Investigating surface binding effects: antibacterial efficacy of bound 8-hydroxyquinoline against Staphylococcus aureus and Escherichia coli.

AIMS: To investigate the binding of the antimicrobial compound 8-hydroxyquinoline (8HQ) to a material interface and to determine whether immobilization affects the antibacterial efficacy. METHODS AND RESULTS: The 8HQ derivative 5-carboxy-8-hydroxyquinoline (5C8HQ) was attached to silica beads through amide bond coupling at the carboxyl moiety of 5C8HQ. Attachment of 5C8HQ was confirmed using a combination of mass spectrometry, thermogravimetric analysis, colorimetric testing and Soxhlet extraction. Computational modelling results indicated that this substitution did not compromise the active sites on the molecule, whereas other positions on the ring system could potentially inhibit antimicrobial activity. The antibacterial effect of 8HQ and the 5C8HQ-modified silica complex against Escherichia coli 15597 (ATCC® 25922) and Staphylococcus aureus (ATCC 25923) was evaluated. CONCLUSIONS: The test results show that the immobilized 8HQ continues to exhibit antibacterial activity, however, quantifying the efficacy compared to free 8HQ bears further investigation. The expected antibacterial mechanism requires that the metal chelation site of 8HQ be retained and available after attachment to a surface. The retention of antibacterial activity after surface bonding represents a novel mechanism of action not previously reported. SIGNIFICANCE AND IMPACT OF THE STUDY: Recent changes in regulations due to environmental concerns prompted many companies and organizations to explore antimicrobial treatments that are chemically bound to the product. Chemically bonding biocidal compounds to a surface limits environmental release; however, molecular mechanisms that drive antibacterial activity when compounds are immobilized are limited. The results reported here demonstrate that the 8HQ reactive site retains antibacterial efficacy even after covalent attachment to a surface. This approach supersedes other antimicrobial treatments where the active component is gradually released from the material surface in order to elicit antimicrobial effects. This specific antibacterial activity of bound 8HQ represents a novel mechanism of action not previously reported, and a potential conduit to a new class of bound antimicrobial materials.

Ascertainment of congenital malformations: a comparative study of two systems.

STUDY OBJECTIVE: The aim was to compare the Office of Population Censuses and Surveys (OPCS) notification system for congenital malformation surveillance and the Liverpool Congenital Malformations Registry (LCMR) with respect to efficiency and uniformity of ascertainment, diagnostic accuracy, and overreporting of minor malformations. DESIGN: Manual matching of computer listings was done, using date of birth, sex, birthweight, and health district of residence. Maternal age was used to confirm the match. SETTING: Data were collected in the English health districts of Liverpool, St Helens and Knowsley, Southport and Formby, South Sefton, and Wirral over the years 1980-1985. SUBJECTS: 1959 malformed children notified to OPCS and 2649 notified to LCMR were assessed. MAIN RESULTS: 35.5% of malformed children ascertained by LCMR within seven days of birth were not notified to OPCS; 35.7% of cases reported to OPCS were exclusions from the LCMR protocol as being trivial malformations according to EUROCAT (European Registry of Congenital Anomalies) guidelines. Misclassification was infrequent but in 11.2% of cases the additional malformations present were not notified to OPCS. Conditions readily diagnosed at birth, such as neural tube defects, exomphalos, and facial clefts, were well ascertained by OPCS but others such as oesophageal atresia and Down's syndrome were not. CONCLUSIONS: It is important that national surveillance of congenital malformations should continue. However, several modifications to the present OPCS monitoring system are necessary, including greater standardisation of data collection, the exclusion of trivial and clinically non-significant malformations, and the inclusion of data on therapeutic abortions performed for fetal abnormality. These issues are being addressed by OPCS.

Stromal Development and Mating System of Balansia epichloë, a Leaf-Colonizing Endophyte of Warm-Season Grasses.

Studies of the stromal development and mating system of Balansia epichloë were conducted. Early development of the stroma consists of both endophytic and epiphytic phases of growth. As development progresses, the epiphytic stromal subiculum on the upper surface of leaves is linked with endophytic mycelium within leaves by hyphal bridges, which may provide carbohydrates for stromal development. Sugar utilization studies suggest that Balansia epichloë is excluded from growth within inflorescence primordia of grasses by the presence of high levels of sugars that are inhibitory to growth of the endophyte. Studies of the mating system of B. epichloë were conducted, and the fungus was shown to be heterothallic, with ephelidial conidia functioning as spermatia. Insect vectoring of ephelidial spermatia is suggested to account for the irregular pattern of perithecial development on stromata.

