Increased expression of the MBP mRNA binding protein HnRNP A2 during oligodendrocyte differentiation.

Heterogeneous nuclear ribonucleoprotein (hnRNP) A2, a trans-acting factor that mediates intracellular trafficking of myelin basic protein (MBP) mRNA to the myelin compartment in oligodendrocytes, is most abundant in the nucleus, but shuttles between the nucleus and cytoplasm. In the cytoplasm, it is associated with granules that transport mRNA from the cell body to the processes of oligodendrocytes. We found that the overall level of hnRNP A2 increased in oligodendrocytes as they differentiated into MBP-positive cells, and that this augmentation was reflected primarily in the cytoplasmic pool of hnRNP A2 present in the form of granules. The extranuclear distribution of hnRNP A2 was also observed in brain during the period of myelination in vivo. Methylation and phosphorylation have been implicated previously in the nuclear to cytoplasmic distribution of hnRNPs, so we used drugs that block methylation and phosphorylation of hnRNPs to assess their effect on hnRNP A2 distribution and mRNA trafficking. Cultures treated with adenosine dialdehyde (AdOx), an inhibitor of S-adenosyl-L-homocysteine hydrolase, or with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), a drug that inhibits casein kinase 2 (CK2), maintained the preferential nuclear distribution of hnRNP A2. Treatment with either drug affected the transport of RNA trafficking granules that remained confined to the cell body.

Quantification of gene transcription and enzyme activity for functionally important proteolytic enzymes during early development in the Pacific oyster Crassostrea gigas.

Gene transcripts and enzyme activities were quantified for a selection of functionally important aminopeptidases at 2-day intervals throughout the first 72 days of development in the Pacific oyster Crassostrea gigas. Leucine aminopeptidase (LAP) and cathepsin B (CathB) gene transcripts were quantified using fluorogenic ('real time') PCR. LAP and CathB gene transcripts were detected at all time points. The proportion of CathB transcripts remained essentially constant and low throughout development (Ct<35). The proportion of LAP transcripts was often similar (Ct<30), but with a distinct peak in transcript abundance at day 19 (Ct approximately 23). CathB and cathepsin D (CathD) enzyme activities were measured biochemically. Whilst CathD activity peaked at day 19, LAP and CathB activities both peaked at day 24. The closely coupled increase in transcript and enzyme activity for LAP indicates regulation at the transcriptional level. Alternatively, the peak in enzyme activity for CathB without enhanced transcriptional activity suggests post-transcriptional regulation. Similar mechanisms of regulation for LAP and CathB have been observed in both plants and mammals, indicating widespread conservation.

Transcript analysis of the genes encoding aminopeptidase N and alanine aminotransferase, two enzymes involved in protein turnover, in the Pacific oyster, Crassostrea gigas.

Molecular probes have been developed to detect aminopeptidase N (ApN) and alanine aminotransferase (ALAT) transcripts in the Pacific oyster Crassostrea gigas. Degenerate primers were designed using ApN and ALAT sequences stored in the EMBL database. Amplification of C. gigas genomic DNA using these primers resulted in amplification of a 344-bp ApN fragment and a 530-bp alanine aminotransferase fragment. The deduced amino acid sequence of the ApN fragment displayed 75 and 73% identities with sequences of ApN from human and mouse, respectively. The deduced amino acid sequence of the ALAT fragment displayed 57% identity both with human and rat ALAT. An ApN transcript of approximately 3.1 kb was detected by northern blotting in larvae and in adult digestive gland and gonadal tissue. No transcript was detected in adult adductor muscle. An ALAT transcript of approximately 2 kb was similarly detected in larvae and in adult gonadal tissue, but was undetectable in adult digestive gland and adductor muscle. Transcript detection employing RT-PCR demonstrated low-level expression of both ApN and ALAT in all studied tissues, in both larvae and adults.

Localisation of GABA(A) receptor subunits in the CNS using RT-PCR.

