Evaluation of a chemiluminescence method for measuring alkaline phosphatase activity in whole milk of multiple species and bovine dairy drinks: interlaboratory study.

Alkaline phosphatase (ALP) is a ubiquitous enzyme in milk with time-temperature destruction similar to that of certain pathogens destroyed in pasteurization. Measurement of ALP to indicate proper pasteurization is a common practice. Recently the public health level for ALP was decreased to 350 mU/L, a level below the sensitivity of older colorimetric ALP methods.

Protective immunity against murine hepatitis virus (MHV) induced by intranasal or subcutaneous administration of hybrids of tobacco mosaic virus that carries an MHV epitope.

Hybrids of tobacco mosaic virus (TMV) were constructed with the use of fusion to the coat protein peptides of 10 or 15 amino acids, containing the 5B19 epitope from the spike protein of murine hepatitis virus (MHV) and giving rise to TMV-5B19 and TMV-5B19L, respectively. The TMV hybrids were propagated in tobacco plants, and the virus particles were purified. Immunogold labeling, with the use of the monoclonal MAb5B19 antibody, showed specific decoration of hybrid TMV particles, confirming the expression and display of the MHV epitope on the surface of the TMV.

Studies of coat protein-mediated resistance to tobacco mosaic tobamovirus: correlation between assembly of mutant coat proteins and resistance.

Coat protein-mediated resistance (CP-MR) has been widely used to protect transgenic plants against virus diseases. To characterize the mechanisms of CP-MR to tobacco mosaic tobamovirus (TMV) we developed mutants of the coat protein that affected subunit-subunit interactions. Mutant CPs were expressed during TMV replication as well as in transgenic Nicotiana tabacum plants.

Virucidal activity of an oral fluid human immunodeficiency virus-1 antibody preservative.

Diagnosis of human immunodeficiency virus-1 (HIV-1) infection requires the collection of either serum or oral fluid that is subsequently tested for the presence of antibodies to HIV-1. The effective use of oral fluid for the detection of HIV antibodies is contingent on stabilization of immunoglobulins in the sample through the use of preservatives. Oral fluid preservatives also contain agents that can disrupt and inactivate viruses.

Future applications of oral fluid specimen technology.

Research has demonstrated that oral mucosal transudate (OMT), a serum-derived fluid that enters saliva from the gingival crevice and across oral mucosal surfaces, can be preferentially concentrated by a novel collecting system to yield detectable levels of immunoglobulins (i.e., IgG and IgM antibodies) against various bacterial and viral diseases.

Evaluation of a system using oral mucosal transudate for HIV-1 antibody screening and confirmatory testing. OraSure HIV Clinical Trials Group.

Objective: To determine accuracy of a human immunodeficiency virus type 1 (HIV-1) antibody testing system using a device to collect and stabilize oral mucosal transudate (OMT), a fluid with increased levels of IgG; an enzyme immunoassay (EIA) screening test optimized for OMT; and a Western blot confirmatory test designed for use with OMT.

Design: The OMT specimens were tested by EIA and, if indicated, confirmatory Western blot according to a standard testing algorithm. The OMT results were compared with true HIV status as determined by serum testing and/or clinical diagnosis.

Studies of coat protein-mediated resistance to tobacco mosaic virus (TMV). II. Challenge by a mutant with altered virion surface does not overcome resistance conferred by TMV coat protein.

Transgenic tobacco plants expressing the coat protein (CP) gene of the U1 strain of tobacco mosaic virus (TMV) exhibit CP-mediated resistance (CP-MR) against some, but not all, tobamoviruses. To investigate the role of the amino acid sequences on the surface of the challenge virus in CP-MR, mutant strains of U1 TMV were constructed to contain the amino or carboxy termini of the CP of Sunn hemp mosaic tobamovirus (SHMV). The modified virus was unable to overcome CP-MR in transgenic plants that contained the TMV CP.

Plant virus expressing hybrid coat protein with added murine epitope elicits autoantibody response.

A modified tobacco mosaic virus (TMV) was used to direct the synthesis of TMV coat protein hybrids containing a 13 amino acid sequence of the murine zona pellucida ZP3 protein. This hybrid protein was found to be robust and accumulate to high concentrations in inoculated plants. Virus-like particles containing the hybrid coat protein could be readily purified from infected plant tissue.

Studies of coat protein-mediated resistance to TMV. I. The PM2 assembly defective mutant confers resistance to TMV.

Tobacco mosaic virus mutant PM2 contains two amino acid changes in coat protein sequence relative to the sequence of the coat protein of TMV U1. This results in unstable infectivity, inability to cause normal systemic infection, and accumulation of elongated open helixes of coat protein. Using site-directed mutagenesis we demonstrated that the characteristics of PM2 are due to the change of Thr28-->Ile, while the second change, Glu95-->Asp, had no apparent effect on virion structure or infectivity.

Molecular cloning and characterization of S-adenosyl-L-methionine:scoulerine-9-O-methyltransferase from cultured cells of Coptis japonica.

