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Biomedical subjects

N Hopkins

Publications and source records attributed to N Hopkins.

At least 19 recordsLinked to original sources
PubMed2006

Anti-inflammatory effect of augmented nitric oxide production in chronic lung infection.

Chronic infection of the lungs with Pseudomonas aeruginosa complicates many long-term lung diseases including cystic fibrosis, bronchiectasis, chronic obstructive lung disease, and mechanical ventilation. In acute inflammatory lung diseases, increased nitric oxide synthase (NOS-2) expression leads to excess nitric oxide (NO) production, resulting in the production of reactive nitrogen intermediates, which contribute to tissue damage. In contrast, the contribution of NO to pulmonary damage in chronic Pseudomonas infection of the lung has not been directly examined and is unclear. Although NOS-2 expression is increased in this condition, NO production is not abnormally elevated. It was hypothesized that chronic infection of the airways does not cause increased NO production but, in contrast, leads to inappropriately low NO concentrations that are pro-inflammatory. A rodent model of chronic airway infection was used to examine the effects on lung damage of augmenting or inhibiting NO production after airway infection with P. aeruginosa was well established. Three days post-infection, L-arginine, which augments NO production, or L-NAME, an inhibitor of NO production, was administered in drinking water. Lung damage was assessed 12 days later. L-arginine treatment reduced tissue damage, inhibited neutrophil recruitment, and reduced the pro-inflammatory cytokine interleukin (IL)-1beta. Treatment with L-NAME caused loss of alveolar walls, greater vascular damage, and increased levels of the pro-inflammatory cytokine IL-6. Thus, in chronic airway infection, inhibition of NO production worsened lung damage, whereas augmenting NO ameliorated this damage. This is the first demonstration that augmenting endogenous NO production in chronic infective lung disease caused by P. aeruginosa is anti-inflammatory. Given that infection with this organism complicates many chronic lung diseases, most notoriously cystic fibrosis, these findings have important clinical implications.

Animals↗
PubMed2004

Discrimination between ricin and sulphur mustard toxicity in vitro using Raman spectroscopy.

A Raman spectroscopy cell-based biosensor has been proposed for rapid detection of toxic agents, identification of the type of toxin and prediction of the concentration used. This technology allows the monitoring of the biochemical properties of living cells over long periods of time by measuring the Raman spectra of the cells non-invasively, rapidly and without use of labels (Notingher et al. 2004 doi:10.1016/j.bios.2004.04.008). Here we show that this technology can be used to distinguish between changes induced in A549 lung cells by the toxin ricin and the chemical warfare agent sulphur mustard. A multivariate model based on principal component analysis (PCA) and linear discriminant analysis (LDA) was used for the analysis of the Raman spectra of the cells. The leave-one-out cross-validation of the PCA-LDA model showed that the damaged cells can be detected with high sensitivity (98.9%) and high specificity (87.7%). High accuracy in identifying the toxic agent was also found: 88.6% for sulphur mustard and 71.4% for ricin. The prediction errors were observed mostly for the ricin treated cells and the cells exposed to the lower concentration of sulphur mustard, as they induced similar biochemical changes, as indicated by cytotoxicity assays. The concentrations of sulphur mustard used were also identified with high accuracy: 93% for 200 microM and 500 microM, and 100% for 1,000 microM. Thus, biological Raman microspectroscopy and PCA-LDA analysis not only distinguishes between viable and damaged cells, but can also discriminate between toxic challenges based on the cellular biochemical and structural changes induced by these agents and the eventual mode of cell death.

Biological Assay↗
PubMed2001

vhnf1, the MODY5 and familial GCKD-associated gene, regulates regional specification of the zebrafish gut, pronephros, and hindbrain.

