Food-entrained rhythmic expression of PER2 and BMAL1 in murine megakaryocytes does not correlate with circadian rhythms in megakaryopoiesis.

BACKGROUND: Circadian rhythms control a vast array of biological processes in a broad spectrum of organisms. The contribution of circadian rhythms to the development of megakaryocytes and the regulation of platelet biology has not been defined. OBJECTIVES: This study tested the hypothesis that murine megakaryocytes exhibit hallmarks of circadian control. METHODS: Mice expressing a PER2::LUCIFERASE circadian reporter protein and C57BI/6 mice were used to establish if megakaryocytes expressed circadian genes in vitro and in vivo. Mice were also subjected to 3 weeks on a restricted feeding regime to separate food-entrained from light-entrained circadian rhythms. Quantitative real time polymerase chain reaction (PCR), flow cytometry and imunohistochemistry were employed to analyse gene expression, DNA content and cell-cycle behavior in megakaryocytes collected from mice over a 24-h period. RESULTS: Megakaryocytes exhibited rhythmic expression of the clock genes mPer2 and mBmal1 and circadian rhythms in megakaryopoiesis. mPer2 and mBmal1 expression phase advanced 8 h to coincide with the availability of food; however, food availability had a more complex effect on megakaryopoiesis, leading to a significant overall increase in megakaryocyte ploidy levels and cell-cycle activity. CONCLUSIONS: Normal megakaryopoiesis requires synchrony between food- and light-entrained circadian oscillators.

Discrimination between bacterial spore types using time-of-flight mass spectrometry and matrix-free infrared laser desorption and ionization.

We demonstrate that molecular ions with mass-to-charge ratios (m/z) ranging from a few hundred to 19 050 can be desorbed from whole bacterial spores using infrared laser desorption and no chemical matrix. We have measured the mass of these ions using time-of-flight mass spectrometry and we observe that different ions are desorbed from spores of Bacillus cereus, Bacillus thuringiensis, Bacillus subtilis, and Bacillus niger. Our results raise the possibility of identifying microorganisms using mass spectrometry without conventional sample preparation techniques such as the addition of a matrix. We have measured the dependence of the ion yield from B. subtilis on desorption wavelength over the range 3.05-3.8 microm, and we observe the best results at 3.05 microm. We have also generated mass spectra from whole spores using 337-nm ultraviolet laser desorption, and we find that these spectra are inferior to spectra generated with infrared desorption. Since aerosol analysis is a natural application for matrix-free desorption, we have measured mass spectra from materials such as ragweed pollen and road dust that are likely to form a background to microbial aerosols. We find that these materials are readily differentiated from bacterial spores.

Expression of alpha- and beta-globin genes occurs within different nuclear domains in haemopoietic cells.

The alpha- and beta-globin gene clusters have been extensively studied. Regulation of these genes ensures that proteins derived from both loci are produced in balanced amounts, and that expression is tissue-restricted and specific to developmental stages. Here we compare the subnuclear location of the endogenous alpha- and beta-globin loci in primary human cells in which the genes are either actively expressed or silent. In erythroblasts, the alpha- and beta-globin genes are localized in areas of the nucleus that are discrete from alpha-satellite-rich constitutive heterochromatin. However, in cycling lymphocytes, which do not express globin genes, the distribution of alpha- and beta-globin genes was markedly different. beta-globin loci, in common with several inactive genes studied here (human c-fms and SOX-1) and previously (mouse lambda5, CD4, CD8alpha, RAGs, TdT and Sox-1), were associated with pericentric heterochromatin in a high proportion of cycling lymphocytes. In contrast, alpha-globin genes were not associated with centromeric heterochromatin in the nucleus of normal human lymphocytes, in lymphocytes from patients with alpha-thalassaemia lacking the regulatory HS-40 element or entire upstream region of the alpha-globin locus, or in mouse erythroblasts and lymphocytes derived from human alpha-globin transgenic mice. These data show that the normal regulated expression of alpha- and beta-globin gene clusters occurs in different nuclear environments in primary haemopoietic cells.

Substantial reservoirs of molecular hydrogen in the debris disks around young stars.

Circumstellar accretion disks transfer matter from molecular clouds to young stars and to the sites of planet formation. The disks observed around pre-main-sequence stars have properties consistent with those expected for the pre-solar nebula from which our own Solar System formed 4.5 Gyr ago. But the 'debris' disks that encircle more than 15% of nearby main-sequence stars appear to have very small amounts of gas, based on observations of the tracer molecule carbon monoxide: these observations have yielded gas/dust ratios much less than 0.1, whereas the interstellar value is about 100 (ref. 9). Here we report observations of the lowest rotational transitions of molecular hydrogen (H2) that reveal large quantities of gas in the debris disks around the stars beta Pictoris, 49 Ceti and HD135344. The gas masses calculated from the data are several hundreds to a thousand times greater than those estimated from the CO observations, and yield gas/dust ratios of the same order as the interstellar value.

