Identification of the essential histidine residue for high-affinity binding of AlbA protein to albicidin antibiotics

The albA gene from Klebsiella oxytoca encodes a protein that binds albicidin phytotoxins and antibiotics with high affinity. Previously, it has been shown that shifting pH from 6 to 4 reduces binding activity of AlbA by about 30%, indicating that histidine residues might be involved in substrate binding. In this study, molecular analysis of the albA coding region revealed sequence discrepancies with the albA sequence reported previously, which were probably due to sequencing errors. The albA gene was subsequently cloned from K oxytoca ATCC 13182(T) to establish the revised sequence. Biochemical and molecular approaches were used to determine the functional role of four histidine residues (His(78), HiS(125), HiS(141) and His(189)) in the corrected sequence for AlbA. Treatment of AlbA with diethyl pyrocarbonate (DEPC), a histidine-specific alkylating reagent, reduced binding activity by about 95%. DEPC treatment increased absorbance at 240-244 nm by an amount indicating conversion to N-carbethoxyhistidine of a single histidine residue per AlbA molecule. Pretreatment with albicidin protected AlbA against modification by DEPC, with a 1 : 1 molar ratio of albicidin to the protected histidine residues. Based on protein secondary structure and amino acid surface probability indices, it is predicted that HiS125 might be the residue required for albicidin binding. Mutation of HiS125 to either alanine or leucine resulted in about 32% loss of binding activity, and deletion of HiS125 totally abolished binding activity. Mutation of HiS125 to arginine and tyrosine had no effect. These results indicate that HiS125 plays a key role either in an electrostatic interaction between AlbA and albicidin or in the conformational dynamics of the albicidin-binding site.

Sexual abuse, antisocial behaviour and substance use: gender differences in young community adolescents

Objective: To investigate gender-specific relationships between self-reported sexual abuse, antisocial behaviour and substance use in a large community sample of adolescents. Method: A cross-sectional study of students aged, on average, 13 (n = 2596), 14 (n = 2475) and 15 years (n = 2290), from 27 schools in South Australia with a questionnaire including sexual abuse, frequency and severity of substance use, depressive symptomatology (CES-D), family functioning (McMaster Family Assessment Device), and antisocial behaviour (an adapted 22-item Self-Report Delinquency Scale). Logistic regression analyses using HLM V5.05 with a population-average model were conducted. Results: In the model considered, reported sexual abuse is significantly independently associated with antisocial behaviour, controlling for confounding factors of depressive symptomatology and family dysfunction, with increased risks of three- to eightfold for sexually abused boys, and two- to threefold for sexually abused girls, compared to nonabused. Increased risks of extreme substance use in sexually abused girls (age 13) and boys (ages 13-15) are more than fourfold, compared to nonabused. Age differences were not statistically significant. Conclusion: Childhood sexual abuse is a risk factor for the development of antisocial behaviour and substance use in young adolescents. Clinicians should be aware of gender differences.

Sexual abuse and suicidality: gender differences in a large community sample of adolescents

Objective: A cross-sectional study of gender specific relationships between self-reported child sexual abuse and suicidality in a community sample of adolescents. Method: Students aged 14 years on average (N = 2,485) from 27 schools in South Australia completed a questionnaire including items on sexual abuse and suicidality, and measures of depression (Centre for Epidemiological Studies Depression Scale), hopelessness (Beck Hopelessness Scale), and family functioning (McMaster Family Assessment Device General Functioning Subscale). Data analysis included logistic regression. Results: In boys, self-report sexual abuse is strongly and independently associated with suicidal thoughts, plans, threats, deliberate self-injury, and suicide attempts, after controlling for current levels of depression, hopelessness, and family dysfunction. In girls, the relationship between sexual abuse and suicidality is mediated fully by depression, hopelessness, and family dysfunction. Girls who report current high distress about sexual abuse, however, have a threefold increased risk of suicidal thoughts and plans, compared to non-abused girls. Boys who report current high distress about sexual abuse have 10-fold increased risk for suicidal plans and threats, and 15-fold increased risk for suicide attempts, compared to non-abused boys. Fifty-five percent (n = 15) of sexually abused boys attempted suicide versus 29% (n = 17) girls. Conclusions: A history of sexual abuse should alert clinicians, professionals and caters in contact with adolescents, to greatly increased risks of suicidal behavior and attempts in boys, even in the absence of depression and hopelessness. Distress following sexual abuse, along with depression and hopelessness indicate increased risk of suicidal behavior in girls, as well as boys. (C) 2004 Published by Elsevier Ltd.

