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Colonial organisms host a large diversity of symbionts (collectively, parasites, mutualists, and commensals) that use vertical transmission (from parent colony to offspring colony) and/or horizontal transmission to disperse between host colonies. The early life of some colonies, characterized by the dispersal and establishment of solitary individuals, may constrain vertical transmission and favor horizontal transmission between large established colonies. We explore this possibility with the miniature cockroach Attaphila fungicola, a symbiont of leaf‐cutter ants and the mutualist fungal gardens they cultivate. The early life of a leaf‐cutter colony is characterized by the dispersal of a female alate (winged “queen”) carrying a fungal pellet, and the subsequent establishment of a foundress (workerless “queen”) raising her incipient fungal garden and colony. Roaches hitchhike on female alates during leaf‐cutter nuptial flights, which strongly suggests that roaches are vertically transmitted to foundresses and their incipient colonies; however, weak compatibility between roaches and incipient gardens may constrain roach vertical transmission. Reciprocally, opportunities for horizontal transmission between large established colonies with abundant fungal gardens may weaken selection against roach‐induced harm (virulence) of incipient gardens. We use a laboratory experiment, behavioral observations, field surveys, and a transmission model to estimate the effect roaches have on the survivorship of incipient gardens and the frequency of roach vertical transmission. Contrary to traditional assumptions, our results indicate that roaches harm incipient gardens and predominantly use horizontal transmission between established leaf‐cutter colonies. Ultimately, “costs of generalism” associated with infecting disparate stages of a host's lifecycle (e.g., incipient vs. established colonies) may constrain the vertical transmission of roaches and a broad range of symbionts., It may be costly for symbionts to infect the "newborn" solitary stages of colonial hosts, constraining vertical transmission and favoring horizontal transmission between larger established host colonies. We explore this possibility using a miniature symbiotic cockroach of leaf‐cutter ants and their fungal gardens. Our study demonstrates that accounting for host colony lifecycle heterogeneity is important for understanding symbiont transmission dynamics and evolution.

Symbionts of ant colonies can hitchhike on winged ant reproductives (alates) during colony nuptial flights. Attaphila fungicola Wheeler, a miniature cockroach that lives in the nests of Tex...

Trypanosoma carassii is a flagellated bloodstream parasite of cyprinid fish with pathogenesis manifesting primarily as anemia in experimentally infected fish. This anemia is characterized by decreases in the number of circulating red blood cells (RBCs) during peak parasitemia. We examined changes in the key blood metrics and expression of genes known to be important in the regulation of erythropoiesis. Increasing parasitemia was strongly correlated with an overall decrease in the total number of circulating RBCs. Gene expression of key erythropoiesis regulators (EPO, EPOR, GATA1, Lmo2, and HIFα) and proinflammatory cytokines (IFNγ and TNFα) were measured and their expressions differed from those in fish made anemic by injections of phenylhydrazine (PHZ). Significant upregulation of pro-erythropoietic genes was observed in PHZ-induced anemia, but not during peak parasitic infection. Previously, we reported on functional characterization of goldfish erythropoietin (rgEPO) and its ability to induce survival and differentiation of erythroid progenitor cells in vitro. Treatment of goldfish during the infection with rgEPO reduced the severity of anemia but failed to fully prevent the onset of the anemic state in infected fish. Proinflammatory cytokines have been implicated in the suppression of erythropoiesis during trypanosomiasis, specifically the cytokines TNFα, IFNγ, and IL-1β. Analysis of key proinflammatory cytokines revealed that mRNA levels of IFNγ and TNFα were upregulated in response to infection, but only TNFα increased in response to PHZ treatment. Synergistic activity of the proinflammatory cytokines may be required to sustain prolonged anemia. These findings provide insight into the relationship between T. carassii and host anemia and suggest that T. carassii may directly or indirectly suppress host erythropoiesis.

Introduction: IL-6 has been ascribed both positive and negative roles in the context of exercise and training. The dichotomous nature of IL-6 signalling appears to be determined by the respective concentration of its receptors (both membrane-bound (IL-6R) and soluble (sIL-6R) forms). The purpose of the present study was to investigate the response of sIL-6R to long-term training, and to investigate the relationship between sIL-6R, self-reported measures of wellbeing, and upper respiratory illness symptoms (URS) in highly-trained endurance athletes. Methods: Twenty-nine athletes provided resting blood samples, and completed wellbeing and illness monitoring questionnaires, on a weekly basis for a period of 18 weeks during a winter training block. Results: URS were not correlated to concentrations of sIL-6R or cortisol, but there was a non-significant trend (P=0.08) for the most illness-prone athletes (as defined by self-reported illness questionnaire data) to exhibit higher average sIL-6R concentrations compared to the least ill (23.7±4.3 Vs 20.1±3.8 ng/ml). Concentrations of sIL-6R were positively correlated to subjective measures of stress (r=0.64, P=0.004) and mood (r=0.49, P=0.02), but were negatively correlated to sleep quality (r=-0.43, P=0.05) and cortisol concentration (r=-0.17, P=0.04). In a sub-group of 10 athletes, weekly training distance was quantified by coaching staff, and this negatively correlated with sIL-6R in the following week (r=-0.74, P

The myrmecophile cockroach Attaphila fungicola lives in the nests of leaf-cutter ants (Atta texana and A. cephalotes) and uses the female winged reproductives (i.e., female alates) of its host as vectors for the first phase of its dispersal. It is unknown whether A. fungicola remain with vectoring A. texana females after mating flights and throughout A. texana nest founding and subsequent colony development, or if the symbiotic cockroaches disperse to established A. texana colonies, either on their own or while still attached to vectoring A. texana females. We captured A. fungicola attached to A. texana female alates as they prepared for mating flights and measured their survivorship in artificial brood chambers with de-alate, recently mated A. texana queens and their incipient gardens, and in a non-natal established fungal garden tended by A. texana workers. After 13 days, 100% of A. fungicola had died in brood chambers with queens, while 100% of A. fungicola remained alive in the fungal garden chamber. We tested the feasibility of alternative modes of dispersal to established colonies by placing A. fungicola attached and unattached to vectoring female alates in the proximity of an established A. texana colony directly after a mating flight, and recorded whether A. fungicola entered the non-natal nest. A significantly higher proportion of A. fungicola attached to vectoring alates entered the nest compared to unattached A. fungicola. We also placed A. fungicola attached to vectoring alates in a foraging chamber of a laboratory colony to determine if, once in the nest, A. fungicola could navigate to the fungal garden chamber; 100% of A. fungicola detached from their vectoring alates and entered the fungal garden chamber. We tested alate preference of A. fungicola by placing A. fungicola separately in containers with one A. texana female alate and one male alate; after 2 h, 71% of A. fungicola were attached to female alates and 0% to male alates. Finally, we report the first record of a male A. fungicola collected during a mating flight of A. texana. These observations accumulated from field and laboratory studies suggest that A. fungicola vectored by A. texana female alates may not remain with foundresses, but instead disperse between established colonies through one or more alternative mechanisms. This study helps elucidate a tripartite ant-fungus-cockroach interaction, and provides a foundation for future research on Attaphila dispersal and reproductive biology.