Presence of a phospholipase D (PLD) distinct from PLD1 or PLD2 in human neutrophils: immunobiochemical characterization and initial purification.

Utilizing the transphosphatidylation reaction catalyzed by phospholipase D (PLD) in the presence of a primary alcohol and the short-chain phospholipid PC8, we have characterized the enzyme from human neutrophils. A pH optimum of 7.8-8.0 was determined. PIP(2), EDTA/EGTA, and ATP were found to enhance basal PLD activity in vitro. Inhibitory elements were: oleate, Triton X-100, n-octyl-beta-glucopyranoside, divalent cations, GTPgammaS and H(2)O(2). The apparent K(m) for the butanol substrate was 0.1 mM and the V(max) was 6.0 nmol mg(-1) h(-1). Immunochemical analysis by anti-pan PLD antibodies revealed a neutrophil PLD of approximately 90 kDa and other bands recognized minimally by anti-PLD1 or anti-PLD2 antibodies. The 90-kDa protein is tyrosine-phosphorylated upon cell stimulation with GM-CSF and formyl-Met-Leu-Phe. Protein partial purification using column liquid chromatography was performed after cell subfractionation. Based on the enzyme's regulatory and inhibitory factors, and its molecular weight, these data indicate an enzyme isoform that might be different from the mammalian PLD1/2 forms described earlier. The present results lay the foundation for further purification of this granulocyte PLD isoform.

Phospholipase D (PLD) is present in Leishmania donovani and its activity increases in response to acute osmotic stress.

We report here that the signaling molecule phospholipase D (PLD) is present in the parasitic protozoan Leishmania donovani. In vitro enzymatic activity is dependent on Ca2+ and Mg2+ ions, its basal activity is stimulated by phosphatidyl-inositol-4,5-bisphosphate (PIP2) and its pH optima are pH 8.0 and pH 6.0. PLD activity increases 3-fold about 5 min after an abrupt decrease in osmolality from 317 mOsm (isosmotic) to 155 mOsm and increases 1.5-fold in response to an abrupt increase in osmolality to 617 mOsM. Cells grown for > 24 h under the anisosmotic conditions showed only marginal changes in activity compared to the controls grown under isosmotic conditions, indicating an adaptation to long-term exposure to hypo- or hyper-osmolarity. Immunologically, two isoforms, PLD1 and PLD2, are present. An analysis of in vitro PLD activity in anti-PLD immunocomplexes revealed that either hypotonic (cell swelling) or hypertonic stress (cell shrinking) causes an increase in PLD1 activation but a reduction in PLD2 activity. The interplay between these two isoforms results in a predominance for PLD1 in the observed increase when measuring total PLD activity. Finally, the increase in enzymatic activity in acute hyposmotic shock is accompanied by tyrosyl phosphorylation of the PLD1 isoform, suggesting a role for protein tyrosine kinase in the control of PLD activity in response to osmotic stress.

Stoolmiller on restriction of range in adoption studies: a comment.

Stoolmiller has recently presented a model featuring an inferred dimension of family quality which, he suggests, is severely truncated in adoption studies such as the Texas Adoption Project, resulting in gross underestimation of the effects of shared family environment. We discuss some potential limitations of his approach in general and as he applies it to the Texas adoption data on IQ.

The influence of rearing order on personality development within two adoption cohorts.

There is an extensive literature on the relationship between birth order and psychological traits, but no previous study has investigated the influence of ordinal position on personality development within adoptive siblings. Such a design is important because it effectively separates the effects of biological birth order and rearing order. Here we report data from two adoption cohorts in which subjects were biological first-borns reared in various ordinal positions. Data were analyzed with reference to Sulloway's (1996) evolutionarily based sibling rivalry theory of birth order effects. Between- and within-family analyses indicated that rearing order's influence on personality was very weak. The only clear difference was for conscientiousness, on which first-reared siblings scored higher. We draw possible implications for Sulloway's theory and speculate upon an alternative, prenatal biological process that may produce birth order differences.

Direct inhibition of in vitro PLD activity by 4-(2-aminoethyl)-benzenesulfonyl fluoride.

