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Hypophosphorylation of the architectural chromatin protein DEK in death-receptor-induced apoptosis revealed by the isotope coded protein label proteomic platform.

During apoptosis nuclear morphology changes dramatically due to alterations of chromatin architecture and cleavage of structural nuclear proteins. To characterize early events in apoptotic nuclear dismantling we have performed a proteomic study of apoptotic nuclei. To this end we have combined a cell-free apoptosis system with a proteomic platform based on the differential isotopic labeling of primary amines with N-nicotinoyloxy-succinimide. We exploited the ability of this system to produce nuclei arrested at different stages of apoptosis to analyze proteome alterations which occur prior to or at a low level of caspase activation. We show that the majority of proteins affected at the onset of apoptosis are involved in chromatin architecture and RNA metabolism. Among them is DEK, an architectural chromatin protein which is linked to autoimmune disorders. The proteomic analysis points to the occurrence of multiple PTMs in early apoptotic nuclei. This is confirmed by showing that the level of phosphorylation of DEK is decreased following apoptosis induction. These results suggest the unexpected existence of an early crosstalk between cytoplasm and nucleus during apoptosis. They further establish a previously unrecognized link between DEK and cell death, which will prove useful in the elucidation of the physiological function of this protein.

Animals↗

Stable isotope-coded proteomic mass spectrometry.

Developing the ability to quantify changes in protein abundance between cells subjected to a variety of physiological and environmental conditions is an extremely active area of proteome research. Although advances in chromatography, mass spectrometry instrumentation, and bioinformatics have contributed to producing a viable method for comparative proteome-wide analyses, the highest precision of quantitation is based, in part, upon improved methods for chemical and metabolic stable isotope labeling of proteins and peptides. The ability to quantify differences in protein expression and post-translational modifications using stable isotope labeling has been achieved, but insights into the biochemical mechanisms that will contribute to the development of new biotechnologies have yet to be realized.

Affinity Labels↗

The role of coding time in estimating and interpreting growth curve models.

The coding of time in growth curve models has important implications for the interpretation of the resulting model that are sometimes not transparent. The authors develop a general framework that includes predictors of growth curve components to illustrate how parameter estimates and their standard errors are exactly determined as a function of receding time in growth curve models. Linear and quadratic growth model examples are provided, and the interpretation of estimates given a particular coding of time is illustrated. How and why the precision and statistical power of predictors of lower order growth curve components changes over time is illustrated and discussed. Recommendations include coding time to produce readily interpretable estimates and graphing lower order effects across time with appropriate confidence intervals to help illustrate and understand the growth process.

Child↗

Phage-induced change of toxigenesis in Vibrio cholerae.

A temperate phage coding for constitutive hypertoxigenicity has been constructed in Vibrio cholerae strain 569B and used to lysogenise the low-toxin-producing strain MAK 757; 18% of lysogens showed 10-100-fold increase in toxin production. This property was also transmitted at low frequency to second generation lysogens. Thus temperate phage can increase toxin production in a low-toxin-producing strain.

Bacteriophages↗

Induction of hepatic metallothionein by feeding zinc to rats of depleted zinc status.

Induction of hepatic metallothionein was investigated in zinc-depleted rats after a single feeding (via stomach tube) of a complete diet with or without supplemental zinc. Subsequent to a maximum elevation in serum zinc (3.25 micrograms/ml) at 3 h, the rate of metallothionein synthesis increased 4.5-fold by 10 h after feeding. Changes in the rate of metallothionein synthesis coincided with similar changes in the level of translatable mRNA coding for metallothionein. Accumulation in liver of Zn2+ as metallothionein rose to a maximum by 12 h after diet administration and thereafter remained constant. Radioactive zinc (65Zn) included in the diet rapidly associated with newly formed metallothionein. Unlike Zn2+, 65Zn exhibited marked fluctuations within the 24-h period following feeding, indicating that zinc associated with metallothionein may be capable of exchange and/or dissociation. Changes in total liver 65Zn were accompanied by concomitant changes in metallothionein-bound 65Zn. This study has related (temporally) the metabolism of dietary zinc to the induction, apparently via transcription of mRNA, of hepatic metallothionein.

Animals↗

The quality of burns coding. Compliance with standards and the effects on clinical data.

