonstrate the contribution of each recombinant antigen to determine clearly the pattern of anti-BVDV antibodies in bovine serum samples.

Animals↗
PubMed2006

New approaches for male fertility control: HE6 as an example of a putative target.

Reversible contraceptive methods for males are still not available. During the last few years several marketing studies have clearly shown that men and women would welcome a situation where men could assume responsibility for family planning. Schering AG and Organon are currently collaborating to develop a hormonal method for male fertility control based on the combination of etonogestrel as gestagenic component and testosterone undecanoate. To further optimize male contraceptives in terms of improved efficiency, rapid onset, reversibility, fewer side effects and a convenient method of application, a search for innovative non-hormonal approaches was started. During the last few years, numerous proteins were identified which play a specific role in male fertility. These proteins have first to fulfil a set of indication-specific criteria before a drug discovery process can be initiated. The most important criteria for a putative target protein are tissue-selective expression, crucial biological function in fertility, drugable properties and feasibility of assay development for high-throughput-screening and lead optimization. The G-protein-coupled receptor HE6 was selected as target and the above selection criteria were applied. HE6 displays a preferred epididymis-specific expression pattern and belongs to the superfamily of GPCRs, which are well known to be drugable with small molecules. A knockout mouse was generated which revealed an infertility phenotype with the onset occurring 6 weeks after initiation of spermatogenesis at the latest. Surprisingly, no epididymis-specific phenotype was observed. Instead, the reabsorption of testicular fluid along the efferent ducts was strongly affected. No further obvious side effects were observed in male or female mice. This study with HE6 exemplifies how targets for male contraception have to be validated before drug development can start.

Animals↗
PubMed2000

Assay for human matrix gla protein in serum: potential applications in the cardiovascular field.

Matrix Gla protein (MGP) is synthesized in a vitamin K-dependent way in smooth muscle cells of the healthy vessel wall, and its mRNA transcription is substantially upregulated in atherosclerotic lesions. Here we report the preparation of a monoclonal antibody against human MGP and its use in an enzyme-linked immunosorbent assay. The intra-assay and interassay coefficients of variation in serum samples were 5.4% and 12.6%, respectively, and the lower detection limit was 8.5% of the normal serum value. Individual within-day variations were <11% and did not show a distinct circadian pattern. Day-to-day variations in fasting morning samples were <8%. In a first explorative survey, serum MGP concentrations were found to be significantly increased in patients with severe atherosclerosis, whereas these values were normal in those with low bone mass and osteoporosis. This finding is consistent with the high MGP mRNA expression observed in atherosclerotic vessels and plaques. More elaborate studies are required to assess the potential clinical utility of this newly developed assay.

Antibodies, Monoclonal↗
PubMed2002

The C-terminal IgI domains of myosin-binding proteins C and H (MyBP-C and MyBP-H) are both necessary and sufficient for the intracellular crosslinking of sarcomeric myosin in transfected non-muscle cells.

