High Degree Of Cross-resistance Research Articles

Structure-activity relationships in a series of auranofin analogues showing remarkable antiproliferative properties

The antiproliferative properties of a series of structurally-related gold(I) and silver(I) linear complexes inspired to the clinically established gold-based drug auranofin were investigated in A2780 ovarian cancer cells and in their auranofin (A2780/AF-R) and cisplatin (A2780/CDDP-R) resistant counterparts. In A2780 cells and in the cisplatin-resistant subline, gold-based analogues manifested a cytotoxicity profile comparable or superior to auranofin, while the silver-based analogues were less active; both gold and silver complexes overcame cisplatin resistance. Yet, a high degree of cross resistance toward gold analogues was noticed in A2780/AF-R cells. In the same cell line cross-resistance for silver analogues was also observed, though lower. All metal complexes were scrutinized for their ability to inhibit thioredoxin reductase (TrxR), the putative primary target for auranofin: overall, gold compounds were more potent TrxR inhibitors than the corresponding silver compounds, probably, as the consequence of the stronger binding of gold to the active site selenocysteine residue. These results highlight that the thiosugar ligand of auranofin is not essential for cytotoxicity while the nature of the metal center (gold/silver) plays a relevant role in its modulation. In addition, a rather clear correlation was found between cytotoxic potency of tested compounds and their ability to inhibit TrxR activity, being gold compounds more effective than silver analogues. However, the residual TrxR activity, measured in A2780 cells treated with the half-maximal inhibitory concentrations of various metal complexes, resulted far higher than expected. These results suggest that additional cytotoxic mechanisms must be operative. The implications of these results are discussed.

Concurrent occurrence of Amp C and Cefotaxime (CTX)-M inclinical isolates of enterobacteriaceae

Enterobacteriaceae producing both Amp C beta lactamases and extended-spectrum beta lactamases (ESBLs) have been increasingly reported worldwide. While the phenotypic tests for ESBL is standardised and used widely, it is not so for Amp C. When they coexist they may mask each other's detection phenotypically. We undertook this study to detect the concurrent occurrence of Cefotaxime (CTX)-M and plasmid Amp C in clinical isolates of Enterobacteriaceae by phenotypic and genotypic methods. One hundred clinically significant isolates of Escherichia coli (E. coli; 43), Klebsiella pneumoniae (K. pneumoniae; 43) and Proteus mirabilis (P. mirabilis; 14) were included in the study. Antibiotic susceptibility testing to various classes of antimicrobials was performed by disc diffusion using Clinical Laboratory Standards Institute (CLSI) guidelines. Isolates were screened for production of ESBL by CLSI method and Amp C beta lactamase by inhibitor based method using boronic acid and cloxacillin. Polymerase chain reaction (PCR) was performed for the detection of plasmid Amp C genes and blaCTX-M . Plasmid Amp C genes were detected in 27 isolates which included CIT (Origin Citrobacter freundii): 14; DHA (Dhahran Hospital in Saudi Arabia): 12; EBC (Origin Enterobacter cloacae): 1. BlaCTX-M was detected in 51 isolates. Both coexisted in one E. coli and two K. pneumoniae. In one of the K. pneumoniae isolate, all phenotypic tests employed were negative. A high degree of cross resistance to other classes of antimicrobials was observed. Carbapenem resistance was noted in 21 isolates. The concurrent occurrence of Amp C and CTX-M is not common in clinical isolates of Enterobacteriaceae. Phenotypic tests perform poorly when these enzymes are coproduced.

Resistencia a telaprevir

Telaprevir is an extremely potent antiviral drug. However, as with other direct-acting antiviral agents against the hepatitis C virus (HCV), this potency can be compromised by the rapid emergence of resistance mutations. This phenomenon is favored by the high rate of HCV replication, the lack of corrective activity to HCV polymerase errors, a low genetic barrier to resistance, and poor treatment adherence. Like other HCV protease inhibitors, telaprevir has a low genetic barrier to resistance. Phase II/III clinical trials (ADVANCE, ILLUMINATE and REALIZE) have characterized resistance to telaprevir in combination with pegylated interferon and ribavirin both genotypically and phenotypically. Mutations are selected mainly in positions 36, 54, 55, 155 and 156. The resistance profile depends on the genetic subtype of HCV, with selection of mutations in positions 36 and 155 for subtype 1a and in positions 36, 54, 55 and 156 for subtype 1b. The V36M+R155K and A156F/T/V variants confer a high degree of resistance (>25-fold increase), while the impact on resistance of other mutations such as V36A/G/M, T54A/S, R155G/K/M/T and A156S is lower (3-25- fold increase). The presence of polymorphisms or mutations associated with telaprevir resistance is very low (< 1%), especially for mutations with a higher impact on resistance. There is a high degree of cross resistance among protease inhibitors. However, the rapid reversion of telaprevir resistance mutations after the end of treatment could allow the use of “recycling” strategies with protease inhibitors.

