The characteristics and expression of laboratory derived chloramphenicol (CM) resistance in P. aeruginosa were examined. Resistant strains exhibiting single cell resistance of 1.5 to 2 mg/mL were readily isolated following one passage in CM at 150 to 1000 (mu)g/mL. Isogenic strains, selected on CM at 150 and 500 (mu)g/mL were chosen for detailed study. Resistance was not a consequence of drug detoxification or altered sensitivity of the target site. The resistant strains exhibited unusual phenotypic properties including pronounced variations in growth rate, CM susceptibility and cell morphology as a function of the composition of the growth medium. Growth in CM also resulted in significant alterations in amino acid transport and respiratory capacity, the extent of which varied with the strain, the growth medium and the concentration of CM. These drug and medium-dependent alterations were determined to reside in an increased and highly specific requirement for Ca('2+), Mg('2+), Mn('2+) or Sr('2+). Manipulation of the divalent cation concentration of a variety of growth media resulted in dramatic alterations in growth rate, resistance and amino acid transport. Ca('2+) was significantly more effective than the latter three ions. The expression of native and plasmid-mediated CM resistance was also modified by the external concentration of divalent cations. In view of the nature and specificity of the cation requirement, it was concluded that (1) divalent cation-mediated alterations of outer membrane permeability are fundamental to the expression of native and acquired CM resistance in P. aeruginosa; (2) laboratory-derived CM resistance involves envelope changes, such that interaction with divalent cations promotes more effective exclusion of CM. The latter conclusion is supported by other divalent cation-dependent alterations in envelope function in the resistant strains.

The characteristics of the effects of chloramphenicol on Pseudomonas aeruginosa were examined. Resistant strains were easily isolated following a single passage in chloramphenicol at 150 $ mu$g/ml to 500 $ mu$g/ml. Drug detoxification or altered sensitivity of the target site could not be the mechanism of resistance. This resistance to chloramphenicol was correlated with the addition of an outer membrane protein with a molecular weight of 49 kDa and the loss of two outer membrane proteins, one with the molecular weight of 19 kDa and the other of about 10 kDa. The highly specific requirement of the resistant strains for Ca$ sp{2+}$, Mg$ sp{2+}$, Mn$ sp{2+}$ or Sr$ sp{2+}$ described by Irvin and Ingram (1982) was confirmed by the observation that the outer membrane of the resistant cells contained twice as much Mg$ sp{2+}$ cation as the sensitive cells. Many other experiments designed to observe the effects of chloramphenicol on the outer membrane of P. aeruginosa failed. It was concluded that the observations made in this study strongly suggested a "re-structuring" of the outer membrane of P. aeruginosa, rendering the resistant cells more impermeable to chloramphenicol.

Glutamate-gated chloride channels (GluCls) are the proposed site of action for macrocyclic lactone anthelminthics, such as IVM, and the milbemycins, such as MOX. The objective of this thesis was to determine whether Haemonchus contortus GluCls are important targets for these anthelminthics. To begin to address this we cloned a full length GluCl alpha-subunit cDNA from H. contortus (HcGluCla). This subunit shares a high homology with GluCl subunits from Caenhorhabditis elegans that have been shown to be important targets for IVM, suggesting that HcGluCla is also an IVM target. However, if HcGluCla is an IVM receptor then it should contain an IVM binding site. To investigate this, the HcGluCla gene was expressed in COS-7 cells. The resulting subunit bound [3H]IVM and [ 3H]MOX with affinities sufficiently high enough to explain their high in vivo potency. Interestingly, glutamate was an allosteric modulator of [ 3H]MOX and [3H]IVM binding where it increased the affinity of these drugs to HcGluCla. To gain further insight into the potentiation of IVM, various glutamatergic and non-glutamatergic ligands were screened for their ability to enhance [3H]IVM binding to HcGluCla. Of the ligands tested, only the GluCl agonists glutamate and ibotenate potentiated [3H]IVM binding. It is possible therefore that if IVM interacts with GluCls in vivo then IVM efficacy may be enhanced by GluCl agonists. To examine this, we tested whether ibotenate could enhance IVM efficacy in gerbils infected with H. contortus. In in vivo efficacy studies, ibotenate (at 1 mg/kg) increased IVM efficacy by 15% (p = 0.048). The enhancement of IVM efficacy in vivo by a GluCl agonist suggests that one of the IVM targets in H. contortus is the GluCl. Finally, to determine the potential physiological response from an interaction between IVM and H. contortus GluCls, we expressed HcGluCla in Xenopus oocytes. HcGluCla expressed in oocytes formed a homomeric channel that responded to

