Haber's Rule: The Search for Quantitative Relationships in Toxicology

Dose (or concentration) and time of exposure are both important in determining the intensity of response to a toxic agent. For a given response intensity, Haber's Rule (c×t=K) has been proposed as a law of toxicology, but this rule is just one special case of a more general relationship c×t ^m =K, where the exponent m is quite variable. For inhaled toxicants m generally has a value between 0 and 1, whereas for carcinogens m is usually between 1 and 5. The absence of a universal value for m, or one that is generally applicable to different classes of toxicants, makes it not yet possible to develop a Haber-type rule with which to extrapolate successfully between exposure scenarios.     

Two new cytotypes reinforce that <Emphasis Type="Italic">Micronycteris hirsuta</Emphasis> Peters, 1869 does not represent a monotypic taxon

Background

The genus Micronycteris is a diverse group of phyllostomid bats currently comprising 11 species, with diploid number (2n) ranging from 26 to 40 chromosomes. The karyotypic relationships within Micronycteris and between Micronycteris and other phyllostomids remain poorly understood. The karyotype of Micronycteris hirsuta is of particular interest: three different diploid numbers were reported for this species in South and Central Americas with 2n?=?26, 28 and 30 chromosomes. Although current evidence suggests some geographic differentiation among populations of M. hirsuta based on chromosomal, morphological, and nuclear and mitochondrial DNA markers, the recognition of new species or subspecies has been avoided due to the need for additional data, mainly chromosomal data.

Results

We describe two new cytotypes for Micronycteris hirsuta (MHI) (2n?=?26 and 25, NF?=?32), whose differences in diploid number are interpreted as the products of Robertsonian rearrangements. C-banding revealed a small amount of constitutive heterochromatin at the centromere and the NOR was located in the interstitial portion of the short arm of a second pair, confirmed by FISH. Telomeric probes hybridized to the centromeric regions and weakly to telomeric regions of most chromosomes. The G-banding analysis and chromosome painting with whole chromosome probes from Carollia brevicauda (CBR) and Phyllostomus hastatus (PHA) enabled the establishment of genome-wide homologies between MHI, CBR and PHA.

Conclusions

The karyotypes of Brazilian specimens of Micronycteris hirsuta described here are new to Micronycteris and reinforce that M. hirsuta does not represent a monotypic taxon. Our results corroborate the hypothesis of karyotypic megaevolution within Micronycteris, and strong evidence for this is that the entire chromosome complement of M. hirsuta was shown to be derivative with respect to species compared in this study.

     

Differential expression of specific sulphate transporters underlies seasonal and spatial patterns of sulphate allocation in trees

Sulphate uptake and its distribution within plants depend on the activity of different sulphate transporters (SULTR). In long‐living deciduous plants such as trees, seasonal changes of spatial patterns add another layer of complexity to the question of how the interplay of different transporters adjusts S distribution within the plant to environmental changes. Poplar is an excellent model to address this question because its S metabolism is already well characterized. In the present study, the importance of SULTRs for seasonal sulphate storage and mobilization was examined in the wood of poplar (Populus tremula × P. alba) by analysing their gene expression in relation to sulphate contents in wood and xylem sap. According to these results, possible functions of the respective SULTRs for seasonal sulphate storage and mobilization in the wood are suggested. Together, the present results complement the previously published model for seasonal sulphate circulation between leaves and bark and provide information for future mechanistic modelling of whole tree sulphate fluxes.     

Pulmonary artery smooth muscle cells from normal subjects and IPAH patients show divergent cAMP-mediated effects on TRPC expression and capacitative Ca2+ entry

Pulmonary vascular remodeling due to overgrowth of pulmonary artery smooth muscle cells (PASMC) is a major cause for the elevated vascular resistance in patients with idiopathic pulmonary arterial hypertension (IPAH). Increased cytosolic Ca(2+) concentration, resulting from enhanced capacitative Ca(2+) entry (CCE) and upregulated transient receptor potential (TRP) channel expression, is involved in stimulating PASMC proliferation. The current study was designed to determine the impact of cAMP, a second messenger that we hypothesized would blunt aspects of PASMC activity, as a possible contributor to IPAH pathophysiology. Short-term (30 min) pretreatment with forskolin (FSK; 10 muM), a direct activator of adenylyl cyclase, in combination with the cyclic nucleotide phosphodiesterase inhibitor isobutylmethylxanthine (IBMX; 200 muM), attenuated CCE in PASMC from normal subjects, patients without pulmonary hypertension (NPH), and patients with IPAH. The FSK-mediated CCE inhibition was independent of protein kinase A (PKA), because the PKA inhibitor H89 negligibly affected the decrease in CCE produced by cAMP. By contrast, longer (4 h) treatment with FSK (with IBMX) attenuated CCE in normal and NPH PASMC but enhanced CCE in IPAH PASMC. This enhancement of CCE was abolished by PKA inhibition and associated with an upregulation of TRPC3. In addition, cAMP increased TRPC1 mRNA expression in IPAH (but not in normal or NPH) PASMC, an effect blunted by H89. Furthermore, iloprost, a prostacyclin analog that increases cAMP, downregulated TRPC3 expression in IPAH PASMC and FSK-mediated cAMP increase inhibited IPAH PASMC proliferation. Although a rapid rise in cellular cAMP decreases CCE by a PKA-independent mechanism, sustained cAMP increase inhibits CCE in normal and NPH PASMC but increases CCE via a PKA-dependent pathway in IPAH PASMC. The divergent effect of cAMP on CCE parallels effects on TRPC expression. The results suggest that the combined use of a PKA inhibitor and cAMP-elevating drugs may provide a novel approach for treatment of IPAH.     

