Modeling of hepatic elimination and organ distribution kinetics with the extended convection-dispersion model

The conventional convection-dispersion (also called axial dispersion) model is widely used to interrelate hepatic availability (F) and clearance (Cl) with the morphology and physiology of the liver and to predict effects such as changes in liver blood flow on F and Cl. An extended form of the convection-dispersion model has been developed to adequately describe the outflow concentration-time profiles for vascular markers at both short and long times after bolus injections into perfused livers. The model, based on flux concentration and a convolution of catheters and large vessels, assumes that solute elimination in hepatocytes follows either fast distribution into or radial diffusion in hepatocytes. The model includes a secondary vascular compartment, postulated to be interconnecting sinusoids. Analysis of the mean hepatic transit time (MTT) and normalized variance (CV2) of solutes with extraction showed that the discrepancy between the predictions of MTT and CV2 for the extended and conventional models are essentially identical irrespective of the magnitude of rate constants representing permeability, volume, and clearance parameters, providing that there is significant hepatic extraction. In conclusion, the application of a newly developed extended convection-dispersion model has shown that the unweighted conventional convection-dispersion model can be used to describe the disposition of extracted solutes and, in particular, to estimate hepatic availability and clearance in booth experimental and clinical situations.

An update on genetic, structural and functional studies of arylamine N-acetyltransferases in eucaryotes and procaryotes

Arylamine N-acetyltransferase (NAT) was first identified as the inactivator of the anti-tubercular drug isoniazid, The enzyme was shown to catalyse the transfer of an acetyl group from acetyl-CoA to the terminal nitrogen of the hydrazine drug. The rate of inactivation of isoniazid was polymorphically distributed in the population and was one of the first examples of pharmacogenetic variation, NAT was identified recently in Mycobacterium tuberculosis and is a candidate for; modulating the response to isoniazid, Genome sequences have revealed many homologous members of this unique family of enzymes. The first three-dimensional structure of a member of the NAT family identifies a catalytic triad consisting of aspartate, histidine and cysteine proposed to form the activation mechanism. So far, all procaryotic NATs resemble the human enzyme which acetylates isoniazid (NAT2), Human NAT2 is characteristic of drug-metabolizing enzymes: it is found in liver and intestine, In humans and other mammals, there are up to three different isoenzymes. If only one isoenzyme is present, it is like human NAT1. Human NAT1 and its murine equivalent specifically acetylate the folate catabolite p-amino-benzoylglutamate. NAT1 and its murine homologue each have a ubiquitous tissue distribution and are expressed early in development at the blastocyst stage, During murine embryonic development, NAT is expressed in the developing neural tube. The proposed endogenous role of NAT in folate metabolism, and its multi-allelic nature, indicate that its role in development should be assessed further.

Conservation of the sequence and temporal expression of let-7 heterochronic regulatory RNA

Two small RNAs regulate the timing of Caenorhabditis elegans development(1,2). Transition from the first to the second larval stage fates requires the 22-nucleotide lin-4 RNA(1,3,4), and transition from late larval to adult cell fates requires the 21-nucleotide let-7 RNA 2. The lin-4 and let-7 RNA genes are not homologous to each other, but are each complementary to sequences in the 3' untranslated regions of a set of protein-coding target genes that are normally negatively regulated by the RNAs1,2,5,6. Here we have detected let-7 RNAs of similar to 21 nucleotides in samples from a wide range of animal species, including vertebrate, ascidian, hemichordate, mollusc, annelid and arthropod, but not in RNAs from several cnidarian and poriferan species, Saccharomyces cerevisiae, Escherichia coli or Arabidopsis. We did not detect lin-4 RNA in these species. We found that let-7 temporal regulation is also conserved: let-7 RNA expression is first detected at late larval stages in C. elegans and Drosophila, at 48 hours after fertilization in zebrafish, and in adult stages of annelids and molluscs. The let-7 regulatory RNA may control late temporal transitions during development across animal phylogeny.

