Biopterin and neopterin in human saliva

Presence of biopterin and neopterin in human saliva was investigated by HPLC after iodine oxidation in acidic medium. Concentrations of biopterin and neopterin (M +/- SEM) were 1.271 +/- 0.254 and 0.358 +/- 0.075 ng per ml, respectively, in saliva of apparently healthy young male adults, ages 20 to 22 years (n = 9). Nearly identical value of the neopterin/biopterin ratio (0.29 +/- 0.07) was obtained for each of these specimens. Monapterin, the L-threo-isomer of neopterin (0.084 +/- 0.022 ng per ml saliva), and other unconjugated pterins such as xanthopterin, 6-hydroxymethylpterin and pterin were also found in the saliva. These pterins were all detectable in saliva of young female adults with similar levels to those of male saliva. Another fluorescent compound which was identical with 7-iso biopterin in retention time on HPLC was observed in all specimens of normal saliva examined.     

Genetic Mapping of a Group of Temperature-Sensitive dna Initiation Mutants in BACILLUS SUBTILIS

Recombination frequencies among temperature-sensitive dna mutants from various laboratories were analyzed, and eleven dna mutants were found to be closely linked. They are classified as group B dna mutants, since these are closely linked with dnaB19, originally isolated and approximately mapped near leuA8 by KARAMATA and GROSS (1970). However, the dnaB19 mutation itself has relatively high recombination frequencies with the other mutations, thus, we propose to subdivide the dnaB group into two subgroups--dnaBI, including ten mutants (dna-1, dna-3, dna-5, dna-17, dna-27, dna-51, dna-60, dna-62, dna-103 and dna-134) and dnaBII, including dnaB19. The map order of dnaB and markers in the vicinity was determined to be argA-citH-citC-phoP-PhoR-polA-dnaBI-dnaBII-citF-leuA-pheA.     

Effect of inhibition of oxygen free radical on ovulation and progesterone production by the in-vitro perfused rabbit ovary

The potential role of oxygen free radicals in hCG-induced ovulation was investigated using the free radical scavenging enzymes superoxide dismutase (SOD) and/or catalase with the in-vitro perfused rabbit ovary preparation. SOD (25 micrograms/ml) and SOD + catalase (25 micrograms/ml) significantly reduced the % of large follicles that ovulated during perfusion (P less than 0.005). Neither maturity nor degeneration of ovulated ova and follicular oocytes was affected by SOD and/or catalase. Progesterone concentration in the perfusate was significantly increased in the SOD + catalase treatment group (P less than 0.01). These results indicate a significant role for oxygen free radicals in the process of ovulation.     

Clonal sublines of rat neurotumor RT4 and cell differentiation: II. A conversion coupling of tumorigenicity and a glial property

RT4 is a neurotumor induced by ethylnitrosourea injection of a newborn BDIX rat. We demonstrated previously that heterogeneity in early cultures of RT4 tumor cells can be regularly reproduced in cultures of clonal stem cells (cell type conversion). Our previous studies included morphology, differentiation of neural properties, and chromosome number of “tumor-derived” and “stem cell-derived” differentiated cells. In this paper, these two sets of differentiated cells were examined further for three additional parameters, all of which are related to malignancy. The stem cell (AC) and one type of differentiated cell (D) cause tumors when subcutaneously injected into syngeneic animals, while the other two types (B and E) do not. The amounts of a 250,000 molecular weight cell surface protein, which is probably equivalent to LETS protein (large external transformation-sensitive protein) of hamster and mouse, and the levels of plasminogen activator were examined as possible markers of malignancy. As anticipated, nontumorigenic cells generally have a large amount of the 250,000 molecular weight cell surface protein and are low in plasminogen activator activities, whereas the reverse is true for tumorigenic cells. This supports the idea that B and E cells are nontumorigenic revertants. The cell type conversion phenomenon of RT4 neurotumor and the differentiation of mouse teratoma and myeloid leukemic cells share a number of similarities, but differ in that differentiated RT4 cells can propagate in vitro even after loss of tumorigenicity. The concomitant expression of tumorigenicity and the S100 protein production of the D cell, or of nontumorigenicity and B and E cell differentiation upon the conversion of the stem cell, may suggest a regulational coupling between the tumorigenicity and the expression of a glial protein (S100 protein) in D cells.     