OSPW is a complex mixture of inorganic and organic substances and its principal toxic components have yet to be fully characterized. Previously, we showed in vitro that the oil sands process-affected water (OSPW) organic fraction (OF) caused a concentration-dependent immunotoxicity in mammals. In the present study we further explore the immunotoxicological properties of OSPW in mammals using a series of in vitro bioassays. Specifically, using the RAW 264.7 mouse macrophage cell line we show that whole OSPW containing naphthenic acid (NA) concentrations ranging from 12 to 18 mg/L, significantly inhibited cell proliferation, reduced cell viability, and was directly cytotoxic, whereas the exposure of cells to equivalent doses of the OSPW-OF had no measurable effects. Whole OSPW exposures also caused morphological changes in RAW 264.7 cells, and at sublethal doses (i.e., 10 mg/L) it induced the early expression of the stress genes hmox1 and gadd45. In addition, at NA concentrations of 10 mg/L, whole OSPW but not the OSPW-OF had significant effects on pro-inflammatory cytokine mRNA levels and cytokine protein secretion activities. Finally, whole OSPW also impaired the ability of RAW 264.7 cells to perform phagocytosis. Overall, we demonstrate that exposure to whole OSPW (at NA doses ranging from 10 to 20 mg/L), but not the OSPW-OF caused both cytotoxic and immunomodulatory changes in mouse macrophages. This suggests that the complex mixture of inorganic and organic components found in whole OSPW are acutely toxic at much lower doses than we previously reported for the OSPW-OF (i.e., 50 mg/L) due to unknown additive and/or synergistic interactions that likely occur between the various components present in whole OSPW.

MiRNAs regulate cardiac development, hypertrophy, and angiogenesis, but their role in cardiac hypertrophy (CH) induced by aerobic training has not previously been studied. Aerobic training promotes physiological CH preserving cardiac function. This study assessed involvement of miRNAs-29 in CH of trained rats. Female Wistar rats ( n = 7/group) were randomized into three groups: sedentary (S), training 1 (T1), training 2 (T2). T1: swimming sessions of 60 min/5 days/wk/10 wk. T2: similar to T1 until 8th wk. On the 9th wk rats swam 2×/day, and on the 10th wk 3×/day. MiRNAs analysis was performed by miRNA microarray and confirmed by real-time PCR. We assessed: markers of training, CH by ratio of left ventricle (LV) weight/body wt and cardiomyocytes diameter, pathological markers of CH (ANF, skeletal α-actin, α/β-MHC), collagen I and III (COLIAI and COLIIIAI) by real-time PCR, protein collagen by hydroxyproline (OH-proline) concentration, CF and CH by echocardiography. Training improved aerobic capacity and induced CH. MiRNAs-1, 133a, and 133b were downregulated as observed in pathological CH, however, without pathological markers. MiRNA-29c expression increased in T1 (52%) and T2 (123%), correlated with a decrease in COLIAI and COLIIIAI expression in T1 (27%, 38%) and T2 (33%, 48%), respectively. MiRNA-29c was inversely correlated to OH-proline concentration (r = 0.61, P < 0.05). The E/A ratio increased in T2, indicating improved LV compliance. Thus, these results show that aerobic training increase miR-29 expression and decreased collagen gene expression and concentration in the heart, which is relevant to the improved LV compliance and beneficial cardiac effects, associated with aerobic high performance training.

The newly excysted juvenile (NEJ) stage of the Fasciola hepatica lifecycle occurs just prior to invasion into the wall of the gut of the host, rendering it an important target for drug development. The cathepsin B enzymes from NEJ flukes have recently been demonstrated to be crucial to invasion and migration by the parasite. Here we characterize one of the cathepsin B enzymes (recombinant FhcatB1) from NEJ flukes. FhcatB1 has biochemical properties distinct from mammalian cathepsin B enzymes, with an atypical preference for Ile over Leu or Arg residues at the P(2) substrate position and an inability to act as an exopeptidase. FhcatB1 was active across a broad pH range (optimal activity at pH 5.5-7.0) and resistant to inhibition by cystatin family inhibitors from sheep and humans, suggesting that this enzyme would be able to function in extracellular environments in its mammalian hosts. It appears, however, that the FhcatB1 protease functions largely as a digestive enzyme in the gut of the parasite, due to the localization of a specific, fluorescently labeled inhibitor with an Ile at the P(2) position. Molecular modelling and dynamics were used to predict the basis for the unusual substrate specificity: a P(2) Ile residue positions the substrate optimally for interaction with catalytic residues of the enzyme, and the enzyme lacks an occluding loop His residue crucial for exopeptidase activity. The unique features of the enzyme, particularly with regard to its specificity and likely importance to a vital stage of the parasite's life cycle, make it an excellent target for therapeutic inhibitors or vaccination.

The medial frontal cortex (MFC) is involved in the temporal organization of behaviour. It receives timing information from the master circadian clock in the suprachiasmatic nucleus (SCN), and exhibits daily oscillations in gene expression itself. In this study, we evaluate various properties of circadian rhythms of locomotor activity following neonatal or adult MFC aspiration lesions. Mice with neonatal lesions were more active during the day than mice with adult lesions and less active during the early night than both mice with adult lesions and control mice. Compared to controls, mice with neonatal lesions exhibited smaller phase delays to an early-night light pulse and marginally larger phase advances to a late-night light pulse. Mice with adult lesions did not differ from controls on either measure. The results suggest that the timing of behaviour is determined by an interaction between the MFC and the SCN and that injury early in life has a significant effect on the ability of animals to organize such behaviours.

Summary The availability of complete genome sequences for a large number of pathogenic organisms has opened the door for large-scale proteomic studies to dissect both protein expression/regulation and function. This review highlights key proteomic methods including two-dimensional gel electrophoresis, reference mapping, protein expression profiling and recent advances in gel-free separation techniques that have made a significant impact on the resolution of complex proteomes. In addition, we highlight recent developments in the field of chemical proteomics, a branch of proteomics aimed at functionally profiling a proteome. These techniques include the development of activity-based probes and activity-based protein profiling methods as well as the use of synthetic small molecule libraries to screen for pharmacological tools to perturb basic biological processes. This review will focus on the applications of these technologies to the field of microbiology.

In summary, the prevailing concept is that brain Ang II increases blood pressure by activating AT1 receptors, and that these have a neuromodulating effect to increase the activity of autonomic nervous system. Pathways for Ang II stimulating thirst and blood pressure, increased vasopressin release and sympathetic activation have been outlined. Brain RAS synthesis, while incompletely understood, is active in the absence of a peripheral RAS. Angiotensin elicits specific receptor mediated signals in neurons, particularly in the hypothalamus and brainstem. These actions are due to neuronal membrane ionic currents and the regulation of transcription factors. The areas to be explored further are characterization and functional roles of the other AT receptor subtypes, such as AT4, AT(1-7) and nuclear AT-R. Their interactions with other peptides and transmitters, and their signaling pathways need to be investigated. The story that began 100 years ago with renin is certainly not ended and will continue to unfold as further investigations with new techniques progress.

