Increased catalytic activity of cytochrome P-450IIE1 in pericentral hepatocytes compared to periportal hepatocytes isolated from pyrazole-treated rats.

Cytochrome P-450IIE1 is induced by a variety of agents, including acetone, ethanol and pyrazole. Recent studies employing immunohistochemical methods have shown that P-450IIE1 was expressed primarily in the pericentral zone of the liver. In order to evaluate whether catalytic activity of P-450IIE1 is preferentially localized in the pericentral zone of the liver acinus, the oxidation of aniline and p-nitrophenol, two effective substrates for P-450IIE1, by periportal and pericentral hepatocytes isolated from pyrazole-treated rats was determined. Periportal and pericentral hepatocytes were prepared by a digitonin-collagenase procedure; the marker enzymes glutamine synthetase and gamma-glutamyl transpeptidase indicated reasonable separation of the two cell populations. Viability, yield and total cytochrome P-450 content were similar for the periportal and pericentral hepatocytes. Pericentral hepatocytes oxidized aniline and p-nitrophenol at rates that were 2-4-fold greater than periportal hepatocytes under a variety of conditions. Carbon monoxide inhibited the oxidation of the substrates with both preparations and abolished the increased oxidation found with the pericentral hepatocytes. Pyrazole or 4-methylpyrazole, added in vitro, effectively inhibited the oxidation of aniline and p-nitrophenol and prevented the augmented rate of oxidation by the pericentral hepatocytes. Western blots carried out using isolated microsomes revealed a more than 2-fold increase in immunochemical staining with microsomes isolated from the pericentral hepatocytes, which correlated to the 2-4-fold increase in the rate of oxidation of aniline or p-nitrophenol by the pericentral hepatocytes. These results suggest that functional catalytic activity of cytochrome P-450IIE1 is preferentially localized in the pericentral zone of the liver acinus, and that most of the induction by pyrazole of P-450IIE1 appears to occur within the pericentral zone.     

A comprehensive analysis of the developmental and tissue-specific expression of the isoactin multigene family in the rat.

The present study represents the first comprehensive analysis of isoactin gene expression in the developing rat. Our results clearly demonstrate that the developmental and tissue-specific expression of the actin multigene family is a highly integrated and complex process involving a variety of regulatory paradigms. The distinct temporal patterns of expression reported in this study indicate that there are three key phases in the regulation of expression of the actin multigene family during development. These include early embryonic development, late fetal development, and early postnatal development. The specific spatial patterns of expression observed in this study demonstrate that the expression of the actin multigene family is much more permissive than previously reported. This permissive expression includes a wide range of "ectopic" expression of the striated muscle isoactins as well as an extended expression of the alpha-smooth muscle isoactin. These findings expand our current understanding of the expression of the actin multigene family in development and provide a fundamental basis for future studies directed at investigating these processes.     

Novel reagents for chemical cleavage at abasic sites and UV photoproducts in DNA.

Hot piperidine is often used to cleave abasic and UV-irradiated DNA at the sites of damage. It can inflict non-specific damage on DNA, probably because it is a strong base and creates significant concentrations of hydroxyl ions which can attack purines and pyrimidines. We show that several other amines can cleave abasic DNA at or near neutral pH without non-specific damage. One diamine, N,N'-dimethylethylenediamine, efficiently cleaves abasic DNA at pH 7.4 by either beta- or beta,delta-elimination, depending on temperature. Using end-labelled oligonucleotides we show that cleavage depends mainly on elimination reactions, but that 4',5'-cyclization is also significant. This reagent also cleaves at photoproducts induced by UVC and UVB, producing the same overall pattern as piperidine, but with no non-specific damage. It should prove valuable in locating low levels of photoproducts in DNA, such as those induced by natural sunlight.     

A high-resolution linkage map of the lethal spotting locus: a mouse model for Hirschsprung disease

Mice homozygous for the lethal spotting (ls) mutation exhibit aganglionic megacolon and a white spotted coat owing to a lack of neural crest-derived enteric ganglia and melanocytes. The ls mutation disrupts the migration, differentiation, or survival of these neural crest lineages during mammalian development. A human congenital disorder, Hirschsprung disease (HSCR), is also characterized by aganglionic megacolon of the distal bowel and can be accompanied by hypopigmentation of the skin. HSCR has been attrrbuted to multiple loci acting independently or in combination. The ls mouse serves as one animal model for HSCR, and the ls gene may represent one of the loci responsible for some cases of HSCR in humans. This study uses 753 N₂ progeny from a combination of three intersubspecific backcrosses to define the molecular genetic linkage map of the ls region and to provide resources necessary for positional cloning. Similar to some cases of HSCR, the ls mutation acts semidominantly, its phenotypic effects dependent upon the presence of modifier genes segregating in the crosses. We have now localized the ls mutation to a 0.8-cM region between the D2Mit113 and D2Mit73/D2Mit174 loci. Three genes, endothelin-3 (Edn3), guanine nucleotide-binding protein -stimulating polypeptide 1 (Gnas), and phosphoenolpyruvate carboxykinase (Pck1) were assessed as candidates for the ls mutation. Only Edn3 and Gnas did not recombine with the ls mutation. Mutational analysis of the Edn3 and Gnas genes will determine whether either gene is responsible for the neural crest deficiencies observed in ls/ls mice.Both authors contributed equally to this research.     

