An assessment of the antibacterial activity in larval excretion/secretion of four species of insects recorded in association with corpses, using Lucilia sericata Meigen as the marker species.

The relative antibacterial activities of excretion/secretion (ES) from two carrion-feeding insects, Calliphora vicina Robineau-Desvoidy and Dermestes maculatus DeGeer, and a detritivore, Tenebrio molitor Linnaeus, were compared to that of Lucilia sericata Meigen, a species with ES of known antibacterial capacity, in order to explore the antimicrobial potential of other carrion and detritivore species. Viable counts were used to assess time-kill of ES against five bacterial species, Staphylococcus aureus, Escherichia coli, Bacillus cereus, Pseudomonas aeruginosa and Proteus mirabilis. Antibacterial activity was recorded in all four insect species although T. molitor and D. maculatus were the most effective in controlling growth of P. mirabilis. The blowflies were more effective in controlling a wider range of both Gram-positive and Gram-negative bacteria. The larval ES from all species was shown to reduce bacterial growth rate although differences in antibacterial spectrum were noted and the degree of potency varied between the four species. These differences may be explained ecologically by the different colonisation times of each insect species on the corpse. Overall, this study demonstrates that research into other carrion-feeding insect species has potential to provide an increased source of antimicrobial chemicals to broaden the range of bacterial species beyond that currently controlled using L. sericata.

Engineering isoflavone metabolism with an artificial bifunctional enzyme.

Plant secondary metabolism has been a focus of research in recent years due to its significant roles in plant defense and in human medicine and nutrition. A protein engineering strategy was designed to more effectively manipulate plant secondary metabolite (isoflavonoid) biosynthesis. A bifunctional isoflavone synthase/chalcone isomerase (IFS/CHI) enzyme was constructed by in-frame gene fusion, and expressed in yeast and tobacco. The fusion protein was targeted to the endoplasmic reticulum (ER) membrane and the individual enzymatic functions of its component fragments were retained when assayed in yeast. Petals and young leaves of IFS/CHI transgenic tobacco plants produced higher levels of the isoflavone genistein and genistein glycosides as a ratio of total flavonoids produced than did plants transformed with IFS alone. Thus, through a combined molecular modeling, in vitro protein engineering and in planta metabolic engineering approach, it was possible to increase the potential for accumulation of isoflavonoid compounds in non-legume plants. Construction of bifunctional enzymes will simplify the transformation of plants with multiple pathway genes, and such enzymes may find broad uses for enzyme (e.g., cytochrome P450 family) and biochemical pathway engineering.

Permeabilizing effects of sub-inhibitory concentrations of microcystin on the growth of Escherichia coli.

Microcystin, a cyanotoxin produced by Microcystis aeruginosa, lacks potent antibacterial activity. When tested in combination, in vitro, inhibitory values for a range of hydrophobic antibiotics were significantly reduced in the presence of at least 1/20 x the minimum inhibitory concentration of microcystin. The degree of inhibition was equivalent to that of a well-characterised permeabilizing agent, polymyxin B nonapeptide. The permeabilizing ability of sub-inhibitory concentrations of microcystin to affect the envelope of Escherichia coli was demonstrated by a rapid and sustained reduction in absorbance values of lysozyme-treated cells and by enhanced uptake of crystal violet in microcystin-treated cultures. Direct effects of appropriate concentrations of microcystin on the integrity of bacterial outer and inner membranes were measured by release of specific enzyme markers. Although the exact mechanism for permeabilizing E. coli with microcystin has not been elucidated, the effects were consistent with permeability changes to the enterobacterial outer membrane caused by polymyxin B nonapeptide.

A study to detect Helicobacter pylori in fresh and archival specimens from patients with interstitial cystitis, using amplification methods.

OBJECTIVES: To detect Helicobacter pylori DNA in fresh and paraffin-embedded bladder biopsy specimens, and thus determine any possible role in interstitial cystitis (IC). MATERIALS AND METHODS: Thirty-three bladder biopsy samples were examined from patients with IC (29 paraffin-embedded and four freshly frozen) diagnosed according to National Institute of Diabetes, Digestive and Kidney Disease criteria. The positive control was a gastric biopsy sample from a patient with a known gastric ulcer caused by H. pylori infection. RESULTS: The anticipated polymerase chain reaction product size of 109 base pairs was obtained with the positive control, whereas none of the other biopsy samples (paraffin embedded or fresh) showed positive amplification specific for H. pylori. CONCLUSIONS: As there was no H. pylori DNA in any of the samples from patients with IC, it is an unlikely candidate in the pathogenesis of IC.