Coded burns cases in two NSW public acute care facilities were reviewed to determine 1. the degree of compliance with Australian Coding Standards (ACS) relating to burns separations 2. the relationship between coding errors and poor quality documentation and 3. the impact of coding errors on ANDRG allocation. Despite high compliance with specific aspects of the ACS, coding errors occurred in 89% of cases. The ANDRG changed in 9% of total cases as a result of recoding. While some cases changed to a higher weighted ANDRG, overall the hospitals would have lost $98,445 after recoding. Significant associations were found between coding errors and poor quality documentation. The results indicate that while the ACS are adhered to, a high degree of coding variation still exists. This is largely due to inadequacies in medical record documentation. Clinicians and coders need to work together to ensure that coding variation is kept to a minimum.

Australia↗

Cochlear mechanisms of frequency and intensity coding. II. Dynamic range and the code for loudness.

Our preceding paper described SPL-dependent changes in the shape of transfer functions recorded from inner and outer hair cells as well as supporting cells, in the 500-2500 Hz regions of the Mongolian gerbil cochlea. As SPL was increased, large shifts were observed in the peak of the transfer function. A strongly compressive nonlinearity was also observed at CF. This paper examines the data from the perspective of intensity coding in the auditory periphery. Based on the data, we offer a new explanation for the mechanisms underlying the different dynamic ranges of low and high threshold auditory neurons. We also find that, for pure tone stimuli, the growth of excitation at the characteristic place saturates rapidly, and cannot encode the wide dynamic range of loudness. The data are analyzed to explore other excitation pattern candidates for loudness coding. The growth of the peak of the IHC transfer function, as well as the growth of the response area, have been found to be linearly related to loudness growth over most of its dynamic range. Implications of the data for auditory intensity coding are discussed.

Animals↗

Electrocutaneous code pairs for artificial sensory communication systems.

Pairs of electrocutaneous codes suitable for dual-channel sensory communication systems were compared using a dual-channel electrocutaneous tracking task. The tracking task required the test subject to dynamically respond to changes in the tactile sensation being modulated by two independent pseudorandom signals, one for each channel. The rule (or method) by which the signals changed the tactile sensations was called an electrocutaneous code. Four frequency variation codes and two intensity variation codes were paired in different combinations and then checked as to their effectiveness for sensory communications. The experimental protocol used a balanced incomplete block design which involved 24 subjects testing 3 of 8 code pairs each. Although the variance in the tracking performances between subjects was larger than the differences between the code pairs, learning rates for the various pairs were significantly different. The easiest one to learn was the Low Pulse Rate Modulation Code paired with itself. other findings included the general superiority of monophasic stimulation code pairs over biphasic stimulation code pairs, the need for placement of the two electrodes on different dermatomes in order to achieve satisfactory dual-channel communications, and the greater sensitivity to electrocutaneous stimulation of the ventral side of the forearm versus its dorsal side.

Biomedical Engineering↗

Germin, a protein marker of early plant development, is an oxalate oxidase.

Germin is a homopentameric glycoprotein, the synthesis of which coincides with the onset of growth in germinating wheat embryos. There have been detailed studies of germin structure, biosynthesis, homology with other proteins, and of its value as a marker of wheat development. Germin isoforms associated with the apoplast have been speculated to have a role in embryo hydration during maturation and germination. Antigenically related isoforms of germin are present during germination in all of the economically important cereals studied, and the amounts of germin-like proteins and coding elements have been found to undergo conspicuous change when salt-tolerant higher plants are subjected to salt stress. In this report, we describe how circumstantial evidence arising from unrelated studies of barley oxalate oxidase and its coding elements have led to definitive evidence that the germin isoform made during wheat germination is an oxalate oxidase. Establishment of links between oxalate degradation, cereal germination, and salt tolerance has significant implications for a broad range of studies related to development and adaptation in higher plants. Roles for germin in cell wall biochemistry and tissue remodeling are discussed, with special emphasis on the generation of hydrogen peroxide during germin-induced oxidation of oxalate.

Amino Acid Sequence↗

'Take the stairs instead of the escalator': effect of environmental prompts on community stair use and implications for a national 'Small Steps' campaign.