Mutations in the homeobox gene vHnf1 are associated with human diseases MODY5 (maturity-onset diabetes of the young, type V) and familial GCKD (glomerulocystic kidney disease). In an insertional mutagenesis screen in zebrafish, we isolated mutant alleles of vhnf1. Phenotypes of these mutants include formation of kidney cysts, underdevelopment of the pancreas and the liver, and reduction in size of the otic vesicles. We show that these abnormalities arise from patterning defects during development. We further provide evidence that vhnf1 regulates the expression of key patterning genes for these organs. vhnf1 is required for the proper expression of pdx1 and shh (sonic hedgehog) in the gut endoderm, pax2 and wt1 in the pronephric primordial, and valentino (val) in the hindbrain. Complementary to the loss-of-function phenotypes, overexpression of vhnf1 induces expansion of the val expression domain in the hindbrain. We propose that vhnf1 controls development of multiple organs through regulating regional specification of organ primordia. The similarity between vhnf1-associated fish phenotypes and human symptoms suggests a correlation between developmental functions of vhnf1 and the molecular etiology of MODY5 and GCKD.

Amino Acid Sequence↗
PubMed2001

Intergroup differentiation: stereotyping as a function of status hierarchy.

The authors explored how negative intergroup comparisons affect intergroup differentiation. More specifically, they tested the prediction that the in-group's negative intergroup comparisons with a high-status group would result in more negative stereotyping of a lower status out-group. The authors elicited stereotypes of a lower status university in 2 conditions. In the 1st, the participants judged only the middle-ranking university in-group and a lower status university. In the 2nd, those judgments followed comparison with a higher status university. In the 2nd condition, there was an increased differentiation between the in-group and the lower status out-group because of the more negative stereotyping of the lower status out-group. This evidence of intergroup differentiation was found only on the dimension judged most important and along which the in-group was negatively compared with the higher status group.

Adult↗
PubMed2001

Chronic airway infection leads to angiogenesis in the pulmonary circulation.

In both pulmonary and systemic hypertension, the walls of the arteriolar vessels are thickened and the lumen size is reduced, leading to increased total vascular resistance. It has been reported previously that chronic airway infection and inflammation lead to increased wall thickness in the pulmonary vasculature, without the development of pulmonary hypertension. The aim of the present study was to examine quantitatively the remodeling of intra-acinar blood vessels in chronically infected rat lungs. Adult rats were anesthetized and inoculated intratracheally with Pseudomonas aeruginosa (n = 10) incorporated into agar beads to induce chronic airway infection. Control groups included rats inoculated with sterile agar beads (n = 8) and rats that were not inoculated (n = 6). Chronic infection caused vascular wall thickening without reduction in mean lumen radius. Furthermore, chronic infection led to increased total length of intra-acinar vessels and increased numbers of branch points, demonstrating that angiogenesis had occurred. Preservation of lumen size and formation of new parallel pathways in the vasculature of chronically infected lungs account for the maintenance of normal PVR despite vessel wall remodeling.

Animals↗
PubMed2001

Analysis of the zebrafish smoothened mutant reveals conserved and divergent functions of hedgehog activity.

Despite extensive studies, there are still many unanswered questions regarding the mechanism of hedgehog signaling and the phylogenic conservation of hedgehog function in vertebrates. For example, whether hedgehog signaling in vertebrates requires smoothened is unclear, and the role of hedgehog activity in zebrafish is controversial. We show that inactivation of smoothened by retroviral insertions in zebrafish results in defects that are characteristic of hedgehog deficiencies, including abnormalities in body size, the central nervous system, adaxial mesoderm, cartilage and pectoral fins. We demonstrate that, as in Drosophila, vertebrate smoothened is essential for hedgehog signaling, and functions upstream of protein kinase A. Further analysis of neural tube defects revealed the absence of lateral floor plate and secondary motoneurons, but the presence of medial floor plate and primary motoneurons in smoothened mutant embryos. Blocking maternal hedgehog signaling by cyclopamine eliminates primary motoneurons, but not medial floor plate. Interestingly, even after inhibition of maternal hedgehog activity, the midbrain dopaminergic neurons still form, and looping of the heart does not randomize in the mutants. We also found decreased proliferation and increased apoptosis in the mutants. Taken together, these data demonstrate the conserved role of vertebrate smoothened in the hedgehog signaling pathway, and reveal similarities and differences of hedgehog function between teleosts and amniotes.

