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Biomedical subjects

David M Donovan

Publications and source records attributed to David M Donovan.

13 recordsLinked to original sources
PubMed2006

Lysis of staphylococcal mastitis pathogens by bacteriophage phi11 endolysin.

The Staphylococcus aureus bacteriophage phi11 endolysin has two peptidoglycan hydrolase domains (endopeptidase and amidase) and an SH3b cell wall-binding domain. In turbidity reduction assays, the purified protein can lyse untreated staphylococcal mastitis pathogens, Staphylococcus aureus and coagulase-negative staphylococci (Staphylococcus chronogenes, Staphylococcus epidermidis, Staphylococcus hyicus, Staphylococcus simulans, Staphylococcus warneri and Staphylococcus xylosus), making it a strong candidate protein antimicrobial. This lytic activity is maintained at the pH (6.7), and the "free" calcium concentration (3 mM) of milk. Truncated endolysin-derived proteins containing only the endopeptidase domain also lyse staphylococci in the absence of the SH3b-binding domain.

Animals↗
PubMed2006

The cell lysis activity of the Streptococcus agalactiae bacteriophage B30 endolysin relies on the cysteine, histidine-dependent amidohydrolase/peptidase domain.

The Streptococcus agalactiae bacteriophage B30 endolysin contains three domains: cysteine, histidine-dependent amidohydrolase/peptidase (CHAP), Acm glycosidase, and the SH3b cell wall binding domain. Truncations and point mutations indicated that the Acm domain requires the SH3b domain for activity, while the CHAP domain is responsible for nearly all the cell lysis activity.

Amidohydrolases↗
PubMed2006

Peptidoglycan hydrolase fusions maintain their parental specificities.

The increased incidence of bacterial antibiotic resistance has led to a renewed search for novel antimicrobials. Avoiding the use of broad-range antimicrobials through the use of specific peptidoglycan hydrolases (endolysins) might reduce the incidence of antibiotic-resistant pathogens worldwide. Staphylococcus aureus and Streptococcus agalactiae are human pathogens and also cause mastitis in dairy cattle. The ultimate goal of this work is to create transgenic cattle that are resistant to mastitis through the expression of an antimicrobial protein(s) in their milk. Toward this end, two novel antimicrobials were produced. The (i) full-length and (ii) 182-amino-acid, C-terminally truncated S. agalactiae bacteriophage B30 endolysins were fused to the mature lysostaphin protein of Staphylococcus simulans. Both fusions display lytic specificity for streptococcal pathogens and S. aureus. The full lytic ability of the truncated B30 protein also suggests that the SH3b domain at the C terminus is dispensable. The fusions are active in a milk-like environment. They are also active against some lactic acid bacteria used to make cheese and yogurt, but their lytic activity is destroyed by pasteurization (63 degrees C for 30 min). Immunohistochemical studies indicated that the fusion proteins can be expressed in cultured mammalian cells with no obvious deleterious effects on the cells, making it a strong candidate for use in future transgenic mice and cattle. Since the fusion peptidoglycan hydrolase also kills multiple human pathogens, it also may prove useful as a highly selective, multipathogen-targeting antimicrobial agent that could potentially reduce the use of broad-range antibiotics in fighting clinical infections.

Amino Acid Sequence↗
PubMed2005

Engineering disease resistant cattle.

Mastitis is a disease of the mammary gland caused by pathogens that find their way into the lumen of the gland through the teat canal. Mammary gland infections cost the US dairy industry approximately $2 billion dollars annually and have a similar impact in Europe. In the absence of effective treatments or breeding strategies to enhance mastitis resistance, we have created transgenic dairy cows that express lysostaphin in their mammary epithelium and secrete the antimicrobial peptide into milk. Staphylococcus aureus, a major mastitis pathogen, is exquisitely sensitive to lysostaphin. The transgenic cattle resist S. aureus mammary gland challenges, and their milk kills the bacteria, in a dose dependent manner. This first step in protecting cattle against mastitis will be followed by introduction of other genes to deal with potential resistance issues and other mastitis causing organisms. Care will be taken to avoid altering milk's nutritional and manufacturing properties. Multi-cistronic constructs may be required to achieve our goals as will other strategies possibly involving RNAi and gene targeting technology. This work demonstrates the possibility of using transgenic technology to address disease problems in agriculturally important species.