Profile of infective microorganisms causing ventilator-associated pneumonia: A clinical study from resource limited intensive care unit

Background:Ventilator-associated pneumonia (VAP) is the most common cause of hospital acquired infection and death among patients admitted in ICU. Microorganisms responsible for VAP vary from place to place. Gram-negative bacteria (GNB) have emerged as a major group of pathogen causing VAP and over the years carbapenem group of antibiotics has emerged as one of the important antibiotics used in the critically ill patients. There have been reports of increased occurrence of infection by carbapenem-resistant bacteria in health care settings in recent times.Aim:The aim of the study was to assess the incidence of VAP, their microbiological profile with reference to carbapenemase producing GNB in the intensive care unit of a tertiary care hospital, their relation to initial emperical antibiotic therapy, sensitivity patterns, and outcome.Materials and Methods:This prospective study was carried out over the period of 1 year (July 2010-June 2011) on 100 randomly selected patients above the age of 18 years admitted in the emergency/ICU and requiring intubation and mechanical ventilation for more than 72 hours. The diagnosis of VAP was established on the basis of clinical and radiological parameters as per Centre of Disease Centres (CDC) guidelines. A baseline sample was obtained after initial endotracheal intubation. Thereafter, the culture sent on the first day of occurrence of clinical sign of VAP. Culture was done on blood agar and MacConkey agar. All imipenem-resistant strains were further confirmed by Modified Hodge test and combined disc for confirmation of respective carbapenemase.Results:Incidence of VAP was found to be 51%. GNB mainly Citrobacter 28 (52.83%) and Klebsiella pneumoniae 7 (13.21%), were the most commonly isolated pathogens. The prevalence of carbapenemase-producing GNB was alarmingly high 24/50 (48%). The entire carbapenemase producers showed high degree of cross resistance to antibiotics with some sensitivity to Polymyxin B (94 %) and Tigecycline (96%)Conclusion:High incidence of VAP and the potential carbapenemase-producing GNB are real threat in our ICU. The emergence of microorganisms known for its inherent resistance among most of the common first-line antibiotics calls for a alarm in all upcoming tertiary care hospitals.

Nonnucleoside Reverse Transcriptase Inhibitor Resistance and the Role of the Second-Generation Agents

To review the current state of nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance, discuss the promising role of second-generation NNRTIs, and provide insight into their clinical utility. Articles were identified through searches of MEDLINE (May 2000-August 2009) and International Pharmaceutical Abstracts (May 1998-August 2009), using the key words etravirine, rilpivirine, TMC125, TMC278, diarylpyrimidine, NNRTI, and resistance. Clinical trials, resistance studies, and pharmacokinetic data were selected for review. NNRTIs are an integral class of antiretroviral agents utilized for the treatment of HIV-1 infection. These agents have become preferred therapy options for treatment-naïve individuals per treatment guideline recommendations and have gained increased popularity over protease inhibitor-based antiretroviral therapy. However, available NNRTIs possess inherent characteristics, such as low genetic barrier to resistance and high degree of cross-resistance, that limit their use in HIV therapy. Due to the growing utilization of this highly efficacious antiretroviral class and the increased capability for resistance development, many HIV-infected patients have experienced treatment failure of an NNRTI. Cross-resistance makes other first-generation NNRTI agents unavailable for future use. Etravirine and rilpivirine are second-generation NNRTIs that are not significantly hampered by cross-resistance and possess potent antiretroviral activity against current NNRTI-resistant viral strains. These agents provide new and important therapy options for many HIV-infected patients. NNRTI resistance is an increasing problem that may impair the chances for therapeutic success in HIV-infected patients. Novel agents such as etravirine and rilpivirine provide new, sensitive options for patients and significantly improve the rate of virologic suppression when appropriately applied.