AIMS: (1) To seek evidence of the existence of Chlamydia pneumoniae in a spectrum of atheromatous lesions in different types of arteries from individuals of different ages, using a polymerase chain reaction (PCR) assay supported by electron microscopy and immunocytochemistry; (2) to use electron microscopy to examine interactions between C pneumoniae and the cells present in the arterial tissue; (3) to assess the extent to which the data fulfil the criteria for causality. METHODS: At necropsy examination, 35 arterial specimens were taken from 25 subjects. The grade of atheroma was determined macroscopically and microscopically and the tissues coded and examined by the three techniques. RESULTS: Of the 35 specimens, 24 had macroscopic or microscopic atheromatous lesions of varying degree. Twenty two of the 35 specimens were examined by electron microscopy, C pneumoniae-like bodies being found in 11 (50%); seven specimens were examined by the immunocytochemical method, positive staining being detected in three; and all specimens were examined by the PCR technique, 15 (43%) being PCR positive. Overall, of the 24 specimens with lesions, 17 (71%) were positive by at least one of the three tests, whereas of the 11 specimens without lesions, only one was positive. The positive specimens comprised 10 of 19 aortas, three of six iliac arteries, and one coronary and one pulmonary artery. C pneumoniae was detected in four of six specimens in which there were early changes and in a 20 year old subject. Concerning the 25 subjects, of 17 who had atheromatous arteries, 14 (82%) were C pneumoniae positive and of the eight who had normal arteries, none was positive. CONCLUSIONS: There is a strong correlation between C pneumoniae and arterial atheromatous lesions. The organism may contribute to the disease process by damaging smooth muscle cells.

Abstract —Angiotensin II (Ang II) promotes vascular smooth muscle growth and may be involved in the initiation and progression of atherosclerosis. To examine whether Ang II receptor expression in vascular tissues is altered in atherosclerosis, male New Zealand White rabbits were fed a high-cholesterol diet (1% cholesterol+4% coconut oil mixed with regular chow; hypercholesterolemic group, n=12) or regular chow (control group, n=8) for 10 weeks. At the end of this period, the serum cholesterol level in the rabbits fed the high-cholesterol diet was higher than that in the control group (3616±144 versus 30±1 mg/dL, P P 1 ) receptor expression (289±38 versus 38±18 fmol/mg, P P =NS). AT 1 receptors were localized primarily in the media and to some extent in the intima of the atherosclerotic aorta, as determined by immunohistochemistry with specific monoclonal and polyclonal AT 1 receptor antibodies. Increased synthesis of AT 1 receptor mRNA in atherosclerotic tissues was confirmed by reverse transcription–polymerase chain reaction. To evaluate the functional significance of increased AT 1 receptor expression, the constrictor response of aortic rings to Ang II was examined and found to be markedly enhanced in atherosclerotic aortic rings ( P P 1 receptor) expression in aortic tissues, which may result in altered vasoreactivity.

Antisense oligodeoxynucleotides have been designed to inhibit the production of specific proteins. In models of hypertension, we have targeted the renin-angiotensin system at the level of synthesis (angiotensinogen) and the receptor (AT 1 receptor). The design of antisense oligonucleotides requires choosing a site to inhibit mRNA processing or translation. The strategy we use is to make three oligonucleotides of antisense sequences, upstream and downstream from the AUG site and over the AUG site. The oligonucleotides are tested in a screening test. Antisense oligonucleotides to AT 1 -receptor mRNA and to angiotensinogen mRNA reduce blood pressure in spontaneously hypertensive rats when injected into the brain. They significantly reduce the concentration of the appropriate protein. The oligonucleotides are also effective when administered systemically. The decrease in blood pressure with antisense oligonucleotides delivered in blood or brain lasts 3 to 7 days. To prolong the action, direct injection of naked DNA and injection of DNA in liposome carriers have been tested. Viral vectors have been developed to deliver antisense DNA. The viral vectors available include retroviruses and adenovirus, but the adeno-associated virus (AAV) vector is the vector of choice for ultimate use in gene therapy. It offers safety because it is nonpathogenic, has longevity because it integrates into the genome, and has sufficient carrying capacity to carry up to 4.5 kb antisense or gene in a recombinant AAV. Using rAAV-antisense to AT 1 mRNA, there is efficient transfection into cells and an inhibition of AT 1 receptor number. In in vivo tests, rAAV-AS AT 1 -receptor when injected into the brains of SHR reduces blood pressure for more than 2 months. In young rats (3 weeks old), rAAV-AS AT 1 -receptor decreases blood pressure and slows the development of hypertension. While further experiments need to be done on dose-response relationships and on the cellular mechanisms of these effects, the results show the feasibility of AAV as a vector for antisense inhibition, which may ultimately be used in gene therapy for hypertension.

The mycoflora of a range of mixed ruminant feed and forage samples collected in Eastern India and Bangladesh were determined. Most fungi isolated from the samples were those associated with low moisture contents. Several potentially mycotoxigenic fungi and thermotolerant species were present particularly in the rice straw. The most frequently isolated fungi were: Aspergillus versicolor (Vuill.) Tiraboschi, Penicillium citrinum Thorn, Eurotium species, Wallemia sebi (Fr.) v. Arx, Aspergillus penicilloides Speg. and yeasts. Fusarium moniliforme Sheldon and Cladosporium species were prevalent in straw but not other feeds. Levels of certain individual fungi, particularly thermotolerant types, were higher in Bangladesh rice straw although overall counts were similar to those of the Indian samples. Mycotoxin analyses for aflatoxins, zearalenone and sterigmatocystin were carried out. Aflatoxins were detected in significant amounts in some feed samples from Eastern India but not in rice straw from India and Bangladesh. Zearalenone occurred in a Paspalum straw sample and three other feed samples from India and sterigmatocystin was detected in three rice straw samples from Bangladesh.

Coagulase-negative staphylococci isolated from patients with endocarditis were divided according to whether the infection was of native or of prosthetic valves and was acquired either in the community or in hospital. Comparisons were made with strains from intravenous line-associated bacteraemias. All strains were examined by direct and indirect adherence tests. Line-associated bacteraemia strains were more likely to produce slime and were more hydrophilic but were less likely to attach HEp2 tissue culture cells than were endocarditis strains, and almost equally likely to adhere to plastic and extracellular matrix proteins. Amongst the endocarditis strains, there was little difference in slime production but hospital-acquired or prosthetic-valve strains were more hydrophobic and more likely to adhere to silicone than were the native-valve or community-acquired strains. Exposure of extracellular matrix proteins on native valves due to a pre-existing non-infective heart condition may account for the selection of strains able to adhere to fibronectin or laminin.

Sixty nine samples of maize were collected from pre-harvest standing crops and on-farm storage facilities from 52 smallholder farms located within 4 regions of Honduras during October 1992 and November 1993. Samples were visually assessed for insect damage and fungal spoilage, and the mycoflora quantified on artificial media. The major components of the ear rot complex were: Fusarium moniliforme, F. moniliforme var. subglutinans, Penicillium species, Stenocarpella maydis, S. macrospora and Acremonium spp. Representative samples were also assayed for mycotoxin content. Fumonisin B1 was detected in all 24 samples tested at levels of between 68-6,555 (micrograms/kg), and aflatoxin was detected in 2 samples heavily contaminated with Aspergillus flavus. Moniliformin and tenuazonic acid were not detected in the samples tested. The implications of these findings for human and livestock health risk are discussed, together with possible strategies for controlling these pathogens.