The US government initiated a national health campaign targeting 100 'small step' lifestyle changes to combat obesity. Small Step #67 advocates stair instead of escalator usage in public settings. The aim of this study is to evaluate the effects of motivational signs prompting stair use over escalator use on pedestrians' stair usage in commuter settings. Eight studies, testing the effects of motivational prompts on stair vs. escalator usage in public settings, were reviewed. Participant and study attributes were descriptively coded. Effect size was calculated as the change in percent units of stair users during the intervention phases vs. the baseline phase. The average study included approximately 45,000 observations that were recorded across an average of 15 weeks of intervention. The mean +/- SD change in percent units of stair users was 2.8% +/- 2.4% (P < 0.001), and effects were twice as large in females (4.8%) as in males (2.4%). The number of stairs/building, baseline stair use, and total intervention weeks predicted change in stair use, although the effects were clinically miniscule. In a hypothetical city intervention, we projected that a 2.8% increase in stair usage would result in a weight loss and/or weight gain prevention of 300 g/person/year among new stair users. In sum, point-of-decision motivational signs may help communities attain Small Step #67. However, the singular impact of this community intervention on correcting energy imbalance may be minimal, having slight impact itself on reducing the national obesity prevalence.

Elevators and Escalators↗

The DNA sequence and comparative analysis of human chromosome 10.

The finished sequence of human chromosome 10 comprises a total of 131,666,441 base pairs. It represents 99.4% of the euchromatic DNA and includes one megabase of heterochromatic sequence within the pericentromeric region of the short and long arm of the chromosome. Sequence annotation revealed 1,357 genes, of which 816 are protein coding, and 430 are pseudogenes. We observed widespread occurrence of overlapping coding genes (either strand) and identified 67 antisense transcripts. Our analysis suggests that both inter- and intrachromosomal segmental duplications have impacted on the gene count on chromosome 10. Multispecies comparative analysis indicated that we can readily annotate the protein-coding genes with current resources. We estimate that over 95% of all coding exons were identified in this study. Assessment of single base changes between the human chromosome 10 and chimpanzee sequence revealed nonsense mutations in only 21 coding genes with respect to the human sequence.

Animals↗

A novel keratin 5 mutation (K5V186L) in a family with EBS-K: a conservative substitution can lead to development of different disease phenotypes.

Epidermolysis bullosa simplex is a hereditary skin blistering disorder caused by mutations in the KRT5 or KRT14 genes. More than 50 different mutations have been described so far. These, and reports of other keratin gene mutations, have highlighted the existence of mutation "hotspots" in keratin proteins at which sequence changes are most likely to be detrimental to protein function. Pathogenic mutations that occur outside these hotspots are usually associated with less severe disease phenotypes. We describe a novel K5 mutation (V186L) that produces a conservative amino acid change (valine to leucine) at position 18 of the 1A helix. The phenotype of this case is unexpectedly severe for the location of the mutation, which lies outside the consensus helix initiation motif mutation hotspot, and other mutations at this position have been associated in Weber--Cockayne (mild) epidermolysis bullosa simplex only. The mutation was confirmed by mismatch-allele-specific polymerase chain reaction and the entire KRT5 coding region was sequenced, but no other changes were identified. De novo K5/K14 (mutant and wild-type) filament assembly in cultured cells was studied to determine the effect of this mutation on filament polymerization and stability. A computer model of the 1A region of the K5/K14 coiled-coil was generated to visualize the structural impact of this mutation and to compare it with an analogous mutation causing mild disease. The results show a high level of concordance between genetic, cell culture and molecular modeling data, suggesting that even a conservative substitution can cause severe dysfunction in a structural protein, depending on the size and structure of the amino acid involved.

Amino Acid Sequence↗

Fine mapping of polymorphic alcohol-related quantitative trait loci candidate genes using interval-specific congenic recombinant mice.

