Reconsideration of the catalytic center and mechanism of mammalian paraoxonase/arylesterase.

For three decades, mammalian paraoxonase (A-esterase, aromatic esterase, arylesterase; PON, EC 3.1.8.1) has been thought to be a cysteine esterase demonstrating structural and mechanistic homologies with the serine esterases (cholinesterases and carboxyesterases). Human, mouse, and rabbit PONs each contain only three cysteine residues, and their positions within PON have been conserved. In purified human PON, residues Cys-41 and Cys-352 form an intramolecular disulfide bond and neither could function as an active-center cysteine. Highly purified, enzymatically active PON contains a single titratable sulfhydryl group. Thus, Cys-283 is the only probable candidate for an active-center cysteine. Through site-directed mutagenesis of the human cDNA, Cys-283 was replaced with either serine (C283S) or alanine (C283A). The expressed C283 (wild-type) enzyme was inactivated by para-hydroxymercuribenzoate, but the C283S and C283A mutant enzymes were not inactivated. C283A and C283S mutant enzymes retained both paraoxonase and arylesterase activities, and the Km values for paraoxon and phenyl acetate were similar to those of the wild type. Clearly, residue Cys-283 is free in active PON, but a free sulfhydryl group is not required for either paraoxonase or arylesterase activities. Consequently, it is necessary to examine other models for the active-site structure and catalytic mechanism of PON.

Transfection of the human heme oxygenase gene into rabbit coronary microvessel endothelial cells: protective effect against heme and hemoglobin toxicity.

Heme oxygenase (HO) is a stress protein and has been suggested to participate in defense mechanisms against agents that may induce oxidative injury such as metals, endotoxin, heme/hemoglobin, and various cytokines. Overexpression of HO in cells might therefore protect against oxidative stress produced by certain of these agents, specifically heme and hemoglobin, by catalyzing their degradation to bilirubin, which itself has antioxidant properties. We report here the successful in vitro transfection of rabbit coronary microvessel endothelial cells with a functioning gene encoding the human HO enzyme. A plasmid containing the cytomegalovirus promoter and the human HO cDNA complexed to cationic liposomes (Lipofectin) was used to transfect rabbit endothelial cells. Cells transfected with human HO exhibited an approximately 3.0-fold increase in enzyme activity and expressed a severalfold induction of human HO mRNA as compared with endogenous rabbit HO mRNA. Transfected and nontransfected cells expressed factor VIII antigen and exhibited similar acetylated low-density lipoprotein uptake (two important features that characterize endothelial cells) with > 85% of cells staining positive for each marker. Moreover, cells transfected with the human HO gene acquired substantial resistance to toxicity produced by exposure to recombinant hemoglobin and heme as compared with nontransfected cells. The protective effect of HO overexpression against heme/hemoglobin toxicity in endothelial cells shown in these studies provides direct evidence that the inductive response of human HO to such injurious stimuli represents an important tissue adaptive mechanism for moderating the severity of cell damage produced by these blood components.

SCT1 mutants suppress the camptothecin sensitivity of yeast cells expressing wild-type DNA topoisomerase I.

Camptothecin is a potent antineoplastic agent that interferes with the action of eukaryotic DNA topoisomerase I; the covalent enzyme-DNA intermediate is reversibly stabilized, leading to G2 arrest and cell death. We used a genetic screen to identify cellular factors, other than DNA topoisomerase I, that participate in the process of camptothecin-induced cell death. Following ethyl methanesulfonate mutagenesis of top1 delta yeast cells expressing plasmid-borne wild-type DNA topoisomerase I, six dominant suppressors of camptothecin toxicity were isolated that define a single genetic locus, sct1. Mutant SCT1 cells expressed DNA topoisomerase I protein of similar specific activity and camptothecin sensitivity to that of congenic, drug-sensitive sct1 cells, yet were resistant to camptothecin-mediated lethality. Moreover, camptothecin-treated SCT1 cells did not exhibit the G2-arrested, terminal phenotype characteristic of drug-treated wild-type cells. SCT1 cell sensitivity to other DNA-damaging agents suggests that alterations in SCT1 function suppress camptothecin-induced DNA damage produced in the presence of yeast DNA topoisomerase I.

