Loss of normal allele of the APC gene in an adrenocortical carcinoma from a patient with familial adenomatous polyposis

Summary Salla disease is a lysosomal storage disorder due to impaired transport of free sialic acid across the lysosomal membrane. The clinical presentation of this autosomal recessive trait is severe psychomotor retardation from early infancy on. In order to determine the gene locus for the disease we have initiated a genetic linkage study using polymorphic gene markers in rep-resentative family material comprising about 60% of all families known to be affected with Salla disease. Here we present an exclusion map based on combined linkage data from 64 informative loci on 19 autosomes. Theoretically, at least 55% of the genome has been excluded as a locus for the disease gene, while some chromosome areas, particularly the long arm of chromosome 2, are highlighted as possible sites for the gene locus.     

Localization of the structural gene for ribosomal protein L19 (rplS) in Escherichia coli

Summary A temperature-sensitive mutant derived from an E. coli K12 strain, PA3092, was found to have an alteration in the ribosomal protein L19 (Isono et al., 1977). This mutant is a double mutant with a temperature-sensitivity mutation and a mutation leading to the structural alteration of L19 protein. Crosses with various Hfr strains and transductions with P1kc have revealed that the latter mutation maps at 56.4 min, between pheA and alaS. From the fact that two other mutations causing different types of alterations in L19 protein also map at this locus, the gene affected by these mutations was concluded to be the structural gene for the ribosomal protein L19 (rplS).     

Conserved protein TTHA1554 from Thermus thermophilus HB8 binds to glutamine synthetase and cystathionine beta-lyase

TTHA1554 was found as a hypothetical protein composed of 95 amino acids in the genome of the extremely thermophilic bacterium, Thermus thermophilus HB8. Proteins homologous to TTHA1554 are conserved in several bacteria and archaea, although their functions are unknown. To investigate the function of TTHA1554, we identified interacting proteins by using a pull-down assay and mass spectrometry. TTHA1329, which is glutamine synthetase, and TTHA1620, a putative aminotransferase, were identified as TTHA1554 binding proteins. The interactions with TTHA1329 and TTHA1620 were validated using in vitro pull-down assays and surface plasmon resonance biosensor assays with recombinant proteins. Since sequence homology analyses suggested that TTHA1620 was a pyridoxal 5'-phosphate-dependent enzyme, such as an aminotransferase, a cystathionine beta-lyase or a cystalysin, putative substrates were investigated. When cystathionine, cystine and S-methylcysteine were used as substrates, pyruvate was produced by TTHA1620. The data revealed that TTHA1620 has cystathionine beta-lyase enzymatic activity. When TTHA1554 was added to the reaction mixtures, the glutamine synthetase and cystathionine beta-lyase enzymatic activities both increased by approximately two-fold. These results indicated that TTHA1554 is a novel protein (we named it GCBP: glutamine synthetase and cystathionine beta-lyase binding protein) that binds to glutamine synthetase and cystathionine beta-lyase.     

A new muscle contractile system composed of a thick filament lattice and a single actin filament

To bridge the gap between the contractile system in muscle and in vitro motility assay, we have devised an A-band motility assay system. A glycerinated skeletal myofibril was treated with gelsolin to selectively remove the thin filaments and expose a single A-band. A single bead-tailed actin filament trapped by optical tweezers was made to interact with the inside or the outer surface of the A-band, and the displacement of the bead-tailed filament was measured in a physiological ionic condition by phase-contrast and fluorescence microscopy. We observed large back-and-forth displacement of the filament accompanied by a large change in developed force. Despite this large tension fluctuation, we found that the average force was proportional to the overlap inside and outside the A-band up to approximately 150 nm and 300 nm from the end of the A-band, respectively. Consistent with the difference in the density of myosin molecules, the average force per unit length of the overlap inside the A-band (the time-averaged force/myosin head was approximately 1 pN) was approximately twice as large as that outside. Thus, we conclude that the A-band motility assay system described here is suitable for studying force generation on a single actin filament, and its sliding movement within a regular three-dimensional thick filament lattice.     

Cloning of the hamster p16 gene 5' upstream region and its aberrant methylation patterns in pancreatic cancer

The hamster model of pancreatic carcinogenesis is useful for understanding the development of human pancreatic cancer. However, there is only a small amount of hamster genetic information available for analyzing the gene alterations in hamster pancreatic cancers. Here, we determined the nucleotide sequence of the 5' upstream region of the hamster p16 gene using a suppression polymerase chain reaction method combined with gene-specific primers. Based on this sequence, we analyzed the methylation status of the 5' region by bisulfite sequencing in three normal pancreatic tissues and five pancreatic duct adenocarcinomas (PDAs). All five PDAs were highly methylated in the 5' upstream region and showed reduced expressions of the p16 gene, while the three normal samples were demethylated. The method described in this study is a highly effective and rapid technique for determining the 5' upstream region, and is applicable to epigenetic studies of the methylation status of this region.     

Prevalence of Herpes Simplex Virus Type 1- and 2- Specific Antibodies among the Acute,Recurrent, and Provoked Types of Female Genital Herpes

Sixty-eight sera from the acute, recurrent, and provoked types of female genital herpes were compared for the seroprevalence of herpes simplex virus (HSV) types 1 and 2 by immunodot assay using HSV glycoprotein G. In the HSV-1-isolated patients, no HSV-2 antibodies were detected, whereas in the HSV-2-isolated patients, HSV-1 seroprevalence was 9% for the acute type, 89% for the provoked type (P< 0.005), and 55% for the recurrent type (P<0.05). The natural history of female genital herpes and the possible protective role of pre-existing antibodies in preventing the acquisition or clinical manifestation of a subsequent HSV infection are discussed.     

Purification and characterization of two forms of chicken liver cytochrome P-450 induced by 3,4,5,3',4'-pentachlorobiphenyl

3,4,5,3',4'-Pentachlorobiphenyl (PenCB), one of the most potent 3-methylcholanthrene (MC)-type inducers of hepatic enzymes in animals, caused a remarkable induction of liver microsomal monooxygenases, particularly 7-ethoxyresorufin (7-ER) O-deethylase, benzo(a)pyrene (BP) 3-hydroxylase, and testosterone 16 alpha-hydroxylase in chickens, but not NADPH-cytochrome c(P-450) reductase and cytochrome b5. Two forms of cytochrome P-450 (P-450) in liver microsomes of PenCB-treated chickens were purified and characterized. The absorption maxima of the CO-reduced difference spectra of both enzymes (chicken P-448 L and chicken P-448 H) were at 448 nm. From the oxidized form of their absolute spectra, chicken P-448 L was a low-spin form and chicken P-448 H was a high-spin form. They had molecular masses of 56 and 54 kDa, respectively. In a reconstituted system, 7-ER O-deethylation, BP 3-hydroxylation, and testosterone 16 alpha-hydroxylation were catalyzed at high rates by chicken P-448 L but not by chicken P-448 H. Chicken P-448 L also catalyzed N-demethylation of aminopyrine, benzphetamine, and ethylmorphine with relatively low activity. On the other hand, chicken P-448 H functioned only in catalyzing estradiol 2-hydroxylation. These results were supported by an inhibition study of microsomal monooxygenases using an antibody against each enzyme. Immunochemical studies revealed that the enzymes differ from each other but are both inducible by PenCB-treatment. Chicken P-448 L and chicken P-448 H respectively comprise about 82 and 7% of the total P-450 content in chicken liver microsomes.