In vitro activity of ceftobiprole against clinical isolates of Pseudomonas aeruginosa obtained from Canadian intensive care unit (ICU) patients as part of the CAN-ICU Study

Sir, In recent years, Pseudomonas aeruginosa isolates resistant to multiple classes of antimicrobial agents have become increasingly common. Several mechanisms may contribute to antimicrobial resistance among P. aeruginosa, including the production of a chromosomally encoded AmpC b-lactamase. Ceftobiprole (BAL9141), an investigational pyrrolidinone cephalosporin, is reported to have activity against a broad spectrum of clinically important Gram-negative bacteria including P. aeruginosa. Additionally, in vitro studies have demonstrated that ceftobiprole is hydrolysed very slowly by AmpC cephalosporinases. The purpose of this report was to describe the in vitro activity of ceftobiprole versus 419 clinical isolates of P. aeruginosa obtained from patients in an intensive care unit (ICU) setting. Cefepime was used as a comparator antimicrobial agent. From September 2005 to June 2006, inclusive, P. aeruginosa isolates were collected as part of the Canadian National Intensive Care Unit (CAN-ICU) Study. The CAN-ICU Study included 19 medical centres from all regions of Canada with active ICUs. Each centre submitted a maximum of 300 consecutive pathogens isolated from blood, urine, tissue/wound and respiratory specimens (one pathogen per cultured site per patient) of ICU patients. Centres were requested to only obtain ‘clinically important’ specimens from patients with a presumed infectious disease. Surveillance swabs, eye, ear, nose and throat swabs were excluded, as were anaerobic bacteria and fungi. Isolates were shipped to the reference laboratory (Health Sciences Centre, Winnipeg, MB, Canada) on Amies charcoal swabs, subcultured onto appropriate media and stocked in skimmed milk at 2808C until MIC testing was performed. The in vitro activities of ceftobiprole and cefepime were determined by microbroth dilution in accordance with the CLSI guidelines. MIC interpretive standards for cefepime were defined according to CLSI breakpoints. At present, susceptibility breakpoints for ceftobiprole do not exist. In total, 419 isolates of P. aeruginosa were collected as part of the CAN-ICU Study. The breakdown of these 419 isolates by specimen source was as follows: respiratory (69.0%), wound (12.4%), urine (10.7%) and blood (7.9%). The MIC distributions of ceftobiprole and cefepime for the isolates were very similar (Table 1). At an antimicrobial concentration of 8 mg/L, 74.7% and 78.7% of our isolates would be inhibited by ceftobiprole and cefepime, respectively. The MIC50 and MIC90 values of ceftobiprole were 4 and 16 mg/L. The corresponding MIC50 and MIC90 values of cefepime were 4 and 32 mg/L. The MIC50/ MIC90 values of ceftobiprole and cefepime were comparable, regardless of specimen source. Cross-resistance between ceftobiprole and cefepime was observed. Of 43 cefepime-resistant isolates (MIC 32 mg/L), only 3 (7.0%) had an MIC of ceftobiprole of 8 mg/L. Similarly, only 6 of 40 isolates (15%) with a ceftobiprole MIC of 32 mg/L were susceptible to cefepime. In agreement with our results, previous studies have reported similar in vitro activity between ceftobiprole and cefepime when evaluated against P. aeruginosa. A recent study by Pillar et al. documented a lower modal MIC of cefepime in comparison with ceftobiprole against P. aeruginosa isolates (1 mg/L versus 2 mg/L). However, the MIC50 and MIC90 values were identical (4 and 16 mg/L respectively) for both antimicrobials. Our data demonstrate a high degree of cross-resistance between ceftobiprole and cefepime. Hebeisen et al. have previously described the finding of cross-resistance between ceftazidime, cefepime and ceftobiprole. These investigators reported the activity of ceftobiprole against 17 ceftazidime-non-susceptible P. aeruginosa isolates (MIC50 of 16 mg/L and MIC90 of .64 mg/L for ceftazidime). The MIC50 and MIC90 values of ceftobiprole against these isolates were 16 and .64 mg/L, respectively. The corresponding MIC50 and MIC90 values of cefepime versus the ceftazidime-non-susceptible isolates were 32 and 32 mg/L. Cross-resistance has also been described in a study by Pillar et al., where the MIC50/MIC90 values of ceftobiprole against 491 ceftazidime-susceptible and 130 ceftazidime-non-susceptible P. aeruginosa isolates were 2/8 and 16/.32 mg/L, respectively. In summary, ceftobiprole and cefepime demonstrated comparable in vitro activity against P. aeruginosa clinical isolates from Canadian ICUs and cross-resistance between these agents was common. Whether ceftobiprole will be clinically useful in the treatment of serious infections caused by P. aeruginosa remains to be determined.