Implementing a new health management information system in Uganda.

This paper reports on research investigating the health management information system (HMIS) implementation process in Uganda, utilizing the diffusion of innovation and dynamic equilibrium organizational change models. Neither perspective guided the HMIS development process. Instead, technological issues, rather than wider organizational issues, dominated the planned change. The need to consider the organizational context when changing information systems arises because the process is more complex than some practitioners have realized, when attempting to understand the causes of information management problems and developing HMIS in low-income countries. In particular, information system developers had not acknowledged that they were promoting an informational approach to management when they promoted a change from a centralized reporting system to a HMIS supporting use of information at the level of collection. Strategies to facilitate this approach were not advocated. Organizational theory can contribute to the diffusion of innovation framework. It has yielded an integration of Rogers's diffusion of innovation framework and Leavitt's concept of organizational forces in equilibrium. The diffusion framework describes the process, but the organizational model has given the context and reason for aspects of the process. The diffusion model does not predict what needs to change within the organization when a particular innovation is introduced, or how much. The addition of the organizational model has helped. These frameworks can facilitate the introduction of future information management innovations and allow practitioners to perceive their introduction as a staged process needing to be managed. Implications for practice are identified.

Rejection of an innovation: health information management training materials in east Africa.

A shift towards decentralization in many low-income countries has meant more skills are demanded of primary health care managers, including data and information handling at all levels of the health care system. Ministries of Health are changing their central reporting health information systems to health management information systems with emphasis on managers utilizing information at the point of collection. This paper reports on a research study to investigate the introduction of new information management strategies intended to promote an informational approach to management at the operational health service level in low-income countries. It aims to understand the process taking place when externally developed training materials (PHC MAP), which are intended to strengthen health management information systems, are introduced to potential users in an east African country. A case study has been undertaken and this research has demonstrated that the dynamic equilibrium approach to organizational change is applicable to the introduction of new information management strategies and management approaches in low-income countries. Although PHC MAP developers envisaged a technical innovation needing implementation, potential users saw the situation as one of organizational change. Contributions to theory have been made and many implications for introducing new information systems or the informational approach to management are identified. This theoretical framework could also facilitate the introduction of future information management innovations and would allow practitioners to perceive the introduction of information management innovations as one of organizational change that needs to be managed. Consequently, issues that may facilitate or inhibit adoption could be identified in advance.

A selectin inhibitor decreases neutrophil infiltration during acute Mannheimia haemolytica pneumonia.

The degree to which the selectin inhibitor TBC1269 reduces neutrophil infiltration in specific microscopic locations of the lung during acute pneumonia of neonates was determined. Neonatal calves were inoculated intrabronchially with Mannheimia (Pasteurella) haemolytica or saline, and lung tissue was collected at 2 and 6 hours postinoculation (PI). One 6-hour group inoculated with M. haemolytica received TBC1269 intravenously before and after inoculation with M. haemolytica. Infiltrates of neutrophils were significantly higher in the alveolar lumen and septae but lower in the bronchial lumen and epithelium at 6 hours PI than at 2 hours PI. Significantly fewer neutrophils (P < 0.05) were present in the alveolar lumen and septae, and the bronchiolar lumen and lamina propria in the lungs of TBC1269-treated calves compared with untreated calves at 6 hours PI. TBC1269 did not alter the infiltration into bronchi and blood vessels or the expression of the selectin-independent adhesion molecule, ICAM-1. This work suggests that during acute pneumonia of neonates 1) neutrophil infiltrates progressively increase in the alveolar lumens and septae but decrease in the bronchial lumen and epithelium with time, 2) TBC1269 reduces neutrophil infiltration into specific regions of alveoli and bronchioles rather than uniformly throughout the lung, and 3) selectin inhibition does not affect the location and intensity of ICAM-1 expression.

Chemical syntheses of caffeoyl and 5-OH coniferyl aldehydes and alcohols and determination of lignin O-methyltransferase activities in dicot and monocot species.

