Enhancing the utility of existing antibiotics by targeting bacterial behaviour?

The discovery of novel classes of antibiotics has slowed dramatically. This has occurred during a time when the appearance of resistant strains of bacteria has shown a substantial increase. Concern is therefore mounting over our ability to continue to treat infections in an effective manner using the antibiotics that are currently available. While ongoing efforts to discover new antibiotics are important, these must be coupled with strategies that aim to maintain as far as possible the spectrum of activity of existing antibiotics. In many instances, the resistance to antibiotics exhibited by bacteria in chronic infections is mediated not by direct resistance mechanisms, but by the adoption of modes of growth that confer reduced susceptibility. These include the formation of biofilms and the occurrence of subpopulations of 'persister' cells. As our understanding of these processes has increased, a number of new potential drug targets have been revealed. Here, advances in our ability to disrupt these systems that confer reduced susceptibility, and in turn increase the efficacy of antibiotic therapy, are discussed.     

β-Adrenoceptor binding correlates with behaviour of rats in the open field

Rats were injected IP once daily for 14 consecutive days with propranolol (5 mg/kg), yohimbine (2.5 mg/kg) or saline vehicle. A fourth group was unhandled during this time. Each rat was then placed in an open field for 4 min and its activity and defaecation recorded. Immediately after this, the animals were killed and cerebral cortices removed for radioligand binding to ₂- and -adrenoceptors and measurement of noradrenaline content. We report two sets of findings. First, -adrenoceptor density correlated positively, and affinity negatively, with the number of movements towards the centre of the field in the final 3 min of the trial. ₂-Adrenoceptor K _d, in contrast, correlated both with movements around the field and those directed towards the centre. Secondly, whereas the only specific drug effect was an increase in defaecation after treatment with propranolol, -adrenoceptor density was increased and affinity decreased in all injected groups, suggesting a non-specific effect of the stress of injection. Movements to and from the centre of the field were also increased in injected groups during the first minute of the trial. In both sets of findings the association of -adrenoceptor density with greater resistance to stress is hard to reconcile with existing theories of the role of -adrenoceptors in behavioural responses to stress.     

Compression behaviour of κ-carrageenan pellets

The compression behavior of high- and low drug strength pellets containing κ-carrageenan as pelletisation aid was investigated. Model drugs and fillers with different compression mechanisms were used and the effects of compression force and turret speed were examined. Regardless of the compression behavior of their starting components, all pellet formulations exhibited minimal to absent fragmentation and underwent compression by deformation, confirmed by increased equivalent diameter and aspect ratio and decreased roundness factor of the pellets retrieved after de-aggregation of tablets prepared from lubricated pellets. The retrieved pellets showed also higher fracture resistance in three of the tested formulations and no statistically significant difference in the remaining one thus excluding significant crack formation. A densification mechanism was suggested by decreased total porosity and reduced median pore radius of the compressed pellets. No effect of the process parameters on the degree of pellet deformation was reported. The tensile strength of the tablets prepared from unlubricated pellets increased slightly with increased compression force. Compression of pellets with high density silicified microcrystalline cellulose (SMCC HD 90) as embedding powder protected them from severe deformation and resulted in tablets with sufficient tensile strength, minimal friability, negligible elastic recovery and short disintegration time. The percentage of the pellets and the compression force affected the tensile strength of the prepared tablets whereas no influence of the turret speed and the pre-compression force was observed.     

Do gender and year of study affect the ability of the theory of planned behaviour to predict binge-drinking intentions and episodes?

Background: The present study tested the utility of the theory of planned behaviour (TPB), augmented with anticipated regret, as a model to predict binge-drinking intentions and episodes among female and male undergraduates and undergraduates in different years of study. Method: Undergraduate students (N?=?180, 54 males, 126 females, 60 per year of study) completed baseline measures of demographic variables, binge-drinking episodes (BDE), TPB constructs and anticipated regret. BDE were assessed one-week later. Results: The TPB accounted for 60% of the variance in female undergraduates' intentions and 54% of the variance in male undergraduates' intentions. The TPB accounted for 57% of the variance in intentions in first-year undergraduates, 63% of the variance in intentions in second-year undergraduates and 68% of the variance in intentions in final-year undergraduates. Follow-up BDE was predicted by intentions and baseline BDE for female undergraduates as well as second- and final-year undergraduates. Baseline BDE predicted male undergraduates’ follow-up BDE and first-year undergraduates’ follow-up BDE. Conclusion: Results show that while the TPB constructs predict undergraduates’ binge-drinking intentions, intentions only predict BDE in female undergraduates, second- and final-year undergraduates. Implications of these findings for interventions to reduce binge drinking are outlined.     