Using the COS cell transfection assay developed previously, we examined which domains of myosin-binding proteins C and H (MyBP-C and MyBP-H) are involved in intracellular interactions with sarcomeric myosin heavy chain (MyHC). Earlier studies demonstrated that overexpression of sarcomeric MyHC in COS cells results in the cytoplasmic assembly of anisotropic, spindle-like aggregates of myosin-containing filaments in the absence of other myofibrillar proteins. When the same sarcomeric MyHC was co-expressed with either MyBP-C or MyBP-H, prominent cable-like co-polymers of MyHC and the MyBPs formed in the cytoplasm instead of the spindle-like aggregates formed by MyHC alone. In vitro binding assays have shown that the C-terminal IgI domain of both MyBP-C (domain C10) and MyBP-H (domain H4) contains the light meromyosin (LMM)-binding sites of each molecule, but this domain cannot explain all of the intracellular properties of the molecules. For example, domains C7-C10 of MyBP-C and domains H1-H4 of MyBP-H are required for the faithful targeting of these proteins to the A-bands of myofibrils in skeletal muscle. Using truncation mutants of both MyBPs tagged with either green fluorescent protein (GFP) or c-myc, we now demonstrate that the last four domains of both MyBP-C and MyBP-H colocalize with the full-length proteins in the MyHC/MyBP cable polymers when co-transfected with MyHC in COS cells. Deletion of the C-terminal IgI domain in either MyBP-C or MyBP-H abrogated cable formation, but the expressed proteins could still colocalize with MyHC-containing filament aggregates. Co-expression of only the C-terminal IgI domain of MyBP-C with sarcomeric MyHC was sufficient for cable formation and colocalization with myosin. We conclude that the C-terminal IgI domains of both MyBP-H and MyBP-C are both necessary and sufficient for inducing MyHC/MyBP cable formation in this COS cell system. However, there must be other myosin-binding sites in MyBP-C and MyBP-H that explain the co-distribution of these proteins with myosin filaments in the absence of cable formation. These latter sites are neither sufficient nor required for cable formation.

Animals↗
PubMed2005

Comparison of ELISA and PCR vis-à-vis cultural methods for detecting Aeromonas spp. in foods of animal origin.

The present study was conducted to assess the best method of the most commonly used methods for detection of aeromonads in foods of animal origin. With this objective an OMP based indirect plate ELISA and a duplex-PCR using primers targeting aerolysin gene and 16S rRNA gene and yielding amplicons of 252 bp and 599 bp, respectively, were standardized. The standardized protocols and the conventional cultural method were then compared for their respective sensitivities and specificities for detecting aeromonads from chicken and milk samples. Both the standardized assays were found to be highly specific for Aeromonas. The efficiency of the standardized indirect-ELISA and duplex-PCR protocols was assessed by artificial inoculation studies with varying concentrations of Aeromonas cells inoculated in chicken and milk samples followed by enrichment in Alkaline Peptone Water supplemented with 10 mg/ml cephalothin (APW-C) for 12 h. The results revealed that indirect-ELISA was able to detect a minimum of 10(3) cells/ml or g of Aeromonas cells in spiked milk and chicken samples, respectively. Whereas, duplex-PCR and cultural method were able to detect as low as 1 cell/ml or g of Aeromonas cells in spiked milk and chicken samples. The developed assays were also tested for their efficiency to detect Aeromonas spp. in naturally contaminated milk and chicken samples. Out of a total 50 milk samples screened for presence of Aeromonas by the three methods viz., indirect-ELISA, duplex-PCR and cultural method only 1 (2%) turned out to be positive showing positive results by all three methods. Similarly, 50 samples of chicken were tested by all three methods. Three samples (6%) turned out to be positive and here again by all the three methods.

Aeromonas↗
PubMed2005

Platelet mitochondrial evaluation during cytochrome c and dichloroacetate treatments of MELAS.

We hypothesized that serial changes in platelet (PLT) mitochondrial enzyme (ME) activities might correspond to the effects of medications for mitochondrial encephalomyopathy and stroke-like episodes (MELAS). Cytochrome c and sodium dichloroacetate (DCA) were given to a 7-year-old girl with MELAS who had an A3243G mitochondrial DNA mutation. The effects were evaluated with whole PLT-ME assays, developed by our group, using a microplate-reader. During cytochrome c treatment, complex II+III (II+III), complex IV (IV) and citrate synthase (CS) activities showed gradual but statistically significant decrease. II+III activity dropped below normal. II+III/CS activity was initially below normal, followed by a transient improvement, then decreased again before the appearance of central nervous system symptoms. II+III, IV, II+III/CS and IV/CS activities reached their lowest levels in association with a stroke-like episode, then increased with DCA treatment. Our results suggest that progressive mitochondrial dysfunction may occur before the stroke-like episodes in MELAS and that DCA treatment may increase mitochondrial activities. Our whole PLT-ME assay system may be useful for serially evaluating mitochondrial functions in relation to clinical symptoms.

