Synthesis and evaluation of delta-lactams (piperazones) as elastase inhibitors.
A series of monocyclic delta-lactams (piperazones) was prepared and analysed as inhibitors of porcine pancreatic elastase and human neutrophil elastase.
Animals↗
Biomedical subjects
David Brown
Publications and source records attributed to David Brown.
Apoptosis in Parkinson's disease: signals for neuronal degradation.
Controversy has surrounded a role for apoptosis in the loss of neurons in Parkinson's disease (PD). Although a variety of evidence has supported an apoptotic contribution to PD neuronal loss particularly in the nigra, two factors have weighed against general acceptance: (1) limitations in the use of in situ 3' end labeling techniques to demonstrate nuclear DNA cleavage; and (2) the insistence that a specific set of nuclear morphological features be present before apoptotic death could be declared. We first review the molecular events that underlie apoptotic nuclear degradation and the literature regarding the unreliability of 3' DNA end labeling as a marker of apoptotic nuclear degradation. Recent findings regarding the multiple caspase-dependent or caspase-independent signaling pathways that mediate apoptotic nuclear degradation and determine the morphological features of apoptotic nuclear degradation are presented. The evidence shows that a single nuclear morphology is not sufficient to identify apoptosis and that a cytochrome c, pro-caspase 9, and caspase 3 pathways is operative in PD nigral apoptosis. BAX-dependent increases in mitochondrial membrane permeability are responsible for the release of mitochondrial factors that signal for apoptotic degradation, and increased BAX levels have been found in a subset of PD nigral neurons. Studies using immunocytochemistry in PD postmortem nigra have begun to define the premitochondrial apoptosis signaling pathways in the disease. Two, possibly interdependent, pathways have been uncovered: (1) a p53-glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-BAX pathway; and (2) FAS receptor-FADD-caspase 8-BAX pathway. Based on the above, it seems unlikely that apoptosis does not contribute to PD neuronal loss, and the definition of the premitochondrial signaling pathways may allow for the development and testing of an apoptosis-based PD therapy.
Apoptosis↗
Polarised secretion of cytokines in primary human microvascular endothelial cells is not dependent on N-linked glycosylation.
Human microvascular endothelial cells (HMVEC) grow in monolayers on Transwell filters and restrict permeability between the apical and basolateral media. We show that these cell monolayers are capable of sorting labelled endogenous proteins, including chemokines, growth factors and cytokines, to either the apical or basolateral media. IL-8 and GMCSF were secreted predominantly into the apical medium, whereas MIC-1 was secreted into the basolateral medium. This polarity did not correlate with glycosylation, as IL-8 and MIC-1 are both N-glycosylated, but were sorted to opposite sides of the cell. IL-6 is not glycosylated and did not display significant polarity in secretion. Similarly, the polarity of secretion of endogenous glycoproteins was not related to their glycosylation.
Adenoviridae↗
The astrocyte/meningeal cell interface is a barrier to neurite outgrowth which can be overcome by manipulation of inhibitory molecules or axonal signalling pathways.
Invading meningeal cells form a barrier to axon regeneration after damage to the spinal cord and other parts of the CNS, axons stopping at the interface between meningeal cells and astrocytes. Axon behavior was examined using an in vitro model of astrocyte/meningeal cell interfaces, created by plating aggregates of astrocytes and meningeal cells onto coverslips. At these interfaces growth of dorsal root ganglion axons attempting to grow from astrocytes to meningeal cells was blocked, but axons grew rapidly from meningeal cells onto astrocytes. Meningeal cells were examined for expression of axon growth inhibitory molecules, and found to express NG2, versican, and semaphorins 3A and 3C. Astrocytes express growth promoting molecules, including N-Cadherin, laminin, fibronectin, and tenascin-C. We treated cultures in various ways to attempt to promote axon growth across the inhibitory boundaries. Blockade of NG2 with antibody and blockade of neuropilin 2 but not neuropilin 1 both promoted axon growth from astrocytes to meningeal cells. Blockade of permissive molecules on astrocytes with N-Cadherin blocking peptide or anti beta-1 integrin had no effect. Manipulation of axonal signalling pathways also increased axon growth from astrocytes to meningeal cells. Increasing cAMP levels and inactivation of rho were both effective when the cultures were fixed in paraformaldehyde, demonstrating that their effect is on axons and not via effects on the glial cells.