A study of adenosine 3'-5' cyclic monophosphate binding sites of human erythrocyte membranes using 8-azidoadenosine 3'-5' cyclic monophosphate, a photoaffinity probe.

An earlier report (1a) has shown the utility of 8-N3cAMP (8-azidoadenosine-3', 5'-cyclic monophosphate) as a photoaffinity probe for cAMP binding sites in human erythrocyte membranes. The increased resolution obtained using a linear-gradient SDS polyacrylamide gel system now shows that: 1) both cAMP and 8-N3cAMP stimulate the phosphorylation by [gamma-32P]-ATP of the same red cell membrane proteins; 2) the protein of approximately 48,000 molecular weight whose phosphorylation by [gamma-32P]-ATP is stimulated by cAMP and 8-N3cAMP migrates at a slower rate than the protein in the same molecular weight range which is heavily photolabeled with [32P]-8-N3cAMP;3) other cyclic nucleotide binding sites exist besides those initially reported; 4) the variation in the ratio of incorporation of [32P]-8-N3cAMP into the two highest affinity binding sites appears to be the result of a specific proteolysis of the larger protein.

Synthesis and utilization of 8-azidoguanosine 3'-phosphate 5'-[5'-32P]phosphate. Photoaffinity studies on cytosolic proteins of Escherichia coli.

A family of guanosine 3',5'-phosphorylated nucleotides have been postulated to have pleiotypic regulatory properties in prokaryotes during the stringent response. To study proteins which may interact with nucleotides of this homologous series, a photoactive analog of guanosine 3',5'-diphosphate has been synthesized. The analog, 8-azidoguanosine 3'-phosphate 5'-[5'-32P]phosphate, proved to be an effective photoaffinity probe for two nucleotide-binding proteins of Escherichia coli sonicates. It predominately photolabels two proteins with approximate molecular weights of 86,000 and 65,000 (p86 and p65, respectively). The Kd for p65 was approximately 10 microM; that for p86 was not determined. The nucleotide-binding sites were characterized by photolabeling in the presence of various nucleotides. The nucleotides guanosine 3',5'-dipyrophosphate, guanosine 3'-monophosphate 5'-diphosphate, and GTP were most effective at decreasing photoincorporation into p86; guanosine 3'-diphosphate 5'-monophosphate was least effective, with guanosine 3',5'-diphosphate and GMP having an intermediate effect. ATP increased photolabeling of p86. However, ATP was one of the best of the nucleotides studied at decreasing photolabeling of p65, although guanosine 3'-monophosphate 5'-diphosphate, guanosine 3',5'-diphosphate, and GMP appeared only slightly less effective. The relative lack of effectiveness of guanosine 3'-diphosphate 5'-monophosphate inhibiting photolabeling of either protein supports observations that this nucleotide does not have a regulatory role in E. coli. The results presented indicate that the 8-azidoguanosine analogs of this homologous series will prove to be effective probes for studying the protein-nucleotide interactions involved in the stringent response.

Use of photoaffinity nucleotide analogs to determine the mechanism of ATP regulation of a membrane-bound, cAMP-activated protein kinase.

Using a radioactively tagged, photoaffinity analog of cAMP, 8-azidoadenosine-3',5'-cyclic monophosphate (8-N3 cAMP) and [gamma32P]ATP, the membrane-binding properties of both the regulatory and catalytic subunits of the cAMP-activated protein kinase of human erythrocyte membranes were investigated. [32P]8-N3 cAMP was used to locate and quantify regulatory subunits. Increased phosphorylation of specific membrane proteins by [gamma32P]ATP was used to determine the presence of the catalytic subunit. The data support a mechanism which operates through a tight membrane-bound regulatory subunit and a catalytic subunit that is released from the membrane when cAMP is present and the Mg.ATP concentration is below approximately 10 micrometer. The catalytic subunit is not required for the Mg.ATP inhibition of 8-N3 cAMP binding. Experiments with a photoaffinity analog of ATP, 8-azidoadenosine triphosphate (8-N3ATP), support the hypothesis that ATP hydrolysis and phosphorylation are not involved in the regulation. The data indicate that the regulatory subunit contains an ATP regulatory site which inhibits 8-N3 cAMP binding and the release of the catalytic subunit. These results indicate that the membrane-bound type I enzyme (type IM) differs significantly from the soluble (type IS) enzyme studied on other tissues. These enzymes are compartmentalized by being in different cellular locations and are regulated differently by Mg.ATP.