Blood Platelets↗
PubMed1978

Quantitation of differential sensitivity of human-tumor stem cells to anticancer drugs.

With a direct in vitro tumor-colony assay developed to measure sensitity of human-tumor stem cells to anticancer drugs, we performed 32 retrospective or prospective clinical studies in nine patients with myeloma and nine with ovarian cancer treated with standard agents that were tested in vitro. The results were clearly correlated (P is less than 0.00001). Unique patterns of sensitivity and resistance to the six drugs tested were observed for individual patients. In eight cases of myeloma and three of obarian carcinoma in vitro sensitivity corresponded with in vivo sensitivity whereas in one case of myeloma it did not. In vitro resistance correlated with clinical resistance in all five comparisons in myeloma and all 15 in ovarian cancer. We conclude that this assay shows sufficient promise to warrant larger-scale testing to determine its efficacy for selection of new agents and individualized cancer chemotherapy regimens.

Antineoplastic Agents↗
PubMed2000

Deficiency of current methods in assaying endochitinase activity.

Since chitin is degraded by a combination of both endo- and exochitinases, it is likely that both enzymes will be present in a crude extract. Currently used substrates for detecting endochitinase activity suffer from the fact that they could easily be digested by the contaminating exochitinase, thus giving either a false-positive or an inaccurate reading of the endochitinase activity. Using Photorhabdus luminescens, a bacterium symbiotically associated with insect-parasitic nematode Heterorhabditis bacteriophora as an exemplary system, we have identified these two chitinases by a simple "fluorimetric zymography" procedure. The exochitinase is a metalloenzyme and its activity is inhibited by 1,10-phenanthroline. Once the exochitinase activity is detected in a crude extract, its contribution must be eliminated before accurate determination of the endochitinase activity can be carried out. Specific properties of these enzymes including the pH activity profile, the requirement of metal ions for activity, and the molecular weight of the enzymes are among the factors to be considered in developing assaying procedures for endochitinase.

Chitinases↗
PubMed1995

Production of a nested set of glycosylphosphatidylinositol structures from a glycosylphosphatidylinositol-anchored protein.

Glycosylphosphatidylinositol (GPI) membrane anchors are synthesized in the endoplasmic reticulum of eukaryotic cells. Synthesis of the core GPI structure is achieved by the sequential transfer of monosaccharides and phosphoethanolamine to phosphatidylinositol. The assembly process can be reproduced in vitro using membrane preparations supplemented with sugar nucleotides. With one exception, however, none of the biosynthetic enzymes involved have been isolated. One impediment to progress in the isolation of these enzymes is the nonavailability of adequate amounts of partially assembled GPI structures for use as assay substrates. In this paper we present procedures to prepare these structures from a GPI-anchored protein. The methods described include selective dephosphorylation of the GPI-anchored variant surface glycoprotein from Trypanosoma brucei variant 118 to generate Man alpha 1-2Man alpha 1-6Man alpha 1-4GlcN alpha 1-6-myo-inositol-P-dimyristoylglycerol (Man3GlcN-PI), followed by exoglycosidase treatments and N-acetylation to produce Man2GlcN-PI, Man1GlcN-PI, GlcN-PI, and GlcNAc-PI. Procedures are also described for the stabilization and purification of these structures. It is anticipated that the convenient preparation of this range of partially assembled GPIs will be useful not only in developing assays for the eventual purification of the GPI biosynthetic enzymes but will also contribute to evaluating the specificity of the phospholipases that hydrolyze GPI anchors.

Animals↗
PubMed2002

Evaluation of tests for anthelmintic resistance in cyathostomes.

