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PubMedDate not supplied

Development and comparison of serologic methods for diagnosing chicken anemia virus infection.

This paper describes the development of an indirect immunoperoxidase assay (IIP) and an indirect enzyme-linked immunosorbent assay (ELISA) for detecting antibodies to chicken anemia virus (VAC). The IIP assay developed used CAV-infected MDCC-MSB1 cells for detecting antibody to CAV, whereas the ELISA utilized gradient-purified immunoadsorbed CAV as the target antigen. The IIP and ELISA were compared with the standard indirect immunofluorescent antibody (IFA) assay, which is more conventionally used to screen chicken serum for antibodies against CAV. Comparative test results of 185 field samples of chicken serum by these three methods were in agreement 84% of the time. Both IFA and IIP assays yielded fewer positive tests than did the ELISA. IFA and IIP assays were in agreement 93% of the time, as compared with 91% agreement of IIP and ELISA results, or 84% agreement for comparative IFA and ELISA results.

Anemia↗

PubMed1991

Radioenzymatic assay for histamine: development and validation.

Radioenzymatic assays are sensitive analytic tools that use an enzyme to quantify a substrate for that enzyme. Purified histamine N-methyltransferase has been used as the basis for an assay for histamine. The sensitivity of the procedure is less than 10 fmol. The specificity of the assay is increased when the transferase reaction is carried out at 0-3 degrees C. Data documenting the precision of the assay, the stability of histamine in human plasma, and the gastric secretory rate of histamine are presented along with a chronologic description of the development of the technique.

Adult↗

PubMed1996

Effect of neuromimetics upon the release of atrial natriuretic peptide granules: are multiple pathways involved in secretion?

The release of atrial natriuretic peptide (ANP) in response to the application of neurohumoral agonists (neuromimetics) is directly demonstrated and quantified at the cellular level, using an ultrastructural assay developed to quantify secretion. The assay uses an in situ tannic acid perfusion technique to arrest the exocytosis of atrial secretory granules in the anesthetized rat. The animal is perfused with the neuromimetic, and secretory granules, which retain the capacity to undergo exocytosis throughout the subsequent 30 min tannic acid perfusion, accumulate at the cell surface in a state of fusion with the plasma membrane. Quantification of arrested granules thus provides a measure of the rate of granule release and allows the responses to different agents to be assessed. The actions of three different agents were investigated: isoproterenol, phenylephrine, and acetylcholine. In previously published studies, investigations of the actions of these agents on ANP release has produced unclear and sometimes contradictory results. Using our ultrastructural assay, it was found that during the 30 min perfusion period neither isoprenaline nor phenylephrine caused a significant change in the rate of secretory granule release, whereas acetylcholine significantly decreased the rate of granule release. A new model of secretion is proposed to integrate these findings with previous results and help clarify the complex picture of atrial natriuretic peptide release.

Acetylcholine↗

PubMed2007

Applications of transcriptional profiling in antibiotics discovery and development.

This chapter will review specific applications of microarray technology and related data analysis strategies in antibacterial research and development. We present examples of microarray applications spanning the entire antibiotics research and development pipeline, from target discovery, assay development, pharmacological evaluation, to compound safety studies. This review emphasizes the utility of microarrays for a systematic evaluation of novel chemistry as antibiotic agents. Transcriptional profiling has revolutionized the process of target elucidation and has the potential to offer substantial guidance in the identification of new targets. Microarrays will continue to be a workhorse of anti-infectives discovery programs ranging from efficacy assessments of antibiotics ('forward pharmacology') to drug safety evaluations ('toxicogenomics').

Anti-Bacterial Agents↗

PubMed1993

Nucleotide sequence of the C-terminal region of the mouse epidermal growth factor receptor and expression in teratoma-derived cell lines with increased tumorigenic properties.