Animals↗
PubMed2004

Identification and characterization of metallothionein-1 and -2 gene expression in the context of (+/-)3,4-methylenedioxymethamphetamine-induced toxicity to brain dopaminergic neurons.

In mice, the recreational drug (+/-)3,4-methylenedioxymethamphetamine [MDMA ("ecstasy")] produces a selective toxic effect on brain dopamine (DA) neurons. Using cDNA microarray technology in combination with an approach designed to facilitate recognition of relevant changes in gene expression, the present studies sought to identify genes potentially involved in murine MDMA-induced toxicity to DA neurons. Of 15,000 mouse cDNA fragments studied, metallothionein (Mt)-1 and Mt2 emerged as candidate genes possibly involved in MDMA-induced toxicity to DA neurons. Northern blot analysis confirmed the microarray findings and revealed a dynamic upregulation of Mt1 and Mt2 mRNA in the ventral midbrain within 4-12 hr after MDMA treatment. Western blot analysis showed a similar increase in MT protein levels, with peak times occurring subsequent to increases in mRNA levels. Mt1-2 double knock-out mice were more vulnerable to MDMA-induced toxicity to DA neurons than corresponding wild-type mice. Stimulation of endogenous expression of MT protein with zinc acetate conferred complete protection against MDMA-induced toxicity to DA neurons, and administration of exogenous MT protein afforded partial protection. Collectively, these results indicate that MDMA-induced toxicity to DA neurons is associated with increased Mt1 and Mt2 gene transcription and translation, possibly as part of a neuroprotective mechanism. The present findings may have therapeutic implications for neuropathological conditions involving DA neurons.

3,4-Methylenedioxyamphetamine↗
PubMed2003

Microarray analysis reveals interleukin-6 as a novel secretory product of the hypothalamo-neurohypophyseal system.

Physiological activation of the hypothalamo-neurohypophyseal system (HNS) by dehydration results is a massive release of vasopressin (VP) from the posterior pituitary. This is accompanied by a functional remodeling of the HNS. In this study we used cDNA arrays in an attempt to identify genes that exhibit differential expression in the hypothalamus following dehydration. Our study revealed nine candidate genes, including interleukin-6 (IL-6) as a putative novel secretory product of HNS worthy of further analysis. In situ hybridization and immunocytochemistry confirmed that IL-6 is robustly expressed in the supraoptic (SON) and the paraventricular (PVN) nuclei of the hypothalamus. By double staining immunofluorescence we showed that IL-6 is largely co-localized with VP in the SON and PVN. In situ hybridization, immunocytochemistry, and Western blotting all revealed IL-6 up-regulation in the SON and PVN following dehydration, thus validating the array data. The same dehydration stimulus resulted in an increase in IL-6 immunoreactivity in the axons of the internal zone of the median eminence and a marked reduction in IL-6-like material in the posterior pituitary gland. We thus suggest that IL-6 takes the same secretory pathway as VP and is secreted from the posterior pituitary following a physiological stimulus.

Animals↗
PubMed2003

Role of micro-opioid receptors in formalin-induced pain behavior in mice.