A highly repeated retropseudogene-like sequence in DNA of the redbreasted merganser (Mergus serrator)

Two highly repeated nucleotide sequences (RBMI and RBMII) cloned from an EcoRI digest of DNA of the redbreasted merganser (Mergus serrator) account for approx. 5 to 10% of the DNA of M. serrator and the closely related Mergus merganser. Complete DNA digestion of seven members of the Mergini with EcoRI produces distinct, relatively species-specific patterns of a few high-Mr (greater than 1.5 kb) fragments of RBMI-like material. In such digests RBMII forms ladder-type patterns with monomers of approx. 200 bp. The sequence of a cloned 2.6-kb RBMI fragment from M. serrator contains several extended (up to 70 bp) and modified poly(dA) sequences, two open reading frames in opposite orientation to the longest poly(dA) sequence and two direct 10-bp repeats suggesting that RBMI is a rearranged retropseudogene-like element.     

Migration of neutrophils across endothelial monolayers is stimulated by treatment of the monolayers with interleukin-1 or tumor necrosis factor-alpha

To study the effects of the cytokines IL-1 and TNF-alpha on the transendothelial migration of neutrophils, human umbilical vein endothelial cells (HUVEC) were grown to confluence on connective tissue prepared from human amniotic membrane. Pretreatment of HUVEC-amnion cultures with rIL-1 beta (7.5 ng/ml) or rTNF-alpha (5 ng/ml) for 4 h resulted in rapid migration of from 20 to 50% of subsequently added neutrophils across the endothelial monolayer. In contrast, only 3 +/- 3% of added neutrophils penetrated the HUVEC monolayer in the absence of any stimulus. The number of neutrophils that migrated across cytokine-treated HUVEC was similar to the number that traversed untreated monolayers in response to gradients of FMLP; in addition, it was only 35% less than the number of neutrophils that migrated in response to leukotriene B4. No consistent additive effect was seen when migration was induced by both cytokine pretreatment of the HUVEC and a chemotactic gradient. The number of neutrophils that migrated across IL-1-treated cultures was proportional to the number added over the range of 2.5 x 10(5) to 4 x 10(6) neutrophils. When used at optimal concentrations, IL-1 and TNF-alpha were equally effective in stimulating neutrophil migration; no additive effect was seen when HUVEC were pretreated with optimal doses of both cytokines together. Direct addition of IL-1 or TNF-alpha to a 1-h migration assay had no effect on neutrophil adhesion to or migration across HUVEC, either in the presence or absence of a chemotactic gradient. Stimulation of neutrophil transendothelial migration in this system did not appear to be caused by adsorption of cytokine by the amniotic tissue, nor was it due to contamination of the cytokine preparations by LPS. These results suggest that IL-1 and TNF-alpha, generated at sites of inflammation, may act upon the endothelium to promote emigration of neutrophils from the vasculature.     

Phylogeographic analysis of invasive Asian earthworms (Amynthas) in the northeast United States

Phylogeographic studies are useful in reconstructing the history of species invasions, and in some instances can elucidate cryptic diversity of invading taxa. This can help in predicting or managing the spread of invasive species. Among terrestrial invasive species in North America, earthworms can have profound ecological effects. We are familiar with the centuries‐old invasions of European earthworms (Lumbricidae) and their impacts on nutrient cycling in soils. More recent invasions by Asian earthworms of the family Megascolecidae are less fully understood. We used data for two mitochondrial gene fragments, cytochrome oxidase I (COI) and 16S rRNA, to examine the relationships among populations of Asian earthworms in the megascolecid genus Amynthas in the northeast United States. Recent reports have indicated that one species in particular, Amynthas agrestis, is having detrimental effects in mixed forest ecosystems, and we were interested in understanding the invasion history for this species. We were surprised to discover three divergent mitochondrial lineages of Amynthas occurring sympatrically in upstate New York. Given the gap between intra‐ and inter‐lineage sequence divergences, we propose that these three lineages represent cryptic species of Amynthas, one of which is A. agrestis. For each of the three lineages of Amynthas, we observed shared haplotypes across broad geographic distances. This may reflect common origins for populations in each lineage, either by direct routes from native ranges or through post‐introduction spread by natural dispersal or human‐mediated transport within North America. Management efforts focused on horticultural imports from Asia, commercial nurseries within the USA, and on prohibition of bait disposal may help to reduce the further invasion success of Amynthas.