Resistance, especially to the anthelmintic benzimidazoles (BZ), has been reported in horse cyathostomes world-wide. Diagnosis of resistance has traditionally been made by faecal egg count reduction (FECR) trials, however, this technique has limitations. Some of the shortcomings may be resolved by refining the test or by using an in vitro test. FECR tests and the larval development assay (LDA) were performed on adult horses held on 15 different horse properties across a wide geographical area of NSW, Australia. FECR were measured before and 10-14 after days treatment with oxibendazole (OBZ), morantel (MOR) or ivermectin (IVM) at recommended dose rates. Eight properties were rejected following low pre-treatment egg counts, leaving seven in the study. On these, the majority of larvae recovered from faecal cultures were cyathostomes. Using a definition of resistance as a FECR of <90%, resistance to OBZ was present on six properties and to MOR on two properties. Resistance to IVM was not detected. An alternative method of calculating FECR based on individual horse egg counts pre- and post-treatment was developed and results from the same properties compared with the results of the LDA. For example, for the BZ, correlation coefficients of values of lethal concentration to kill 50% of population (LC50) on LDA and FECR percentages were -0.536 before and -0.704 after OBZ treatment. We conclude that the LDA has the potential to be a single visit test for detection of anthelmintic resistance in horse cyathostomes but requires further investigation and standardisation.

Animals↗
PubMed1992

Comparative evaluation of a chemiluminescent DNA probe and an exoantigen test for rapid identification of Histoplasma capsulatum.

A chemiluminescent DNA probe (Accuprobe) assay developed by Gen Probe, Inc., for the rapid identification of Histoplasma capsulatum was evaluated and compared with the exoantigen test by using 162 coded cultures including Histoplasma capsulatum var. capsulatum, Histoplasma capsulatum var. duboisii, Histoplasma capsulatum var. farciminosum, Blastomyces dermatitidis, Coccidioides immitis, Paracoccidioides brasiliensis, and morphologically related saprobic fungi. Each test uses a chemiluminescent, acridinium ester-labeled, single-stranded DNA probe that is complementary to the rRNA of the target organism. Lysates of the test cultures were prepared by sonication with glass beads and heat treated. After the rRNA was released from the target organism, the labeled DNA probe combined with the target H. capsulatum rRNA to form a stable DNA-RNA hybrid. A hybridization protection assay was used, and the chemiluminescence of hybrids was measured initially with a Leader 1 luminometer as relative light units and later during the investigation with a probe assay luminometer as probe light units. Of the 162 coded mycelial cultures tested by the Accuprobe assay, 105 were identified as H. capsulatum. The test could be performed with an inoculum of a few square millimeters (1 to 2 mm2) of growth. In the primary evaluation, the Accuprobe identified 103 of the 105 cultures as H. capsulatum within 2 h. The remaining two cultures, contaminated with bacteria, had to be purified before the Accuprobe assay identified them correctly as H. capsulatum. Since each coded culture was concurrently tested for H. capsulatum, B. dermatitidis, and C. immitis exoantigens, the identification of all three dimorphic pathogens was provided simultaneously. Of the 162 coded cultures tested, 105 were identified by the exoantigen test as H. capsulatum, 12 were identified as B. dermatitidis, 13 were identified as C. immitis, and 32 were negative for H. capsulatum, B. dermatitidis, and C. immitis. The bacterial contamination in two isolates did not interfere with the exoantigen testing. The exoantigen test required 7- to 10-day-old colonies and required 48 to 72 h of incubation before definitive identification was obtained.

Antigens, Fungal↗
PubMed2000

Development and validation of sensitive assays to quantitate gene expression after p53 gene therapy and paclitaxel chemotherapy using in vivo dosing in tumor xenograft models.