Characterization of the guanosine-3'-diphosphate-5'-diphosphate binding site on E. coli RNA polymerase using a photoprobe, 8-azidoguanosine-3'-5'-bisphosphate.

Nucleotide binding sites on DNA-dependent RNA polymerase from E. coli have been studied by photoaffinity labeling with a GTP analog [gamma-32P]-8-AzidoGTP and a guanosine-3'-diphosphate-5'-diphosphate analog, 8-Azidoguanosine-3'-phosphate-5'-85'-32P]phosphate. The guanosine diphosphate photoprobe labeled the beta, beta', and sigma subunits with the sigma subunit being most heavily labeled. The GTP photoprobe also labeled the beta, beta', sigma subunits but the beta' subunit was most heavily labeled. In competition experiments guanosine-3'-diphosphate-5'-diphosphate decreased photolabeling by 8-Azidoguanosine-3'-phosphate-5'-[5'-32P]phosphate better than GTP, while the opposite was true for photolabeling with [gamma-32P]8- AzidoGTP. The guanosine diphosphate photoprobe inhibited transcription on E. coli DNA with Ki of ca. 150 microM. Present studies suggest a unique ppGpp binding site distinct from substrate binding site(s) and this photoprobe may be used to localize this binding site(s).

Epidemiology of facial clefting.

An analysis was performed of patients with facial clefts notified between 1960 and 1982 to the Liverpool Congenital Malformations Registry. From 1960-82 there were 325 727 births in the area surveyed and 544 cases of facial clefting were notified. When 88 patients with recognised syndromes and multiple congenital anomalies were excluded, the overall prevalence of facial clefts alone was 1.4 per 1000 total births. This group was then classified further into 137 cases of cleft lip alone, 166 cases of cleft lip and palate, and 153 cases of cleft palate alone. The prevalence of these groups per 1000 total births is 0.42, 0.51, and 0.47 respectively. There were some fluctuations in annual prevalence with rises being observed in the mid and late 1960s and mid and late 1970s. There was a noticeable male predominance in the cleft lip and cleft lip and palate groups of 1.52:1 and 1.98:1 respectively, with a 1:1 ratio in the cleft palate group. There were no significant differences in birthweight and mean maternal age in the three groups. In the cleft palate group, however, there was a significant trend towards an increase in the frequence of conception in the second half of the year. There was a maternal history of epilepsy in 4.4% of the cleft lip and 3% of the cleft lip and palate groups but only in 1 patient (0.6%) in the cleft palate group. The study illustrates the importance of environmental factors in the aetiology of facial clefting.

The Liverpool Congenital Malformations Registry.

Data collection and validation for the Liverpool Congenital Malformations Registry (LCMR) are described. Founded in 1960, the LCMR increased its area of surveillance in 1979 to include five health districts in Liverpool and its environs with approximately 20,000 births per annum. The LCMR is now one of the members of the European Congenital Anomalies Register (EUROCAT). Multiple sources of ascertainment are employed, the most useful of these being OPCS notifications, hospital discharge letters and data from specialised paediatric units. In spite of several difficulties encountered in data collection the data base is an invaluable tool both for routine monitoring of prevalence rates and as a starting point for epidemiological research.

The incidence of Down's syndrome over a 19-year period with special reference to maternal age.

The incidence of Down's syndrome in the Liverpool and Bootle areas from 1961 to 1979 was investigated. A total of 319 liveborn cases was ascertained over this period. Using 3-year moving averages, the incidence of the condition fell gradually from 1.62 per 1000 livebirths for 1961 to 1963 to 1.09 per 1000 livebirths for 1977 to 1979. This trend is significant at the 0.1% level. Over the same period the mean maternal age of Down's syndrome births fell gradually from 36.7 years in 1961 to 29.0 years in 1979. This trend is significant at the 1% level. There was a contemporaneous decrease in the proportion of total births to women over 35 years in the study area. Cytogenetic analysis was performed on 175 out of the 319 index cases (54.9%). Of these, there were 161 trisomies (92%), 11 translocations (6.3%), and three mosaics (1.7%). Between 1969 and 1979 four terminations of pregnancy for Down's syndrome were performed, all for trisomy. Quinquennial age specific incidences for Down's syndrome were calculated for the years 1960 to 1964, 1965 to 1969, 1970 to 1974, and 1975 to 1979. There have been no statistically significant changes over this time. It is suggested that the fall in incidence of Down's syndrome can be explained by the fall in mean maternal age.