Animals↗
PubMed2001

Group variability judgments: investigating the context-dependence of stereotypicality and dispersal judgments.

This study explores the context-dependent nature of perceptions of group variability by examining how ingroup and outgroup ratings are affected by asking participants (N = 237) to rate these groups either on their own or together. In key respects, it replicates the design utilized by Haslam, Oakes, Turner, and McGarty (1995). However, several features of the present study's design were distinctive and intended to address methodological issues raised in the original. First, an alternative to the Katz-Braly procedure was adopted such that the measure of 'stereotypicality' referred to the applicability of both stereotypic and counter-stereotypic attributes. Second, a measure of 'dispersion' was also employed. The data show that ingroup stereotypicality was, as predicted, affected by a manipulation of context, while outgroup stereotypicality was not. No effects of context on the dispersion measures were obtained for either the ratings of the ingroup or the outgroup. The relationship between stereotypicality and dispersal measures of variability is discussed.

Adolescent↗
PubMed2000

Proviral insertions in the zebrafish hagoromo gene, encoding an F-box/WD40-repeat protein, cause stripe pattern anomalies.

The zebrafish, Danio rerio, has three types of pigment cells (melanophores, xanthophores and iridophores) and, in adult fish, these cells are organized into a stripe pattern. The mechanisms underlying formation of the stripe pattern are largely unknown. We report here the identification and characterization of a novel dominant zebrafish mutation, hagoromo (hag), which was generated by insertional mutagenesis using a pseudotyped retrovirus. The hag mutation caused disorganized stripe patterns. Two hag mutant alleles were isolated independently and proviruses were located within the fifth intron of a novel gene, which we named hag, encoding an F-box/WD40-repeat protein. The hag gene was mapped to linkage group (LG)13, close to fgf8 and pax2.1. Amino acid sequence similarity, conserved exon-intron boundaries and conserved synteny indicated that zebrafish hag is an ortholog of mouse Dactylin, the gene mutated in the Dactylaplasia (Dac) mouse [1]. The Dac mutation is dominant and causes defects in digit formation in fore- and hindlimbs. This study revealed that the hag locus is important for pattern formation in fish but is involved in distinct morphogenetic events in different vertebrates.

Amino Acid Sequence↗
PubMed2000

Expression of murine adenosine deaminase (ADA) in transgenic maize.

A murine adenosine deaminase (ADA) gene, driven by the maize ubi-1 promoter and intron region, was transformed into embryogenic maize callus, along with a bar and gusA gene-containing plasmid, using microparticle bombardment. Selection in the presence of either the herbicide Basta or the adenosine analogue 2'-deoxyadenosine resulted in transgenic cultures that expressed GUS and accumulated a 41-kD protein that immunoprecipated with an ADA-specific polyclonal antibody. ADA enzyme activity was observed in extracts from transgenic callus as well as regenerated plants and progeny. Culltures expressing ADA grew in the presence of 200 mg/l 2'-deoxyadenosine, a concentration which completely inhibited the growth of non-transgenic cultures. ADA activity appeared to segregate in progency of regenerated plants as a single, dominant Mendelian trait. These results suggest that ADA, in combination with adenosine analogue selection, represents a potentially viable selectable marker system for transgenic maize production.

Adenosine↗
PubMed1999

A large-scale insertional mutagenesis screen in zebrafish.