Animals↗
Comparative pharmacophore development for inhibitors of human and rat 5-alpha-reductase.
There are a number of diseases where the 5-alpha-reductase (5AR) enzyme is of therapeutic interest as a drug target. Currently the crystal structure for 5-alpha-reductase is unavailable, thus ligand-based pharmacophore techniques are beneficial in the drug development process. We have developed pharmacophores to aid inhibitor design for both human types I (preliminary) and II 5-alpha-reductase isozymes and also the rat type II isozyme. To our knowledge, these are the first published pharmacophores for inhibitors of the human type I and rat type II enzymes. A comparison between isozymes and the previously published human type II isozyme pharmacophore is also presented.
Animals↗
Development of a measles specific IgM ELISA for use with serum and oral fluid samples using recombinant measles nucleoprotein produced in Saccharomyces cerevisiae.
In order to develop sensitive assays for detecting measles antibodies in oral fluid specimens, we have produced recombinant measles virus nucleoprotein (rMVN) in a yeast expression system and prepared monoclonal antibodies to the protein. Measles nucleoprotein gene from the Schwarz vaccine strain was cloned into a yeast expression vector, pFX7 under the control of the hybrid GAL10-PYK1 promoter. High levels of rMVN (20 mg/litre of yeast culture) were generated. Electron microscopy showed that the purified rMVN assembled into typical herring-bone structures. Monoclonal antibodies produced to the rMVN also reacted with native measles virus N in immunofluorescence tests. The purified rMVN and a monoclonal antibody to the rMVN conjugated to horseradish peroxidase were used to develop a measles specific IgM capture EIA (MACEIA) in both serum and oral fluid specimens. Evaluations of the MACEIA were performed by testing a) serum samples (n=80) and b) paired oral fluid/serum samples from measles cases (n=50, representing 16 cases) and oral fluids from controls with non-measles rash (n=59, representing 48 cases). The samples were also tested for measles IgM, using a reference radioimmunoassay (MACRIA). The sensitivity and specificity of the MACEIA compared with MACRIA for a) the serum samples were 100 and 96.6% respectively and b) for paired serum/oral fluids samples 100 and 100%, respectively.
Animals↗
A longitudinal study of United Kingdom pharmacists' misdemeanours--trials, tribulations and trends.
BACKGROUND: Standards of UK pharmacy practice are maintained by the Royal Pharmaceutical Society of Great Britain, which has the power to take a range of sanctions, including removal of the right to practice, against those found guilty of malpractice. This function is currently under review. OBJECTIVE: To conduct a longitudinal study in order to define trends and identify areas where remedial or preventative support could be focused. METHOD: Case analysis of reports of individuals' misdemeanours published in the British Pharmaceutical Journal over a 12-year period (September 1988-October 2000). Professional and personal misdemeanours were considered. MAIN OUTCOME MEASURE: Nature of misdemeanour, conviction or disciplinary proceedings against individual, practising pharmacists in the study period. Reasons offered for committing the misdemeanour and penalties applied. RESULTS: 344 cases, involving a wide range of personal (162) and professional (590) misdemeanours were found. On an annual basis, the maximum incidence of pharmacists found guilty of any misdemeanour was extremely low (< 0.1 of 1% on the pharmaceutical register). The most common professional misdemeanour was failure to keep adequate written records. The most common personal misdemeanour was fraud. The most common reason cited for committing any misdemeanour was financial gain. Numbers in individual offence categories were persistent but low and there were few obvious trends with time. The odds of involvement ratio for male versus female pharmacists was 7.36 (CI: 5.23-10.35) and for ethnic minority versus Caucasian pharmacists was 3.8 (CI: 3.06-4.72). The most stringent penalties (either imprisonment or removal of the right to practice and frequently both) were applied to cases involving personal use or trafficking of drugs subject to abuse. CONCLUSIONS: The current self-regulation of pharmacy practice in the UK involves a wide range of misdemeanours of varying severity; but the incidence of reports of pharmacists found guilty of malpractice was extremely low. The nature of misdemeanours appeared to change little over the period of the study; this study therefore indicates the spectrum of misdemeanours likely to be encountered by a regulating board in the immediate to medium-term future. If regulatory changes such as competence-based practice rights are introduced, the spectrum may change.