Loperamide in the treatment of acute gastroenteritis in early childhood. Report of a two centre, double-blind, controlled clinical trial.

Loperamide at a dose of 0 . 2 mg/kg/day was compared with placebo in the treatment of acute infantile gastro-enteritis in hospital-based double-blind clinical trials carried out in parallel in Liverpool, England and Benghazi, Libya. Fifty patients aged one month to four years entered the study in each centre. Rotavirus was the predominant pathogen isolated in both centres. Pathogenic Escherichia coli was cultured from five children in the Liverpool study only. No statistically significant differences were observed in the duration of diarrhoea, length of stay in hospital or weight gain during the first 48 h after admission, between loperamide and placebo groups in either centre. Loperamide, in the dosage used in this study, appears to have no significant effect on the course of acute gastro-enteritis in early childhood. The possibility that these results may reflect specifically on rotavirus infection is discussed. No toxic effects of loperamide were observed.

Modulation of adrenal cell functions by cadmium salts. 4. Ca(2+)-dependent sites affected by CdCl2 during basal and ACTH-stimulated steroid synthesis.

In previous studies, nonlethal CdCl2 concentrations apparently inhibited basal Y-1 mouse adrenal tumor cell endogenous mitochondrial cholesterol conversion to pregnenolone. In addition, CdCl2 inhibited all agents stimulating both plasma membrane-dependent cAMP synthesis and 20 alpha-hydroxy-4-pregnen-3-one (20DHP) secretion. Bypassing the plasma membrane using dibutyryl-cAMP (dbcAMP) stimulated cytoplasmic cholesterol metabolism and 20DHP secretion in the presence of CdCl2. Since CdCl2 competed at metabolic steps requiring Ca2+ in other tissues, experiments were designed to examine Cd2+ competition with Ca2+ during steroidogenesis. Sets of cells incubated with either medium or adrenocorticotropin (ACTH) with or without CdCl2 were also treated with 0, 1.0, 5.0 or 10.0 mmol/L CaCl2 in the presence or absence of EGTA, a relatively specific Ca2+, but not Cd2+, chelating agent. Another experimental cell set incubated with either medium or ACTH, with or without CdCl2, was treated with or without 1 mmol/L A23187, an ionophore specifically facilitating extracellular Ca2+ transfer across plasma membranes. Besides determining Ca2+ involvement in steroidogenesis using steroid secretion as an endpoint, we directly measured Ca2+ concentrations using intracellular fura-2 fluorescence. Following loading with 2 mumol/L fura-2, cells remained untreated or medium was infused with CdCl2, ACTH, ACTH/CdCl2 or ACTH followed after 50 s by CdCl2. Using Ca(2+)-supplemented media, we observed that Cd2+ inhibition of ACTH-stimulated 20DHP secretion was completely reversed. Standard Ca(2+)-containing medium supplemented with Ca2+ also enhanced maximally stimulated 20DHP secretion by ACTH. 20DHP secretion by ACTH-treated and ACTH/Cd(2+)-treated cells was only reduced by EGTA, when Ca2+ was not supplemented. The ionophore A23187 increased basal and ACTH-stimulated 20DHP secretion by Cd(2+)-treated cells, suggesting that extracellular Ca2+ resources may compete against Cd2+ effects on plasma membrane cAMP synthesis and on basal cholesterol metabolism by mitochondria. No time-dependent change in Ca2+ concentrations occurred within untreated cell suspensions. ACTH stimulation caused a 25 s burst in Ca2+ concentrations before returning to basal, steady-state levels. Cd2+ also stimulated intracellular fura-2 fluorescence. Untreated cell suspensions infused with Cd2+ exhibited a continuous rise in intracellular fluorescence. ACTH/CdCl2-treated cells exhibited a hyperbolic rise in intracellular fluorescence over the 300 s study period. Cells treated with Cd2+ 50 s after ACTH treatment initially exhibited the 25 s fluorescence burst followed by a Cd(2+)-induced hyperbolic rise in intracellular Cd2+. These fluorescence measurements suggested that cytoplasmic Ca2+ changes do not appear to be necessary for basal 20DHP synthesis and secretion; only a 25 s burst in intracellular Ca2+ is necessary to a slightly higher plateau level for stimulated 20DHP synthesis and secretion. Cd2+ freely enters the cell under basal conditions and Cd2+ entry is accelerated by ACTH stimulation. Data were consistent with Ca2+ being required for optimal stimulated steroid production and Cd2+ probably competing with Ca2+ during basal mitochondrial cholesterol metabolism and plasma membrane ACTH-stimulated cAMP generation.