Different cellular backgrounds confer a marked advantage to either mutant or wild-type mitochondrial genomes.

After the introduction of mitochondria with a mixture of mutant and wild-type mitochondrial DNA (mtDNA) into a human rho degree cell line (143B.206), Yoneda et al. [Yoneda, M., Chomyn, A., Martinuzzi, A., Hurko, O. & Attardi, G. (1992) Proc. Natl. Acad. Sci. USA 89, 11164-11168] observed a shift in the proportion of the two mitochondrial genotypes in a number of cybrid clones. In every case where a shift was observed, there was an increase in the proportion of mutant mtDNA. By using the same cell line (143B.206 rho degree), we also generated cybrids that were either stable in their mitochondrial genotype or showed an increase in the proportion of mutant mtDNA. However, temporal analysis of the same mutant mtDNA type in another rho degree cell line revealed a quite distinct outcome. Those clones that showed a change shifted toward higher levels of wild-type rather than mutant mtDNA. These results indicate that the nuclear genetic background of the recipient (rho degree) cell can influence the segregation of mutant and wild-type mitochondrial genomes in cell cybrids.

The weediness of wild plants: molecular analysis of genes influencing dispersal and persistence of johnsongrass, Sorghum halepense (L.) Pers.

Many major weeds rely upon vegetative dispersal by rhizomes and seed dispersal by "shattering" of the mature inflorescence. We report molecular analysis of these traits in a cross between cultivated and wild species of Sorghum that are the probable progenitors of the major weed "johnsongrass." By restriction fragment length polymorphism mapping, variation in the number of rhizomes producing above-ground shoots was associated with three quantitative trait loci (QTLs). Variation in regrowth (ratooning) after overwintering was associated with QTLs accounting for additional rhizomatous growth and with QTLs influencing tillering. Vegetative buds that become rhizomes are similar to those that become tillers--one QTL appears to influence the number of such vegetative buds available, and additional independent genes determine whether individual buds differentiate into tillers or rhizomes. DNA markers described herein facilitate cloning of genes associated with weediness, comparative study of rhizomatousness in other Poaceae, and assessment of gene flow between cultivated and weedy sorghums--a risk that constrains improvement of sorghum through biotechnology. Cloning of "weediness" genes may create opportunities for plant growth regulation, in suppressing propagation of weeds and enhancing productivity of major forage, turf, and "ratoon" crops.

Inactivation of the superior cerebellar peduncle blocks expression but not acquisition of the rabbit's classically conditioned eye-blink response.

The localization of sites of memory formation within the mammalian brain has proven to be a formidable task even for simple forms of learning and memory. Recent studies have demonstrated that reversibly inactivating a localized region of cerebellum, including the dorsal anterior interpositus nucleus, completely prevents acquisition of the conditioned eye-blink response with no effect upon subsequent learning without inactivation. This result indicates that the memory trace for this type of learning is located either (i) within this inactivated region of cerebellum or (ii) within some structure(s) efferent from the cerebellum to which output from the interpositus nucleus ultimately projects. To distinguish between these possibilities, two groups of rabbits were conditioned (by using two conditioning stimuli) while the output fibers of the interpositus (the superior cerebellar peduncle) were reversibly blocked with microinjections of the sodium channel blocker tetrodotoxin. Rabbits performed no conditioned responses during this inactivation training. However, training after inactivation revealed that the rabbits (trained with either conditioned stimulus) had fully learned the response during the previous inactivation training. Cerebellar output, therefore, does not appear to be essential for acquisition of the learned response. This result, coupled with the fact that inactivation of the appropriate region of cerebellum completely prevents learning, provides compelling evidence supporting the hypothesis that the essential memory trace for the classically conditioned eye-blink response is localized within the cerebellum.