To investigate the substrate preferences of O-methyltransferases in the monolignol biosynthetic pathways, caffeoyl and 5-hydroxy coniferyl aldehydes were synthesized by a new procedure involving a Wittig reaction with the corresponding hydroxybenzaldehydes. The same procedure can also be used to synthesize caffeoyl and 5-hydroxyconiferyl alcohols. Relative O-methyltransferase activities against these substrates were determined using crude extracts and recombinant caffeic acid O-methyltransferase from alfalfa (Medicago sativa), and crude extracts from the model legume Medicago truncatula, tobacco, wheat and tall fescue. Extracts from all these species catalyzed methylation of the various monolignol aldehydes and alcohols more effectively than the corresponding hydroxycinnamic acids.

Elicitor-induced association of isoflavone O-methyltransferase with endomembranes prevents the formation and 7-O-methylation of daidzein during isoflavonoid phytoalexin biosynthesis.

The bioactive isoflavonoids of the Leguminosae often are methylated on the 4'-position of their B-rings. Paradoxically, reverse genetic evidence implicates alfalfa isoflavone O-methyltransferase (IOMT) in the biosynthesis of 4'-O-methylated isoflavonoids such as the phytoalexin medicarpin in vivo, whereas biochemical studies indicate that IOMT has strict specificity for methylation of the A-ring 7-hydroxyl of daidzein, the presumed substrate for O-methylation, in vitro. Radiolabeling and isotope dilution studies now confirm that daidzein is not an intermediate in isoflavonoid phytoalexin biosynthesis in alfalfa. Furthermore, protein gel blot analysis and confocal microscopy of a transiently expressed IOMT-green fluorescent protein fusion in alfalfa leaves show that the operationally soluble IOMT localizes to endomembranes after elicitation of the isoflavonoid pathway. We propose that IOMT colocalizes with the endoplasmic reticulum-associated isoflavone synthase cytochrome P450 to ensure rapid B-ring methylation of the unstable 2,4',7-trihydroxyisoflavanone product of isoflavone synthase, thereby preventing its dehydration to daidzein and subsequent A-ring methylation by free IOMT. In this way, metabolic channeling at the entry point into isoflavonoid phytoalexin biosynthesis protects an unstable intermediate from an unproductive metabolic conversion.

Improvement of in-rumen digestibility of alfalfa forage by genetic manipulation of lignin O-methyltransferases.

Lignin inhibits forage digestibility by ruminant animals, and lignin levels and the proportion of dimethylated syringyl (S) lignin monomers increase with progressive maturity in stems of forage crops. We generated transgenic alfalfa (Medicago sativa L.) with reduced lignin content and altered lignin composition. Down-regulation of caffeic acid 3-O-methyltransferase (COMT) reduces lignin content, accompanied by near total loss of S lignin, whereas down-regulation of caffeoyl coenzyme A 3-O-methyltransferase (CCoAOMT) reduces lignin content without reduction in S lignin. These changes are not accompanied by altered ratios of cell wall polysaccharides. Analysis of rumen digestibility of alfalfa forage in fistulated steers revealed improved digestibility of forage from COMT down-regulated plants, but a greater improvement in digestibility following down-regulation of CCoAOMT. The results indicate that both lignin content and composition affect digestibility of alfalfa forage, and reveal a new strategy for forage quality improvement by genetic manipulation of CCoAOMT expression.

Yesterday's hair--human hair in archaeology.

Hair removed from archaeological burials can tell us a lot about the diet and lifestyle of our ancestors--information that may survive because of the unique biology of hair formation. But hair is also biodegradable and the effects of time and burial conditions can result in conflicting evidence of past lives.

Mycolic acids and ancient DNA confirm an osteological diagnosis of tuberculosis.