Intraplantar formalin injection is widely used as an experimental model of tonic pain. We investigated the role of endogenous micro-opioid receptor mechanisms in formalin-induced nocifensive behavior in mice. The flinching response induced by formalin (2%, 20 microl) was studied in mice with normal (wild type, n = 8) and absent (homozygous micro-opioid receptor knockout, n = 8) micro-opioid receptor levels. The flinch responses were counted every 5 min for 60 min post-formalin injection. Lumbar spinal cord (L4, 5) was harvested 2 h post-formalin injection to examine c-Fos expression using immunohistochemistry. The effects of naloxone (5 mg/kg, sc) administered 30 min before the intraplantar formalin injection on the flinching response of wild-type mice (n = 7) were also recorded. The second-phase formalin response (10-60 min after formalin) was higher in homozygous micro-opioid receptor knockout mice compared to the wild-type mice (P < 0.01). Naloxone administration in wild-type mice before formalin injection resulted in pain behavior similar to that observed in homozygous micro-opioid receptor knockout mice (P > 0.05). The c-Fos expression induced by formalin injection in the knockout mice was not different from that observed in wild-type mice. Our results suggest that the endogenous micro-opioid system is activated by intraplantar formalin injection and exerts a tonic inhibitory effect on the pain behavior. These results suggest an important modulatory role of endogenous micro-opioid receptor mechanisms in tonic pain states.

Animals↗
PubMed2002

Double round hybridization of membrane based cDNA arrays: improved background reduction and data replication.

Microarray analysis provides the ability to obtain gene expression data on thousands of gene targets in parallel and is being applied to an increasing variety of neurobiological samples. Nylon filter based cDNA arrays often provide an alternative to oligo-based or glass-based gene expression analysis systems. cDNA arrays regardless of the platform, are usually quite expensive to purchase and equally expensive to produce. As such, obtaining both high quality and replicated data, as well as the ability to recover from an apparently failed hybridization experiment are important factors in microarray experimental design. Using a collection of 1152 neuronally relevant cDNAs, this report describes simple modifications in experimental design of radioactive microarray experiments that, (1) improves reproducibility; (2) allows recovery from a failed experiment and; (3) provides a type of replicate data set with no requirement for additional arrays, no additional probe labelings, and little extra cost.

DNA, Complementary↗
PubMed2002

Murine peripherin gene sequences direct Cre recombinase expression to peripheral neurons in transgenic mice.

Spatially and temporally regulated somatic mutations can be achieved by using the Cre/loxP recombination system of bacteriophage P1. To develop a cell type-specific system of gene targeting in the peripheral nervous system, we generated the transgenic mouse lines expressing Cre recombinase under the control of the mouse peripherin gene promoter. The activity of the Cre recombinase during embryonic development was examined by mating the peripherin-Cre transgenic mice to the knock-in Cre-mediated recombination reporter strain, R26R. Analysis of F1 embryos from this cross showed specific excision of loxP-flanked sequences in the dorsal root ganglia, trigeminal ganglia, and olfactory epithelium, in a pattern very similar to the expression of the endogenous mouse peripherin gene, and the previously reported peripherin-lacZ transgenic mice. Thus, the peripherin-Cre mouse described here will provide a valuable tool for Cre-loxP-mediated conditional expression in the peripheral nervous system.

Animals↗
PubMed2002

Changes in gene expression linked to methamphetamine-induced dopaminergic neurotoxicity.

The purpose of these studies was to examine the role of gene expression in methamphetamine (METH)-induced dopamine (DA) neurotoxicity. First, the effects of the mRNA synthesis inhibitor, actinomycin-D, and the protein synthesis inhibitor, cycloheximide, were examined. Both agents afforded complete protection against METH-induced DA neurotoxicity and did so independently of effects on core temperature, DA transporter function, or METH brain levels, suggesting that gene transcription and mRNA translation play a role in METH neurotoxicity. Next, microarray technology, in combination with an experimental approach designed to facilitate recognition of relevant gene expression patterns, was used to identify gene products linked to METH-induced DA neurotoxicity. This led to the identification of several genes in the ventral midbrain associated with the neurotoxic process, including genes for energy metabolism [cytochrome c oxidase subunit 1 (COX1), reduced nicotinamide adenine dinucleotide ubiquinone oxidoreductase chain 2, and phosphoglycerate mutase B], ion regulation (members of sodium/hydrogen exchanger and sodium/bile acid cotransporter family), signal transduction (adenylyl cyclase III), and cell differentiation and degeneration (N-myc downstream-regulated gene 3 and tau protein). Of these differentially expressed genes, we elected to further examine the increase in COX1 expression, because of data implicating energy utilization in METH neurotoxicity and the known role of COX1 in energy metabolism. On the basis of time course studies, Northern blot analyses, in situ hybridization results, and temperature studies, we now report that increased COX1 expression in the ventral midbrain is linked to METH-induced DA neuronal injury. The precise role of COX1 and other genes in METH neurotoxicity remains to be elucidated.