Study of the complexation behaviour of gliclazide with partially methylated β-cyclodextrin in solution and solid state

The complexation of Gliclazide (GL) with a partially methylated β-cyclodextrin was studied. Phase-solubility and ¹H NMR spectroscopy were employed to investigate the complexation behaviour in solution and to demonstrate the complexation in liquid medium with the participation of both azabicyclooctyl and tolyl moieties of GL in the inclusion process. Solid systems prepared by kneading, co-grinding and spray drying have also been checked, using DSC and HSM, for assessing the formation of the inclusion compound. Experimental evidence of the complexation between drug and cyclodextrin was reported for the co-ground and spray-dried systems.     

Effect of serotonergic lesion on “anxious” behaviour measured in the elevated plus-maze test in the rat

5,7-Dihydroxytryptamine (250 µg) was administered intracerebroventricularly to rats to lesion central serotonergic neurones. Fourteen days later the rats were tested in the elevated plus-maze anxiety model in comparison to sham lesioned animals. Twenty-four hours later, the rats were killed and serotonin levels and [³H]paroxetine binding measured in cortical and hippocampal membranes. The lesion destroyed 81% of the serotonergic innervation in the cortex and 99% in the hippocampus as determined by endogenous serotonin levels. Lesioned rats had an increased ratio of open/total arm entries in the elevated plus-maze, reflecting a decreased level of anxiety. These results are compatible with the implication of serotonin in the control of anxiety and suggest that an anxiolytic effect may be induced by lowering the level of serotonergic activation.     

Dopaminergic and serotonergic modulation of persistent behaviour in the reinforced spatial alternation model of obsessive–compulsive disorder

RATIONALE: We have proposed rewarded T-maze alternation as a model of obsessive-compulsive disorder (OCD): the serotonin agonist m-chlorophenylpiperazine (mCPP) increments persistence therein, while chronic pretreatment with selective serotonin reuptake inhibitor (SSRI fluoxetine) but not benzodiazepine or desipramine abolishes mCPP effects. However, we noted that acute SSRI administration also causes transient persistence increase, counteracted by mCPP pretreatment. OBJECTIVES: This study (a) further explores the cross-tolerance between fluoxetine and mCPP and (b) extends the model by investigating its sensitivity to dopaminergic manipulations (D2, 3 agonism--quinpirole). MATERIALS AND METHODS: In both experiments, baseline and drug testing were carried out under daily T-maze alternation training. Exp. 1: Matched group (n = 8) pairs of rats received one of the following 20-day pretreatments (daily intraperitoneal administration): (1) saline, (2) low-dose fluoxetine (2.5 mg/kg), (3) low-dose mCPP (0.5 mg/kg) or (4) combined fluoxetine + mCPP. One group per pretreatment then received a 4-day challenge with high-dose fluoxetine (10 mg/kg), the other with high-dose mCPP (2.5 mg/kg). Exp. 2: One group (n = 12) of rats received 20-day treatment with saline, another with quinpirole (0.5 mg/kg). RESULTS: Exp. 1: Saline and low-dose mCPP- or fluoxetine-pretreated animals showed significant persistence increases under both challenges, while combined low-dose fluoxetine + mCPP pretreatment afforded full protection from either challenge. Exp. 2: Quinpirole significantly increased directional persistence after 13 administration days. CONCLUSIONS: These results establish the sensitivity of the rewarded alternation OCD model to D2, 3 receptor activation, thereby extending its profile of pharmacological isomorphism with OCD. Furthermore, they suggest a common mechanism of action of an SSRI and a serotonin agonist in the control of directional persistence.     

Reliability of heroin users’ reports of drug use behaviour using a 24 month timeline follow-back technique to assess the impact of the australian heroin shortage

Background: In early 2001 Australia experienced a dramatic disruption to heroin supply. In order to investigate the impact of this phenomenon, heroin users were interviewed retrospectively to determine drug use behaviour over a two year period.

Aim: To examine the reliability of the timeline follow-back (TLFB) technique in determining heroin users’ patterns of drug use and related behaviours over a 24 month period.

Method: 27 current heroin users were recruited through drug use services and interviewed about their drug use behaviour retrospectively using the calendar method of the TLFB. Test–retest reliability was measured over seven days.

Results: Recall of regular drug use was generally reliable. Recall was poorest during January–April 2001, the peak period of the heroin shortage. Recall of criminal activity and weekly expenditure on drugs was variable, though generally poor. Recall of treatment entry and drug related health problems such as overdose was also variable.