SCH58500 (ACN53) is a replication-deficient, type 5 adenovirus (Ad) expressing human wild-type p53 tumor suppressor. It is currently undergoing clinical trials as a cancer therapeutic. Many SCH58500 clinical trials incorporate an arm comparing traditional chemotherapy against chemotherapy combined with SCH58500. Paclitaxel was chosen for combination therapy in the preclinical study reported here due to its extensive use as a first-line therapy in ovarian cancer, its synergy with SCH58500 in preclinical cancer models, and its activation of p53-independent apoptosis, which might result in a "lowered threshold" for tumor cell death. SCID mice bearing human tumor xenografts were dosed with intratumoral vehicle, control Ad vector, or SCH58500, with or without paclitaxel. Real-time quantitative reverse transcriptase polymerase chain reaction assays were developed and validated to quantitate expression of p53, the p53 downstream effector gene p21, and the apoptosis-related genes, bax, bcl-2, and survivin. Protein expression was confirmed using immunohistochemical assays for p53 and p21. Only tumors injected with SCH58500 had detectable levels of exogenous p53 DNA and mRNA. After SCH58500 treatment, 3-11-fold elevations of p21 expression were observed in tumor xenografts containing nonfunctional p53 (MDA-MB-468, MDA-MB-231, MIAPaCa2, DU-145, and SK-OV-3), but no change in p21 mRNA in wild-type p53 PA-1 tumors. Immunohistochemical assays confirmed induction of p21 protein in MDAMB-468 and SK-OV-3 cells, but not in PA-1 cells. Ad vector alone or paclitaxel alone had no effect on p21 mRNA levels in most tumors. However, paclitaxel suppressed p21 expression induced by SCH58500 4-fold in DU-145 and SK-OV-3 tumors. Paclitaxel also affected expression of the housekeeping gene gapdh. There was no consistent pattern to the changes in bax, bcl-2, or survivin after SCH58500 treatment with or without paclitaxel between tumor types, although there were consistent responses within individual tumor lines. The mRNA ratios for bax/bcl-2 and bax/survivin were also not informative across tumor types. Of the genes examined, only p21 gave a predictable response 24 hours after p53 gene therapy and therefore, p21 expression may be useful for confirming SCH58500 activity in human tumor biopsies.

Adenoviridae↗
PubMed2005

Absence of V599E BRAF mutations in desmoplastic melanomas.

BACKGROUND: Desmoplastic melanoma is an uncommon variant of cutaneous melanoma that mimics soft tissue sarcoma both clinically and morphologically. An activating point mutation of the BRAF oncogene has been identified in a high proportion of conventional cutaneous melanomas, but its frequency in the desmoplastic subtype is not known. METHODS: The authors tested 12 desmoplastic melanoma specimens for the thymine (T)-->adenine (A) missense mutation at nucleotide 1796 of the BRAF gene using a newly developed assay that employs a novel primer extension method. They also tested 57 vertical growth phase cutaneous nondesmoplastic melanoma specimens. RESULTS: The 1796 T-->A mutation was detected in 23 of the 57 conventional cutaneous melanoma specimens but in none of the 12 desmoplastic melanoma specimens (40% vs. 0%; P=0.0006, Fisher exact 2-tailed test). CONCLUSIONS: The relative importance of BRAF mutational activation in melanocytic tumorigenesis clearly was not the same across the various subtypes of melanoma, even for melanomas of cutaneous origin that are associated with sun exposure. In contrast to conventional cutaneous melanomas, the desmoplastic variant frequently did not harbor an activating mutation of BRAF. Accordingly, patients with melanomas should not be collectively regarded as a uniform group as new therapeutic strategies are developed that target specific genetic alterations.

Female↗
PubMed2006

A purified Bacillus thuringiensis crystal protein with therapeutic activity against the hookworm parasite Ancylostoma ceylanicum.