The susceptibility of 173 TEM-1-producing isolates of Escherichia coli was assessed by determination of MICs by the agar dilution method. MICs of amoxicillin, mezlocillin, cephaloridine, and, to a smaller extent, amoxicillin-clavulanic acid (but not cephalexin, cefuroxime, cefotaxime, ceftazidime, or imipenem) were higher for isolates that produced large amounts of beta-lactamase than for isolates that produced smaller amounts. The effect of fixed concentrations of clavulanic acid on resistance to amoxicillin was assessed for 34 selected TEM-1-producing isolates. Low concentrations of the inhibitor (0.5 to 1 microgram/ml) reduced the amoxicillin MICs substantially for almost all the isolates, although the reductions were not sufficient to render any of the isolates amoxicillin susceptible. Higher concentrations of clavulanic acid had progressively greater effects on amoxicillin MICs, but even at 8 micrograms/ml some of the isolates with high beta-lactamase activities remained resistant or only moderately susceptible to amoxicillin. All the isolates were inhibited by clavulanic acid (in the absence of amoxicillin) at concentrations of 16 to 32 micrograms/ml. TEM-1 beta-lactamase activity was inhibited in vitro by clavulanic acid, but not totally, with approximately 2% of the initial activity remaining at 2 micrograms/ml and 0.4% remaining at 8 micrograms/ml. These findings suggest that the amount of beta-lactamase activity is a major determinant of the degree of resistance to several beta-lactam antibiotics and can make the difference between susceptibility and resistance to some compounds, notably the combination of amoxicillin with clavulanic acid.

Summary. Experiments were performed to determine whether a modern flow cytometer could be used to study bacterial populations in suspension, with particular reference to their morphological characteristics and their responses to antibiotics. The FACScan, a commercial benchtop flow cytometer fitted with an air-cooled laser, designed primarily for the study of eukaryotic peripheral blood mononuclear cells, yielded reproducible data relating to bacterial shape and internal architecture. It was sensitive enough to detect changes in bacterial morphology on entry into the growth cycle and after exposure to antibiotics. Antibioticinduced morphological changes affecting subpopulations of bacteria were sufficiently specific to allow differentiation between antibiotics with different cell-wall enzyme targets. Simultaneously, the effect of such antibiotics on the integrity of the outer cell membrane of Escherichia coli was assessed by measurement of the association of the nucleic acid-binding dye propidium iodide with the bacteria. These experiments demonstrated complex patterns of probable cell-wall leakage, related to the modes of action of the antibiotics. The FACScan is a useful and sensitive tool for the study of the morphology and physiology of bacterial populations in suspension, and is especially applicable to the study of antibiotic action.

Angiotensin type-1 receptor subtypes (AT1) are implicated in the physiological actions of angiotensin II in the brain. In the present study we used an AT1 receptor antibody and a polymerase chain reaction--synthesized AT1 receptor complementary DNA to show that the hypothalamus expresses significantly higher levels of AT1 receptor messenger RNA and protein compared with the brain stem. Intracerebroventricular injections of AT1-specific antibody blocks the dipsogenic and blood pressure responses induced by centrally injected angiotensin II. These results demonstrate the expression of AT1 receptor gene in the brain and that the AT1 receptor antibody is able to inhibit the physiological responses of angiotensin II mediated by the brain.

OBJECTIVE--To examine the penicillinase producing N. gonorrhoeae (PPNG) collected at St Thomas' Hospital from 1976-1990 and, by determination of antibiotic susceptibility pattern and application of three typing methods, examine the prevalence of different gonococcal types. Also to determine whether there is any relationship between antibiotic sensitivity, plasmid profile, auxotype and serovar. MATERIALS AND METHODS--A total of 665 isolates of PPNG from patients attending the Department of Genitourinary Medicine at St Thomas' Hospital were characterised by antibiotic MIC, plasmid profile, auxotyping and serotyping. RESULTS--Penicillin MICs for 85% of all isolates were between 0.25 and 32 mg/l. The MIC of tetracycline for 60-80% of the isolates was < 1 mg/l. A small number of isolates had tetracycline MICs of 32 mg/l but MICs > 32 mg/l were not seen. Over 90% of the isolates were sensitive to the remaining three antibiotics tested, erythromycin, cefuroxime and spectinomycin. The 3.2 or 4.4 MDa plasmid with or without the 24.5 MDa conjugal plasmid was seen in all isolates until 1989/90 when a 2.9 MDa beta-lactamase encoding plasmid and the 25.2 MDa plasmid mediating tetracycline resistance were also recognised. Ninety-nine percent of all isolates belonged to one of four auxotypes, prototrophic, arginine, proline or proline/arginine requiring. An initial predominance of isolates with the 1A outer membrane protein was reversed in 1982 and 1B has remained predominant. Thirty two different serovars were identified among the 665 isolates, 14 belonged to serogroup 1A and 18 to 1B, and the eight most common accounted for 83.9% (554) of all isolates. Analysis of the results of combined typing methods showed there was an association between antibiotic resistance, plasmid profile and serogroup. The number of auxotypes and serovars detected in the collection, indicates the possibility that PPNG have been introduced from abroad or outside our local population. CONCLUSION--Temporal trends in the distribution of auxotype/serovar classes show that the total population of PPNG isolates is formed by a heterogenous mixture in which certain auxotype/serovar classes appear, disappear and may re-emerge. Others were present throughout in small numbers.

European Starlings (Sturnus vulgaris) are often pests in commercial fruit crops in North America and Europe. Because starlings lack the digestive enzyme sucrase and cannot digest sucrose, they may develop an aversion to high-sucrose fruits. In water- tube drinking trials, we tested captive starlings with aqueous solutions of 15% (mass/ volume) mixed sugars to identify the level of sucrose required to develop a conditioned feeding aversion when digestible sugars are present. In one-tube tests, starlings decreased intake of 1 1.25 and 15% sucrose solutions relative to their pretest intake of a 15% glucose- fructose mixture. In two-tube tests with sucrose solutions paired against a digestible glucose- fructose solution, starlings decreased preferences for the sucrose solutions as sucrose con- centrations increased. These data suggest that the presence of digestible nutrients mitigates the effect of sucrose in sucrase-deficient birds and that a fruit cultivar would require -11.25% sucrose to repel starlings.

To test the hypothesis that brain angiotensin II (Ang II) may be involved in the preovulatory release of LH on proestrus, we evaluated the pattern of changes in hypothalamic Ang II levels and release in ovariectomized (ovx) rats treated sequentially with estrogen and progesterone. This is an experimental paradigm that reliably evokes dynamic changes in hypothalamic LHRH levels in association with LH hypersecretion, simulating the LH surge on proestrus. Rats were ovx and after 4 weeks received estradiol benzoate followed by progesterone 2 days later at 1000 h. We observed that in these progesterone-treated rats, serum LH levels were low until 1400 h, but thereafter, the rate of LH secretion increased and remained elevated at 1600 h when the experiment was terminated. In these rats, hypothalamic Ang II levels increased abruptly at 1330 h and returned rapidly to baseline levels before the onset of LH surge. Also, a similar pattern in hypothalamic Ang II levels occurred at 1500 h with the rise and peak serum LH levels in the late afternoon. In the second experiment, Ang II levels in the cerebrospinal fluid (CSF) of rats similarly pretreated with ovarian steroids were evaluated. Again, CSF Ang II levels rose abruptly to a peak at 1330 h and returned to baseline range preceding the expected rise in serum LH. Thereafter, no further change in CSF Ang II levels was detected during the period of LH hypersecretion. In the third experiment, perfusates were collected from a push-pull cannula aimed at the paraventricular nucleus in ovx rats similarly treated with ovarian steroids. A peak of Ang II was observed at 1330 h and a later peak at 1430 h. A comparison with LH profiles indicated that these peaks in Ang II levels were evident before and during the LH surge. Thus, in three separate experiments, the results showed that rapid dynamic changes in hypothalamic Ang II levels and release occur in association with the progesterone-induced LH surge in estrogen-primed ovx rats. These findings support the previous observations that Ang II can stimulate LHRH and LH release. Since similar, temporally correlated changes occur in hypothalamic neuropeptide Y and LHRH, the peptides involved in the induction of LH surge, these results are in agreement with the hypothesis that Ang II-expressing neurons may play an important role in the hypothalamic circuitry responsible for stimulation of LH surge in ovarian steroid-treated ovx rats.