3,4-Dihydroxyphenylacetic Acid↗
PubMed2002

Studies of the biogenic amine transporters. 10. Characterization of a novel cocaine binding site in brain membranes prepared from dopamine transporter knockout mice.

Previous work suggested that the cocaine analog [(125)I]RTI-55 labels a novel binding site in rat brain membranes, which is not associated with the dopamine (DA), serotonin (5-HT), or norepinephrine (NE) transporters [Rothman et al. 1995 J Pharmacol Exp Ther 274:385-395]. Here, we tested whether this site is a product of the DA transporter (DAT) gene. We used a T-antigen knock-in at the DAT gene that results in an effective DAT knock-out (KO) confirmed by Southern blot, DAT immunohistochemistry, and [(125)I]RTI-55 ligand binding. Brain membranes were prepared from frozen whole brain minus caudate of wild-type (WT) B6/Sv129, +/+ and minus sign/minus sign (KO) mice. KO mice were used at approximately 23 days of age. Binding surface analysis of [(125)I]RTI-55 binding to membranes prepared from the brains of WT mice, with 100 nM citalopram to block binding to the 5-HT transporter (SERT), revealed two binding sites: the DAT and a second site, replicating previous studies conducted with rat brains. In the absence of the DAT (minus sign/minus sign mice), binding surface analysis demonstrated that [(125)I]RTI-55 labeled two sites: the NET and a second site called site "X." Structure-activity studies of site "X" demonstrated that high-affinity ligands for the DAT, NET, and SERT have low or negligible affinity for site "X." The relatively high density of site "X" in brain membranes and the fact that the K(i) values of cocaine and cocaethylene for site "X" are in the range achieved in the brain following cocaine administration suggests that site "X" could contribute to the pharmacological or toxicological effects of cocaine. Further progress in delineating the function of site "X" will depend on developing potent and selective agents for this site.

Algorithms↗
PubMed2002

Intravenous cocaine induced-activity and behavioural sensitization in norepinephrine-, but not dopamine-transporter knockout mice.

Previously, it was reported that both norepinephrine transporter (NET) and dopamine transporter (DAT) knockout (KO) mice were sensitive to the reinforcing effects of cocaine. However, assessing the locomotor-stimulant effects of cocaine in these subjects has proven difficult due to significant differences in their baseline activity compared to wild-type controls. The present studies were designed to clarify the role of NET and DAT in the stimulant effects of acute and repeated cocaine utilizing these knockout mice, and thereby assess the role of these substrates in the locomotor stimulant effects of cocaine. Mice were habituated to the test environment for sufficient time to ensure equal baselines at the time of cocaine administration. Mice then received cocaine (3-25 mg/kg) intravenously according to a within-session cumulative dose-response design. Cocaine dosing was repeated at 48-h intervals for four sessions to assess behavioural sensitization. NET-KO mice exhibited a reduced response to acute cocaine administration compared to wild-type (WT) controls. However, comparable sensitization developed in NET-KO and WT mice. The DAT-KO and DAT-heterozygote (HT) mice displayed no locomotor activation following either acute or repeated cocaine administration. These data suggest a role for the NET in the acute response to cocaine, but no involvement in sensitization to cocaine. In contrast, DAT appears to be necessary for both the acute locomotor response to cocaine and the subsequent development of sensitization. In addition to existing data concerning the reinforcing effects of cocaine in DAT-KO mice, these data suggest a dissociation between the reinforcing and locomotor stimulant effects of cocaine.

Animals↗