The isolation of a cDNA corresponding to a portion (amino acid 943 to 1073) of the cytoplasmic domain of the mouse EGF receptor surrounding the auto phosphorylation sites was obtained by using the reverse transcriptase polymerase chain reaction (RT-PCR) approach. Deduced amino acid sequence of mouse EGF receptor (EGFr) shows a 92% and 76% homology to corresponding regions in the human and the chicken EGFr, respectively. This cDNA was used to develop a sensitive RNase protection assay to investigate EGF receptor mRNA expression in mouse C3H teratoma derived cell lines with increased tumorigenic properties which display a progressive decrease of EGF binding and response. The results show that increased tumorigenicity was not accompanied by a change in EGF receptor mRNA expression. Moreover, they indicate that the RNase protection assay developed using the probe described here is a sensitive approach to investigate EGF receptor expression in murine cells.

Amino Acid Sequence↗

PubMed1991

Development of pyrroline-5-carboxylate synthase and N-acetylglutamate synthase and their changes in lactation and aging.

Using newly developed assay procedures, we studied the development of pyrroline-5-carboxylate synthase (PCS) and N-acetylglutamate synthase (AGAS) activity in rat tissues. PCS in the small intestine of fetuses was 1/5 that of adults and reached an adult level as early as postnatal Day 1. The highest peak was observed at Day 14, and then activity decreased to the adult level. However, PCS in the brain was highest at birth and quickly inactivated in a few days. AGAS in the fetus small intestine was 1/3 that of adults and became higher than the adult level by 40% at Day 1 but was reduced to 1/2 that of adults at Day 3. Subsequently activity increased gradually to the adult level at Day 24. On the contrary, AGAS in the fetus liver was only 1/20 that of adults, and activity increased slowly up to 10 weeks and more. Pregnancy and lactation reduced liver AGAS markedly up to Day 8 and intestinal PCS considerably up to Day 14 after parturition. PCS in the small intestine of senescent rats was almost halved compared to young controls on a whole tissue basis. AGAS in the small intestine was also halved on a gram wet weight basis. Nonetheless the liver AGAS of 430-day-old rats was higher than that of the controls, although that of 630-day rats was lower. The results indicate that the arginine synthesizing enzymes in the small intestine are highly activated in suckling and weaning, and raise a question whether arginine remains fully dispensable in pregnancy, lactation, and senescence.

Acetyltransferases↗

PubMed1989

Comparison of three in vitro techniques to estimate benzimidazole resistance in Haemonchus contortus of sheep.

Three in vitro assays to detect benzimidazole resistance, namely, the egg-hatch assay, tubulin-binding assay, and a larval-development assay, were evaluated by estimating the level of benzimidazole resistance in three field isolates of Haemonchus contortus compared with a susceptible reference strain. Comparisons were also made with estimates of benzimidazole resistance of the three field strains obtained from an in vivo controlled anthelmintic efficacy test. All three in vitro tests showed similar, consistent results which also suggested greater sensitivity than the in vivo assay. These results indicate that selection of an in vitro technique to determine benzimidazole resistance should therefore be based on considerations other than precision, such as technical expertise, availability of equipment, cost and speed in which diagnosis is required.

Analysis of Variance↗

PubMed2006

Capillary electrophoresis-mass spectrometry as a characterization tool for therapeutic proteins.

With the increasing use of capillary electrophoresis (CE) in the biotechnology industry, there is a demand for analytical tools and methodology that can be used to characterize CE profiles. This article describes the implementation and optimization of a robust online CE-mass spectrometry (CE-MS) system used for the characterization of several CE assays developed at Genentech Inc. These assays include CE as a complement to reverse-phase peptide mapping for the identification of small peptides eluting in the void volume, profiling N-linked glycopeptide heterogeneity, and determining O-linked site occupancy. In addition, CE-MS was used to confirm major 8-aminopyrene-1,3,6-trisulfonate (APTS)-labeled glycans released from recombinant antibodies that are routinely profiled by CE-laser-induced fluorescence (CE-LIF). For each study, CE-MS was able to successfully identify components seen in UV or LIF electropherograms, thereby expanding the capability of CE and CE-MS for profiling biomolecules.

Antibodies, Monoclonal↗

PubMed2006

Measurement of hepatitis B virus core-related antigen as predicting factor for relapse after cessation of lamivudine therapy for chronic hepatitis B virus infection.