The in vitro activity of MDL 62,879, a new peptide antibiotic that inhibits protein synthesis through an interaction with elongation factor Tu, against a wide range of recent clinical isolates of common aerobic gram-positive and anaerobic organisms was determined. MDL 62,879 was highly active against staphylococci (MIC for 90% of isolates [MIC90], 0.125 microgram/ml), streptococci (MIC90, 1 microgram/ml), and enterococci (MIC90, 0.03 microgram/ml). All isolates of peptostreptococci and Mobiluncus spp. were susceptible, as were most isolates of clostridia. MDL 62,879 was not active against isolates of fusobacteria or Bacteroides spp., but some isolates of Prevotella spp. and Porphyromonas asaccharolytica were susceptible.

Monoclonal antibodies (M Abs) have proved useful in the development of immunoassays to detect the mycelium of specific fungi in foods and feeds. Development of M Ab-ELISA (enzyme-linked immunosorbent assay) and ‘user-friendly’ dip-stick assays for the detection of Humicola lanuginosa and Penicillium islandicum, involved in undesirable post-harvest discoloration of rice grains, is described briefly, as is a M Ab-ELISA for the detection of Botrytis cinerea in extracts from infected strawberries and grapes. These assays are sensitive, rapid, easily replicated and do not require surface sterilisation. The antigens, which are located on the surfaces and within the walls of the hyphae, are not present in the ungerminated spores of P. islandicum and are masked in aleurospores of H. lanuginosa, thus immunoassays for these fungi have the added advantage of detecting only mycelial growth. The most sensitive fungal M Abs are those that recognise heat-stable carbohydrate epitopes on carbohydrate or glycoprotein molecules. Difficulties in raising species-specific M Abs to fungi and particularly those related to choice of immunogen, are discussed.

Induction of an enzyme is a temporary phenomenon in which the rate of enzyme synthesis is greatly increased in response to the presence of an inducer in the environment. Induction of beta-lactamase synthesis is important for the resistance of staphylococci to penicillins since the drug both induces synthesis of the enzyme and is hydrolysed by it. Similarly, some compounds both strongly induce and are hydrolysed by the chromosomally-determined beta-lactamases of gram-negative bacilli (e.g. amoxicillin and cefoxitin for Enterobacter cloacae). Other compounds (e.g. piperacillin and cefotaxime) although labile are poor inducers, so that in the case of these drugs the phenomenon of induction is not important but the presence of the enzyme is, since resistant mutants with genetically derepressed synthesis of the enzyme can emerge. Induction can also be important when a strong inducer is present with a poor inducer and antagonises the activity of the latter.

One hundred and forty-two coagulase-negative staphylococci (CNS) isolated from dialysate effluent or skin of patients receiving continuous ambulatory peritoneal dialysis (CAPD) were typed by extended antibiogram (16 antibiotics) and biotype (26 reactions). These isolates were then typed by supplementary methods to determine the most suitable typing method for an epidemiological study of antibiotic resistance. These included phage typing, reverse phage typing, plasmid typing, whole-cell protein typing by SDS-PAGE with analysis by densitometry, and immunoblotting. The percentage of isolates typed successfully by the supplementary methods were: phage typing 20%, reverse phage typing 0%, plasmid typing 66%, SDS-PAGE 100%, immunoblotting 100%. The discrimination of each method was: phage typing 20%, plasmid typing 37%, SDS-PAGE 69%, immunoblotting 57%. Reproducibility was 88% for phage typing and 97% for plasmid typing. The reproducibility of the whole-cell protein typing was 83% if the same extracts were used but only 43% when separate protein extracts were analysed on separate occasions. However, strain relatedness was highly reproducible. The determination of an antibiogram-biotype profile was not a sufficiently accurate typing method for an epidemiological study of antibiotic resistance. Whole-cell protein typing by SDS-PAGE or immunoblotting was technically demanding but was the most effective of the supplementary methods for detecting erroneous discrimination and false matching produced by antibiogram-biotype combinations.

Rates of resistance to 12 antibiotics were determined for 1,289 isolates of theBacteroides fragilis group submitted in 1988–1989 by 22 laboratories in 15 European countries. There was no resistance to metronidazole (breakpoint 8 mg/l) and only one isolate was resistant to chloramphenicol (breakpoint 8 mg/l). Resistance was uncommon for imipenem (0.3 % at > 4 mg/l), amoxicillin/clavulanate (1 % at > 8 mg/l), cefoxitin (3 % at > 32 mg/l), mezlocillin (6 % at > 64 mg/l) and clindamycin (9 % at > 4 mg/l). Resistance was the rule for ampicillin (93 % at > 4 mg/l), ciprofloxacin (56 % at > 4 mg/l) and tetracycline (64 % at > 4 mg/l).Bacteroides fragilis, the commonest species, was generally the most sensitive: resistance of this organism was uncommon for cefotetan (4 % at > 32 mg/l) and ceftazidime (12 % at > 32 mg/l) to which the other species were more often resistant. There were small but significant differences between laboratories and countries for many of the antibiotics. Regionally the most striking differences were for clindamycin where resistance inBacteroides fragilis was most common in the South and for tetracycline where resistance inBacteroides fragilis, Bacteroides thetaiotaomicron andBacteroides uniformis was least common in the North.

Induction by the 4-quinolone group of antibacterial drugs of the umuC gene, the SOS function most involved in error-prone DNA repair (together with umuD), was assessed in a strain of Escherichia coli harbouring a umuC::lacZ gene fusion. All 4-quinolones tested induced this umuC::lacZ fusion, with maximum induction at 4-quinolone concentrations close to their minimum inhibitory concentrations for this strain, and the SOS Inducing Potential (SOSIP) was closely related to antibacterial activity. Mitomycin C, a known mutagen, was a slightly better inducer (in terms of SOSIP) than any of the quinolones. In contrast, induction by 4-quinolones of the sfiA (sulA) gene, an SOS function involved in cell division inhibition, was better than induction by mitomycin C in an E. coli strain harbouring an sfiA::lacZ gene fusion. The umuC gene fusion was induced at lower concentrations of 4-quinolone than was the sfiA gene fusion.

Two plasmids containing the cloned Escherichia coli wild-type gyrA gene were used to transform ciprofloxacin-resistant Gram-negative clinical isolates to screen for DNA gyrase A-mediated quinolone resistance. The results show that the technique is simple and applicable to a wide range of Gram-negative species including E. coli, Enterobacter cloacae, Klebsiella aerogenes, Morganella morganii, Proteus mirabilis, Pseudomonas aeruginosa, Campylobacter jejuni and Neisseria gonorrhoeae. The use of an arithmetical MIC series of dilutions (as opposed to standard geometrical ones) was found to be essential during screening for the detection of altered gyrase A. The observations were consistent with the suggestion that DNA gyrase is highly conserved among different species of bacteria and that gyrase A-mediated resistance can occur in all.