Non-nucleoside reverse transcriptase inhibitor resistance among patients failing a nevirapine plus protease inhibitor-containing regimen

To determine the rate of nevirapine resistance in patients failing a nevirapine plus protease inhibitor (PI)-based regimen, and whether these isolates remain susceptible to other non-nucleoside reverse transcriptase inhibitors (NNRTI). A retrospective cohort study in two tertiary university hospitals. Eighty-eight HIV-infected, NNRTI-naive patients receiving nevirapine plus PI as a rescue regimen after PI treatment failure. Genotypic and phenotypic resistance data at inclusion (73 and 60 plasma samples, respectively) and after 24 weeks (53 and 42 samples). Baseline phenotypic susceptibility to nevirapine was found in 70% of patients, and similar data were observed for efavirenz (91%) and delavirdine (71%). NNRTI resistance-associated mutations were found in 11 patients (12.5%). At 24 weeks, resistant isolates to nevirapine were found in 92% of patients, and correlated with similar resistance to efavirenz (68%) and delavirdine (73%). In the genotypic analysis, the Y181 C mutation was observed in 76% of mutants, and the most common changes were a combination of mutations at positions Y181C/K103N (23%) and the single mutation Y181C (15%). The development of nevirapine resistance was associated with baseline resistance to PI included in the regimen (P= 0.01). For isolates containing the single amino acid substitution Y181C, 29% remained fully susceptible to efavirenz, whereas 14% showed intermediate resistance to efavirenz and delavirdine. The failure of a nevirapine plus PI-containing regimen is associated with nevirapine resistance in most patients, with the most common mutation occurring at amino acid residue 181. Although there is a high degree of cross-resistance among NNRTI, nearly one third of resistant isolates carrying the single Y181C mutation remain susceptible to efavirenz.

Common resistance mechanisms to deoxynucleoside analogues in variants of the human erythroleukaemic line K562.

Resistant variants of the human leukaemic line K562 were developed using selection with the deoxynucleoside analogues cytosine arabinoside, 2-chlorodeoxyadenosine, fludarabine and gemcitabine. The resistant lines displayed a high degree of cross resistance to all deoxynucleoside analogues, with little or no cross resistance to other agents. There was a profound accumulation defect of all nucleoside analogues in the resistant variants but no significant defect in nucleoside transport in any of the variants. 5' nucleotidase activity was strongly increased and deoxycytidine kinase activity was moderately reduced in all of the resistant variants, resulting in reduced accumulation of triphosphate analogues. In addition a deletion in one of the alleles of the deoxycytidine kinase was detected in the fludarabine-resistant line. Ribonucleotide reductase activity was found to be strongly increased in the gemcitabine-selected line and purine nucleoside phosphorylase was increased in the 2-chlorodeoxyadenosine-selected line. Free nucleotide pools were increased in the 2-chlorodeoxyadenosine-selected line. There was no expression of the mdr1 gene by the resistant lines. Karyotypic analysis and FISH experiments using a 6q21 specific probe showed alterations in the 6(q16-q22) region which contains the 5'-nucleotidase gene. Early events in the activation and degradation of deoxynucleoside analogues appear to constitute common mechanisms of resistance to these compounds.

Common resistance mechanisms to nucleoside analogues in variants of the human erythroleukemic line K562.

Variants of the human K562 were developed against the nucleoside analogues cytosine arabinoside, 2 chlorodeoxyadenosine, fludarabine and gemcitabine. The resistant lines displayed a high degree of cross-resistance to all nucleoside analogues, with little or no cross resistance to other agents. There was a profound accumulation defect of the different nucleoside analogues in all of the variants. There was a strong overexpression of 5'nucleotidase, measured by rt-PCR and enzyme activity, in all resistant variants. There was a two fold increase of ribonucleotide reductase in the fludarabine resistant line and increased expression of purine nucleoside phosphorylase in the 2 chlorodeoxyadenosine selected line. Karyotypic analysis revealed the loss of a 6(q16;q22) deletion present in the parental line in all of the resistant lines. This portion of chromosome 6 has been shown to contain the gene for 5'nucleotidase. Early events in the transport and metabolism appear to be involved in the resistance mechanisms to nucleoside analogues and are responsible for broad cross resistance to this family of compounds.