BACKGROUND: Prolonged lamivudine therapy has two major problems: breakthrough hepatitis during treatment and relapse of aminotransferase (ALT) after cessation of the therapy. The aim of this study was to examine factors that could predict ALT flare after stopping lamivudine therapy. METHODS: We analyzed 22 Japanese patients with chronic hepatitis B infection, in whom lamivudine therapy was stopped after HBV DNA level had been gone undetectable (<3.7LGE/ml) during at least six consecutive months. The post-treatment followed up was carried for 28 months in median (range 9-41). HBV core-related antigen (HBcrAg) assay was assessed using newly developed assay. RESULTS: After cessation of lamivudine therapy, 11 patients (50%) had relapsed (reactivation of serum ALT >80IU/l, relapsers) and remaining 11 (50%) did not relapse (non-relapsers). In the univariate comparison of relapsers versus non-relapsers, HBcrAg level at lamivudine cessation point (4.5+/-1.0 versus 3.4+/-0.9; p=0.0145) has been shown as a significant predictive factor for non-relapse. All patients with HBcrAg <3.0logU/ml at the cessation point had no ALT flares. Multivariate analysis on effects of 10 factors (age, sex, cirrhosis, pretreatment ALT level, HBV DNA level, HBcrAg level, mean months till undetectable HBV DNA, duration of undetectable HBV DNA and HBcrAg level at lamivudine cessation point), indicated that HBcrAg level at lamivudine cessation point <3.4log U/ml was the only independent predictive factor for absence of the post-treatment relapse. CONCLUSIONS: HBcrAg level at lamivudine cessation point might be useful as a prognostic predictor of response to lamivudine therapy cessation. The measurement of HBcrAg is a useful additional test for monitoring chronic HBV infection.

Journal Article↗

PubMed1998

Resistance to benzimidazoles and levamisole in nematode parasites of sheep in Nyandarua District of Kenya.

The occurrence of anthelmintic resistance on 25 sheep farms in the Nyandarua District of Kenya was investigated, using the faecal egg count reduction test (FECRT), the egg hatch assay (EHA) and a larval development assay (LDA). In the FECRT, resistance to both benzimidazoles (BZs) and levamisole (LEV) was detected on four farms, resistance to LEV only on three farms and to BZs, only on two farms. Haemonchus contortus was the predominant nematode species in both pre-treatment and post-treatment faecal cultures. Out of the six farms where BZ resistance was detected in the FECRT, only isolates from one farm had an LD50 value higher than 0.5 microM thiabendazole (TBZ) (0.1 microgram TBZ/ml) in the EHA indicating resistance. Isolates from three other farms, where susceptibility to BZs was detected and four with suspected BZ resistance in the FECRT, had LD50 values higher than 0.5 microM TBZ in the EHA. The LD50 values for TBZ in the LDA for four of the six isolates with BZ resistance in the FECRT were higher than 0.5 microM (0.59-2.07) TBZ. There were disagreements in ascribing resistance for various farms, between methods of calculating and interpreting the faecal egg count reduction percentages (FECR%) based on the arithmetic mean, and those where the geometric mean eggs per gram (EPG) of faeces are used. Inclusion of pre-treatment EPG or control group EPG in the calculation of FECR% resulted in similar variations.

Animals↗

PubMed1988

Modulation of antifolate cytotoxicity by metabolites from dying cells in a lymphocyte clonal assay.

A lymphocyte clonal assay developed to quantitate in vivo somatic cell mutations at the hypoxanthine-guanine phosphoribosyltransferase locus was modified in order to study resistance to methotrexate. Even though nucleoside-free culture conditions were used methotrexate was not lethal to lymphocytes plated into micro-wells at greater than 10(2) cells/well. HPLC analysis of supernatants from wells plated initially with 10(4) cells/well in 100 microM methotrexate revealed the presence of micro-molar levels of hypoxanthine and thymidine by the 5th and 8th day of culture respectively. When lymphocytes were plated at less than or equal to 10(2) cells/well in nucleoside free medium, methotrexate was cytotoxic and micro-molar levels of thymidine together with hypoxanthine protected lymphocytes cultured under these conditions from toxicity. Modulation of nucleic acid antimetabolite cytotoxicity by nucleosides and bases has been recognised for some years. Nucleoside free culture conditions have been advocated for studying cellular sensitivity to antifolates to avoid such interfering factors. However our results indicate that metabolites from dying or damaged cells can prevent methotrexate cytotoxicity, further complicating the development of a suitable clonogenic assay for investigating antifolate sensitivity.