Plasmodium falciparum is a protozoan parasite that causes malaria, a disease that is widely spread in the tropical world. Chloroquine has been very effective against malaria since it was introduced into the field until the emergence of chloroquine resistant malaria. Chloroquine resistant malaria has become widely spread in the endemic area. In addition, cross resistance to other antimalarial drugs that are different in structure and function has been reported, even though some of these drugs had not been previously used in that particular region. The objective of this study was to determine the molecular mechanism of this resistance. Actinomycin D resistant Plasmodium falciparum was selected in vitro from the drug sensitive parental clone, 3D7. Interestingly, we found that the selected strain is resistant to chloroquine, mefloquine, antimalarial drugs, and Rhodamine 123. Comparison between 3D7 parental and 3D7R/act-D2 resistant P. falciparum did not show a difference in the level of expression of pfmdr1 previously implicated in the drug resistance. In addition we found that the level of accumulation of two drugs actinomycin D is reduced in the resistant parasite as compared with the sensitive one. Further studies indicated that the reduction in the drug accumulation was due to the increase in drug efflux. Furthermore, to identify if other P-glycoprotein homologues are involved in the resistance, oligonucleotide primers to conserved sequences in ABC domains have been used. An ABC protein homologous to subunit 4 of the 26S proteasome complex has been cloned. In vitro transcription, translation and immunoprecipitation analysis were done using reticulocytes lysate and polyclonal antibodies generated against peptide sequence in the P. falciparum S4 subunit. Surprisingly a decrease in the expression of this gene was found in the resistant clone, 3D7R/act-D2, compared to its parental cell line as determined by Northern blot analysis. Studies are in progress to determine

Bacteriological survey of the fish pathogens in Greek mariculture between 1994- 1997 was followed by analysis of prevalence in sea bass, sea bream, sharpsnout bream and common Dentex and discussion of the impact of various fish pathogens. In addition antibiotic resistance profiles and frequencies were studied using quantitative antibiogram and MIC analysis for the two most commonly used antibiotics Oxolinic acid and Oxytetracycline and clinically relevant MIC breakpoints were extrapolated for different fish species and main fish pathogens. The kinetics of the above antimicrobials were analysed in eight experiments where two fish species namely sea bass and sea bream as well as two water temperatures were employed. Muscle, liver, serum, skin samples were analysed by two HPLC methods and two bioassay methods were developed. The relative importance and significance of these findings was evaluated in the general context of pharmacokinetic studies in fish. Kinetic data were compared to clinical data and practical implications were evaluated. Issues like antibiotic resistance and its implications, the implications of residues and resistance in human health and the environment were analysed in order to put this study in context. Conclusions tackled important aspects of antimicrobial chemotherapy and future work was suggested.

The abundant nuclear enzyme poly (ADP-ribose)polymerase (P ARP) catalyses the formation of long homopolymeric chains of ADP-ribose, utilising NAD+ as a substrate, as the immediate cellular response to DNA damage. PARP recognises a damaged section of DNA and initiates polymer synthesis, which is believed to act as a signal to effect the repair of the lesion. A selective, potent PARP inhibitor could block the recognition, and hence repair, of DNA damage induced by cancer chemotherapy. Since increased DNA repair is regarded as a mechanism whereby tumour cells can become resistant to treatment, PARP inhibitors have therapeutic potential as resistance modifying agents. From a study of PARP inhibitors such as 3-hydroxybenzarnide (A), benzimidazole derivatives (B) were proposed as inhibitors of the enzyme, and the synthesis and biological evaluation of a series of such molecules has been achieved. Substituted 2-aryl benzirnidazoles have proved to be highly potent PARP inhibitors (B;R= 4'NO2Ph, IC5o= 59 nM), under a permeabilised cell assay the nitro phenyl derivative (B; R= 4'N02Ph) is the most potent compound reported to date (IC50= 19 nM). 2-Methyl benzirnidazole-4-carboxamide (B; R= Me) has been shown to potentiate the in vitro cytotoxicity of the antitumour agent temozolomide in L1210 cells, and the synthesis of benzimidazole inhibitors suitable for pre-clinical in vivo eluation has also been investigated, This thesis demonstrates that benzimidazole PARP inhibitors have promising potential for clinical development as resistance modifying agents.

No description available.