Clonal sublines of rat neurotumor RT4 and cell differentiation: I. Isolation and characterization of cell lines and cell type conversion

A neurotumor of a peripheral nerve origin, RT4, was induced by subcutaneous injection of a newborn BDIX strain rat with ethylnitrosourea. The tumor cells were adapted to cell culture and, after about 2 months, were found to consist of cells displaying four distinct morphologies (AC, B, D, and E). Clonal lines of the four cell types were established. Their morphological stability suggested that there was a stem cell type (AC) which differentiated into other types of cells (B, D, and E). Presumptive stem cell lines were established from single cells. In 10 of the 12 clonal stem cell lines, colonies of differentiated cells appeared in approximately 40 days. One stem cell culture was maintained for 55 days, and all three differentiated cell types resulted in the same culture. The differentiated cells are morphologically indistinguishable from the cells isolated from the original tumor. We are thus able to demonstrate a stem cell in the RT4 neurotumor apparently capable of multipotential differentiation in vitro. We call this phenomenon cell type conversion. The stem cells (AC) and one type (D) of the differentiated cells produce a nervous system specific protein, S100 protein, while its production is arrested when the stem cells differentiate into two other cell types (B and E). No appreciable levels of neurotransmitter synthesizing enzymes (choline acetyltransferase, tyrosine hydroxylase, and glutamic acid decarboxylase) are detected in any cells. The chromosome number of each line is predominantly that of normal diploid rat cells, which is 42.     

The complete amino acid sequence of the mature form of rat sepiapterin reductase

The partial amino acid sequence of rat sepiapterin reductase was determined using peptides generated by cleavage of the S-carboxyamidomethylated protein with Achromobacter protease I, cyanogen bromide, chymotrypsin or BNPS-skatole. The protein began with N-acetyl methionyl residue at the N-terminus and ended with isoleucyl residue at the C-terminus. The present results essentially coincided with the amino acid sequence predicted from the nucleotide sequence of the cDNA recently reported by Citron et al. (Proc. Natl. Acad. Sci. USA 87, 6436-6440 (1990)), clarified the processing event during the biosynthesis and provided the complete amino acid sequence of the mature form of the enzyme.     

Rapid mutation screening of phenylketonuria by polymerase chain reaction-linked restriction enzyme assay and direct sequence of the phenylalanine hydroxylase gene: clinical application in northern Japan and northern China

We describe a simple and technically feasible method for mutation screening of the phenylalanine hydroxylase (PAH) gene and its application to Japanese and Chinese patients with hyperphenylalaninemia. The strategy is based on the identification of a nucleotide substitution by restriction enzyme analysis, coupled with PCR and direct sequencing of exon 7 of the PAH gene. Because the detection of various mutations can proceed simultaneously using the same technique, it is quite rapid and reproducible, making it possible to perform effective molecular diagnosis and carrier screening in most laboratories. Using this procedure, we found that the most common molecular defects were R413P in Hokkaido, Japan (35 %) and R243Q in Heilongjiang, China (50%). R111X, IVS4nt-1, and five mutations in exon 7 (R241C, R243Q, R252W, A259T, and S273P) accounted for 55% of phenylketonuria (PKU) alleles in Hokkaido. In Heilongjiang, the R111X, Y356X, and R408W mutations accounted for 35% of PKU alleles. Clinically, homozygotes or compound heterozygotes of null alleles, which express nonfunctional enzyme activity, were all associated with classic PKU. On the other hand, patients heterozygous for the R241C allele had a benign phenotype of mild hyperphenylalaninemia. The DNA diagnosis in early infancy can predict various PKU phenotypes, and can prove useful in decision-making concerning dietary therapy.     

Development of Lymphoproliferative Diseases by Hypoxia Inducible Factor-1alpha Is Associated with Prolonged Lymphocyte Survival

Hypoxia-inducible factor-1alpha (HIF-1 alpha) plays an essential role in the regulation of various genes associated with low oxygen consumption. Elevated expression of HIF-1alpha has been reported to be associated with tumor progression, invasion and metastasis in many cancers. To investigate the role of HIF-1alpha in tumor development and metastasis, we established transgenic mice constitutively expressing HIF1A gene under regulation of the cytomegalovirus gene promoter. Although HIF-1alpha protein levels varied among organs, expression of HIF1A mRNA in most organs gradually increased in an age-dependent manner. The transgenic mice showed no gross morphological abnormality up to 8 weeks after birth, although they subsequently developed tumors in the lymphoid, lung, and breast; the most prominent tumor was lymphoma appearing in the intestinal mucosa and intra-mesenchymal tissues. The prevalence of tumors reached 80% in 13 months after birth. The constitution of lymphocyte populations in the transgenic mice did not differ from that in wild-type mice. However, lymphocytes of the transgenic mice revealed prolonged survival under long-term culture conditions and revealed increased resistance to cytotoxic etoposide. These results suggest that HIF-1alpha itself is not oncogenic but it may play an important role in lymphomagenesis mediated through the prolonged survival of lymphocytes in this transgenic mouse model.