It is estimated that approximately 2500 genes are essential for the normal development of a zebrafish embryo. A mutation in any one of these genes can result in a visible developmental defect, usually followed by the death of the embryo or larva by days 5-7 of age. We are performing a large-scale insertional mutagenesis screen in the zebrafish with the goal of isolating approximately 1000 embryonic mutations. We plan to clone a significant fraction of the mutated genes, as these are the genes important for normal embryogenesis of a vertebrate. To achieve this goal, we prepared approximately 36, 000 founder fish by injecting blastula-stage embryos with one of two pseudotyped retroviruses. We estimate that together these fish harbor between 500,000-1,000,000 proviral insertions in their germ lines. The protocol we have devised and the size of our facility allow us to breed approximately 80,000-150,000 of these insertions to homozygosity within 2 years. Because a pilot screen conducted earlier in our laboratory revealed that the frequency of mutations obtained with this type of insertional mutagen is 1 embryonic lethal mutation per 70-100 proviral insertions, screening 100,000 insertions should yield at least 1000 mutants. Here we describe the protocol for the screen and initial results with the first of the two retroviral vectors used, a virus designated F(5). We screened an estimated 760 insertions among F(3) progeny from 92 F(2) families and obtained 9 recessive embryonic lethal mutations. Thus, the efficiency of mutagenesis with this viral vector is approximately one-ninth that observed with the chemical mutagen ENU in zebrafish. We have also obtained two dominant mutations, one of which is described here. As expected, mutated genes can be readily identified. So far, genes mutated in four of the nine recessive mutants and one of the two dominant mutants have been cloned. Further improvements to this technology could make large-scale insertional mutagenesis screening and rapid gene cloning accessible to relatively small zebrafish laboratories.

Alleles↗
PubMed1999

Enhanced expression of inducible nitric oxide synthase without vasodilator effect in chronically infected lungs.

We hypothesized that abnormal ventilation-perfusion matching in chronically infected lungs was in part due to excess nitric oxide (NO) production after upregulation of inducible NO synthase (iNOS) expression. Rats were anesthetized and inoculated intratracheally with Pseudomonas aeruginosa incorporated into agar beads (chronically infected) or with sterile agar beads (placebo inoculated) and killed 10-15 days later. Immunohistochemistry demonstrated increased expression of iNOS and reduced expression of endothelial NOS (eNOS) in chronically infected compared with placebo-inoculated or noninoculated lungs. In isolated lungs from chronically infected rats, NOS inhibition with N(omega)-nitro-L-arginine methyl ester increased the mean perfusion pressure (14.4 +/- 2.7 mmHg) significantly more than in the placebo-inoculated (4.8 +/- 1.0 mmHg) or noninoculated (5.3 +/- 0.8 mmHg) lungs (P < 0.01). Although the chronically infected lungs were more sensitive to NOS inhibition, further evidence suggested that the increased iNOS expression was not associated with enhanced iNOS activity. Selective inhibitors of iNOS did not produce an increase in vascular resistance similar to that produced by nonselective inhibitors. Accumulation of nitrate/nitrite in the perfusate of isolated lungs was unchanged by chronic infection. Thus although iNOS expression was increased in chronic pulmonary infection, iNOS activity in the intact lung was not. Nonetheless, endogenous NO production was essential to maintain normal vascular resistance in these lungs.

Animals↗
PubMed1998

Angiotensin II induces diverse signal transduction pathways via both Gq and Gi proteins in liver epithelial cells.

Angiotensin II stimulates a biphasic activation of Raf-1, MEK, and ERK in WB liver epithelial cells. The first peak of activity is rapid and transient and is followed by a sustained phase. Angiotensin II also causes a rapid activation of p21ras in these cells. Moreover, two Src family kinases (Fyn and Yes) were activated by angiotensin II in a time- and concentration-dependent manner. Microinjection of antibodies against Fyn and Yes blocked angiotensin II-induced DNA synthesis and c-Fos expression in WB cells, indicating an obligatory involvement of these tyrosine kinases in the activation of the ERK cascade by angiotensin II. Finally, substantial reduction of the angiotensin II-stimulated activation of Fyn, Raf-1, ERK, and expression of c-Fos by pertussis toxin pretreatment argues that G proteins of the Gi family as well as the Gq family are involved in angiotensin II-mediated mitogenic pathways in WB cells.

Angiotensin II↗
PubMed1998

Endothelium-dependent relaxation and hyperpolarization in guinea-pig coronary artery: role of epoxyeicosatrienoic acid.