Cell Division↗

PubMed1992

Effects of petrochemicals and ultraviolet radiation on epidermal IA expression in vitro.

We previously demonstrated that combined treatment of mice with crude oil and longwave ultraviolet radiation (UVA) led to the depletion of IA-positive cells from the epidermis. In the present study, we have developed an in vitro screening assay for combined effects of purified petrochemicals and UVA on epidermal IA and Thy-1 expression. This method involves removal of skin from donor mice prior to treatment with chemicals and UVA (20,000 J/m2), followed by in vitro culture and subsequent immunoperoxidase staining. In this study, a complete correlation was observed in terms of IA-positive cell density among similarly treated cultured skin and live mice. In vivo and in vitro studies both indicated that anthracene but not phenanthrene or benzo[a]pyrene led to significant depletion of both epidermal Langerhans cells and Thy-1-positive dendritic cells when followed by UVA treatment. The in vitro assay developed for this study should prove to be a valuable tool for the screening of a wide variety of chemicals for contact photosensitizing activity.

Animals↗

PubMed1999

Molecular analysis of riboflavin synthesis genes in Bartonella henselae and use of the ribC gene for differentiation of Bartonella species by PCR.

The biosynthesis pathway for riboflavin (vitamin B(2)), the precursor of the essential cofactors flavin mononucleotide and flavin adenine dinucleotide, is present in bacteria and plants but is absent in vertebrates. Due to their conservation in bacterial species and their absence in humans, the riboflavin synthesis genes should be well suited either for detection of bacterial DNA in human specimens or for the differentiation of pathogenic bacteria by molecular techniques. A DNA fragment carrying the genes ribD, ribC, and ribE, which encode homologues of riboflavin deaminase (RibD) and subunits of riboflavin synthetase (RibC and RibE), respectively, was isolated from a plasmid-based DNA library of the human pathogen Bartonella henselae by complementation of a ribC mutation in Escherichia coli. Sequence analysis of the ribC gene region in strains of B. henselae, which were previously shown to be genetically different, revealed that the ribC gene is highly conserved at the species level. PCR amplification with primers derived from the ribC locus of B. henselae was used to isolate the corresponding DNA regions in B. bacilliformis, B. clarridgeiae, and B. quintana. Sequence analysis indicated that the riboflavin synthesis genes are conserved and show the same operon-like genetic organization in all four Bartonella species. Primer oligonucleotides designed on the basis of localized differences within the ribC DNA region were successfully used to develop species-specific PCR assays for the differentiation of B. henselae, B. clarridgeiae, B. quintana, and B. bacilliformis. The results obtained indicate that the riboflavin synthesis genes are excellent targets for PCR-directed differentiation of these emerging pathogens. The PCR assays developed should increase our diagnostic potential to differentiate Bartonella species, especially B. henselae and the newly recognized species B. clarridgeiae.

Amino Acid Sequence↗

PubMed2006

Development of a quantitative real-time PCR method to enumerate total bacterial counts in ready-to-eat fruits and vegetables.

A newly developed real-time PCR assay rapidly quantifies the total bacterial numbers in contaminated ready-to-eat vegetables and fruits compared with the standard plate count method. Primers targeting the rpoB gene, which encodes for the beta subunit of the bacterial RNA polymerase and which is common to most bacterial species, was used instead of the 16S rRNA gene, which has multiple copies and varies among bacterial species. A primer pair specific for rpoB was confirmed to amplify rpoB in a wide range of bacterial species after we assessed 49 strains isolated from five kinds of fruits and vegetables. We purchased fruits and vegetables from retail shops and enumerated the bacteria associated with them by use of real-time PCR and compared this to the number found by the culture method. We found a high correlation between the threshold PCR cycle number when compared with the plate count culture number. The real-time PCR assay developed in this study can enumerate the dominant bacterial species in ready-to-eat fruits and vegetables.

