Isolation of Expressed Sequences Encoded by the Human Xq Terminal Portion Using Microclone Probes Generated by Laser Microdissection

The genes that cause a variety of neurologic and neuromuscular disorders have been mapped to the distal region of Xq. In an effort to isolate genes from this area, a regional genomic library of the distal 30% of Xq was constructed from a single metaphase spread by means of laser microdissection and single unique primer-polymerase chain reaction. Using pooled probes of 1000 clones from the genomic library, human brain cDNA libraries were screened for expressed sequences encoded by this region. From the 250,000 cDNA clones screened so far, 10 nonoverlapping sequences that mapped back to the target portion were isolated. The complete nucleotide sequences of these cDNA clones have been determined. Analysis of the sequences indicates that none has significant similarity to previously characterized primate genes. One sequence mapping to Xq27.3-qter contained an open reading frame of 281 amino acids and was expressed in every tissue tested. This gene, as well as others isolated in this manner, may prove to be a candidate gene for heritable disorders mapping to this region.     

Effect of selenium and protein deficiency on selenium and glutathione peroxidase in rats

Twenty-four weanling male Wistar rats were divided into four groups fed diets containing adequate or deficient levels of selenium (0.5 ppm [+ Se] or <0.02 ppm [−Se] and protein (15% [+Pro] or 5% [−Pro]), but adequate levels of all other nutrients for 4 wk to determine the effects of Se deficiency and protein deficiency on tissue Se and glutathione peroxidase (GSHPx) activity in rats. Plasma, heart, liver, and kidney Se and GSHPx were significantly lower in Se-deficient groups in relation to Se-sufficient groups. In Se-deficient groups, Se and GSHPx were significantly higher in −Se−Pro rats in heart, liver, and kidney. Data analysis showed that there were significant interaction effects between dietary Se and protein on Se and GSHPx of rats. It is assumed that under the condition of Se deficiency. a low level of protein may decrease Se and GSHPx utilization, increase GSHPx synthesis, and result in Se redistribution. This could account for high levels of Se and GSHPx in the −Se−Pro rats compared to −Se+Pro rats.     

Iodothyronine deiodinase activity in methionine-deficient rats fed selenium-deficient or selenium-sufficient diets

We examined the effect of methionine deficiency on iodothyronine 5’-deiodinase activity in selenium-deficient rats or selenium-sufficient rats fed sodium selenate or selenomethionine. Forty-two weanling male Wistar rats were divided into six groups and pair fed the respective purifiedl-amino acid-based diets for 4 wk.l-methionine concentrations in the diet were 8.0 g/kg for sufficient rats, and 2.0 g/kg for deficient rats. Selenium concentrations in the diet were 0.5 mg/kg (as sodium selenate or selenomethionine) for selenium-sufficient rats and less than 0.005 mg/kg for selenium-deficient rats. Type I 5’-deiodinase activities were significantly lower in liver and higher in kidney of methionine-deficient rats than in those of methionine-sufficient rats fed either the selenium-sufficient or the selenium-deficient diets. The type I 5’-deiodinase activity in brain was significantly lower in the methionine-deficient rats than in the methionine-sufficient rats fed the selenium-deficient diet. Type II 5’-deiodinase activity in brain was significantly higher in the methionine-deficient rats than in the methionine-sufficient rats fed selenium-sufficient diet as sodium selenate. Both thyroxine and 3,3’,5-triiodothyronine concentrations in plasma were significantly higher in the methionine-deficient rats than in the methionine-sufficient rats. It is suggested that the methionine deficiency affects the 5’-deiodinase activity and thyroid hormones level in the rats.     

Prostaglandin D2 lowers nuclear DNA polymerase activity in cultured mastocytoma cells

Prostaglandin D₂ strongly inhibited growth of cultured mastocytoma P-815, 2-E-6 cells, which were established and cloned from mouse mast tumor cells. The inhibition was dose-dependent (IC₅₀ = 2.09 × 10⁻⁵ M). Prostaglandin D₂ also inhibited the DNA synthesizing activity of the cells dose-dependently. We next measured the activities of endogenous DNA polymerases extracted from untreated and prostaglandin D₂-treated cells. Prostaglandin D₂ pretreatment reduced DNA polymerase α activity by 52%. The sedimentation coefficients of the enzymes from untreated and prostaglandin D₂-treated cells were the same suggesting there was no gross change in the size of the enzyme. Prostaglandin D₂ pretreatment of the cells reduced endogenous DNA polymerase β activity to 68% of the control value; the sedimentation coefficients of the enzymes from treated and untreated cells were both 3.5 S. Interestingly, prostaglandin D₂ had no direct inhibitory effect on the activity of either DNA polymerase α or β. Our results indicate that the activities of DNA polymerase α and β are lower in prostaglandin D₂-treated mastocytoma cells. This finding account for the lower level of DNA synthesis in these cells.     