Conclusion: The 24 month TLFB did not obtain information reliable enough to examine sporadic drug use or overall changes in the patterns of drug use associated with the heroin shortage. To avoid this situation cohorts of injecting drug users need to be established as these phenomena cannot be accurately studied retrospectively.     

Wettability parameters and deformational behaviour of powder–liquid mixes in the funicular agglomeration phase

Wettability of two powdered materials (lactose and sulfadiazine) of similar particle size is quantified as contact angle, spreading coefficients and work of adhesion. On the basis of overwetting tests, performed in a specially-constructed wet-kneading rheometer, the agglomeration phases are distinguished by recording the resistance to mixing as torque exerted on the mixing vessel with increasing quantities of different granulating fluids (water, isopropanol, water/isopropanol mixture and liquid paraffin). Separate powder–liquid mixing experiments are carried out, with selected amounts of liquids, and the tensile strength of the resulting wet masses is measured at different consolidation states together with their resistance to densification. It was found that both the amount of granulating liquid, corresponding to the mean funicular agglomeration state, and the deformational behaviour of wet masses with certain liquid volume fraction, quantified as tensile strength and resistance to densification are related to the work of adhesion but only for liquids of similar viscosity. No correlation was observed between the torque and the work of adhesion or the viscosity of the granulating liquid.     

Effects of apomorphine and haloperidol on “spontaneous” stereotyped licking behaviour in the Cebus monkey

Three recently arrived drug naive Cebus apella monkeys with spontaneous stereotyped oral movements were treated with apomorphine and haloperidol using a wide dose range. Low doses of apomorphine (0.05–0.1 mg/kg) suppressed the oral stereotypies without affecting normal behaviour such as grooming and scratching. Higher doses of apomorphine (0.25–1.0 mg/kg) and haloperidol (0.01–0.1 mg/kg) also decreased or abolished the oral stereotypies, but induced generalized stereotypies (apomorphine) or dystonia/parkinsonism (haloperidol), suppressing normal behaviour. The findings indicate that dopamine is involved in these presumably stress-induced (not drug-induced) stereotypies.     

Does informing people who inject drugs of their hepatitis C status influence their injecting behaviour? Analysis of the Networks II study

BackgroundPeople who inject drugs (PWID) are at risk of hepatitis C virus (HCV). It is plausible that PWID who receive a diagnosis of HCV will reduce their injecting risk out of concern for their injecting partners, although evidence for this is currently limited. The aim of this study was to investigate whether informing PWID of their HCV diagnosis was associated with a change in injecting behaviour.MethodsProspective, longitudinal study of PWID recruited from street drug markets across Melbourne, Australia. Interviews and HCV testing were conducted at 3-monthly intervals. The association between receiving a diagnosis of HCV and (i) injecting frequency and (ii) injecting equipment borrowing, was examined using generalized estimating equations (GEE) analysis.ResultsThirty-five individuals received a diagnosis of HCV during the study period. Receiving a diagnosis of HCV was associated with a decrease of 0.35 injections per month (p = 0.046) but there was no change in injecting equipment borrowing (p = 0.750).ConclusionsA small reduction in injecting frequency was observed in PWID who received a diagnosis of HCV. This finding should be investigated further in larger studies examining a wider range of injecting risk behaviours.     

Identification of a novel snake peptide toxin displaying high affinity and antagonist behaviour for the ��2-adrenoceptors

BACKGROUND AND PURPOSE

Muscarinic and adrenergic G protein-coupled receptors (GPCRs) are the targets of rare peptide toxins isolated from snake or cone snail venoms. We used a screen to identify novel toxins from Dendroaspis angusticeps targeting aminergic GPCRs. These toxins may offer new candidates for the development of new tools and drugs.

EXPERIMENTAL APPROACH

In binding experiments with ³H-rauwolscine, we studied the interactions of green mamba venom fractions with α₂-adrenoceptors from rat brain synaptosomes. We isolated, sequenced and chemically synthesized a novel peptide, ρ-Da1b. This peptide was pharmacologically characterized using binding experiments and functional tests on human α₂-adrenoceptors expressed in mammalian cells.