Codes of Ethics↗
Hydroxyurea-induced pneumonitis in a patient with chronic idiopathic myelofibrosis after prolonged drug exposure.
We describe a 63-yr-old patient diagnosed with chronic idiopathic myelofibrosis whose disease was treated with hydroxyurea for 2 yr. He developed respiratory symptoms that were extensively investigated including lung biopsy. Clinical, radiological and histological features were compatible with a diagnosis of hydroxyurea-induced pneumonitis. Following drug withdrawal there was remarkable improvement in clinical and radiological findings.
Diagnosis, Differential↗
Incidence and risk factors of paediatric rotavirus diarrhoea in northern Ghana.
We measured the type-specific incidence of paediatric rotavirus diarrhoea in an area of northern Ghana. Over 1 year, diarrhoea 1717 episodes were identified, of which 677 (39%) were positive for rotavirus. Risk factors for rotavirus infection included old age, wasting, high Vesikari score and the episode occurring in the dry season. Rotavirus-positive episodes tended to be more acute, causing vomiting and greater dehydration, and were more likely to require hospitalization. The incidence was 0.089 episodes per person-year for all diarrhoea, and 0.035 for rotavirus diarrhoea. The observed incidence decreased markedly with distance from the nearest health centre, suggesting a large unobserved burden. G2P[6], G3P[4] and G9P[8] made up more than half the genotypes detected, but the remainder were diverse. There is a large burden of rotavirus diarrhoea, but the effectiveness of future vaccines could be diluted by the high polymorphism of the virus, and the difficulty of reaching remote populations.
Child, Preschool↗
International collaborative study to compare reverse transcriptase PCR assays for detection and genotyping of noroviruses.
To allow more rapid and internationally standardized assessment of the spread of noroviruses (previously called Norwalk-like viruses [NLVs]) as important food-borne pathogens, harmonization of methods for their detection is needed. Diagnosis of NLVs in clinical diagnostic laboratories is usually performed by reverse transciptase PCR (RT-PCR) assays. In the present study, the performance of five different RT-PCR assays for the detection of NLVs was evaluated in an international collaborative study by five laboratories in five countries with a coded panel of 91 fecal specimens. The assays were tested for their sensitivity, detection limit, and ease of standardization. In total, NLVs could be detected by at least one RT-PCR assay in 69 (84%) of the samples that originally tested positive. Sensitivity ranged from 52 to 73% overall and from 54 to 100% and 58 to 85% for genogroup I and II viruses, respectively. In all, 64% of the false-negative results were obtained with a set of diluted stools (n = 20) that may have lost quality upon storage. Sensitivity was improved when these samples were excluded from analysis. No one single assay stood out as the best, although the p1 assay demonstrated the most satisfactory overall performance. To promote comparability of data, this assay will be recommended for newly starting groups in future collaborative studies.
Caliciviridae Infections↗
Changing patterns of rotavirus genotypes in ghana: emergence of human rotavirus G9 as a major cause of diarrhea in children.
Genotyping of human rotaviruses was performed on 312 rotavirus-positive samples collected from 2,205 young children with diarrhea in the Upper East District of Ghana, a rural community. Of the 271 (86.9%) rotavirus strains that could be VP7 (G) or VP4 (P) characterized, 73 (26.9%) were of G9 specificity. The predominant G9 genotype was G9P[8], which constituted 79.5% of all G9 strains detected, followed by G9P[6] (12.3%), G9P[10] (2.7%), and G9P[4] (1.3%). G9 strains with mixed P types constituted 2.7% of all G9 strains found in the study. All the G9P[8] strains had a long RNA electrophoretic pattern with VP6 subgroup II specificity. Four G9 isolates, GH1319, GH1416, GH3550, and GH3574, which were selected based on the abundance of stool material and were representative of the three electropherotypes observed, were cloned and sequenced. The Ghanaian isolates shared more than 98% sequence nucleotide homology with other G9 strains from the United States (US1205), Malawi (MW69), Brazil (R160), Japan (95H115), and Nigeria (Bulumkutu). However, they showed only 95% nucleotide homology with the Thai G9 strain Mc345. Phylogenetic analysis of the nucleic acid sequence revealed the existence of at least three clusters, with Ghanaian strains forming one cluster, Nigerian and Brazilian strains forming a second cluster, and U.S., Malawian, and Japanese strains forming a third.