Interleukin-2 receptor regulates activation of phosphatidylinositol 3-kinase.

Interleukin-2 (IL-2) stimulates proliferation of T lymphocytes and is involved in the activation of both natural killer and lymphokine-activated killer precursor cells. The intracellular messengers which mediate IL-2-dependent events have not yet been identified. IL-2 receptor is not a protein-tyrosine kinase. Activation of a cellular protein-tyrosine kinase and direct association of a protein-tyrosine kinase activity with the IL-2 receptor occurs within minutes of IL-2 stimulation. We investigated the activation of phosphatidylinositol 3-kinase (PI 3-kinase) in IL-2-mediated signal transduction using the IL-2-dependent murine T-cell line, CTLL-2, and human phytohemagglutinin-stimulated peripheral blood lymphocytes (phytohemagglutinin blasts). Within a minute following stimulation of these cells with IL-2, PI 3-kinase activity could be detected in antiphosphotyrosine (anti-P-Tyr) antibody immunoprecipitates. IL-2 triggered a direct association of PI 3-kinase with the IL-2 receptor as detected in immunoprecipitates using anti-IL-2 receptor beta chain antibody. In vivo labeled CTLL-2 cells have a time-dependent increase in D-3-phosphorylated polyphosphoinositides following stimulation with IL-2. This is the first group of second messengers identified in IL-2-mediated signal transduction.     

Mapping fungal ion channel locations

Ion channel mapping techniques are described and the results for two fungal organisms, Saprolegnia ferax and Neurospora crassa, are presented. In these species, two channel types have been characterized, stretch-activated channels exhibiting significant calcium permeability and spontaneous channels having significant potassium permeability. Two distinct analyses of patch clamp data, analysis of channel self-clustering and association between different channel types, and localization along the hyphae, reveal significant differences between the two organisms. S. ferax maintains a tip-high gradient of both channel types which is lost after disruption of the actin cytoskeleton. There is significant self-clustering of the channels, as well as interactions between channel types. N. crassa on the other hand does not maintain tip-high gradients, and clustered distributions are observed only for the stretch-activated channels. In terms of physiological roles, evidence is quite strong that the stretch-activated channels function as a growth sensor in S. ferax, but have an unknown function in N. crassa. In both organisms, the potassium permeable channels presumably function in potassium uptake. The differences between these two organisms may be due, in part, to differences in their normal environment: aquatic versus terrestrial. Copyright 1998 Academic Press.     

Changes to the HIV long terminal repeat and to HIV integrase differentially impact HIV integrase assembly, activity, and the binding of strand transfer inhibitors

Human immunodeficiency virus (HIV) integrase enzyme is required for the integration of viral DNA into the host cell chromosome. Integrase complex assembly and subsequent strand transfer catalysis are mediated by specific interactions between integrase and bases at the end of the viral long terminal repeat (LTR). The strand transfer reaction can be blocked by the action of small molecule inhibitors, thought to bind in the vicinity of the viral LTR termini. This study examines the contributions of the terminal four bases of the nonprocessed strand (G(2)T(1)C(-1)A(-2)) of the HIV LTR on complex assembly, specific strand transfer activity, and inhibitor binding. Base substitutions and abasic replacements at the LTR terminus provided a means to probe the importance of each nucleotide on the different functions. An approach is described wherein the specific strand transfer activity for each integrase/LTR variant is derived by normalizing strand transfer activity to the concentration of active sites. The key findings of this study are as follows. 1) The G(2):C(2) base pair is necessary for efficient assembly of the complex and for maintenance of an active site architecture, which has high affinity for strand transfer inhibitors. 2) Inhibitor-resistant enzymes exhibit greatly increased sensitivity to LTR changes. 3) The strand transfer and inhibitor binding defects of a Q148R mutant are due to a decreased affinity of the complex for magnesium. 4) Gln(148) interacts with G(2), T(1), and C(-1) at the 5' end of the viral LTR, with these four determinants playing important and overlapping roles in assembly, strand transfer catalysis and high affinity inhibitor binding.