KEY RESULTS

ρ-Da1b, a 66-amino acid peptide stabilized by four disulphide bridges, belongs to the three-finger-fold peptide family. Its synthetic homologue inhibited 80% of ³H-rauwolscine binding to the three α₂-adrenoceptor subtypes, with an affinity between 14 and 73 nM and Hill slopes close to unity. Functional experiments on α_2A-adrenoceptor demonstrated that ρ-Da1b is an antagonist, shifting adrenaline activation curves to the right. Schild regression revealed slopes of 0.97 and 0.67 and pA₂ values of 5.93 and 5.32 for yohimbine and ρ-Da1b, respectively.

CONCLUSIONS AND IMPLICATIONS

ρ-Da1b is the first toxin identified to specifically interact with α₂-adrenoceptors, extending the list of class A GPCRs sensitive to toxins. Additionally, its affinity and atypical mode of interaction open up the possibility of its use as a new pharmacological tool, in the study of the physiological roles of α₂-adrenoceptor subtypes.     

Potential of the octanol–water partition coefficient (log P) to predict the dermal penetration behaviour of amphiphilic compounds in aqueous solutions

Aqueous amphiphilic compounds may exhibit enhanced skin penetration compared with neat compounds. Conventional models do not predict this percutaneous penetration behaviour. We investigated the potential of the octanol–water partition coefficient (log P) to predict dermal fluxes for eight compounds applied neat and as 50% aqueous solutions in diffusion cell experiments using human skin. Data for seven other compounds were accessed from literature. In total, seven glycol ethers, three alcohols, two glycols, and three other chemicals were considered. Of these 15 compounds, 10 penetrated faster through the skin as aqueous solutions than as neat compounds. The other five compounds exhibited larger fluxes as neat applications. For 13 of the 15 compounds, a consistent relationship was identified between the percutaneous penetration behaviour and the log P. Compared with the neat applications, positive log P were associated with larger fluxes for eight of the diluted compounds, and negative log P were associated with smaller fluxes for five of the diluted compounds. Our study demonstrates that decreases or enhancements in dermal penetration upon aqueous dilution can be predicted for many compounds from the sign of log P (i.e., positive or negative). This approach may be suitable as a first approximation in risk assessments of dermal exposure.     

α-Melanocyte stimulating hormone stimulates sexual behaviour in the female rat

Administration of -melanocyte stimulating hormone into the third ventricle increased lordosis behaviour in ovariectomized female rats primed with either oestradiol benzoate and progesterone or oestradiol benzoate alone. -Melanocyte stimulating hormone had a similar effect in oestradiol benzoate-primed females when administered subcutaneously. These data suggest that -melanocyte stimulating hormone may be important in controlling sexual behaviour in the female rat.     

LC–MS profiling of N-alkylamides in Spilanthes acmella extract and the transmucosal behaviour of its main bio-active spilanthol

N-Alkylamides are a promising group of naturally occurring bio-actives, with evidence for immune stimulating properties, which find applications i.a. in buccal preparations. In Spilanthes extracts, these properties are mainly ascribed to the most abundant N-isobutylamide, spilanthol. Yet, other N-alkylamides present in these extracts may contribute to this effect, as well as to its potential toxicity and physiologic interactions. Therefore, N-alkylamide profiling of an ethanolic Spilanthes extract was performed using a gradient reversed phase high performance liquid chromatography/electrospray ionization ion trap mass spectrometry (HPLC/ESI-MS) method on an embedded polar column. MS¹ and MS² fragmentation data were used for identification purposes. Moreover, the transmucosal behaviour of spilanthol, formulated in this ethanolic extract and in two commercially available oral gels, was evaluated using porcine buccal mucosa in a Franz diffusion cell experimental set-up. A high-throughput HPLC-UV method was used for the quantification of spilanthol in the receptor phase. Fundamental permeation characteristics of spilanthol in a solvent-independent way (100% aqueous dose solution) were obtained using different propylene glycol/water ratios.     

Are there pseudophototropic reactions in biology? Part 3: The pseudophototropic behaviour of the cell membrane of halobacteria (author's transl)

The behaviour of coloured membrane portions of "halobacteria" is studied with respect to spectral response and reversibility. The results are in good correlation with colour generating effects of pseudophototropic reactions. The disclosed and dicussed systems are in the range from purely synthetic to purely biological.     

Neonatal phencyclidine administration and post-weaning social isolation as a dual-hit model of ‘schizophrenia-like’ behaviour in the rat

Rationale

Schizophrenia is a debilitating disorder comprising positive, negative and cognitive deficits with a poorly defined neurobiological aetiology; therefore, animal models with greater translational reliability are essential to develop improved therapies.

Objectives

This study combines two developmental challenges in rats, neonatal phencyclidine (PCP) injection and subsequent rearing in social isolation from weaning, to attempt to produce more robust behavioural deficits with greater translational relevance to schizophrenia than either challenge alone.