This paper presents an RT-PCR in situ histochemistry (RT-ISH) method for the detection and localisation of isoforms of the GABA(A) receptor in formalin-fixed, paraffin-embedded brain sections. RT-ISH was performed using PCR conditions already established in our laboratory for the amplification of the alpha(1-3) and beta(1-3) subunits of the GABA(A) receptor [2,5]. Initial experiments determined whether mRNA isolated from such sections was suitable for use in RT-PCR. Transcripts encoding both the alpha- and beta-subunits of the GABA(A) receptor were successfully amplified. RT-ISH, a one-step RT-PCR method, was used to amplify the transcripts and digoxigenin-labeled nucleotides were directly incorporated into the amplified products during cycling. RT-PCR products were detected using anti-digoxigenin antibody conjugated to alkaline-phosphatase and signal was visualised using light microscopy. This protocol may be used to study the expression of GABA(A) receptor isoforms in vivo and examine alterations in receptor composition during development and disease states.

Comparison of genetic variability and parentage in different ploidy classes of the Japanese oyster Crassostrea gigas.

Chemical treatments with cytochalasin B were used to induce triploidy in the progeny of a mass fertilization of 3 male and 7 female Crassostrea gigas parents. Triploids were produced either by retention of the first (meiosis I (MI) triploids) or the second (meiosis II (MII) triploids) polar bodies. These animals, together with their diploid siblings, were divided for two experiments. One set was used to compare physiological performance, and the other set deployed to compare growth in two different natural environments. For both experiments, genetic variability in different ploidy classes was estimated using three microsatellite loci and eight allozyme loci. The microsatellite loci were highly polymorphic, allowing independent confirmation of ploidy status and the unambiguous identification of parentage for each oyster. Significant differences in parentage were found between ploidy classes, despite the fact they originated from the same mass fertilization. This indicates that the assumptions of a common genetic background among random samples of animals taken from the same mass fertilization may not be generally valid. Knowledge of parentage also allowed the more accurate scoring of allozyme loci. As expected, triploids were found to be significantly more polymorphic than diploids. However, MI triploids were not significantly more polymorphic than MII triploids. MII triploid genotypes were used to estimate recombination rates between loci and their centromeres. These rates varied between 0.29 and 0.71, indicating only moderate chiasma interference.

Separate effects of triploidy, parentage and genomic diversity upon feeding behaviour, metabolic efficiency and net energy balance in the Pacific oyster Crassostrea gigas.

Triploid oysters were induced using cytochalasin B upon retention of either the first (meiosis I triploids) or the second (meiosis II triploids) polar body in embryos from a single cohort derived from mixed parentage. Allozyme and microsatellite assays enabled the confirmation of both parentage and triploidy status in each oyster. Comparison of meiosis I triploids, meiosis II triploids and diploid siblings established that improved physiological performance in triploids was associated with increased allelic variation, rather than with the quantitative dosage effects of ploidy status. An unidentified maternal influence also interacted with genotype. Among full sibs, allelic variation measured as multi-locus enzyme heterozygosity accounted for up to 42% of the variance in physiological performance; significant positive influences were identified upon feeding rate, absorption efficiency, net energy balance and growth efficiency (= net energy balance divided by energy absorbed). Whilst allelic variation was greater in both meiosis I and meiosis II triploids than in diploid siblings, both allelic variation and net energy balance were highest in triploids induced at meiosis I. This suggests that it may be preferable to induce triploidy by blocking meiosis I, rather than meiosis II as has traditionally been undertaken during commercial breeding programmes.

Population responses to contaminant exposure in marine animals: influences of genetic diversity measured as allozyme polymorphism.

Current understanding of the genetic and metabolic basis of relations between heterozygosity and animal performance under non-polluted conditions is relevent to interpreting apparently inconsistent findings concerning the relative advantage of allozyme polymorphism during contaminant exposure. Many interrelated factors which may influence and even compromise those relations include species (lifestyle, reproductive behaviour etc), lifestage, environmental influences and a variety of background genetic effects (limited parentage, null alleles, aneuploidy, genomic imprinting etc). Nevertheless, there is some promise that single-locus responses may be diagnostic for specific pollutants. In addition, limited evidence to date supports the a priori expectation that reduced energy requirements for maintenance metabolism may facilitate longer survival of multiple-locus heterozygotes during exposure to contaminants with toxic effects that result either in the reduced acquisition or availability of metabolizable energy, and/or a reduction in the efficiency with which metabolizable energy is used to fuel metabolic processes. More work is required to fully establish this trend in response to specific contaminant types, to assess any direct consequences of underlying differences in protein metabolism, and to resolve the interactive effects of contaminant mixtures. But the functional value of genetic variation within populations is confirmed. Reduced genetic diversity may not only compromise the capacity of an impacted population for genetic adaptation in the face of further environmental challenge, but may also result in increased energy requirements, lower production efficiency and reduced reproductive output. These metabolic consequences of reduced genetic polymorphism would further lower that population's potential for survival under lethal conditions of contaminant exposure, and also affect the genetic makeup of populations through differential reproduction under conditions of sublethal stress.