S-Adenosyl-L-methionine:scoulerine-9-O-methyltransferase (SMT) catalyzes the transfer of the S-methyl group of S-adenosyl-L-methionine to the 9-hydroxyl group of scoulerine during the biosynthesis of berberine. We have isolated functionally active cDNA clones (pCJSMTs) from a cDNA library prepared from cultured cells of Coptis japonica. The longest cDNA insert (pCJSMT1) had an open reading frame that encoded 351 amino acids, but the calculated molecular mass (38,364 Da) of the deduced product was slightly lower than the experimentally determined molecular mass of purified SMT.

Theophylline in oral mucosal transudate. A practical method for monitoring outpatient therapy.

We tested the utility of a novel collection system that allows measurement of theophylline in oral mucosal transudate (OMT) to calculate serum theophylline concentrations. In 25 adult patients, theophylline levels in OMT correlated better than expectorated saliva with serum theophylline (r = 0.927 for OMT versus r = 0.

Genetically engineered protection against viruses in transgenic plants.

Transgenic plants carrying nucleotide sequences derived from plant viruses can exhibit increased resistance to viral disease. Many viral sequences confer some level of either resistance to infection or suppression of disease symptoms (tolerance). These include segments of viral genomes encoding capsid or coat proteins, sequences encoding proteins that are or may be subunits of the viral replicase, sequences incapable of encoding proteins, entire genomes of defective interfering viruses and satellite viruses, and complete genomes of mild strains of virus.

Synergistic activity of 5-trifluoromethylthioribose and inhibitors of methionine synthesis against Klebsiella pneumoniae.

5-Methylthioribose (MTR) is an intermediate in the methionine recycling pathway of organisms containing the enzyme MTR kinase. Analogs of MTR have been proposed as a new class of antimicrobial agents because of their ability to perturb the growth of MTR kinase-containing pathogens through inhibition of methionine salvage or by conversion to toxic products. One such analog, 5-trifluoromethylthioribose (TFMTR), has demonstrated potent inhibitory effects on the growth of Klebsiella pneumoniae (A.

Residues in three conserved regions of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase are required for quaternary structure.

To explore the role of individual residues in the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.

Selective killing of Klebsiella pneumoniae by 5-trifluoromethylthioribose. Chemotherapeutic exploitation of the enzyme 5-methylthioribose kinase.

5'-Deoxy-5'-methylthioadenosine (MTA), an important intermediate in methionine recycling, can be metabolized by one of two mechanisms that appear to be mutually exclusive. In human cells, MTA is degraded in one step to adenine and 5-methylthioribose 1-phosphate (MTR-1-P) via MTA phosphorylase. In contrast, certain microbes metabolize MTA in two steps: first to 5-methylthioribose (MTR) followed by conversion to MTR-1-P.

Methionine recycling as a target for antiprotozoal drug development.

The development of new and effective ontiprotozool drugs has been difficult because of the close metabolic relationship between protozoa and mammalian cells. In this article, Michael Riscoe, Al Ferro and john Fitchen present their hypothesis for chemotherapeutic exploitation of methylthioribose (MTR) kinase, an enzyme critical to methionine salvage in certain protozoa. They propose that analogues of MTR if properly designed, would be converted to toxic products in organisms that contain MTR kinase but not in mammalian cells, which lack this enzyme.

Purine metabolism as a target for leukemia chemotherapy.

This article focuses on the chemotherapeutic agents which alter purine metabolism as a means to achieve selective killing of leukemic cells. We present an overview of purine metabolism in order to highlight enzymatic steps which are targeted by antileukemic drugs. Purine antimetabolites used in the treatment of leukemia can be grouped into three classes: (1) structural analogs of normal purines (6-mercaptopurine and 6-thioguanine); (2) inhibitors of de novo purine biosynthesis (methotrexate and hydroxyurea); and (3) inhibitors of purine salvage (2'-deoxycoformycin).

Analogs of 5-methylthioribose, a novel class of antiprotozoal agents.

Since drug resistance and toxicity limit the use of available antiprotozoal agents, it is important that new drugs be developed as soon as possible. In this study, the method by which several protozoa degrade 5'-methylthioadenosine (MTA) was shown to differ from MTA catabolism in human cells. To exploit this metabolic difference, two analogs of methylthioribose (MTR), an MTA catabolite, were synthesized and found to be cytocidal to Plasmodium falciparum, Giardia lamblia, and Ochromonas malhamensis in vitro.

Methylthioadenosine phosphorylase deficiency in acute leukemia: pathologic, cytogenetic, and clinical features.

Blast cells from 100 cases of acute leukemia were evaluated for the presence of methylthioadenosine phosphorylase (MTAase), an enzyme important in polyamine metabolism. Ten cases (10%) had undetectable levels of MTAase activity. Of the 10, 5 had acute lymphoblastic leukemia (ALL), 3 had acute myeloblastic leukemia (AML) and 2 expressed mixed lineage markers as determined by immunophenotyping.

Cytogenetic evidence for involvement of B lymphocytes in acquired idiopathic sideroblastic anemias.

We studied the cellular distribution of an unusual chromosomal abnormality, an interstitial deletion of the long arm of chromosome 13, in the peripheral blood lymphocytes of two patients with acquired idiopathic sideroblastic anemia (AISA). We found no metaphases containing the 13q- abnormality in preparations of phytohemagglutinin (PHA)-stimulated lymphocytes from either patient. In both cases, however, some metaphases from Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines contained the clonal karyotypic abnormality.