Glutamate binding studies on membrane preparations from unselected and ivermectin-selected strains of the parasitic nematode of ruminants, Haemonchus contortus, indicated a single class of saturable, high affinity binding sites which are sensitive to ivermectin and exhibit different pharmacological characteristics from any known mammalian glutamate receptor. These studies showed that H. contortus larvae possess substantially more glutamate binding sites with lower affinity than adults. Moreover, selection with ivermectin was associated with an increase in the number of glutamate binding sites in adults and an increase in the affinity for glutamate binding in larvae. When investigating the effects of ivermectin on glutamate binding kinetics, it was discovered that ivermectin decreased Bmax values in unselected, but not in ivermectin-selected, parasites. Inulin intake studies were performed in unselected and ivermectin-selected H. contortus worms to relate the glutamate binding results to the biological activity, pharyngeal pumping. These studies showed that glutamate, ivermectin and the structurally similar anthelmintic, moxidectin, inhibit pharyngeal pumping, and that glutamate influences the effects of ivermectin and of moxidectin, on pharyngeal pumping. Lastly, selection with ivermectin was associated with an alteration of the effects of ivermectin, but not moxidectin, on pharyngeal pumping. The results of this work suggest that a novel ivermectin-sensitive, glutamate receptor, which influences pharyngeal function, is involved in the development of ivermectin resistance in H. contortus.

Plasmodium falciparum is a protozoan parasite and the causative agent of the most lethal form of malaria, a major disease in the tropical world. Chloroquine has been very effective in treatment of this disease, however the emergence of chloroquine-resistant strains in most geographical regions where malaria is endemic has made difficult the control of malaria. In addition, resistance to other antimalarials has been observed in these regions. The objective of this study was to determine the molecular mechanisms of multidrug resistance in P. falciparum. We have selected in vitro a P. falciparum strain resistant to actinomycin D from a parental drug sensitive clone, 3D7. Interestingly, we found that the actinomycin D resistant clone is less sensitive to chloroquine and mefloquine (antimalarial drugs) and rhodamine123. Comparison between parental 3D7 and resistant P. falciparum did not show differences in the copy number or level of expression of pfmdr1 previously implicated in chloroquine or mefloquine resistance. Furthermore, to identify if other P-glycoprotein homologues are involved in resistance, we used oligonucleotide primers to conserved sequences in ABC domains. An ABC protein, a homologue to the subunit 4, of the 26S proteasome complex has been cloned. To determine if this gene was involved in resistance to actinomycin D, a Northern blot was done. Surprisingly it was found a decreased in the expression of this gene in the resistant cell line, 3D7R/actD2, in comparison with its parental cell line, 3D7. Studies are in progress to determine the role of the PFS4 subunit in the resistance phenotype of 3D7R/actD2.

The mechanism of benzimidazole (BZ) anthelmintic resistance in Haemonchus contortus was investigated. The total binding (TB), low-affinity binding (LAB) and high-affinity (specific) binding (HAB) of ($ sp3$H) BZs (mebendazole (MBZ), oxibendazole (OBZ), albendazole (ABZ) and oxfendazole (OFZ)) in supernatants derived from BZ-susceptible (S) and BZ-resistant (R) strains were examined and compared. The TB of all ($ sp3$H) BZs was reduced for the R strain. The TB of OBZ, MBZ and ABZ was separated into LAB and HAB. However, OFZ bound with low-affinity. The binding affinity, K$ sb{ rm a},$ and maximum binding, B$ sb{ rm max},$ for the HAB of OBZ and MBZ were calculated using computer programs. Compared with the S strain, the B$ sb{ rm max}$ of the R strain was reduced but the K$ sb{ rm a}$ was not affected. LAB to parasite preparations devoid of tubulin was observed but HAB occurred to preparations containing tubulin only. The HAB per mg protein decreased from egg through larva to adult stage. It was shown by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blot and enzyme-linked immunosorbent assay (ELISA) analysis that the tubulin content per mg protein decreased from egg, through larva to adult worm. The ability of various BZs--OBZ, MBZ, ABZ, OFZ, fenbendazole (FBZ), albendazole sulphoxide (ABZSO), albendazole sulphone (ABZSO$ sb2),$ and thiabendazole (TBZ)--to bind tubulin was compared by displacement analysis and their IC$ sb{50}$ ( (BZ) required to inhibit 50% of the ($ sp3$H) BZ binding) and K$ sb{ rm a}$ values were determined. The IC$ sb{50}$ and K$ sb{ rm a}$ values approximately correlated with the known anthelmintic potency (recommended therapeutic doses) of the BZs except for OFZ and ABZSO. Tubulin bound BZs at 4$ sp circ$C with lower K$ sb{ rm a}$ than at 37$ sp circ$C. Western blot of tubulin separated by 2-dimensional electrophoresis showed that the $ beta$-tubulin isoform pattern of the S and R strains were dissimilar whil