Crystal (Cry) proteins produced by the soil bacterium Bacillus thuringiensis (Bt) are harmless to vertebrates, but they are highly toxic to insects and nematodes. Their value in controlling insects that destroy crops and transmit human diseases is well established. Although it has recently been demonstrated that a few individual Bt Cry proteins, such as Cry5B, are toxic to a wide range of free-living nematodes, the potential activity of purified Cry proteins against parasitic nematodes remains largely unknown. We report here studies aimed at characterizing in vitro and in vivo anthelminthic activities of purified recombinant Cry5B against the hookworm parasite Ancylostoma ceylanicum, a bloodfeeding gastrointestinal nematode for which humans are permissive hosts. By using in vitro larval development assays, Cry5B was found to be highly toxic to early stage hookworm larvae. Exposure of adult A. ceylanicum to Cry5B was also associated with significant toxicity, including a substantial reduction in egg excretion by adult female worms. To demonstrate therapeutic efficacy in vivo, hamsters infected with A. ceylanicum were treated with three daily oral doses of purified Cry5B, the benzimidazole anthelminthic mebendazole, or buffer. Compared with control (buffer-treated) animals, infected hamsters that received Cry5B showed statistically significant improvements in growth and blood hemoglobin levels as well as reduced worm burdens that were comparable to the mebendazole-treated animals. These data demonstrate that Cry5B is highly active in vitro and in vivo against a globally significant nematode parasite and that Cry5B warrants further clinical development for human and veterinary use.

Ancylostoma↗
PubMed2006

Determination of N-desmethyl- and N-bisdesmethyl metabolites of Sibutramine in doping control analysis using liquid chromatography-tandem mass spectrometry.

Since January 2006, the list of prohibited substances established by the World Anti-Doping Agency includes the antidepressant / anti-obesity drug Sibutramine. Due to its rapid degradation to its active metabolites N-desmethyl and N-bisdesmethyl Sibutramine, reference compounds were synthesized and included into an existing screening assay to allow the unambiguous determination of these metabolic products in human urine using liquid-liquid extraction followed by liquid chromatography/tandem mass spectrometry. Characteristic product ions, obtained after electrospray ionization and collision-induced dissociation, were elucidated using high resolution/high accuracy mass measurements with a hybrid linear ion trap/orbitrap mass analyzer. Based on diagnostic product ions, the extended screening procedure was validated for both Sibutramine metabolites using a triple quadrupole mass spectrometer. Items such as lower limits of detection (6-40 ng mL(-1)), recoveries (39-42%), intraday precision (low: 5.5-10.6%, medium: 4.9-5.9%), high: 12.8-16.4%) and interday precision (low: 15.0-22.8%, medium: 17.7-18.6%), high: 16.5-25.6%) were evaluated, and a clinical spot urine sample was analyzed to demonstrate the applicability of the developed assay in sports drug testing.

Chromatography, Liquid↗
PubMed2004

Measurement of barbed ends, actin polymerization, and motility in live carcinoma cells after growth factor stimulation.

Motility is associated with the ability to extend F-actin-rich protrusions and depends on free barbed ends as new actin polymerization sites. To understand the function and regulation of different proteins involved in the process of generating barbed ends, e.g., cofilin and Arp2/3, fixed cell approaches have been used to determine the relative barbed end concentration in cells. The major disadvantages of these approaches are permeabilization and fixation of cells. In this work, we describe a new live-cell time-lapse microscopy assay to determine the increase of barbed ends after cell stimulation that does not use permeabilization and provides a better time resolution. We established a metastatic carcinoma cell line (MTLn3) stably expressing GFP-beta-actin at physiological levels. Stimulation of MTLn3 cells with epidermal growth factor (EGF) causes rapid and transient lamellipod protrusion along with an increase in actin polymerization at the leading edge, which can be followed in live cell experiments. By measuring the increase of F-actin at the leading edge vs. time, we were able to determine the relative increase of barbed ends after stimulation with a high temporal resolution. The F-actin as well as the barbed end concentration agrees well with published data for this cell line. Using this newly developed assay, a decrease in lamellipod extension and a large reduction of barbed ends was documented after microinjecting an anti-cofilin function blocking antibody. This assay has a high potential for applications where rapid changes in the dynamic filament population are to be measured.

Actin Depolymerizing Factors↗