Analysis of Mouse Keratin 6a Regulatory Sequences in Transgenic Mice Reveals Constitutive, Tissue-Specific Expression by a Keratin 6a Minigene

The analysis of keratin 6 expression is complicated by the presence of multiple isoforms that are expressed constitutively in a number of internal stratified epithelia, in palmoplantar epidermis, and in the companion cell layer of the hair follicle. In addition, keratin 6 expression is inducible in interfollicular epidermis and the outer root sheath of the follicle, in response to wounding stimuli, phorbol esters, or retinoic acid. In order to establish the critical regions involved in the regulation of keratin 6a (the dominant isoform in mice), we generated transgenic mice with two different-sized mouse keratin 6a constructs containing either 1.3 kb or 0.12 kb of 5' flanking sequence linked to the lacZ reporter gene. Both constructs also contained the first intron and the 3' flanking sequence of mouse keratin 6a. Ectopic expression of either transgene was not observed. Double-label immunofluorescence analyses demonstrated expression of the reporter gene in keratin 6 expressing tissues, including the hair follicle, tongue, footpad, and nail bed, showing that both transgenes retained keratinocyte-specific expression. Quantitative analysis of beta -galactosidase activity verified that both the 1.3 and 0.12 kb keratin 6a promoter constructs produced similar levels of the reporter. Notably, both constructs were constitutively expressed in the outer root sheath and interfollicular epidermis in the absence of any activating stimulus, suggesting that they lack the regulatory elements that normally silence transcription in these cells. This study has revealed that a keratin 6a minigene contains critical cis elements that mediate tissue-specific expression and that the elements regulating keratin 6 induction lie distal to the 1.3 kb promoter region.

An object-oriented neural network approach to short-term traffic forecasting

This paper discusses an object-oriented neural network model that was developed for predicting short-term traffic conditions on a section of the Pacific Highway between Brisbane and the Gold Coast in Queensland, Australia. The feasibility of this approach is demonstrated through a time-lag recurrent network (TLRN) which was developed for predicting speed data up to 15 minutes into the future. The results obtained indicate that the TLRN is capable of predicting speed up to 5 minutes into the future with a high degree of accuracy (90-94%). Similar models, which were developed for predicting freeway travel times on the same facility, were successful in predicting travel times up to 15 minutes into the future with a similar degree of accuracy (93-95%). These results represent substantial improvements on conventional model performance and clearly demonstrate the feasibility of using the object-oriented approach for short-term traffic prediction. (C) 2001 Elsevier Science B.V. All rights reserved.

Perturbed upper limb movements cause short-latency postural responses in trunk muscles

Addition of a load to a moving upper limb produces a perturbation of the trunk due to transmission of mechanical forces. This experiment investigated the postural response of the trunk muscles in relation to unexpected limb loading. Subjects performed rapid, bilateral shoulder flexion in response to a stimulus. In one third of trials, an unexpected load was added bilaterally to the upper limbs in the first third of the movement. Trunk muscle electromyography, intra-abdominal pressure and upper limb and trunk motion were measured. A short-latency response of the erector spinae and transversus abdominis muscles occurred similar to 50 ms after the onset of the limb perturbation that resulted from addition of the load early in the movement and was coincident with the onset of the observed perturbation at the trunk. The results provide evidence of initiation of a complex postural response of the trunk muscles that is consistent with mediation by afferent input from a site distant to the lumbar spine, which may include afferents of the upper limb.