While conducting a purification protocol of phospholipase D (PLD) from human granulocytes, we observed that PLD activity was inhibited by a commonly-used protease inhibitor cocktail. Of the six inhibitors present in the cocktail, the serine protease inhibitor, 4-(2-aminoethyl)-benezensulfonyl fluoride (AEBSF), was found to be the sole inhibitor of PLD. AEBSF caused a loss of neutrophil and purified plant PLD activities in vitro, but not in intact cells at the concentrations used, nor did it affect the related phospholipases A(2) and C, that were utilized as specificity controls. The compound AEBSNH(2), which has the fluoride replaced by an -NH(2) group, failed to affect PLD activity as did other compounds structurally related to AEBSF with known protease inhibitory capabilities. Finally, basal- and agonist-stimulated PLD activity was inhibited in phosphatidylcholine-specific anti-PLD immunoprecipitates (IC(50) = 75 microM). These results suggest that AEBSF, in an effect probably unrelated to its anti-proteolytic ability, directly interferes with PLD enzymatic activity, making it a significant compound to begin analyzing the role of PLD in mammalian cell signaling.

Xist has properties of the X-chromosome inactivation centre.

X-chromosome inactivation is the process by which female mammals (with two X chromosomes) achieve expression of X-chromosomal genes equivalent to that of males (one X and one Y chromosome). This results in the transcriptional silencing of virtually all genes on one of the X chromosomes in female somatic cells. X-chromosome inactivation has been shown to act in cis and to initiate and spread from a single site on the X chromosome known as the X-inactivation centre (Xic). The Xic has been localized to a 450-kilobase region of the mouse X chromosome. The Xist gene also maps to this region and is expressed exclusively from the inactive X chromosome. Xist is unusual in that it appears not to code for a protein but produces a nuclear RNA which colocalizes with the inactive X chromosome. The creation of a null allele of Xist in embryonic stem cells has demonstrated that this gene is required for X inactivation to occur in cis. Here we show that Xist, introduced onto an autosome, is sufficient by itself for inactivation in cis and that Xist RNA becomes localized close to the autosome into which the gene is integrated. In addition, the presence of autosomal Xist copies leads to activation of the endogeneous Xist gene in some cells, suggesting that elements required for some aspects of chromosome counting are contained within the construct. Thus the Xist gene exhibits properties of the X-inactivation centre.

A member of the caudal family of homeobox genes maps to the X-inactivation centre region of the mouse and human X chromosomes.

X-inactivation is the process which allows equalization of the dosage of X chromosomal genes between males and females. A specific region of the X chromosome, called the X-inactivation centre (XIC), is thought to have a key role in regulating this process. A gene, XIST, has been identified which maps to the XIC and so far has the unique property of being expressed exclusively from the inactive X chromosome. Although XIST is a good candidate for a gene involved in regulating X-inactivation there is as yet no formal proof it has such a role. Here we describe another gene, Cdx4, a member of the caudal-related family of homeobox genes, which is located within the minimal region assigned to XIC in humans. Furthermore, this gene is the closest known gene to XIST in both mouse and human. Unlike Xist, Cdx4 appears to be normally X-inactivated in mice. Although it is not clear whether the location of this gene within the XIC region is of any significance in X-inactivation, the isolation of the gene will allow further definition of the region of inactive X-specific expression surrounding XIST.

APC mutation analysis by chemical cleavage of mismatch and a protein truncation assay in familial adenomatous polyposis.

Overall, the causative APC mutation has been identified in only 30% of the patients with familial adenomatous polyposis (FAP) who have been included in studies reported in the literature. In order to determine the true frequency of detectable APC mutations, we set out to search exhaustively the entire coding region of APC for causative mutations in ten patients with classical FAP from Scottish kindreds shown to be linked to 5q markers. Chemical cleavage of mismatch analysis was employed as the initial screening technique. Mutations were confirmed by direct DNA sequencing and shown to generate a premature stop codon by an in vitro protein synthesis assay. Mutations resulting in a premature stop codon either by base substitution or by frameshift were identified in nine families. Although the remaining kindred was linked to intragenic APC markers with a lodscore of 1.69 at Zmax = 0.0, further analysis of DNA, RNA and chromosome spreads from the proband failed to detect any abnormality. This was despite employing single-strand conformation polymorphism (SSCP) analysis, heteroduplex analysis, DNA sequencing, reverse transcription-polymerase chain reaction (RT-PCR) analysis for splicing defects, a protein truncation test encompassing the entire APC gene and fluorescent in situ hybridisation chromosome analysis (FISH). These data show that 90% of these FAP kindreds had APC mutations detectable by chemical cleavage of mismatch and that none of the numerous other techniques employed could detect the mutation in the remaining kindred. This study shows the value of screening the APC gene using a combination of chemical cleavage of mismatch analysis and an in vitro protein truncation test.