BACKGROUND: The inbred long-sleep (ILS) and inbred short-sleep (ISS) strains of mice are widely studied as a model of initial sensitivity to alcohol. Recently, a large comparative DNA sequencing study of candidate genes located within the four Lore quantitative trait loci (QTLs) associated with the ethanol-induced loss of righting reflex in ILS and ISS mice has identified eight genes that contain coding region differences corresponding to amino acid changes. Here, recently developed interval-specific congenic recombinant mice (ISCRs) have been used to map these genes in relationship to newly narrowed QTL regions. METHODS: Regions of candidate genes containing DNA differences corresponding to previously identified amino acid changes between ISS and ILS mice were amplified from either genomic DNA or complementary DNA from ISCR mice using polymerase chain reaction. The products were purified and directly sequenced to determine the genotypes for each polymorphism. On the basis of these genotypic data, each candidate gene was determined to be located either within or outside of recently narrowed Lore QTL intervals. RESULTS: Of these eight candidates with protein-coding differences, five are now excluded from their respective Lore intervals. The other three (Znf142, Ptprn, and Znf133) have been localized to the narrowed QTL intervals. CONCLUSIONS: These three central nervous system genes (Znf142, Ptprn, and Znf133) represent promising candidates for involvement in the differential sensitivity to alcohol exhibited between ILS and ISS mice. This study also demonstrates how the combination of high-throughput comparative gene sequencing and concomitant genetic fine mapping of QTL regions with ISCRs can be an effective tool for accelerating the process of moving from QTL to gene.

Alcoholism↗

Selective activation of the N-glycosylation apparatus in uteri by estrogen.

Estrogen rapidly, preferentially and markedly enhances the rate of N-linked glycoprotein synthesis in mouse uteri. In contrast, the rate of glycoprotein turnover is unaffected by the hormone. Estrogen's effect on the expression of mRNA coding for glycoproteins was studied using an in vitro translation-glycosylation system as well as by Northern/slot blot analyses. Both approaches indicated that estrogen did not have a preferential stimulatory effect on the general expression of glycoprotein mRNA. Neither was there a significant change in the relative levels of specific mRNA coding for several N-linked glycoproteins, i.e. laminin B1 and B2, fibronectin, and uvomorulin, as a function of estrogen treatment. Immunoprecipitation studies also demonstrated no change in the relative rates of synthesis of the corresponding core proteins for laminin or fibronectin. Taken together, these results suggested that estrogen primarily stimulated glycoprotein synthesis by stimulating the glycosylation apparatus, and not by increasing synthesis of protein acceptors. Previous studies have indicated that of a variety of potential regulatory points in the pathway of N-linked glycoprotein assembly, only expression of mannosylphosphoryldolichol synthase (MPDS) increases sufficiently to account for the increase in glycoprotein expression observed in response to estrogen. Consistent with these observations, it was found that injection of uterine poly(A+) RNA from estrogen-treated uteri into Xenopus oocytes markedly stimulated MPDS activity in the oocytes. In contrast, injection of RNA from non-estrogen-treated uteri did not stimulate MPDS activity in oocytes. Collectively, these results indicate that steroid hormones can modulate glycoprotein expression by preferentially stimulating the glycosylation apparatus. Nonetheless, one of estrogen's effects on the glycosylation apparatus, induction of MPDS activity, appears to occur at a transcriptional level.

Animals↗

Marked elevation in serum apolipoprotein E in a case of heterozygous cholesteryl ester transfer protein deficiency.

The subject was a 57-year-old Japanese woman with a body mass index of 21.2 kgm(-2). Her serum total cholesterol (TC), triglycerides (TG) and HDL-cholesterol levels were 7.11 mmoll(-1), 0.53 mmoll(-1) and 2.05 mmoll(-1), respectively. She had a marked increase of serum apolipoprotein (Apo) E concentration of 25 mgdl(-1) with normal concentrations of serum Apo A-I, A-II, B, C-II and C-III. Polymerase chain reaction-restriction fragments length polymorphism analysis of the cholesteryl ester transfer protein (CETP) gene from this subject revealed the heterozygous nucleotide change causing a Asp442 to Gly substitution (D442G) in the CETP protein. For comparison, 11 unrelated female subjects with this mutation (age, 57+/-5.1 years; BMI, 22+/-1.5 kgm(-2); TC, 7.23+/-1.16 mmoll(-1); TG, 1.44+/-0.80 mmoll(-1); HDL-C, 2.47+/-0.53 mmoll(-1)) were found to have a serum Apo E concentration of 7+/-1.5 mgdl(-1), about a third of the patient's concentration. The lipoprotein profile of the proband's serum analyzed by disk polyacrylamide gel electrophoresis showed a trace amount of VLDL. A vitamin A fat-loading test showed little increase in serum triglycerides and retinyl palmitate levels compared with control subjects at 2, 4 and 6 h after fat loading. Ultracentrifugation analysis of her serum revealed no detectable Apo E in the VLDL fraction but showed a large amount of Apo E in the HDL fraction, in contrast to a normal control, who had Apo E in the VLDL fraction as well as in the HDL fraction. Sequence analysis of the Apo E gene from the subject showed no nucleotide changes in exon 3 and exon 4, which code the mature Apo E protein, indicating there is no structural abnormality in the Apo E protein. Direct sequence analysis of the LDL receptor gene also did not show any nucleotide change. Based on these findings, it was hypothesized that the marked increase of Apo E in the patient's serum was caused by a decreased transfer of Apo E from HDL particles to TG-rich lipoproteins or impaired uptake of Apo E-containing HDL by LDL receptor or remnant receptor, due presumably to a dysfunction of these receptors in the patient.