Ethics and intervention: The 'humanitarian exception' and the problem of abuse in the case of Iraq

This article investigates the ethics of intervention and explores the decision to invade Iraq. It begins by arguing that while positive international law provides an important framework for understanding and debating the legitimacy of war, it does not cover the full spectrum of moral reasoning on issues of war and peace. To that end, after briefly discussing the two primary legal justifications for war (implied UN authorization and pre-emptive self-defence), and finding them wanting, it asks whether there is a moral 'humanitarian exceptions to this rule grounded in the 'just war' tradition. The article argues that two aspects of the broad tradition could be used to make a humanitarian case for war: the 'holy war' tradition and classical just war thinking based on natural law. The former it finds problematic, while the latter it argues provides a moral space to justify the use of force to halt gross breaches of natural law. Although such an approach may provide a moral justification for war, it also opens the door to abuse. It was this very problem that legal positivism from Vattel onwards was designed to address. As a result, the article argues that natural law and legal positivist arguments should be understood as complementary sets of ideas whose sometimes competing claims must be balanced in relation to particular cases. Therefore, although natural law may open a space for justifying the invasion of Iraq on humanitarian terms, legal positivism strictly limits that right. Ignoring this latter fact, as happened in the Iraq case, opens the door to abuse.

Ethical issues in using a cocaine vaccine to treat and prevent cocaine abuse and dependence

A cocaine vaccine'' is a promising immunotherapeutic approach to treating cocaine dependence which induces the immune system to form antibodies that prevent cocaine from crossing the blood brain barrier to act on receptor sites in the brain. Studies in rats show that cocaine antibodies block cocaine from reaching the brain and prevent the reinstatement of cocaine self administration. A successful phase 1 trial of a human cocaine vaccine has been reported. The most promising application of a cocaine vaccine is to prevent relapse to dependence in abstinent users who voluntarily enter treatment. Any use of a vaccine to treat cocaine addicts under legal coercion raises major ethical issues. If this is done at all, it should be carefully trialled first, and only after considerable clinical experience has been obtained in using the vaccine to treat voluntary patients. There will need to be an informed community debate about what role, if any, a cocaine vaccine may have as a way of preventing cocaine addiction in children and adolescents.

Ecosystem response to antibiotics entering the aquatic environment

Awareness of antibiotics in wastewaters and aquatic ecosystems is growing as investigations into alternate pollutants increase and analytical techniques for detecting these chemicals improve. The presence of three antibiotics (ciproffoxacin, norfloxacin and cephalexin) was evaluated in both sewage effluent and environmental waters downstream from a sewage discharge. Bacteria cultured from the sewage bioreactor and receiving waters were tested for resistance against six antibiotics (ciprofloxacin, tetracycline, ampicillin, trimethoprim, erythromycin and trimethoprim/sulphamethoxazole) and effects of short term exposure (24h) to antibiotics on bacterial denitrification rates were examined. Antibiotics were detected entering the sewage treatment plant with varying levels of removal during the treatment process. Antibiotics were also detected in effluent entering receiving waters and detectable 500m from the source. Among the bacteria cultured from the sewage bioreactor, resistance was displayed against all six antibiotics tested and bacteria cultured from receiving waters were resistant against two of the antibiotics tested. Rates of denitrification were observed to decrease in response to some antibiotics and not to others, though this was only observed at concentrations exceeding those likely to be found in the environment. Findings from this preliminary research have indicated that antibiotics are entering our aquatic systems and pose a potential threat to ecosystem function and potentially human health. (c) 2004 Elsevier Ltd. All rights reserved.