SETTING: The underlying trends in the past epidemiology of tuberculosis (TB) are obscure, requiring recourse to the archaeological record. It would therefore be of value to develop methods for reliable TB diagnosis in ancient populations. OBJECTIVE: To test the capability of two biomarkers, Mycobacterium tuberculosis complex mycolic acids and a DNA target (IS6110), for confirming an osteological diagnosis of TB in medieval individuals, based on the presence of Pott's disease and/or rib lesions. DESIGN: Osteological examination of three archaeological individuals (Medieval: approximately 1000 years old) revealed a Pott's disease case, one with no changes consistent with TB and one with rib lesions. Rib samples from these individuals were examined for the presence of Mycobacterium tuberculosis complex mycolic acids and mycobacterial DNA. RESULTS: Mycobacterium tuberculosis complex mycolic acids and the DNA target were detected in the Pott's disease case, whilst mycolic acids (insufficient for confirmation) alone were detected in the rib lesion case. CONCLUSIONS: Biomarkers provide a sensitive tool to detect ancient TB. Mycobacterium tuberculosis DNA is not distributed homogeneously, making multiple sampling essential. Mycolic acids seem more reliable for ancient TB diagnosis than IS6110. The demonstrated stability of mycolic acids show that they may be of value in tracing the palaeoepidemiology of tuberculosis back into antiquity.

Natural products and plant disease resistance.

Plants elaborate a vast array of natural products, many of which have evolved to confer selective advantage against microbial attack. Recent advances in molecular technology, aided by the enormous power of large-scale genomics initiatives, are leading to a more complete understanding of the enzymatic machinery that underlies the often complex pathways of plant natural product biosynthesis. Meanwhile, genetic and reverse genetic approaches are providing evidence for the importance of natural products in host defence. Metabolic engineering of natural product pathways is now a feasible strategy for enhancement of plant disease resistance.

The biosynthesis of monolignols: a "metabolic grid", or independent pathways to guaiacyl and syringyl units?

Lignin is a complex polymer formed by the oxidative polymerization of hydroxycinnamyl alcohol derivatives termed monolignols. The major monolignols in dicotyledonous angiosperm lignin are monomethylated guaiacyl (G) units derived from coniferyl alcohol, and dimethylated syringyl (S) units derived from sinapyl alcohol. The biochemical pathways leading to the formation of monolignols feature successive hydroxylation and O-methylation of the aromatic ring and conversion of the side chain carboxyl to an alcohol function. The current view of the monolignol biosynthetic pathway envisages a metabolic grid leading to G and S units, through which the successive hydroxylation and O-methylation reactions may occur at different levels of side chain oxidation. The present article assesses biochemical and genetic evidence for and against such a model, including recent data on the methylation reactions of monolignol biosynthesis in alfalfa. We draw attention to portions of the currently accepted monolignol pathway that may require revision, and suggest an alternative model in which metabolic channeling allows for independent pathways to G and S lignin.

Comparison of bronchoalveolar lavage fluid obtained from Mannheimia haemolytica-inoculated calves with and without prior treatment with the selectin inhibitor TBC1269.

OBJECTIVES: To determine effects of selectin inhibitor TBC1269 on neutrophil infiltration, and neutrophil-associated injury during pneumonia induced by Mannheimia haemolytica and concentration of antimicrobial anionic peptide (AAP) in bronchoalveolar lavage fluid (BALF) as well as antimicrobial activity of BALF from healthy (control) neonatal calves, neonatal calves with M haemolytica-induced pneumonia, neonatal calves with prior treatment with TBC1269, and adult cattle. ANIMALS: Eighteen 1- to 3-day-old calves and 9 adult cattle. PROCEDURE: Calves were inoculated with M haemolytica or pyrogen-free saline (0.14M NaCl) solution into the right cranial lung lobe, and BALF was collected 2 or 6 hours after inoculation. Thirty minutes before and 2 hours after inoculation, 4 calves received TBC1269. The BALF collected from 9 adult cattle was used for comparison of BALF AAP concentration and antimicrobial activity. Protein concentration and neutrophil differential percentage and degeneration in BALF were determined. An ELISA and killing assay were used to determine BALF AAP concentration and antimicrobial activity, respectively. RESULTS: Total protein concentration was significantly decreased in BALF from calves receiving TBC1269. Similar concentrations of AAP were detected in BALF from all calves, which were 3-fold higher than those in BALF from adult cattle. However, BALF from neonates had little or no anti-M haemolytica activity. CONCLUSIONS AND CLINICAL RELEVANCE: These results suggest that TBC1269 decreases pulmonary tissue injury in neonatal calves infected with M haemolytica. Although AAP is detectable in neonatal BALF at 3 times the concentration detected in adult BALF, neonatal BALF lacks antimicrobial activity for M haemolytica.