Bacteria↗

PubMed1999

[Molecular mechanisms of nephro-protective action of enalapril in experimental chronic renal failure].

Locally increased synthesis of angiotensin II (ANG II) in the kidney has been linked to glomerular hypertrophy, glomerulosclerosis and tubulo-interstitial fibrosis observed in chronic renal failure after subtotal nephrectomy. This action of ANG II is thought to be mediated mainly by transforming growth factor-beta (TGF-beta), which stimulates the synthesis and decreases the degradation of extracellular matrix (ECM) components, including various collagen types and fibronectin. Some recent reports indicate that reduced ANG II activity diminishes TGF-beta overexpression, and in consequence renal injury. However, no studies in SNx models concerning the influence of ANG II on gene expression regulated by TGF-beta have so far been performed. Therefore, the present study has been initiated with the following aims: 1. To develop a RT-PCR assay for evaluating gene expression concerning renin (REN), angiotensinogen (ATG) and the following ECM components: transforming growth factor-beta 1 (TGF-beta 1), fibronectin (FN), matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinases-2 (TIMP-2); 2. To assess the influence of renal mass reduction (RMR) caused by subtotal (5/6) or partial (2/6) nephrectomy on gene expression for TGF-beta 1, FN, MMP-2 and TIMP-2; 3. To evaluate the correlation between expression of these genes and activity of the circulatory or renal renin-angiotensin systems; 4. To assess the influence of treatment with enalapril (angiotensin-converting enzyme inhibitor) on renal expression of these genes, renal morphology and function in rats, relative to duration of treatment and RMR. The study consisted of two independent experiments performed in adult male Sprague-Dawley rats. Ten days prior to surgery, the animals were matched for body weight and systolic blood pressure (SBP) values and subsequently were distributed into untreated (control) and enalapril treated groups. Treatment with enalapril (EN) (50 mg/l in drinking water) was started 9 days prior to surgery. The first (short-term) experiment was performed in rats with chronic renal failure caused by subtotal nephrectomy. Remnant kidneys were taken for molecular studies at the day of SNx and 3, 7 and 21 days thereafter. Blood samples collected at the time of sacrifice served to determine plasma renin activity and plasma concentration of angiotensinogen and angiotensin II. The second (long-term) experiment was done in subtotally (5/6) and partially (2/6) nephrectomized rats. Remnant kidneys were taken for molecular and morphological studies at the day of surgery and 1 or 16 weeks thereafter. 24-hour proteinuria, hematocrit, serum creatinine and creatinine clearance values were also measured. Quantitation of renal gene expression for REN, ATG, TGF-beta 1, FN, MMP-2 and TIMP-2 was performed using RT-PCR assay and comparing amounts of respective gene mRNA with house-keeping gene mRNA encoding L19 ribosomal protein. The results obtained have led to the following conclusions: 1. The RT-PCR assay developed here ensures a reliable quantitation of gene expression for renin, angiotensinogen, transforming growth factor-beta 1, fibronectin, matrix metalloproteinase-2 and tissue inhibitor of metalloproteinases-2. 2. Renin gene expression in the kidney depends on renal synthesis of angiotensin II. In contrast, regulation of angiotensinogen mRNA expression seems to be independent of ANG II. 3. Long-term treatment with enalapril prevents an early increase in renal TGF-beta 1 and FN gene expression, retards the progression of chronic renal failure caused by critical renal mass reduction, and prolongs survival. 4. Intrarenal activity of the renin-angiotensin system is not a principal factor in the regulation of gene transcription for matrix metalloproteinase-2 and tissue inhibitor of metalloproteinases-2.

Angiotensins↗

PubMed2000

Mutation analysis and description of sixteen RSH/Smith-Lemli-Opitz syndrome patients: polymerase chain reaction-based assays to simplify genotyping.