1. Acetylcholine (ACh) elicits an endothelium-dependent relaxation and hyperpolarization in the absence of nitric oxide (NO) and prostaglandin synthesis in the guinea-pig coronary artery (GPCA). This response has been attributed to a factor termed endothelial-derived hyperpolarizing factor (EDHF). Recently it has been suggested that EDHF may be a cytochrome P450 product of arachidonic acid (AA) i.e., an epoxyeicosatrienoic acid (EET). The present study investigated whether this pathway could account for the response to ACh observed in the GPCA in the presence of 100 microM N(omega)-nitro-L-arginine and 10 microM indomethacin. 2. ACh, AA and 11,12-EET each produced concentration-dependent relaxations in arteries contracted with the H1-receptor agonist AEP (2,2-aminoethylpyridine). The AA-induced relaxation was significantly enhanced in the presence of the cyclo-oxygenase/lipoxygenase inhibitor, eicosatetranynoic acid (30 microM). 3. The cytochrome P450 inhibitors proadifen (10 microM) and clotrimazole (10 microM) inhibited ACh, lemakalim (LEM) and AA-induced relaxation, whereas 17-octadecynoic acid (100 microM) and 7-ethoxyresorufin (10 microM) were without effect on all three vasodilators. Proadifen and clotrimazole also inhibited ACh (1 microM) and LEM (1 microM)-induced hyperpolarization. 4. The ability of various potassium channel blockers to inhibit relaxation responses elicited with ACh, AA and 11,12-EET was also determined. Iberiotoxin (IBTX; 100 nM) was without effect on responses to ACh but significantly reduced responses to both AA and 11,12-EET. In contrast, 4-aminopyridine (4-AP; 5 mM) significantly reduced response to ACh but not responses to AA and 11,12-EET. Combined IBTX plus (4-AP) inhibited the ACh-induced relaxation to a greater extent than 4-AP alone. Apamin (1 microM), glibenclamide (10 microM) and BaCl2 (50 microM) had no significant effect on responses to ACh, AA and 11,12-EET. 5. IBTX (100 nM) significantly reduced both 11,12-EET (33 microM) and AA (30 microM) hyperpolarization without affecting the ACh (1 microM)-induced hyperpolarization. In contrast, 4-AP significantly reduced the ACh-induced hyperpolarization without affecting either AA or 11,12-EET-induced hyperpolarizations. 6. In summary, our results suggest that the coronary endothelium releases a factor upon application of AA which hyperpolarizes the smooth muscle. The similarity of pharmacology between AA and 11,12-EET suggests that this factor is an EET. However, the disparity of pharmacology between responses to ACh versus responses to 11,12-EET do not support the hypothesis that EETs represent the predominant factor which ACh releases from the endothelium that leads to NO- and prostaglandin-independent hyperpolarization and relaxation in the GPCA.

1-Methyl-3-isobutylxanthine↗
PubMed1998

Ribozymes targeted to stearoyl-ACP delta9 desaturase mRNA produce heritable increases of stearic acid in transgenic maize leaves.

Ribozymes are RNAs that can be designed to catalyze the specific cleavage or ligation of target RNAs. We have explored the possibility of using ribozymes in maize to downregulate the expression of the stearoyl-acyl carrier protein (Delta9) desaturase gene. Based on site accessibility and catalytic activity, several ribozyme constructs were designed and transformed into regenerable maize lines. One of these constructs, a multimer hammerhead ribozyme linked to a selectable marker gene, was shown to increase leaf stearate in two of 13 maize lines. There were concomitant decreases in Delta9 desaturase mRNA and protein. The plants with the altered stearate phenotype were shown to express ribozyme RNA. The ribozyme-mediated trait was heritable, as evidenced by stearate increases in the leaves of the R1 plants derived from a high-stearate line. The increase in stearate correlated with the presence of the ribozyme gene. A catalytically inactive version of this ribozyme did not produce any significant effect in transgenic maize. This is evidence that ribozymes can be used to modulate the expression of endogenous genes in maize.

Amino Acid Sequence↗