A sensitive, specific, quantitative enzyme‐linked immunosorbent assay (ELISA) has been developed that can be used to determine the extent of mycelial growth of a sporulating thermophilic fungus, Humicola lanuginosa on the surface of rice grains. The assay employs a monoclonal antibody EC6, developed in a previous study, which does not recognize spores of the fungus. Using antigen‐coated wells, a direct linear relationship was established between dilutions of extracts from freeze‐dried mycelium (0.5 to 3 μg/ml) and absorbance values but to eliminate day‐to‐day variations it was found to be necessary to run a dilution series, prepared from stock freeze‐dried mycelium, with every test sample. The ELISA method was compared with conventional quantitative methods. Estimates of total mycelial length in freeze‐dried material by ELISA were found to be in the same order of magnitude as those determined by ergosterol and a theoretical calculation. The ELISA method also compared favourably with direct linear measurem...

Monoclonal antibodies (mAbs) were raised against Penicillium islandicum, a fungus commonly isolated from stored rice grains in South-East Asia. Mice were immunized by a direct, simple method; fresh cell-free surface washings from a solid agar slant culture were injected directly into the peritoneum without prior concentration. Hybridoma supernatants were screened by ELISA. Most of the mAbs raised cross-reacted with other storage fungi and/or uninfected rice grains but there were species-specific. One of these, PI01, was used to develop ELISA and DIP-STICK assays for the detection of P. islandicum in individual grains. All inoculated grains and approximately 90% of grains in natural infected samples from Indonesia tested positively, by ELISA, for P. islandicum. This result and those obtained for discoloured grains from both Indonesia and the Philippines, 32% and 14% respectively, are higher than those obtained by direct plating of surface-sterilized grains. Heat and periodate treatment of the PI01 antigen and binding on Western blots indicate that it is a glycoprotein of Mr greater than 90,000. Hyphae of all ages stained uniformly by immunofluorescence using the PI01 antibody but mature conidia stained only weakly.

Neuronal and glial cultures from the hypothalamic-brain stem areas of 1-day-old normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive (SH) rat brains stained positively with atrial natriuretic peptide (ANP)-specific antibodies. The endogenous levels of the ANP immunoreactivity in WKY neuronal and glial cultures were 17.0 +/- 2.2 and 14.3 +/- 2.7 pg/mg, respectively. Comparable neuronal and glial cultures from SH rat brains contained a 48 to 70% decrease in the endogenous ANP immunoreactivity levels. Culture media from both brain cell types also contained ANP immunoreactivity, the levels of which are significantly higher than those found in the cells. However, similar to endogenous levels, the media levels of immunoreactive ANP in SH neuronal and glial cultures were significantly reduced compared with WKY brain cultures. These observations demonstrate that endogenous ANP-like immunoreactivity is found in neuronal and glial cells and is released into the media. The levels of peptide are reduced in cultures of SH compared with WKY cultures, suggesting a genetically controlled difference between the hypertensive and normotensive rat strains long before hypertension develops.

Newly replicated Plasmodium falciparum parasites escape from host erythrocytes through a tightly regulated process that is mediated by multiple classes of proteolytic enzymes. However, the identification of specific proteases has been challenging. We describe here a forward chemical genetic screen using a highly focused library of more than 1,200 covalent serine and cysteine protease inhibitors to identify compounds that block host cell rupture by P. falciparum. Using hits from the library screen, we identified the subtilisin-family serine protease PfSU B1 and the cysteine protease dipeptidyl peptidase 3 (DPAP3) as primary regulators of this process. Inhibition of both DPAP3 and PfSUB1 caused a block in proteolytic processing of the serine repeat antigen (SERA) protein SERA5 that correlated with the observed block in rupture. Furthermore, DPAP3 inhibition reduced the levels of mature PfSUB1. These results suggest that two mechanistically distinct proteases function to regulate processing of downstream substrates required for efficient release of parasites from host red blood cells.

This hypothesis is readily testable by replicating in additional well-characterized diabetic populations. Interestingly, raised fasting triglycerides and slightly increased fasting insulin have recently been associated with an mtDNA polymorphism at position 16 517 in a native American population[15xHegele, R. et al. Am. J. Hum. Genet. 1997; 60: 1552–1555Abstract | Full Text PDF | PubMedSee all References[15]. As this polymorphism is closely associated with the 16 189 variant in this group[16xTorroni, A. et al. Am. J. Hum. Genet. 1993; 53: 591–608PubMedSee all References[16], it is likely that the 16 189 variant and fasting insulin levels are associated in this population. Investigation of the effect of variation in the lengths of the homopolymeric tract on mitochondrial function might establish a mechanistic basis for this association.If correct, this hypothesis has important implications for clinicians and scientists in three separate fields of endeavour: diabetes research, biological anthropology and mitochondrial genetics. Firstly, if the 16 189 variant is a significant genetic predisposing factor for Type 2 diabetes, measures for preventing obesity could be targeted to individuals with the variant. Secondly, the variant might be a genetic determinant of metabolic thrift and, hence, have contributed to the successful colonization of Polynesia despite the adversity of the long sea voyages involved. Thirdly, by predisposing to Type 2 diabetes, the 16 189 variant might be responsible for orders of magnitude more morbidity than all other mitochondrial diseases put together.

Corticosteroid-binding globulin (SERPINA6) deficiency is an extremely rare hereditary disorder characterized by reduced corticosteroid-binding capacity with normal or low plasma corticosteroid-binding globulin concentration, and normal or low basal cortisol levels associated with hypo-/hypertension and muscle fatigue. Here, we present a patient with severe muscle fatigue, normal blood pressure, and abnormal high saliva cortisol levels following a standardized stress test. This patient was found heterozygous for a de novo 367 asparagine-encoding variant of the corticosteroid-binding globulin gene, previously described as "transcortin Lyon". Both parents were homozygous for the ("wildtype") 367 aspartate-encoding allele. To the best of our knowledge, this case represents the first de novo mutation reported for corticosteroid-binding globulin deficiency, implicating a pathogenic role of variants of SERPINA6 in some cases of muscle fatigue.

SV40 has been implicated in the etiology of 40% to 60% of human mesotheliomas. These studies could have important medical implications concerning possible sources of human infection and potential therapies if human tumors are induced by this agent. We did PCR-based analysis to detect SV40 large T antigen DNA in human mesotheliomas. None of 69 tumors in which a single copy gene was readily amplified contained detectable SV40 large T antigen sequences. Under these conditions, it was possible to detect one copy of integrated SV40 DNA per cell in a mixture containing a 5,000-fold excess of normal cells using formalin-fixed preparations. Kidney, a known reservoir of SV40 in monkeys, from some of these individuals were also negative for SV40 large T antigen sequences. A subset of mesotheliomas was analyzed for SV40 large T antigen expression by immunostaining with a highly specific SV40 antibody. These tumors as well as several human mesothelioma cell lines previously reported to contain SV40 large T antigen were negative for detection of the virally encoded oncoprotein. Moreover, mesothelioma cell lines with wild-type p53 showed normal p53 function in response to genotoxic stress, findings inconsistent with p53 inactivation by the putative presence of SV40 large T antigen. Taken together, these findings strongly argue against a role of SV40 by any known transformation mechanism in the etiology of the majority of human malignant mesotheliomas.