Wild-type p53 protein undergoes cytoplasmic sequestration in undifferentiated neuroblastomas but not in differentiated tumors.

Neuroblastoma (NB), a tumor arising from the sympathetic nervous system, is one of the most common malignancies in childhood. Several recent reports on the p53 genotype found virtually exclusive wild-type status in primary tumors, and it was postulated that p53 plays no role in the development of NB. Here, however, we report that the vast majority of undifferentiated NBs exhibit abnormal cytoplasmic sequestration of wild-type p53. This inability of p53 to translocate to the nucleus presumably prevents the protein from functioning as a suppressor. Thirty of 31 cases (96%) of undifferentiated NB showed elevated levels of wild-type p53 in the cytoplasm of all tumor cells concomittant with a lack of nuclear staining. p53 immunoprecipitation from tumor tissues showed a 4.5- to 8-fold increase over normal protein levels. All of 10 tumors analyzed harbored wild-type p53 by direct sequencing of full-length cDNA and Southern blot. In addition, no MDM-2 gene amplification was seen in all 11 tumors analyzed. In contrast, no p53 abnormality was detected in 14 differentiated ganglioneuroblastomas and 1 benign ganglioneuroma. We conclude that loss of p53 function seems to play a major role in the tumorigenesis of undifferentiated NB. This tumor might abrogate the transactivating function of p53 by inhibiting its access to the nucleus, rather than by gene mutation. Importantly, our results suggest that (i) this could be a general mechanism for p53 inactivation not limited to breast cancer (where we first described it) and that (ii) it is found in a tumor previously not thought to be affected by p53 alteration.

Receptor usage and differential downregulation of CD46 by measles virus wild-type and vaccine strains.

Recently, two cell surface molecules, CD46 and moesin, have been found to be functionally associated with measles virus (MV) infectivity of cells. We investigated the receptor usage of MV wild-type, subacute sclerosing panencephalitis, and vaccine strains and their effect on the down-regulation of CD46 after infection. We found that the infection of human cell lines with all 19 MV strains tested was inhibitable with antibodies against CD46. In contrast, not all strains of MV led to the downregulation of CD46 following infection. The group of CD46 non-downregulating strains comprised four lymphotropic wild-type isolates designated AB, DF, DL, and WTF. Since the downregulation of CD46 is caused by interaction with newly synthesized MV hemagglutinin (MV-H), we tested the capability of recombinant MV-H proteins to downregulate CD46. Recombinant MV-H proteins of MV strains Edmonston, Halle, and CM led to the down-regulation of CD46, whereas those of DL and WTF did not. This observed differential downregulation by different MV strains has profound consequences, since lack of CD46 on the cell surface leads to susceptibility of cells to complement lysis. These results suggest that lymphotropic wild-type strains of MV which do not downregulate CD46 may have an advantage for replication in vivo. The relatively weak immune response against attenuated vaccine strains of MV compared with wild-type strains might be related to this phenomenon.

Pancreatic digestive enzyme secretion in the rabbit: rapid cyclic variations in enzyme composition.

The role and mechanism of nonparallel pancreatic secretion of digestive enzymes, in which enzyme proportions change in rapidly regulated fashion, remain controversial. Secretion was collected from male 2.2-kg New Zealand rabbits in 5-min intervals for 3 h under basal conditions or constant stimulation with cholecystokinin (CCK; 0.1 microgram per kg per h i.v.) or methacholine chloride (MCh; 40 micrograms per kg per h i.v.). Both CCK and MCh produced an 8-fold stimulation of protein output. Enzymes were separated by SDS/PAGE and quantitated by densitometry of Coomassie blue-stained gels. Under both basal conditions and constant MCh infusion, rapid neurosecretory-like 12-min cyclic changes occurred in the proportions of amylase, lipase I, chymotrypsinogen, and trypsinogen. During constant infusion their percentages changed as much as 10-fold, and their ratios cycled by as much as 30-fold. The mean percentage for the entire infusion period for lipase I declined > 25% with CCK or MCh, for amylase it rose approximately 30%, and for chymotrypsinogen and trypsinogen it doubled (for all, P < 0.05). CCK and MCh elicited subtly but significantly different mean enzyme percentages and enzyme ratios (P < 0.05) for amylase, chymotrypsinogen, and trypsinogen; these differences were also confirmed by regression and correlation analyses. The changes in enzyme percentages and ratios were explicitly consistent with secretagogue-caused shifts in the intrapancreatic enzyme secretory sources. Nonparallel secretion of digestive enzymes occurs routinely, even during constant stimulation, and is due to cyclic neurosecretory-like secretion from heterogeneous intrapancreatic sources.

