Nonallelic histone gene clusters of individual sea urchins (Lytechinus pictus): mapping of homologies in coding and spacer DNA

The linear arrangement and lengths of the spacers and coding regions in the two nonallelic histone gene variant clusters of L. pictus are remarkably homologous by R loop analysis and are similar in general topography to the histone gene repeat units of other sea urchins examined to date. No interventing sequences were detected. The coding regions of these two histone gene variants share considerable sequence homology; however, there are areas of nonhomology in every spacer region and the lengths of the nonhomologous spacers between the H2A and H1 genes are not the same for the two repeat unit classes (inter-gene heterogeneity). Combining length measurements obtained with both R loops and heteroduplexes suggests that the DNA sequences of the analogous leader regions for the two H1 mRNAs are nonhomologous. Similar observations were made for the H4 leader sequences, as well as the trailer region on H2B. S. purpuratus spacer DNA segments share little sequence homology with L. pictus; however, the analgous coding (and possibly flanking) regions have conserved their sequences. The various coding and spacer regions within a repeat unit do not share DNA sequences. Thus certain areas in the sea urchin histone gene repeat units have been highly conserved during evolution, while other areas have been allowed to undergo considerable sequence change not only between species but within a species.     

Delay and failure in treatment seeking after first onset of mental disorders in the World Health Organization's World Mental Health Survey Initiative

Data are presented on patterns of failure and delay in making initial treatment contact after first onset of a mental disorder in 15 countries in the World Health Organization (WHO)''s World Mental Health (WMH) Surveys. Representative face-to-face household surveys were conducted among 76,012 respondents aged 18 and older in Belgium, Colombia, France, Germany, Israel, Italy, Japan, Lebanon, Mexico, the Netherlands, New Zealand, Nigeria, People''s Republic of China (Beijing and Shanghai), Spain, and the United States. The WHO Composite International Diagnostic Interview (CIDI) was used to assess lifetime DSM-IV anxiety, mood, and substance use disorders. Ages of onset for individual disorders and ages of first treatment contact for each disorder were used to calculate the extent of failure and delay in initial help seeking. The proportion of lifetime cases making treatment contact in the year of disorder onset ranged from 0.8 to 36.4% for anxiety disorders, from 6.0 to 52.1% for mood disorders, and from 0.9 to 18.6% for substance use disorders. By 50 years, the proportion of lifetime cases making treatment contact ranged from 15.2 to 95.0% for anxiety disorders, from 7.9 to 98.6% for mood disorders, and from 19.8 to 86.1% for substance use disorders. Median delays among cases eventually making contact ranged from 3.0 to 30.0 years for anxiety disorders, from 1.0 to 14.0 years for mood disorders, and from 6.0 to 18.0 years for substance use disorders. Failure and delays in treatment seeking were generally greater in developing countries, older cohorts, men, and cases with earlier ages of onset. These results show that failure and delays in initial help seeking are pervasive problems worldwide. Interventions to ensure prompt initial treatment contacts are needed to reduce the global burdens and hazards of untreated mental disorders.     

Involvement of Threonine Dehydratase in Biosynthesis of the α-Ketobutyrate Prosthetic Group of Urocanase

Seventeen mutants of Pseudomonas putida that were unable to grow on threonine as nitrogen source owing to a lack of threonine dehydratase were isolated, and all were found to be unable to synthesize active urocanase. Spontaneous revertants selected for urocanase production concomitantly regained threonine dehydratase. Mutants that were unable to utilize urocanate as carbon source were also isolated, and these were defective in urocanase formation but were normal in threonine dehydratase levels. Since alpha-ketobutyrate is the prosthetic group for urocanase, these results are consistent with the proposal that threonine dehydratase is necessary for urocanase prosthetic group biosynthesis. However, the lack of urocanase activity in threonine dehydratase-negative mutants was shown not to be the result of reduced levels of endogenous free alpha-ketobutyrate, nor to the participation of threonine dehydratase in the initiation of urocanase biosynthesis through the conversion of threonyl-tRNA(Thr) to alpha-ketobutyryl-tRNA(Thr). Other alternatives for the participation of threonine dehydratase in urocanase biosynthesis are discussed.     

In vivo or in vitro selection for resistance to natural cytotoxic cell lysis selects for variants with increased tumorigenicity

Experiments were designed to test the hypothesis that transformed cells that are NC sensitive must escape NC activity if they are to grow as tumors in normal individuals. NC-resistant variants were selected either in vivo or in vitro from NC-sensitive cell lines that grow as tumors in immunodeficient mice but not in syngeneic normal mice. The tumorigenicity of cloned NC-resistant variants was compared with the parental cell lines and to cell lines that went through the selection procedure, but after cloning remained NC sensitive. Cloned NC-resistant cell lines derived from tumors that developed in x-irradiated nude mice after the injection of an NC-sensitive cell line are tumorigenic in normal mice, whereas cloned NC-sensitive cell lines derived from the same tumors are unable to grow as tumors in normal mice. Similarly, six of seven NC-resistant cloned cell lines independently isolated after in vitro selection for NC-resistance are tumorigenic in normal mice, whereas cloned NC-sensitive cell lines isolated from the same in vitro selected populations are not tumorigenic in normal mice. Thus, either the in vivo or in vitro selection of NC-resistant cells selects for cells tumorigenic in normal mice; these findings, along with our previous observations that selection for cells tumorigenic in normal mice selects for NC resistance, provide compelling evidence that escape from NC activity is required before some transformed cells can grow as tumors in normal mice.     