Genetics and molecular biology of mouse pigmentation.

The formation of mouse coat color is a relatively complex developmental process that is affected by a large number of mutations, both naturally occurring and induced. The cloning of the genes in which these mutations occur and the elucidation of the mechanisms by which these mutations disrupt the normal pigmentation pattern is leading to an understanding of the way interactions between gene products lead to a final phenotype.

Pseudomonas putida Strains Which Constitutively Overexpress Mercury Resistance for Biodetoxification of Organomercurial Pollutants.

Improved biocatalysts for mercury (Hg) remediation were generated by random mutagenesis of Pseudomonas putida with a minitransposon containing merTPAB, the structural genes specifying organomercury resistance. Subsequent selection for derivatives exhibiting elevated resistance levels to phenylmercury allowed the isolation of strains that constitutively express merTPAB at high levels, conferring the ability to cleave Hg from an organic moiety and reduce the freed Hg(II) to the less toxic elemental form, Hg, at greater rates. Constitutive overexpression of merTPAB had no apparent effect on culture growth rates, even when Hg(II) was initially present at otherwise toxic concentrations. These properties were also combined with benzene and toluene catabolism, allowing detoxification of the metal component of phenyl mercuric acetate, as well as degradation of its aromatic moiety.

Microbial retention of mercury from waste streams in a laboratory column containing merA gene bacteria.

The microorganisms used for the mercury retention experiments were natural isolates and genetically engineered bacteria. All mercury-resistant strains contained the merA gene. Column experiments with these strains were carried out by immobilizing them on different support materials. To obtain kinetic data of the reductase activity for whole cells and the crude extract, batch experiments were carried out under different conditions.

An adoption and a cross-fostering study of the Minnesota Multiphasic Personality Inventory (MMPI) Psychopathic Deviate Scale.

The first of two complementary studies compared biological and adoptive parents of teenage adoptees with either higher (n = 21) or low (n = 51) MMPI Psychopathic Deviate (Pd) scale scores. In comparison to biological mothers of the low-Pd adoptees, biological mothers of the high-Pd adoptees obtained significantly higher MMPI scores on six of eight clinical scales. Fewer differences existed between the corresponding groups of adoptive mothers, but adoptive mothers of the high Pd's did obtain significantly higher scores on the Pd and Hypomania scales. Substantial genetic correspondences also existed for Harris-Lingoes content subscales, with fewer correspondences between adoptees and their adoptive mothers. There were indications that adoptive mothers of the high-Pd children had personality traits which may have made them less effective in attenuating early signs of antisocial behavior. The second study employed a cross-fostering design dividing all biological and adoptive mothers (n = 138 each) by their respective median Pd raw scores to examine effects on offspring. Results confirmed the effect of biological mother Pd score, but only a trend suggested an adoptive mother effect, with no hint of an interaction.

DNA sequence determination of the TOL plasmid (pWWO) xylGFJ genes of Pseudomonas putida: implications for the evolution of aromatic catabolism.

The meta operon of the Pseudomonas putida TOL plasmid (pWWO) encodes all enzymes of a meta-cleavage pathway for the metabolism of benzoic acids to Krebs-cycle intermediates. We have determined and analysed the nucleic acid sequence of a 3442 bp region of the meta operon containing the xyl-GFJ genes whose products are involved in the post meta-ring fission transformation of catechols. Homology analysis of the xylGFJ gene products revealed evidence of biochemical relatedness, suggested enzymatic mechanisms, and permitted us to propose evolutionary events which may have generated the current variety of aromatic degradative pathways. The xylG gene, which specifies 2-hydroxymuconic semialdehyde dehydrogenase (HMSD), was found to encode a protein of 51.7 kDa. The predicted protein sequence exhibits significant homology to eukaryotic aldehyde dehydrogenases (ADHs) and to the products of two other Pseudomonas catabolic genes, i.e. xylC and alkH. Expansion of the ADH superfamily to include these prokaryotic enzymes permitted a broader analysis of functionally critical ADH residues and phylogenetic relationships among superfamily members. The importance of three regions of these enzymes previously thought to be critical to ADH activity was reinforced by this analysis. However glutamine-487, also thought to be critical, is less well conserved. The revised ADH phylogeny proposed here suggests early catabolic ADH divergence with subsequent interkingdom gene exchange. The xylF gene, which specifies 2-hydroxymuconic semialdehyde hydrolase (HMSH), was delineated by N-terminal sequence analysis of the purified gene product and is shown to encode a protein of 30.6 kDa. Homology analysis revealed sequence similarity to a chromosomally encoded serine hydrolase, especially in the region of the previously identified active-site serine residue, suggesting that HMSH may also possess a serine hydrolytic enzymatic mechanism. Likewise, the xylJ gene, which specifies 2-hydroxy-pent-2,4-dienoate hydratase (HPH), was delineated by N-terminal sequence analysis of purified HPH, and was found to encode a 23.9 kDa protein. Sequence comparisons revealed that both HMSH and HPH have analogues in the tod gene cluster, which specifies a toluene/benzene degradative pathway. Although the newly identified todF and todJ genes had been at least partially sequenced (Zylstra and Gibson, 1989), the open reading frames had not been positively identified. The presence of todJ provides strong evidence that the reactions following ring fission in the tod pathway are identical to those of the TOL pathway.(ABSTRACT TRUNCATED AT 400 WORDS)