The longest 18S ribosomal RNA ever known. Nucleotide sequence and presumed secondary structure of the 18S rRNA of the pea aphid, Acyrthosiphon pisum.

An EMBL4 recombinant phage which encodes one of the full length of the aphid ribosomal DNA has been isolated from the aphid genomic library. Determination of the complete nucleotide sequence of the aphid 18S rRNA gene revealed that it is 2469 bp with a G + C content of 59%. The aphid 18S rRNA gene studied here is the longest and has the highest G + C content among the 18S rRNA genes examined so far. Evidence provided by the S1 nuclease assay suggests that the aphid 18S rRNA gene examined in this study is not a pseudogene containing an insertion sequence. Based on the nucleotide sequence of the 18S rRNA gene, we constructed a presumed secondary-structure model of the aphid 18S rRNA. In the aphid 18S rRNA, the eucaryote-specific E21 and 41 region are supposed to be longer and more complex than the counterparts of other 18S rRNA.     

Prostaglandin E1 receptor from mouse mastocytoma P-815 cells couples to 60 kDa GTP-binding protein.

The stable [3H]prostaglandin E1 (PGE1)-bound receptor, which couples to 60 kDa GTP-binding protein, from membranes of mouse mastocytoma P-815 cells has been purified and characterized. When the membranes were preincubated with [3H]PGE1 for 60 min at 37 degrees C, the dissociation of the ligand from the receptor was remarkably decreased, even in the presence of GTP gamma S. The stable [3H]PGE1-bound receptor complex was solubilized with 6% digitonin. The solubilized [3H]PGE1 receptor was eluted with [35S]GTP gamma S bindings activity from an Ultrogel AcA44 column. The fractions containing activities of both [3H]PGE1 and [35S]GTP gamma S bindings were further purified by column chromatographies on wheat germ agglutinin (WGA)-agarose and phenyl-Sepharose CL-4B. The partially purified [3H]PGE1-bound receptor was affinity-labeled with [14C]5'-p-fluorosulfonylbenzoylguanosine and a protein with a molecular mass of 60 kDa was detected. These results suggest that the ligand-bound PGE1 receptor of P-815 cells associates with a novel GTP-binding protein with a molecular mass of 60 kDa.     

Synthetic sialyl glycolipids (sialo-cholesterol and sialo-diglyceride) induce granulocytic differentiation of human myelogenous leukemia cell line HL-60

When HL-60 cells were cultivated with synthetic sialyl glycolipids, sialo-cholesterol and sialo-diglyceride, the cells were found to be differentiated into mature granulocytes on morphological and functional criteria. The differentiation of cells was accompanied by inhibition of cell proliferation. The differentiation-inducing activity of sialo-cholesterol was greater than that of sialo-diglyceride on a molar basis, and the alpha-anomer of each compound was more potent than the beta-anomer, suggesting that the stereospecific structure of the compounds is important for the differentiation-inducing activity.     

Comparison of 4 serological tests--complement fixation, neutralization, fluorescent antibody to membrane antigen and immune adherence hemagglutination--for assay of antibody to varicella-zoster (V-Z) virus.

Four tests for antibody to varicella-zoster (V-Z) virus were compared; these were tests of complement fixation (CF), neutralization (NT), fluorescent antibody to membrane antigen (FAMA) and immune adherence hemagglutination (IAHA). Fifty-two sera from patients with varicella and zoster and from recipients of live varicella vaccine were examined by the 4 tests. The CF test was least sensitive, but the antibody titers by the NT, FAMA and IAHA tests were roughly comparable. The IAHA test was the simplest and fastest to perform, and appeared suitable for routine serological assay to V-Z virus. The correlation between the IAHA antibody titer and susceptibility of individuals to clinical varicella was investigated retrospectively using sera obtained during 2 outbreaks of varicella in an institution for children, where all the unvaccinated children had developed varicella symptoms. Most of the 25 pre-exposure sera from unvaccinated children examined by the IAHA test had tiers of less than 1:2. In contrast, all the 23 sera from vaccinated children who did not develop varicella had detectable antibody titers of 1:2 to 1:64. These results indicate that the IAHA titer reflects the susceptibility or resistance of individuals to clinical varicella.     

Studies on live rubella vaccine. VI. Seroepidemiological surveillance and immunization of adolescents in high school.

The rubella seroimmunity status of a total of 1,204 students aged 12 to 19 in a junior and a senior high school in Osaka district was surveyed. Among these, 487 students (40.5%) were found to be seronegative (less than 1 : 8) by the hemagglutination inhibition (HI) test. A total of 287 students were immunized with live rubella vaccine, Biken lot No.7233. This caused an increased titer in all except one of the 262 seronegative students, while among 25 students with an initial HI antibody titer of 1 : 8 it caused more than 4-fold increase in 20 and 2-fold increase in 5. The vaccine caused no clinical manifestations, such as fever, rash or arthralgia.