Methods

Forty-two male Lister-hooded rat pups received the N-methyl-d-aspartate (NMDA) receptor antagonist, phencyclidine (PCP, 10 mg/kg, s.c.), or vehicle on post-natal day (PND) 7, 9 and 11 and were weaned on PND 23 into group housing (saline-treated n?=?11 or PCP-treated n?=?10) or isolation (saline n?=?10 or PCP n?=?11). Six weeks post-weaning, novelty- and PCP-induced (3.2 mg/kg) locomotor activity, novel object discrimination, prepulse inhibition of acoustic startle and contextual memory in a conditioned emotion response (CER) were recorded.

Results

Isolation rearing alone significantly elevated baseline locomotor activity and induced visual recognition memory impairment in novel object discrimination. Neonatal PCP treatment did not induce locomotor sensitisation to a subsequent acute PCP injection, but it impaired prepulse inhibition when combined with isolation rearing. CER freezing behaviour was significantly reduced by isolation rearing but an even greater effect occurred when combined with neonatal PCP treatment.

Conclusions

Neonatal PCP and isolation rearing both produce behavioural deficits in adult rats, but combined treatment caused a wider range of more severe cognitive impairments, providing a more comprehensive preclinical model to determine the neurobiological aetiology of schizophrenia than either treatment alone.     

Distinct periods of developmental sensitivity to the effects of 3,4-(±)-methylenedioxymethamphetamine (MDMA) on behaviour and monoamines in rats

Previous findings showed allocentric and egocentric learning deficits in rats after MDMA treatment from postnatal days (PD) 11-20 but not after treatment from PD 1-10. Shorter treatment periods (PD 1-5, 6-10, 11-15, or 16-20) resulted in allocentric learning deficits averaged across intervals but not for any interval individually and no egocentric learning deficits individually or collectively. Whether this difference was attributable to treatment length or age at the start of treatment was unclear. In the present experiment rat litters were treated on PD 1-10, 6-15, or 11-20 with 0, 10, or 15 mg/kg MDMA q.i.d. at 2-h intervals. Two male/female pairs/litter received each treatment. One pair/litter received acoustic startle with prepulse inhibition, straight channel swimming, Cincinnati water maze (CWM), and conditioned fear in a latent inhibition paradigm. The other pair/litter received locomotor activity, straight channel swimming, Morris water maze (MWM), and locomotor activity retest with MK-801 challenge. MDMA impaired CWM learning following PD 6-15 or 11-20 exposure. In MWM acquisition, all MDMA-treated groups showed impairment. During reversal and shift, the PD 6-15 and PD 11-20 MDMA-treated groups were significantly impaired. Reductions in locomotor activity were most evident after PD 6-15 treatment while increases in acoustic startle were most evident after PD 1-10 treatment. After MK-801 challenge, MDMA-treated offspring showed less locomotion compared to controls. Region-specific changes in brain monoamines were also observed but were not significantly correlated with behavioural changes. The results show that PD 11-20 exposure to MDMA caused the largest long-term cognitive deficits followed by PD 6-15 exposure with PD 1-10 exposure least affected. Other effects, such as those upon MK-801-stimulated locomotion showed greatest effects after PD 1-10 MDMA exposure. Hence, each effect has a different window of developmental susceptibility.     

Does the effect of morphine challenge change on maternal behaviour of dams chronically treated with morphine during gestation and further on during lactation?

Opioids impair the maternal behaviour of rats. The effect of morphine on maternal behaviour in dams treated chronically with morphine during the whole pregnancy and lactation has not been analysed systematically. The aim of the present study was to investigate the possible differences in the disruptive effect of morphine on maternal behaviour following morphine challenges between dams treated chronically with saline or morphine during gestation and postpartum. The antinociceptive action of morphine was also studied in dams. The disruptive effect of morphine on maternal behaviour was not changed as the postpartum period passed. The duration of this effect of morphine lasted for about 2 h. The dose-dependent disruptive effect of acute doses of morphine on maternal behaviour was more marked in the morphine-treated dams, than in the saline-treated ones, indicating a tendency for sensitisation to this effect. A trend for tolerance was observed to the antinociceptive effect of morphine in animals treated daily with morphine during the entire gestational and lactation periods; however, this difference did not reach statistical significance. Our experimental protocol might be a predictive model of human opioid abuse. Sensitisation to the impairing effect of opiates on maternal behaviour may explain why a mother abusing heroin neglects her baby even if she does not experience euphoria.