Heredity, environment, and personality change: evidence from the Texas Adoption Project.

Personality changes over time can be analyzed by the same twin and adoption methods used to analyze the genetic and environmental influences on a trait at a given time. Composite parent rating measures of Extra-version, Socialization, and Stability made on two occasions approximately 10 years apart on 229 adopted and 83 nonadopted children from the Texas Adoption Project were used to illustrate this point in two ways. The first was based on correlations among family members, from which it appeared that by far the chief source of individual change was neither the genes nor shared family environment, but individual experience (and/or measurement error). The second was via a path-analytic approach to changes in the means of adopted and natural children, from which it appeared that, nonetheless, the children were tending to change on the average in the direction of their genetic parents' personalities.

Modeling IQ change: evidence from the Texas Adoption Project.

An analysis of genetic and environmental contributions to intellectual change was carried out by means of a path model applied to IQ data from the Texas Adoption Project, an adoption study in which children were measured on 2 occasions approximately 10 years apart. Included in the model were assortative mating, selective placement, genotype-environment correlation, a measure of socioeconomic status, and alternative hypotheses about cross-generation environmental transmission and the persistence of a trait over time. Some form of environmental transmission across generations was necessary, but either of the 2 forms tested was sufficient. The data were best fit by considering persistence over time to occur at the level of the developed trait. The effect of both genes and family environment was significant at the time of the first measurement, but only the genes made an additional contribution between the first and the second, suggesting the necessity of revising some popular stereotypes about development.

Autogenous regulation and kinetics of induction of Pseudomonas aeruginosa recA transcription as analyzed with operon fusions.

A promoterless chloramphenicol acetyltransferase gene (cat) was used to construct recA-cat operon fusions to quantitatively examine the transcriptional regulation of the Pseudomonas aeruginosa recA gene in P. aeruginosa PAO. Wild-type P. aeruginosa containing the recA8-cat fusion was treated with methyl methanesulfonate (MMS) and showed immediate induction of chloramphenicol acetyltransferase (CAT) specific activity, whereas a recA::Tn501 mutant of P. aeruginosa containing recA8-cat showed no induction with MMS. This indicated that a functional copy of recA was required for derepression of recA transcription and that P. aeruginosa recA protein was a positive regulatory factor promoting its own expression. Compared with that in the wild type, the uninduced level of CAT in recA8-cat-containing cells was reduced by approximately one-half in the recA::Tn501 mutant, indicating that recA+-dependent spontaneous induction contributes to the uninduced levels of recA expression in P. aeruginosa. MMS (0.012%) caused recA-directed CAT synthesis to increase almost immediately, with maximum CAT activity, fourfold higher than uninduced levels, attained at 60 min postinduction. The kinetics of recA8-cat fusion activity were shown to be directly related to the MMS doses used. Another fusion called recAa1-cat, where cat was located between the two transcriptional terminators of the P. aeruginosa recA gene, also showed dose-dependent induction by MMS, but the CAT activity from recAa1-cat was only one-half of that obtained with recA8-cat under the same conditions. Treatment of recA+ P. aeruginosa containing recA8-cat with UV irradiation produced an immediate effect on recA8-cat transcription and showed little UV dose dependency at doses of 5 J/m2 or greater. Treatment with 10 J/m2 produced peak levels of recA-directed CAT activity, fivefold higher than background levels, by 60 min postirradiation; CAT activity remained at peak levels during the 120 min of the experiment. In contrast, nalidixic acid had a weak effect on recA8-cat expression in P. aeruginosa, although the response was dose dependent. Nalidixic acid (800 micrograms/ml) produced maximal CAT activity that was only twofold higher than background levels.