Antigens, Viral↗
Diarylurea compounds inhibit caspase activation by preventing the formation of the active 700-kilodalton apoptosome complex.
The release of mitochondrial proapoptotic proteins into the cytosol is the key event in apoptosis signaling, leading to the activation of caspases. Once in the cytosol, cytochrome c triggers the formation of a caspase-activating protein complex called the apoptosome, whereas Smac/Diablo and Omi/htra2 antagonize the caspase inhibitory effect of inhibitor of apoptosis proteins (IAPs). Here, we identify diarylurea compounds as effective inhibitors of the cytochrome c-induced formation of the active, approximately 700-kDa apoptosome complex and caspase activation. Using diarylureas to inhibit the formation of the apoptosome complex, we demonstrated that cytochrome c, rather than IAP antagonists, is the major mitochondrial caspase activation factor in tumor cells treated with tumor necrosis factor. Thus, we have identified a novel class of compounds that inhibits apoptosis by blocking the activation of the initiator caspase 9 by directly inhibiting the formation of the apoptosome complex. This mechanism of action is different from that employed by the widely used tetrapeptide inhibitors of caspases or known endogenous apoptosis inhibitors, such as Bcl-2 and IAPs. Thus, these compounds provide a novel specific tool to investigate the role of the apoptosome in mitochondrion-dependent death paradigms.
Apoptosis↗
Strategies for setting a national research agenda that is responsive to community needs.
Setting a national environmental health research agenda requires broad public input, including that from leading scientists, health care professionals, and communities. Contributions from these diverse constituencies are essential to formulating a research and education strategy that both advances our understanding of the causes and mechanisms of environmentally related diseases and translates such findings into effective prevention and clinical applications to protect those most affected by adverse environmental exposures. Given the increasing number of individual researchers working with communities to address environmental health needs during the past decade, it is also essential for research institutions to foster relationships with communities to understand and respond to their unique public health needs, as well as to communicate research advances in a manner that is both understandable and culturally appropriate. To achieve broad public input and to foster community-university partnerships, the National Institute of Environmental Health Sciences (NIEHS) supports various workshops, roundtables, and advisory groups. In particular, the NIEHS finds Town Meetings to be a successful model for bringing academic researchers together with community residents, state and local departments of health, and community-based organizations to foster greater awareness of community needs, public health needs, and environmental health science research. Since 1998, the NIEHS has supported 16 Town Meetings across the country. In this article we highlight the major outcomes of these meetings to demonstrate the effectiveness of this mechanism for enhancing cooperation among researchers, community residents, and public health officials with the goal of improving public health and setting a national research agenda.
Community Health Planning↗
Viral gastroenteritis outbreaks in Europe, 1995-2000.
To gain understanding of surveillance and epidemiology of viral gastroenteritis outbreaks in Europe, we compiled data from 10 surveillance systems in the Foodborne Viruses in Europe network. Established surveillance systems found Norovirus to be responsible for >85% (N =3,714) of all nonbacterial outbreaks of gastroenteritis reported from 1995 to 2000. However, the absolute number and population-based rates of viral gastroenteritis outbreaks differed markedly among European surveillance systems. A wide range of estimates of the importance of foodborne transmission were also found. We review these differences within the context of the sources of outbreak surveillance information, clinical definitions, and structures of the outbreak surveillance systems.
Data Collection↗
Early identification of common-source foodborne virus outbreaks in Europe.