The success of gene therapy requires specific targets. For the potential treatment of cardiovascular disease by gene therapy, the renin-angiotensin system offers targets that are know to be effective based on pharmaceutical drugs that have targeted angiotensin-converting enzyme (ACE) angiotensin type I receptors (AT1R). Other targets include renin and angiotensinogen. It is clear that lowering overactive amounts of angiotensin II has positive effects on reducing high blood pressure, decreasing left ventricular hypertrophy, and lowering the risk of heart attacks, stroke, and kidney failure. To inhibit the RAS system we have used antisense (AS) inhibitors which can be delivered either as oligonucleotides and used like small molecule drugs or in vectors with a sequence of DNA in the antisense direction. This review summarizes the effects of these two approaches on reducing high blood pressure. Gene therapy with antisense offers long-lasting, highly specific inhibition of gene products such as the components of RAS when their overactivity needs to be reduced in order to ameliorate disease.

Comparison of MIC results obtained in different parts of the world is currently difficult because of variations in methods. In this study, cation-adjusted Mueller-Hinton broth, the NCCLS-recommended medium, was compared with Iso-Sensitest broth, which is widely used in Europe. Microbroth dilution testing, using the NCCLS procedure, was performed on 124 Gram-positive (staphylococci and enterococci) and Gram-negative (Enterobacteriaceae and Pseudomonas aeruginosa) isolates from the CDC reference set, with the only variable being the medium used. Twelve antimicrobial agents were tested: amoxycillin-clavulanic acid, ampicillin, ciprofloxacin, erythromycin, gentamicin, imipenem, levofloxacin, oxacillin, gemifloxacin, trimethoprim- sulphamethoxazole, tetracycline and vancomycin. Vancomycin, erythromycin and oxacillin were only evaluated for the Gram-positive organisms. Trimethoprim-sulphamethoxazole was only evaluated for a subset of Gram-negative organisms because of off-scale results. The 124 isolates were tested in one American and one UK laboratory with two batches of cation-adjusted Mueller-Hinton broth and two of Iso-Sensitest broth. A statistical evaluation of the data used a 24 fully specified factorial analysis to determine if there were significant differences in results owing to Gram reaction, site of testing and type and/or batch of broth. In addition, the cumulative results for each antimicrobial agent in each broth were plotted against the range of MIC dilutions tested. MICs of ciprofloxacin, levofloxacin, gemifloxacin, gentamicin and tetracycline were slightly higher (half a doubling dilution) with Iso-Sensitest broth than with Mueller-Hinton broth. MIC results for the other antimicrobial agents were equivalent. Essential and category agreement rates were comparable for all agents (88.4-100% and 88.2-99.0%, respectively).

Angiotensin-converting enzyme (ACE) activity in the myocardium and angiotensin-II (Ang-II) levels in plasma increase after myocardial ischemia, which lead to exacerbation of myocardial injury and cardiac dysfunction. We examined the protective role of novel antisense-oligodeoxynucleotide (AS-ODN) directed at ACE mRNA in myocardial ischemic injury. Sprague-Dawley rats were treated with ACE-AS-ODN (200 microg per rat, n = 8, i.v.) or inverted-ODN (IN-ODN, 200 microg per rat, n = 8, i.v.), given with 600 microg per rat of liposome DOTAP/DOPE. Hearts from AS-ODN- or IN-ODN-treated rats were excised, perfused in vitro, and subjected to 25 min of global ischemia followed by 30 min of reperfusion. Parallel groups of rats were given ACE inhibitor captopril (5 mg/kg, n = 8) or saline (n = 8) before excising the hearts. Ischemia/reperfusion resulted in myocardial dysfunction (increase in coronary perfusion pressure and LV end-diastolic pressure and a decrease in developed LV pressure) in the saline-treated rats. Myocardial dysfunction was associated with evidence of lipid peroxidation and enzyme leakage (MDA and LDH levels in the myocardium) and up-regulation of ACE protein expression. Administration of AS-ODN or captopril, but not IN-ODN, reduced Ang-II levels in the plasma, decreased ischemia/reperfusion-mediated cardiac functional deterioration and lipid peroxidation, and preserved LDH in the myocardium (all P < 0.05 versus the saline group). AS-ODN and captopril had equipotent effects on cardiac dynamics. ACE protein expression (western blot) was decreased in the hearts of the AS-ODN-treated group, but not in IN-ODN-treated rat hearts. In contrast, ACE protein expression was significantly increased in captopril-treated rat hearts. These observations suggest that AS-ODN directed at ACE mRNA can ameliorate myocardial dysfunction and injury after ischemia/reperfusion, and its use is associated with decreased expression of ACE protein in the ischemic myocardium.

Publisher Summary Vectors (plasmid or viruses) can be used for delivery of the gene of interest in sense or antisense orientation. The sense orientation is to express, as a final product, active protein capable of performing physiological function. The antisense orientation is to produce antisense messenger ribonucleic acid (mRNA) that interferes with normally produced sense mRNA and, as a result, decreases the amount of translated protein. Sense or antisense deoxyribonucleic acid (DNA) is usually at least a few hundred bases long and is subcloned into the plasmid vector containing the following: a bacterial origin of replication, so that the plasmid can be multiplied in the bacteria; an antibiotic resistance gene to select bacteria containing plasmid; a mammalian-type promoter to drive expression of cyclic DNA (cDNA) in mammalian cells; a splicing signal for efficient RNA processing; and a polyadenylation signal for translation of mRNA. If the plasmid is packaged into the virus later on, the cassette should also contain the specific virus sequences required in cis configuration, for instance, in terminal repeats. The presence of a reporter or selection gene, which can be driven by a separate mammalian-type promoter or separated from the gene of interest by an internal ribosome entry site (IRES), is optional.

Publisher Summary Antisense (AS) inhibition has developed, particularly in cell culture applications, to the point where it is being tested in clinical trials for human immunodeficiency virus (HIV) and cancer. Before 1992, antisense had not been applied in vivo with any success. There was much concern about the efficiency of the cellular uptake of oligonucleotides (ODNs). In 1992-1993, three or four laboratories simultaneously and independently made and tested AS-ODNs in the brain. Cellular uptake was not a limiting factor in the central nervous system. Receptor binding reduction in the brain with antisense to the angiotensin AT 1 receptor, neuropeptide Y, and NMDA receptors showed that AS delivered centrally was an effective inhibitior. AS-ODNs have many potential attractive features as a new class of therapeutic agents. They can be studied in the same way as drugs for dose-response effects, length of time of activity, and pharmacokinetics. They have longer lasting actions than current drugs and, because of their molecular specificity, fewer side effects. To prolong the effect of antisense inhibition for weeks or months, DNA (partial or full-length) can be inserted in the antisense direction in viral vectors.

