2-(5--chloro-2--phosphoryloxyphenyl)-6-chloro-4-(3h)-quinazolinone and 5-bromo-4-chloro-3-indoxyl-phosphate

To determine the frequency of mutation in different cell types of mammals, transgenic mice that allow mutant cells to be visualized in situ were used. These mice carry a defective allele of the human placental alkaline phosphatase (PLAP) gene. The allele does not produce enzyme because the reading frame is shifted by an insertion of 7 G:C basepairs. The insertion is adjacent to four existing G:C basepairs, so the allele has a tract of 11Gs. The G11 PLAP allele was studied in wildtype mice and in mice deficient in mismatch-repair (MMR) due to lack of either Pms2 or Mlh1. PLAP(+) cells were counted in brain, heart, kidney, and liver. In wildtype mice, there was an average of between 5 and 30 PLAP(+) events per million cells. No cells with alkaline phosphatase activity were detected in tissues from mice lacking the PLAP gene. In MMR-deficient mice, the number of PLAP(+) allele was increased by at least three-order of magnitude in brain, heart and kidney, but <10-fold in liver. These data show that MMR is vital to maintaining repeat stability in brain, heart and kidney cells. The reason for the different results in the liver is not clear. Cells in the liver were shown to be capable of expressing of PLAP enzyme and PLAP mRNA was present in this organ.

Topics: Adaptor Proteins, Signal Transducing; Adenosine Triphosphatases; Alkaline Phosphatase; Alleles; Animals; Base Pair Mismatch; Benzothiazoles; Carrier Proteins; Diamines; DNA Repair; DNA Repair Enzymes; DNA-Binding Proteins; Female; Fluorescent Dyes; GPI-Linked Proteins; Humans; Indoles; Isoenzymes; Liver; Male; Mice; Mice, Knockout; Mice, Transgenic; Microsatellite Repeats; Microscopy, Fluorescence; Mismatch Repair Endonuclease PMS2; Mosaicism; Mutagenesis, Insertional; Mutation; MutL Protein Homolog 1; Neoplasm Proteins; Nitroblue Tetrazolium; Nuclear Proteins; Organ Specificity; Organic Chemicals; Organophosphorus Compounds; Quinazolines; Quinazolinones; Quinolines; RNA, Messenger; Staining and Labeling; Transgenes

Alkaline phosphatase activity was histochemically localized in adult whiteflies (Bemisia tabaci B biotype, syn. B. argentifolii) with a chromogenic substrate (5-bromo-4-chloro-3-indolylphosphate) and a fluorogenic substrate (ELF-97). The greatest amount of staining was in the basal regions of adult salivary glands with additional activity traced into the connecting salivary ducts. Other tissues that had alkaline phosphatase activity were the accessory salivary glands, the midgut, the portion of the ovariole surrounding the terminal oocyte, and the colleterial gland. Whitefly nymphs had activity in salivary ducts, whereas activity was not detected in two aphid species (Rhodobium porosum and Aphis gossypii). Whitefly diet (15% sucrose) was collected from whitefly feeding chambers and found to have alkaline phosphatase activity, indicating the enzyme was secreted in saliva. Further studies with salivary alkaline phosphatase collected from diet indicated that the enzyme had a pH optimum of 10.4 and was inhibited by 1 mM cysteine and to a lesser extent 1 mM histidine. Dithiothreitol, inorganic phosphate, and ethylenediaminetetraacetic acid (EDTA) also inhibited activity, whereas levamisole only partially inhibited salivary alkaline phosphatase. The enzyme was heat tolerant and retained approximately 50% activity after a 1-h treatment at 65 degrees C. The amount of alkaline phosphatase activity secreted by whiteflies increased under conditions that stimulate increased feeding. These observations indicate alkaline phosphatase may play a role during whitefly feeding.

Topics: Alkaline Phosphatase; Animals; Fluorescent Dyes; Hemiptera; Indoles; Organophosphorus Compounds; Pilocarpine; Quinazolines; Quinazolinones; Saliva; Salivary Glands; Sucrose