We report the clinical and molecular data of 16 patients with RSH/Smith-Lemli-Opitz syndrome (RSH/SLOS) with varying phenotypic severity, for which we have identified mutations in both alleles. RSH/SLOS is an autosomal recessive malformation syndrome caused by mutations in the gene encoding the sterol Delta(7)-reductase. This protein catalyzes the reduction of 7-dehydrocholesterol to cholesterol in the last step of cholesterol biosynthesis via the Kandutsch-Russell pathway. In addition to previously reported mutations (T93M, L109P, G147D, W151X, T154M, R242C, A247V, T289I, IVS8-1G-->C, Y408H, and E448K), we have identified six previously undescribed mutations (321G-->C, W177R, R242H, Y318N, L341P, and C444Y). We also report rapid polymerase chain reaction (PCR)-based assays developed to detect four of the recurring mutations (T93M, W151X, V326L, and R404C) and six other RSH/SLOS mutations (321G-->C, L109P, T154M, T289I, Y318N, and L341P). The purpose of this article is to correlate detailed clinical information with molecular data in order to improve our understanding of the genotype-phenotype correlation of RSH/SLOS and to report the development of PCR-based assays that will allow more rapid mutation analysis.

Alleles↗

PubMed1980

Influences of the host and local conditions on the in vivo clonogenic expression of subcutaneously inoculated R-1,M tumour cells.

In order to study possible variations in the expression of the clonogenic capacity of cultured R-1,M tumour cells due to different conditions of the growth substrate, assays were performed by employing the in vitro plating, technique described by Puck & Marcus (1956) and the in vivo TD50 assay developed by Hewitt & Wilson (1959). Assays were performed with cell suspensions containing R-1,M cells alone or admixed with either heavily irradiated R-1,M cells designated as F(R-1,M) cells or normal, syngeneic MER-1 cells that have a phagocytic capacity. In vitro assays demonstrated a maximal capacity for colony formation of 80 to 100% of the R-1,M cells plated. TD50 assays performed with the syngeneic WAG/Rij rat and the allogeneic BALB/c.nu mouse revealed that R-1,M cells can express their clonogenic capacity in both strains equally well, with a TD50 of 6,000 cells. From results of assays performed with admixed cells, it was concluded that, in the BALB/c.nu mouse, MER-1 cells are capable of reducing the TD50 by a factor of 600, while admixture with both MER-1 and F(R-1,M) cells in the WAG/Rij rat resulted in a reduction by a factor of only 3-4. The intrinsic radiosensitivity of R-1,M cells grown in single cultures, and mixed cultures with MER-1 cells, was studied by the in vitro assay after in vitro irradiation. For R-1,M cells DQ and D0 values of 2.5 and 1.3 Gy, respectively, were obtained. However, in vivo assays for survival of in vitro irradiated R-1,M cells in single culture provided data which cannot be correlated in a simple manner with data obtained by the in vitro assay.

Animals↗

PubMed2003

Anti-Müllerian hormone levels in serum from human foetuses and children: pattern and clinical interest.

Serum anti-Müllerian hormone (AMH) determination has been used to investigate gonadal development and abnormal sexual differentiation, but until recently, it was based on assays developed by specialized laboratories. A short time ago, a sensitive assay kit was developed commercially (Immunotech-Beckman Coulter) for clinical use. With this method, we established usual levels of serum AMH in fetuses, newborns, and pre-pubertal children, and evaluated the clinical value of this assay. AMH measurement required only 25 microl of sample and could be performed within 3 h. In females, AMH emerged after birth at low levels (median: 4 ng/ml). In males, AMH levels remained stable during fetal life (median: 44.4 ng/ml), peaked in the first months of life to reach a median of 124.7 ng/ml, then fell with wide individual variations. Cord blood AMH levels at birth may be useful to investigate ambiguous genitalia suspected prenatally. In children with isolated microphallus or hypospadias, decreased AMH values are in favor of testis dysfunction. When testes cannot be palpated, a single determination of serum AMH levels can distinguish between anorchia and cryptorchidism.

Age Factors↗