Protein metabolism, the costs of growth, and genomic heterozygosity: experiments with the mussel Mytilus galloprovincialis Lmk.

A single cohort of small individuals (31 mm mean shell length, 112 mg mean dry flesh weight) of the marine bivalve mollusc Mytilus galloprovincialis Lmk. was held sequentially for 2 wk at each of four food levels equivalent to ingested rations of less than 0.1%, 2.6%, 3.1%, and 7.4% of dry body weight per day. Growth rate reached a maximum at the highest ration level and was strongly correlated, amongst individuals, with mean heterozygosity measured across nine enzyme loci. Rates of energy expenditure were analysed separately as maintenance metabolic rate and the energy costs of growth (J mg-1 dry tissue). The maintenance metabolic rate correlated with traits of protein metabolism (protein synthesis, deposition, and breakdown), and the separate energy costs of both maintenance and growth correlated with the efficiency of protein deposition (protein growth as a proportion of synthesis). The energy costs of growth also varied in negative relation to mean individual heterozygosity. In a multiple regression analysis, the energy allocation to the costs of growth, body size, mean heterozygosity, and the efficiency of protein deposition together explained 90% of the variance amongst individuals in observed rates of growth. The results support the hypothesis that individual variability in the energy costs of protein turnover and in the efficiency of protein deposition during rapid growth are significant factors providing a link between individual genotype and its phenotypic expression as growth.

Patterns of adult male coresidence among young children of adolescent mothers.

This article uses data from the National Longitudinal Survey of Youth to examine the coresidence patterns of children and adult males during the first three years of a child's life, with special attention to the children of adolescent mothers. Overall, the most common experience was for the children to have an adult male present over the full period. However, there were differences by race and the mother's age when she gave birth. For example, 83 percent of white children and 47 percent of black children born to mothers aged 20 or older lived with an adult male during their entire early childhood, while three quarters of white children and fewer than one-third of black children born to mothers younger than 18 had a male present in their household over their first three years. Among both races, children of older mothers were significantly more likely than others to be born into a household where an adult male was present. The stability of male coresidence varied significantly by the mother's age among white children, but not among blacks. Overall, black children experienced more changes in male coresidence than whites. Finally, the likelihood that the adult male would be married to the mother was positively associated with white race and the age of the mother when she gave birth.

Disposition of 3H-enisoprost, a gastric antisecretory prostaglandin, in healthy humans.