Abstract —Angiotensin I–converting enzyme (ACE) inhibitors have been proven to be highly effective and are for the most part the drugs of choice in the treatment and control of hypertension, congestive heart failure, and left ventricular dysfunction. Despite this, questions regarding side effects and compliance with this traditional pharmacological strategy remain. In view of these observations, coupled with recent advances in gene-transfer technology, our objective in this study was to determine whether the expression of ACE could be controlled on a permanent basis at a genetic level. We argued that the introduction of ACE antisense to inhibit the enzyme would be a prerequisite in considering the antisense gene therapy for the control of hypertension and other related pathological states. Retroviral vectors (LNSV) containing ACE sense (LNSV-ACE-S) and ACE antisense (LNSV-ACE-AS) sequences were constructed and were used in rat pulmonary artery endothelial cells (RPAECs) to determine the feasibility of this approach. Infection of rat RPAECs with LNSV-ACE-S and LNSV-ACE-AS resulted in a robust expression of transcripts corresponding to ACE-S and ACE-AS, respectively, for the duration of these experiments, ie, 8 consecutive passages. The expression of ACE-AS but not of ACE-S was associated with a permanent decrease of ≈70% to 75% in ACE expression and a 50% increase in the B max for the AT 1 s. Although angiotensin II caused a concentration-dependent stimulation of intracellular Ca 2+ levels in both ACE-S– and ACE-AS–expressing cells, the stimulation was significantly higher in ACE-AS–expressing RPAECs. In vivo experiments demonstrated a prolonged expression of ACE-AS transcripts in cardiovascularly relevant tissues of rats. This was associated with a long-term reduction in blood pressure by ≈15 mm Hg, exclusively in the spontaneously hypertensive rat. These observations demonstrate that delivery of ACE-AS by retroviral vector results in a permanent inhibition of ACE and a long-term reduction in high blood pressure in the spontaneously hypertensive rat.

Introduction: A knowledge of Helicobacter pylori status is likely to be important in the management of patients presenting in general practice with dyspepsia. Near-patient testing kits for the detection of the H. pylori antibodies in whole blood, which provide results very quickly, are now available. Aim: To determine the test characteristics of the Helisal Rapid Blood test in general practice and to describe the ways in which general practitioners incorporate the results of H. pylori testing into their management of patients with dyspepsia. Setting: Nine general practices in the United Kingdom. Patients: Consecutive patients presenting with dyspepsia in whom further investigation or referral was considered. Methods: A whole blood fingerstick sample was tested for H. pylori antibodies using the Helisal Rapid Blood test and a venous sample was obtained for subsequent laboratory testing using Helisal Serum and Helico G (ELISA) methods. Results of the three tests were compared and analysed. Clinical data were recorded by the general practitioners. Results: Full serological data were obtained on 123 patients, mean age 44.8 years. The sensitivity and specificity of the Helisal Rapid Blood test, compared with concordant results of the laboratory tests, were 83% and 78%, respectively. The prevalence of H. pylori-positive patients was 57%, with a positive predictive value of 83% and a negative predictive value of 78%. The results of the test had a significant impact on the clinical management of the patients in the study. Comment: The Helisal Rapid Blood test is a useful and convenient method of improving the clinical care of patients with dyspepsia in general practice.

The brain peptides atrial natriuretic peptide (ANP) and angiotensin II (AngII) have antagonistic actions centrally that have evolved to play an important role in maintaining the homeostasis of fluid volume and electrolytes. This paper discusses the possible evolution of these functions as viewed through studies on fish and rats. In the euryhaline teleost fish, the major form of ANP is CNP. CNP is important for the adaptation of fish in sea water to water with lower salinity (50% SW). The concentration of CNP in the hypothalamus (HTS-CNP) of toadfish is significantly increased when the fish moves from SW to 50% SW. Interestingly, the plasma CNP concentrations of these fish go in the opposite direction to the brain CNP. Plasma CNP is reduced 24 hrs and 10 days after the fish has been in 50% SW. We postulate that the increased hypothalamic CNP is correlated with an increase in hypothalamic dopamine turnover. Dopamine is the main inhibitory factor for the release of prolactin. In sea-water-adapted fish, prolactin plasma levels are low. In 50% SW, the levels are increased. Extracts of fish-brain CNP caused an increase in urinary sodium and volume, indicating the natriuretic action of CNP. In contrast to CNP, hypothalamic AngII was decreased during adaptation to 50% SW. Thus, CNP and AngII in the brain have opposite actions in fish which aid in their survival in adapting to different salinities. This implies that the hormones evolved as sodium/osmoregulatory peptides, not volume-regulatory peptides. In moving from an aquatic to a terrestrial environment, ANP further evolved in mammals as a volume-regulating hormone while retaining its sodium-regulating properties. In the brain the antagonism between the peptides is apparent in many actions, but when volume is changed both peptides are increased. This is in contrast to plasma ANP and plasma AngII, which have opposite actions during hemorrhage. Plasma AngII is increased and plasma AnP is decreased after blood volume loss. Brain ANP counteracts the thirst-inducing and volume-restoring roles of brain AngII in mammals. By these actions, the brain peptides play significant roles in maintaining volume and electrolyte homeostasis.

A multicentre, double-blind, randomised study comparing the efficacy and safety of oral sparfloxacin (200 mg on day 1 followed by 100 mg daily for 2 or 6 additional days) with doxycycline (200 mg once daily for 7 days) in the treatment of non-gonococcal urethritis in men was conducted. The overall success rates for the three treatment groups were statistically equivalent both at the end of treatment and at the follow-up visits. For chlamydial urethritis, the rates of relapse or possible reinfection were similar in the 7-day sparfloxacin and doxycycline groups. For ureaplasmal urethritis and urethritis of unknown aetiology, the rates of relapse or possible reinfection were lower in the 7-day sparfloxacin group than in the other two treatment groups. Sparfloxacin was well tolerated. A 7-day sparfloxacin regimen appears to be a useful alternative to doxycycline for the treatment of non-gonococca l urethritis in men.

The mechanisms by which quinolones rapidly kill are ill defined. We have investigated the action of ciprofloxacin on Escherichia coli KL16 with a combination of traditional and flow cytometric methods and have analyzed cells for changes in membrane potential, membrane integrity, oxidative metabolism, morphology, and viability. Log-phase cultures were exposed to various concentrations (0.1, 1, 10, and 100 times the MIC) of ciprofloxacin and analyzed at regular intervals over 120 min. We also measured protein synthesis in the related strain PQ37 cultured under the same conditions over 300 min, using a colorimetric assay for beta-galactosidase release. Despite a 3-log order decrease in CFU after 60-min exposure to 10 and 100 times the MIC of ciprofloxacin, there was no equivalent decrease in bacterial numbers as determined by both light microscopy and flow cytometry. Furthermore, while these bacteria showed concentration-dependent morphological changes, most were capable not only of excluding the fluorescent nucleic acid-binding dye propidium iodide, but also of reducing the tetrazolium dye cyanoditodyl tetrazolium chloride. Over 90% of the bacteria maintained a membrane potential [as determined by exclusion of bis-[1,3-dibutylbarbituric acid) trimethine oxonol] when exposed to ciprofloxacin for 120 min, except at 100 times the MIC, when this figure fell to < 10%. Finally, protein synthesis was either maintained or induced at all concentrations of ciprofloxacin up to 5 h postexposure. Taken together, these results demonstrate the continuing physical and metabolic survival of ciprofloxacin-exposed bacteria; we suggest parallels with the concept of the viable nonculturable state.

SUMMARYPlasmids belonging to the FImeincompatibility group were found in seven different serogroups of multiply antimicrobial-resistantEscherichia coliisolated from patients with urinary tract infection (UTI) and living in south-east London. Although widespread inSalmonellaspp., FImeplasmids have only previously been described inE. coliin a strain of serogroup O15 K52 H1 responsible for an extensive and protracted outbreak of invasive community-acquired infection in south-east London in 1986. Our findings suggest either a wider background occurrence of FImeplasmids inE. coliassociated with UTI than previously reported or alternatively, the dissemination and subsequent molecular diversification of the FImeplasmid associated with the epidemic strain of serogroup O15 K52 H1.