Cholera toxin ADP-ribosylates the islet-activating protein substrate in adipocyte membranes and alters its function.

In adipocyte membranes, cholera toxin may ADP-ribosylate the islet-activating protein (IAP) substrate, under certain conditions. Covalent modification is maximal in the absence of a guanosine triphosphate; in the presence of 5'-guanylylimidodiphosphate, incorporation of [32P]ADP-ribose is markedly reduced. ADP-ribosylation by cholera toxin has similar functional consequences as does IAP-mediated modification, i.e. the biphasic response of isoproterenol-stimulated adenylate cyclase to GTP and the inhibition by N6-phenylisopropyladenosine is abolished, and only the stimulatory phase remains. In contrast, membranes treated with cholera toxin in the presence of GTP display both the stimulatory and inhibitory responses to GTP. The binding of the adenosine analog [3H]N6-phenylisopropyladenosine is increased in the presence of GTP. Treatment of the membranes with IAP, but not with cholera toxin in the absence of GTP, reverses this GTP effect on [3H]N6-phenylisopropyladenosine binding. However, [3H]N6-phenylisopropyladenosine binding is still sensitive to GTP in membranes treated with cholera toxin in the presence of GTP. In adipocyte and cerebral cortical membranes, the IAP substrate appears as a 39,000/41,000-Da doublet which does not appear to reflect protease activity. On two-dimensional polyacrylamide gels, these two proteins migrate with approximate pI values 6.0 and 5.6, respectively. Although both behave similarly under all conditions explored in this study, it is unknown whether both, or only one, are involved in inhibition of adenylate cyclase activity. These results extend the already striking homology between the adenylate cyclase complex and the visual system. Ni, as well as transducin, may be ADP-ribosylated by cholera toxin and by IAP, and, in all cases, there are functional consequences.

Novel actinomycete isolated from bulking industrial sludge.

A novel actinomycete was the predominant filamentous microorganism in bulking activated sludge in a bench-scale reactor treating coke plant wastewater. The bacterium was isolated and identified as an actinomycete that is biochemically and morphologically similar to Amycolatopsis orientalis; however, a lack of DNA homology excludes true relatedness. At present, the isolate (NRRL B 16216) cannot be assigned to the recognized taxa of actinomycetes.

Surgical complications of intraoperative radiation therapy: the Radiation Therapy Oncology Group experience.

Intraoperative radiotherapy (IORT) is being used with increasing frequency in many institutions in the United States but little is known about the surgical complication rates. The Radiation Therapy Oncology Group initiated three prospective studies in IORT in 1986 and we report here the experience in advanced malignancies of the stomach, pancreas, and rectum. The incidence and nature of major surgical complications were reviewed and presented with their implications in regard to future IORT trials. Two hundred twenty-seven patients were entered on three studies by 20 participating institutions between 1985 and 1989. One hundred twenty-nine patients received IORT while 98 patients were found to have too advanced disease to be benefited by IORT and underwent palliative surgical procedures only. IORT doses ranged from 12-22 Gy and bowel anastomoses were not irradiated. Wound infection in the IORT group was 6% vs. 2% in the non-IORT patients but this was not significant at the P = 0.05 level. Other complications included anastomotic leak (n = 5), operative bleeding (n = 10), pancreatitis (n = 2), and were not statistically different in the IORT and non-IORT groups. The mortality rate for the IORT and non-IORT groups combined was 1.8%. This large multi-institutional experience in patients with advanced malignancy demonstrates that patients receiving IORT do not have a higher surgical complication rate than those not receiving IORT. Long-term survival data await the implementation of Phase III trials in advanced intra-abdominal malignancy.