The importance of foodborne viral infections is increasingly recognized. Food handlers can transmit infection during preparation or serving; fruit and vegetables may be contaminated by fecally contaminated water used for growing or washing. And the globalization of the food industry mean that a contaminated food item may not be limited to national distribution. International outbreaks do occur, but little data are available about the incidence of such events and the food items associated with the highest risks. We developed a combined research and surveillance program for enteric viruses involving 12 laboratories in 9 European countries. This project aims to gain insight into the epidemiology of enteric viruses in Europe and the role of food in transmission by harmonizing (i.e., assessing the comparability of data through studies of molecular detection techniques) and enhancing epidemiologic surveillance. We describe the setup and preliminary results of our system, which uses a Web-accessible central database to track viruses and provides the foundation for an early warning system of foodborne and other common-source outbreaks.
Clinical Laboratory Techniques↗
Exuberant cellular reaction of the optic nerves in experimental Creutzfeldt-Jakob disease.
We report here on the exuberant glial reaction in the optic nerves affected by prion diseases. Optic nerves from CJD- and GSS-, and scrapie-infected mice and hamsters showed severe pathology. These lesions were qualitatively indistinguishable from each other but were more intense in the Fujisaki model than in the hamsters inoculated with Echigo-1. Exuberant cellular reaction comprised of macrophages containing numerous mitochondria, abundant rough endoplasmic reticulum, and secondary lysosomes filled with digested myelin debris, electron-dense material and occasionally, entire myelin-bound vacuoles were readily observed in both models. Macrophages actively digesting myelin fragments and containing lyre-like bodies and paracrystalline inclusions were frequently noted. Some macrophages extended long filopodia to form labyrinth-like structures, and within a few macrophages, concentric arrays of cisterns and channels sequestrated part of the cytoplasm. An analogous network of narrow cisterns was seen to surround whole segments of the myelinated fibers.
Animals↗
Macrophage inhibitory cytokine-1: a novel biomarker for p53 pathway activation.
The p53 tumor suppressor protein plays a key function in the cellular response to stress by activating a subset of genes responsible for cell cycle arrest and apoptosis. Activation of the p53 pathway in tumor cells has been proposed as a novel approach to cancer therapy and substantial efforts have been dedicated to the discovery of pharmacological p53 activators. Here, we show that the transforming growth factor-beta superfamily cytokine, macrophage inhibitory cytokine-1 (MIC-1), can serve as a secreted biomarker for activation of p53 in both cellular and xenograft models of human cancer. Using doxorubicin treatment in the HCT116 colon cancer cell line, we have shown that MIC-1 secretion into culture media is correlated with p53 pathway activation as measured by the up-regulation of its downstream transcriptional target p21. When transplanted into nude mice, HCT116 cells continued to secret human MIC-1 and mouse plasma levels correlated well with tumor volume. Treatment of these animals with a single dose of doxorubicin led to activation of the p53 pathway and a nearly 4-fold elevation of the plasma MIC-1 level, which was paralleled by p21 induction in the tumor xenografts. Estimation of MIC-1 concentration, both in vivo and in vitro, represents a novel tool for the study of p53 pathway and development of p53-activating therapeutics.
Animals↗
[A survey on the dioxin level in breast milk in coastal and inland region].
OBJECTIVE: To study the dioxin level of breast milk among Chinese mothers, and to assess the dioxin intake of new-born babies from mother's milk and compare with the Tolerable Daily Intake (TDI) of dioxin. METHODS: The CALUX bioassay was used to detect the dioxin concentration of the first time mother's milk among the inland samples (Shenyang region; 32 cases) and the coastal city samples (Dalian region; 47 cases). RESULTS: The median value of the dioxin Toxic Equivalence (TEQ) in breast milk in the Dalian region was 15.84 pg TEQs.g(-1) fat, which was significantly higher than that in the Shenyang region 7.21 pg TEQs.g(-1) fat (P < 0.01). CONCLUSION: The dioxin level in breast milk in Chinese is at the world's average level. The dioxin intake of the new-born babies during the period of lactation was higher than the lowest limit of the Tolerable Daily Intake (TDI) proposed by WHO. This situation should be noticed by the related authorities.
China↗