Storage of human plasma samples leads to alterations in the lipoprotein distribution of apoC-III and apoE

The effect of frozen storage on lipoprotein distribution of apolipoprotein C-III (apoC-III) and apoE was investigated by measuring apoC-III and apoE by ELISA in HDL and apoB-containing lipoproteins of human plasma samples (n = 16) before and after 2 weeks of frozen storage (-20 degrees C). HDLs were separated by heparin-manganese precipitation (HMP) or by fast-protein liquid chromatography (FPLC). Total plasma apoC-III and apoE levels were not affected by frozen storage. HDL-HMP apoC-III and apoE levels were significantly higher in frozen versus fresh samples: 7.7 +/- 0.7 versus 6.7 +/- 0.7 mg/dl (P < 0.05) and 2.0 +/- 0.1 versus 1.2 +/- 0.1 mg/dl (P < 0.001), respectively. HDL-FPLC apoC-III and apoE, but not triglyceride (TG) or cholesterol, levels were also higher in frozen samples: 12.0 +/- 1.2 versus 7.5 +/- 0.6 mg/dl (P < 0.001) and 2.7 +/- 0.2 versus 1.6 +/- 0.2 mg/dl (P < 0.001), respectively. Frozen storage led to a decrease in apoC-III (-17 +/- 9%) and apoE (-19 +/- 9%) in triglyceride-rich lipoprotein. Redistribution of apoC-III and apoE was most evident in samples with high TG levels. HDL apoC-III and apoE levels were also significantly higher when measured in plasma stored at -80 degrees C. Our results demonstrate that lipoprotein distribution of apoC-III and apoE is affected by storage of human plasma, suggesting that analysis of frozen plasma should be avoided in studies relating lipoprotein levels of apoC-III and/or apoE to the incidence of coronary artery disease.     

Comparison of deuterated leucine, valine, and lysine in the measurement of human apolipoprotein A-I and B-100 kinetics

The production rates of apolipoprotein (apo)B-100 in very low density lipoprotein and in low density lipoprotein and apolipoprotein A-I in high density lipoprotein were determined using a primed-constant infusion of [5,5,5,-2H3]leucine, [4,4,4,-2H3]valine, and [6,6-2H2,1,2-13C2]lysine. The three stable isotope-labeled amino acids were administered simultaneously to determine whether absolute production rates calculated using a stochastic model were independent of the tracer species utilized. Three normolipidemic adult males were studied in the constantly fed state over a 15-h period. The absolute production rates of very low density lipoprotein apoB-100 were 11.4 +/- 5.8 (leucine), 11.2 +/- 6.8 (valine), and 11.1 +/- 5.4 (lysine) mg per kg per day (mean +/- SDM). The absolute production rates for low density lipoprotein apoB-100 were 8.0 +/- 4.7 (leucine), 7.5 +/- 3.8 (valine), and 7.5 +/- 4.2 (lysine) mg per kg per day. The absolute production rates for high density lipoprotein apoA-I were 9.7 +/- 0.2 (leucine), 9.4 +/- 1.7 (valine, and 9.1 +/- 1.3 (lysine) mg per kg per day. There were no statistically significant differences in absolute synthetic rates of the three apolipoproteins when the plateau isotopic enrichment values of very low density lipoprotein apoB-100 were used to define the isotopic enrichment of the intracellular precursor pool. Our data indicate that deuterated leucine, valine, or lysine provided similar results when used for the determination of apoA-I and apoB-100 absolute production rates within plasma lipoproteins as part of a primed-constant infusion protocol.     

Peptidyl 3-hydroxyproline binding properties of type I collagen suggest a function in fibril supramolecular assembly

Proline residues in collagens are extensively hydroxylated post-translationally. A rare form of this modification, (3S,2S)-l-hydroxyproline (3Hyp), remains without a clear function. Disruption of the enzyme complex responsible for prolyl 3-hydroxylation results in severe forms of recessive osteogenesis imperfecta (OI). These OI types exhibit a loss of or reduction in the level of 3-hydroxylation at two proline residues, α1(I) Pro986 and α2(I) Pro707. Whether the resulting brittle bone phenotype is caused by the lack of the 3-hydroxyl addition or by another function of the enzyme complex is unknown. We have speculated that the most efficient mechanism for explaining the chemistry of collagen intermolecular cross-linking is for pairs of collagen molecules in register to be the subunit that assembles into fibrils. In this concept, the exposed hydroxyls from 3Hyp are positioned within mutually interactive binding motifs on adjacent collagen molecules that contribute through hydrogen bonding to the process of fibril supramolecular assembly. Here we report observations on the physical binding properties of 3Hyp in collagen chains from experiments designed to explore the potential for interaction using synthetic collagen-like peptides containing 3Hyp. Evidence of self-association was observed between a synthetic peptide containing 3Hyp and the CB6 domain of the α1(I) chain, which contains the single fully 3-hydroxylated proline. Using collagen from a case of severe recessive OI with a CRTAP defect, in which Pro986 was minimally 3-hydroxylated, such binding was not observed. Further study of the role of 3Hyp in supramolecular assembly is warranted for understanding the evolution of tissue-specific variations in collagen fibril organization.