Prior calcium channel blockade and short-term survival following acute myocardial infarction

There is concern over the safety of calcium channel blockers (CCBs) in acute coronary disease. We sought to determine if patients taking calcium channel blockers (CCBs) at the time of admission with acute myocardial infarction (AMI) had a higher case-fatality compared with those taking beta-blockers or neither medication. Clinical and drug treatment variables at the time of hospital admission predictive of survival at 28 days were examined in a community-based registry of patients aged under 65 years admitted to hospital for suspected AMI in Perth, Australia, between 1984 and 1993. Among 7766 patients, 1291 (16.6%) were taking a CCB and 1259 (16.2%) a betablocker alone at hospital admission. Patients taking CCBs had a worse clinical profile than those taking a beta-blocker alone or neither drug (control group), and a higher unadjusted 28-day mortality (17.6% versus 9.3% and 11.1% respectively, both P < 0.001). There was no significant heterogeneity with respect to mortality between nifedipine, diltiazem, or verapamil when used alone, or with a beta-blocker. After adjustment for factors predictive of death at 28 days, patients taking a CCB were found not to have an excess chance of death compared with the control group (odds ratio [OR] 1.06, 95% confidence interval [CI]; 0.87, 1.30), whereas those taking a beta-blocker alone had a lower odds of death (OR 0.75, 95% CI; 0.59, 0.94). These results indicate that established calcium channel blockade is not associated with an excess risk of death following AMI once other differences between patients are taken into account, but neither does it have the survival advantage seen with prior beta-blocker therapy.

Chimeric 16S rDNA sequences of diverse origin are accumulating in the public databases

A significant number of chimeric 16S rDNA sequences of diverse origin were identified in the public databases by partial treeing analysis. This suggests that chimeric sequences, representing phylogenetically novel non-existent organisms, are routinely being overlooked in molecular phylogenetic surveys despite a general awareness of PCR-generated artefacts amongst researchers.

Myb-binding Protein 1a is a Nucleocytoplasmic Shuttling Protein that Utilizes CRM1-dependent and Independent Nuclear Export Pathways

Myb-binding protein 1a (Mybbp1a) is a novel nuclear protein localized predominantly, but not exclusively, in nucleoli. Although initially isolated as a c-Myb interacting protein, Mybbp1a is expressed ubiquitously, associates with a number of different transcription factors, and may play a role in both RNA polymerase I- and II-mediated transcriptional regulation. However, its precise function remains unclear. In this study we show that Mybbp1a is a nucleocytoplasmic shuttling protein and investigate the mechanisms responsible for both nuclear import and export. The carboxyl terminus of Mybbp1a, which contains seven short basic amino acid repeat sequences, is responsible for both nuclear and nucleolar localization, and this activity can be transferred to a heterologous protein. Deletion mapping demonstrated that these repeat sequences appear to act incrementally, with successive deletions resulting in a corresponding increase in the proportion of protein localized in the cytoplasm. Glutathione S-transferase pulldown experiments showed that the nuclear receptor importin-alpha/beta mediates Mybbp1a nuclear import. Interspecies heterokaryons were used to demonstrate that Mybbp1a was capable of shuttling between the nucleus and the cytoplasm. Deletion analysis and in vivo export studies using a heterologous assay system identified several nuclear export sequences which facilitate Mybbp1a nuclear export of Mybbp1a by CRM1-dependent and -independent pathways. (C) 2003 Elsevier Science (USA). All rights reserved.

Molecular evidence for definition of genera in the Oxylobium group (Fabaceae : Mirbelieae)

The circumscription of Oxylobium and related genera has been problematic for nearly 200 years. Traditional definitions of genera in the group have relied on morphological features of the leaves, flower, and fruit that overlap extensively between genera. Therefore sequences of cpDNA (trnL-F intron and spacer) and nrDNA (ITS) were used to estimate the phylogeny of the group in an attempt to redefine the genera as monophyletic groups. Oxylobium sens. str. was found to be a well supported clade in both data sets, with the inclusion of Mirbelia oxylobioides. No other genus in the group was supported by these data, except Gastrolobium sens. lat. Some species groups within Chorizema, Mirbelia, and Podolobium were supported but relationships among these, Oxylobium and Gastrolobium differed significantly between the chloroplast and nuclear data sets. No group supported by the molecular data had a morphological synapomorphy, not even Oxylobium or Gastrolobium. Therefore it may be necessary to adopt a much broader generic concept in this group than has been done previously. Incongruence between the two molecular data sets, and very short internal basal branches in both, suggest a rapid early radiation in this group, possibly combined with hybridization and lineage sorting.