Apolipoproteins E↗

Microarray analysis of genes expressed in the frontal cortex of rats chronically treated with morphine and after naloxone precipitated withdrawal.

Opioid dependence may be associated with adaptive changes in gene expression in the brain. In the present study we used DNA microarrays (U34A; Affymetrix) to analyze the expression of about 8000 genes in the frontal cortex of rats chronically treated with morphine and in rats after naloxone precipitated withdrawal. Chronic treatment for 10 days with ascending doses of morphine (10-50 mg/kg twice daily) resulted in a more than twofold induction of 14 genes after the last injection of morphine. The majority of these genes code for heat shock proteins (hsp70, hsp 27, hsp 40, hsp105, GRP78, etc.). The expression of the heat shock genes in the morphine-treated animals was reversed by naloxone (10 mg/kg). The opioid antagonist, in turn, precipitated withdrawal and increased the expression of a set of genes which are predominantly transcription factors (krox20, CREM, NGFI-B, IkappaB, etc). Only a few genes remained increased after naloxone application. Such persistently changed genes code for arc, a cytoskeleton-associated protein which is induced by synaptic activity, ania-3, a splice variant of the Homer 1 protein which is critically involved in activity-dependent alterations of synaptic function and rPer2, a protein regulating circadian rhythms. For selected genes the changes in gene expression were confirmed by real time PCR and by in situ hybridization. These findings indicate that the persistent changes in long-lasting plasticity during opiate dependence do not primarily depend on the increased expression levels of genes encoding for neurotransmitter, receptor and/or ion channel proteins, but rather on altered pattern of synaptic connectivity.

Animals↗

Epigenetics and airways disease.

Epigenetics is the term used to describe heritable changes in gene expression that are not coded in the DNA sequence itself but by post-translational modifications in DNA and histone proteins. These modifications include histone acetylation, methylation, ubiquitination, sumoylation and phosphorylation. Epigenetic regulation is not only critical for generating diversity of cell types during mammalian development, but it is also important for maintaining the stability and integrity of the expression profiles of different cell types. Until recently, the study of human disease has focused on genetic mechanisms rather than on non-coding events. However, it is becoming increasingly clear that disruption of epigenetic processes can lead to several major pathologies, including cancer, syndromes involving chromosomal instabilities, and mental retardation. Furthermore, the expression and activity of enzymes that regulate these epigenetic modifications have been reported to be abnormal in the airways of patients with respiratory disease. The development of new diagnostic tools might reveal other diseases that are caused by epigenetic alterations. These changes, despite being heritable and stably maintained, are also potentially reversible and there is scope for the development of 'epigenetic therapies' for disease.

Arginine↗

Coordinate regulation of RNAs encoding two isoforms of the rat muscle nicotinic acetylcholine receptor beta-subunit.

The nicotinic acetylcholine receptor (nAchR) mediates communication between nerve and skeletal muscle. The properties, levels and distribution of these receptors change during development of the neuromuscular junction. These changes may be due, in part, to expression of different gene products. We are using nuclease protection experiments and cDNA cloning to identify the RNA transcripts that encode nAchRs in rat muscle. This analysis has identified two beta-subunit mRNAs. Complementary DNAs corresponding to these two RNAs have been isolated from a rat skeletal muscle cDNA library. Based on nucleotide sequence analysis, these RNAs differ by 9 bases in their 5' coding sequence. The levels of both mRNAs change similarly during muscle development and upon denervation of adult skeletal muscle. These two beta-subunit-RNAs probably result from the use of different exon/intron splice sites in the beta-subunit gene.

Animals↗