1. After oral administration of 3H-enisoprost (450 micrograms) to five healthy men, as a solution in capsules, peak 3H levels of 5624 +/- 566 pg equiv./ml (mean +/- S.E.M.) were reached within one hour. No unchanged drug was detected in plasma. 2. Enisoprost was rapidly de-esterified to SC-36067 [(+/-)11 alpha, 16 zeta-dihydroxy-16-methyl-9-oxoprost-4Z, 13E-dien-1-oic acid], a pharmacologically active analogue, which reached peak concentrations of 651 +/- 200 pg/ml within 20 min of dosing. SC-36067 was eliminated metabolically, with a half-life of 1.61 h, by a combination of beta-oxidation, omega-oxidation and 9-keto-reduction. 3. After nine days 59.0 +/- 2.98% and 17.4 +/- 1.57% of the dose was excreted in urine and faeces respectively. The majority of this excretion was complete in two days. 4. Five urinary metabolites were identified by GC-MS. These were (+/-)3-[2 beta-(4-hydroxy-4-methyl-1E-octenyl)-3 alpha-hydroxy-5-oxo-1 alpha-cyclopentanyl]propanoic acid (SC-41411; 3.6% dose), (+/-)3-[3 alpha,5-dihydroxy-2 beta-(4-hydroxy-4-methyl-1E-octenyl)-1 alpha-cyclopentanyl]propanoic acid (SC-41411 PGF analogue; 4.8% dose), (+/-)3-[2 beta-(8-carboxy-4-hydroxy- 4-methyl-1E-octenyl)-3 alpha-hydroxy-5-oxo-1 alpha-cyclopentanyl] propanoic acid (SC-41411-16-carboxylic acid; 22% dose), (+/-)3-[2 beta-(8-carboxy-4- hydroxy-4-methyl-1E-octenyl)-3 alpha,5-dihydroxy-1 alpha-cyclopentanyl] propanoic acid (SC-41411 PGF analogue-16-carboxylic acid; 8.5% dose) and its gamma lactone (2.6% dose). 5. These metabolites were also identified chromatographically in plasma, as were SC-36067, (+/-)3-[2 beta-(4-hydroxy-4-methyl-1E-octenyl)-5-oxo-1 alpha- cyclopent-3-enyl]propanoic acid and (+/-)3-[2 beta-(4-hydroxy-4-methyl-1E-octenyl)-5-oxo-cyclopent-1- propanoic acid. 6. Some 5-10% of the dose was excreted in urine as tritiated water, indicating that oxidation of the 11 alpha-hydroxy group in SC-36067 or its metabolites also occurred.

Studies on the disposition of a 5-nitroimidazole in laboratory animals.

The disposition and metabolism of a 5-nitroimidazole compound (SC 28538) was investigated in the rat, beagle dog and rhesus monkey. The absorption of [14C]-SC 28538 was rapid and essentially complete after oral dosage in male animals, and also after intravaginal dosage in the female rat. Peak plasma levels of radioactivity occurred within 2 h of dosage. In the rat and dog the radioactivity was excreted predominantly in the faeces (greater than 60%) but in the monkey more than 60% was excreted in the urine. In both the male and pregnant female rat radioactivity was concentrated in the gastro-intestinal tract, liver and harderian gland and the concentrations of radioactivity in other tissues was generally lower than in plasma. Radioactivity was cleared more rapidly from plasma than from the majority of tissues. SC 28538 was extensively metabolised to form glucuronide and amino acid conjugates. The half-life of SC 28538 was of the order of 1 h in the dog and 3.7 h in the monkey.

The development of harmonic distortion in pulsed finite-amplitude ultrasound passing through liver.

The progressive development of finite-amplitude distortion of ultrasonic pulses has been investigated in excised bovine liver using pulsed focused ultrasonic beams at nominal frequencies of 2.5 and 3.5 MHz. Both the transducers and the powers used were those which may be encountered with clinical imaging equipment. Significant distortion of the waveform was observed to occur, particularly at higher powers. For example, at 2.5 MHz, with a mean input pressure (p0) of 0.58 MPa, the second harmonic in the pulse spectrum showed a maximum value of 10.5 dB below the fundamental and the highest third harmonic component was 19 dB below the fundamental. These particular observations illustrate that finite-amplitude distortion may be of considerable significance in the transmission through tissue of ultrasonic pulses during diagnostic scanning.

The output of pulse-echo ultrasound equipment: a survey of powers, pressures and intensities.

A survey of the powers, pressures and intensities generated by ultrasonic pulse-echo equipment in clinical use has been carried out. Three conventional B-scanners, four linear-array scanners and four mechanically sectored scanners were included in the study. Measurements were made on a total of 22 transducers covering the nominal frequency range 2.25-7.5 MHz. On those instruments where an output power control was provided, two measurements were made: one at the maximum available power and a second at a lower power. On arrays with a variable transmit focus control, measurements were made at all available focus settings. In all, measurements were made on 38 separate focused pulsed ultrasonic fields. The measurements were carried out using a calibrated ultrasonic force balance, and a calibrated polyvinylidene difluoride (PVdF) membrane hydrophone. A very wide range of maximum powers, pressures and intensities were found. Powers from 0.5-80 mW were measured; spatial-average temporal-peak positive pressures at the transducer varied between 30 kPa and 1.15 MPa, and spatial-peak pulse-average intensities were in the range 3.6 X 10(3)-1.1 X 10(7) Wm-2.