Stimulating dormancy release and emergence of annual ryegrass (Lolium rigidum) seeds using short-term hydrated storage in darkness

Experiments were performed to determine whether the dormancy release effect of hydrated storage in darkness (dark-stratification) is common amongst annual ryegrass populations and has the potential to occur under field conditions. Dormant seeds from all populations tested (22) became sensitive to light during dark-stratification, enabling them to germinate when subsequently exposed to light. Under controlled temperature (25/15degreesC), light (12-h photoperiod), and hydration (solidified agar-water) conditions, more seeds germinated by 28 days if the first 14 days were in darkness followed by exposure to light for 12 h per day than if they were exposed to light throughout or darkness throughout. Constraint over the conditions imposed during dark-stratification and germination was gradually reduced to investigate whether the dormancy release effect was diminished. Dark-stratification was effective in promoting germination when performed under natural diurnal temperatures, and burial in moist soil provided suitable conditions for dark-stratification to occur. The surface of moist soil, with natural diurnal temperatures and sunlight, was suitable for germination of dark-stratified seeds. Dark-stratification is a quick and effective means to enhance the sensitivity of dormant annual ryegrass seeds to light, enabling the majority of the population to germinate. However, large quantities of light are required to promote germination of dark-stratified seeds, so buried seeds must be moved to the soil surface to allow exposure to adequate light for germination.

Short- and long-term, salinity-induced modulation of V-ATPase activity in the posterior gills of the true freshwater crab, Dilocarcinus pagei (Brachyura, Trichodactylidae)

To better understand the biochemical mechanisms underlying anisosmotic extracellular regulation in the freshwater Brachyura, we kinetically characterized the V-ATPase from the posterior gills of Dilocarcinus pagei, acclimated for 10 days to salinities up to 21%.. Specific activity was highest in fresh water (26.5 +/- 2.1 U mg(-1)), decreasing in 5 parts per thousand to 21 parts per thousand, attaining 3-fold less at 15 parts per thousand. Apparent affinities for ATP and Mg(2+) respectively increased 3.2- and 2-fold at 10 parts per thousand, suggesting expression of different isoenzymes. In a 240-h time-course study of exposure to 21%., maximum specific activity decreased 2.5- to 4-fold within 1 to 24 h while apparent affinities for ATP and Mg(2+) respectively increased by 12-fold within 24 h and 2.4-fold after 1 h, unchanged thereafter. K(I) for bafilomycin A(1) decreased 150-fold after 1 h, remaining constant up to 120 h. This is the first kinetic analysis of V-ATPase specific activity in crustacean gills during salinity acclimation. Our findings indicate active gill Cl(-) uptake by D. pagei in fresh water, and short- and long-term down-regulation of V-ATPase-driven ion uptake processes during salinity exposure, aiding in comprehension of the biochemical adaptations underpinning the establishment of the Brachyura in fresh water. (C) 2011 Elsevier Inc. All rights reserved.

Intra- and interspecific divergence in the nuclear sequences of the clock gene period in species of the Drosophila buzzatii cluster

We have measured nucleotide variation in the CLOCK/CYCLE heterodimer inhibition domain (CCID) of the clock X-linked gene period in seven species belonging to the Drosophila buzzatii cluster, namely D. buzzatii, Drosophila koepferae, Drosophila antonietae, Drosophila serido, Drosophila gouveai, Drosophila seriema and Drosophila borborema. We detected that the purifying selection is the main force driving the sequence evolution in period, in agreement with the important role of CCID in clock machinery. Our survey revealed that period provides valuable phylogenetic information that allowed to resolve phylogenetic relationships among D. gouveai, D. borborema and D. seriema, which composed a polytomic clade in preliminary studies. The analysis of patterns of intraspecific variation revealed two different lineages of period in D. koepferae, probably reflecting introgressive hybridization from D. buzzatii, in concordance with previous molecular data.