Acyl substitution at the ortho position of anilides enhances oral bioavailability of thiophene sulfonamides: TBC3214, an ETA selective endothelin antagonist.

Sitaxsentan (3, TBC11251) (Wu et al. J. Med. Chem. 1997, 40, 1690) is an orally active ET(A) selective endothelin antagonist that attenuates pulmonary vascular hypertension and cardiac hypertrophy in rats (Tilton et al. Pulm. Pharmacol. Ther. 2000, 13, 87). It has demonstrated efficacy in a phase II clinical trial for congestive heart failure (Givertz et al. Circulation 2000, 101, 2922). During the discovery of 3, we observed several structure-oral bioavailability relationships. To investigate whether there is any generality in these trends, we synthesized some similar pairs of compounds in the latest series (Wu et al. J. Med. Chem. 1999, 42, 4485) and evaluated their oral properties. In both series, an acyl group at the 2-position of the anilide of these thiophene sulfonamides improved oral bioavailability. As a result of this exercise, TBC3214 (17) was identified as a sitaxsentan follow-on candidate. It is very potent (IC(50) for ET(A) = 40 pM) and highly selective for ET(A) vs ET(B) receptors (400 000-fold), with a half-life of >4 h and oral bioavailability of 25% in rats, 42% in cats, and 70% in dogs.

Well-being as a moving target: measurement equivalence of the Bradburn Affect Balance Scale.

Although the Bradburn Affect Balance scale (ABS) is a frequently used two-factor indicator of well-being in later life, its measurement and invariance properties are not well documented. We examined these issues using confirmatory factor analyses of cross-sectional (adults ages 54-87 years) and longitudinal data from the Victoria Longitudinal Study. Stability of the positive and negative affect factors was moderate across a 3-year period. Overall, factor loadings for positive affect items were invariant over time with the exception of the pleased item. Negative affect items were time invariant. However, age-group comparisons between young-old and old-old groups revealed age differences in loadings for the upset item at Time 1. Finally, gender groups differed in loadings for the top of the world and going your way items. Thus a pattern of partial measurement equivalence characterized item response to the ABS. Our results suggest that group comparisons and longitudinal change in ABS scale scores of positive and negative affect should be interpreted with caution.

Effects of the synthetic selectin inhibitor TBC1269 on tissue damage during acute Mannheimia haemolytica-induced pneumonia in neonatal calves.

OBJECTIVE: To determine effects of the selectin inhibitor TBC1269 on neutrophil-mediated pulmonary damage during acute Mannheimia haemolytica-induced pneumonia in newborn calves. ANIMALS: Eighteen 1- to 3-day-old colostrum-deprived calves. PROCEDURE: Mannheimia haemolytica or saline (0.9% NaCl) solution was inoculated in both cranial lung lobes of 12 and 6 calves, respectively. Calves were euthanatized 2 (saline, n = 3; M haemolytica, n = 4) or 6 hours (saline, n = 3; M haemolytica, n = 8) after inoculation. Four M haemolytica-inoculated calves euthanatized at 6 hours also received TBC1269 (25 mg/kg, IV) 30 minutes before and 2 hours after inoculation. Conjugated diene (CD) concentrations, inducible nitric oxide synthase (iNOS) expression, and apoptotic cell counts were determined in lung specimens collected during necropsy. RESULTS: Conjugated diene concentrations were significantly increased in all M haemolytica-inoculated groups, compared with saline-inoculated groups. Calves treated with TBC1269 had decreased concentrations of CD, compared with untreated calves, although the difference was not significant. Number of apoptotic neutrophils and macrophages increased significantly inTBC1269-treated calves, compared with untreated calves. Inducible nitric oxide synthase was expressed by epithelial cells and leukocytes. However, iNOS was less abundant in airway epithelial cells associated with inflammatory exudates. Degree of iNOS expression was similar between TBC1269-treated and untreated calves. CONCLUSIONS: Mannheimia haemolytica infection in neonatal calves resulted in pulmonary tissue damage and decreased epithelial cell iNOS expression. The selectin inhibitor TCB1269 altered, but did not completely inhibit, neutrophil-mediated pulmonary damage.