The Decline of Wild Pacific Salmon

As threatened and endangered species, wild Pacific salmon are in peril. This paper discusses the differences of the five species of wild Pacific salmon. As salmon go through several stages of their lifecycles, they face a myriad of threats to their existence. Threats from humans in the form of hydropower dams, habitat destruction, harvesting issues, and hatcheries are explained. A draft recovery plan for salmon in the Puget Sound area of Washington State is used as a case study. Strengths and weaknesses of this plan are discussed. The paper then discusses the need for growth management laws supporting salmon habitat and a change in individual behaviors if wild Pacific salmon sustainability is to become a reality.

Electroretinographical and histological study of mouse retina after optic nerve section: a comparison between wild-type and retinal degeneration 1 mice

Background: Retinal ganglion cell death underlies the pathophysiology of neurodegenerative disorders such as glaucoma or optic nerve trauma. To assess the potential influence of photoreceptor degeneration on retinal ganglion cell survival, and to evaluate functionality, we took advantage of the optic nerve section mouse model. Methods: Surviving retinal ganglion cells were double-stained by exposing both superior colliculi to fluorogold, and by applying dextran-tetramethylrhodamine to the injured optic nerve stump. To assess retinal function in wild-type animals, electroretinograms were recorded on the injured eyes and compared with the contralateral. Similar labelling experiments were carried out on retinal degeneration 1 mice. Surviving retinal ganglion cells were counted 21 days after axotomy and compared with wild-type mice. No functional experiments were performed on retinal degeneration 1 animals because they do not develop normal electroretinographical responses. Results: A significant decrease in retinal ganglion cell density was observed 6 days after axotomy in the wild type. Functional studies revealed that, in scotopic conditions, axotomy induced a significant amplitude decrease in the positive scotopic threshold response component of the electroretinogram. Such decrease paralleled cell loss, suggesting it may be an appropriate technique to evaluate functionality. When comparing retinal ganglion cell densities in wild-type and retinal degeneration 1 mice, a significant greater survival was observed on the latter. Conclusions: After optic nerve section, electroretinographical recordings exhibited a progressive decrease in the amplitude of the positive scotopic threshold response wave, reflecting ganglion cell loss. Interestingly, rod degeneration seemed, at least initially, to protect from axotomy-driven damage.

Potential impact of Wild boar (Sus scrofa) on Pheasant (Phasianus colchicus) nesting success

In recent decades wild boar have greatly increased in Europe due a combination of environmental and human factors. Because of a high reproduction rate, wild boar cause conflicts with human activities and in particularly agriculture. However, there are concerns also about environmental impact and in particular on ground nesting birds. Our study aim to evaluate the potential impact of wild boar on pheasants nests. Predation rates were compared between two areas with different wild boar abundance and between nests placed in fenced plots with no wild boar access (but accessible to other predators) and open plots in which the wild boar gains access. Predation rate by wild boar in the area with higher wild boar abundance was 61.5% whereas in the area with lower abundance was 16.0%. In plots with free access, wild boar predation was 34.2% whereas in exclusion plots we did not registered predation by wild boar. Moreover, wild boar resulted the main nest predator (total loss 22.8%). Our findings suggest that wild boar could have e strong impact on pheasant reproductive success.