Relationships between the synthesis and breakdown of protein, dietary absorption and turnovers of nitrogen and carbon in the blue mussel, Mytilus edulis L.

Seasonal and nutritionally induced changes of whole body protein metabolism have been studied in 45 to 57 mm shell-length Mytilus edulis from Whitsand Bay, southwest England. The subtraction of measured net protein balances from coincident rates of protein synthesis, determined in vivo by supplying 15N-labelled alga and monitoring the enrichment of excreted ammonia, enabled computation of protein breakdown rates. Over the range of protein absorption from zero to 0.58% of total soft tissue protein 24 h-1, fractional rates of protein breakdown decreased from 0.41 to 0.03%, whereas protein synthesis and net protein balance both increased from 0.25% to 0.39% and from-0.16% to 0.36%, respectively. The progressive reduction in fractional protein degradation with elevated net protein balance represented a "protein sparing" effect, whereby the efficiency of protein synthesis (defined as net synthesis/overall synthesis) confirmed theoretical predictions of as much as 92% during periods of maximal growth. In addition, 38% of breakdown products were recycled directly to synthesis under conditions of zero net balance, with an increasing contribution evident upon further decreases of protein absorption. The overall response was characterized by a consistently conservative elemental turnover of nitrogen relative to carbon, so that as a fraction of each element absorbed, between 1.2 and 1.9 times as much nitrogen was incorporated within structural materials. Such conservation was most pronounced among mussels starved prior to experimentation, indicating nutritionally related efficiencies in the utilization of resources for synthesis. The changing balance between individual processes also effected large alterations in proportional size of the metabolic pool of free amino acids (0.2 to 14.5% of total soft tissue nitrogen). Finally, it is suggested that adjustments of protein synthetic rate may be significant in the regulation of energy expenditure, accounting for at least 16% of basal energy requirements. Results throughout have been compared and contrasted with those for mammals, and whole-body measurements of both protein synthesis and breakdown proposed as a valuable index for environmental effects on instantaneous growth and metabolism.

Propantheline bromide plasma level, urinary excretion and pharmacological data in a comparison of the bioavailability of three oral formulations of Pro-Banthine.

To determine the comparative bioavailability of three oral formulations of propantheline bromide (PB) by both pharmacokinetic and pharmacodynamic parameters, six normal men received three standard Pro-Banthine 15 mg tablets, two prolonged acting (PA) Pro-Banthine 30 mg tablets or one developmental PA Pro-Banthine 45 mg capsule, in a study of balanced random crossover design. Plasma concentrations and urinary excretion of the unchanged drug were measured after each treatment using a stable isotope dilution assay. Salivary secretion rate and heart rate measurements were also made at intervals after each medication. The standard Pro-Banthine formulation was significantly more bioavailable, weight for weight, than either the developmental PA capsule (45 mg), p less than 0.05, or the two 30 mg PA tablets (60 mg), p less than 0.01, based on urinary excretion and plasma levels of PB and on salivary secretion and heart rate data. There was no evidence of significant prolonged action for the PA formulations.

Plasma levels and urinary excretion of orally administered propantheline bromide in man.

After a single oral dose of 30 or 60 mg of propantheline bromide peak plasma levels of the drug were reached within 2 h in six healthy men. Mean peak plasma concentrations were 20.6 and 53.1 ng/ml after 30 mg and 60 mg respectively. The mean apparent absorption and elimination half-lives after 30 mg dose were 0.22 and 1.57 h respectively, and similar half-lives were found at the higher dose level. There was a dose related change in plasma levels and AUCinfinity of the drug, and some 3% to 4% of the administered dose of propantheline bromide was excreted unchanged in urine at each dose level. Comparison of the plasma levels and urinary excretion of the drug with those seen after i.v. administration in an earlier study indicated an apparently low systemic availability of orally administered propantheline bromide. There was tentative evidence of a qualitative relationship between the oral dose administered, plasma concentrations and the effects of propantheline bromide on salivary excretion.