Detection and Quantification of Resistance of Venturia inaequalis Populations to Sterol Demethylation Inhibitors

ABSTRACT Monoconidial isolates of Venturia inaequalis were collected in 1990 and 1991 from orchards in New York, Michigan, and Nova Scotia that had never or only sporadically been treated with fungicides acting as sterol demethylation inhibitors (DMIs). Sensitivities of isolates to two representative DMIs (fenarimol and myclobutanil) were determined by a sensitivity test based on the relative growth (RG) of mycelial colonies at one discriminatory dose. Mean isolate sensitivities were not significantly different (P > 0.2) for the majority of the populations tested, and all sensitivity data obtained from these sites were combined to provide a baseline distribution of isolate sensitivities for both fenarimol and myclobutanil. The baseline distributions were compared with isolate sensitivities determined for an experimental orchard in Nova Scotia with a documented case of DMI resistance and for a commercial orchard in Michigan with a long history of DMI use and first evidence of practical DMI resistance. For both DMIs tested and in both treated orchards, frequencies of isolates with RG values <80 had decreased or only slightly increased compared to the baseline population. In contrast, frequencies of isolates with RG values >80 had increased more than 20-fold over baseline levels. Thus, isolates with RG values >80 were rated DMI resistant. The validity of a qualitative isolate classification was tested in controlled infection studies. At doses of fenarimol and myclobutanil recommended for commercial control of apple scab, reproduction of a typical sensitive isolate on treated apple leaves was suppressed completely. Lesions caused by a resistant isolate continued to expand and produced abundant conidia. Statistical analysis of orchard sensitivities revealed that the analysis of isolate counts grouped into the categories DMI sensitive or resistant was most indicative in comparisons of orchard sensitivities aimed at detection of practical DMI resistance. A high degree of cross-resistance between fenarimol and myclobutanil indicated that sensitivity tests with one of the DMIs employed as the diagnostic tool in this study can serve as a test for other DMIs.

RTS gene expression is associated with altered cell sensitivity to thymidylate synthase inhibitors

rTS is a recently discovered gene, phylogenetically conserved and found to be expressed in a wide variety of cell lines. rTS has also been found to be overexpressed in two cell lines resistant to FU and to MTX. The MTX-resistant cell line was found to have a high degree of cross resistance to several TS inhibitors. An apparent paradox to this correlation of rTS overexpression and resistance to TS inhibitors is the observation that expression of transfected rTSα results in enhanced sensitivity of cells to the TS inhibitor prodrug TFT and a modest increase in resistance to FUdR. Since immunoprecipitation of TS leads to the co-immunoprecipitation of two proteins within the expected molecular weight range of the two rTS proteins, it may be that both proteins bind to TS in vivo and modify its activity. Preliminary data substantiate this conclusion. It is conceivable that the ratio of the two rTS proteins associated with TS in vivo may differentially alter TS activity depending upon their stoichiometry or possibly posttranslational modification. Thus it may be possible for rTS to confer greater sensitivity to one pyrimidine analog (e.g., TFT) which is a product analog but to increase resistance or have a minor effect on a substrate analog (e.g., FdUMP) by stabilizing different conformations of TS. The structure of the rTS proteins suggests they are expected to have catalytic activity which involves proton abstraction from an α-carbon of a carboxyl group. Whether this enzyme activity is functional and related to pyrimidine metabolism awaits futher study.

Imazaquin and chlorsulfuron resistance and cross resistance in mutants of Chlamydomonas reinhardtii.

Chlorsulfuron and/or imazaquin resistant mutants of Chlamydomonas reinhardtii strain CW15 have been obtained and shown to have actolactate synthase (ALS) with altered sensitivity to one or both of these herbicides. Herbicide resistance in the three mutants described is allelic, and resistance appears to result from a dominant or semidominant mutation in a single, nuclear gene. Imazaquin and chlorsulfuron resistant ALS from imazaquin and chlorsulfuron resistant mutants, together with single-gene Mendelian inheritance of these phenotypes, suggests that ALS is the sole site of action of the two herbicides in Chlamydomonas. A high degree of cross resistance between the two herbicides was found in only one mutant. This mutant (IMR-13) was selected for resistance to imazaquin and has a high level of in vitro resistance to both imazaquin (270-fold increased I50) and chlorsulfuron (900-fold increased I50). In another mutant selected for resistance to imazaquin (IMR-2), hyper-sensitivity to chlorsulfuron was found. A mutant selected for resistance to chlorsulfuron (CSR-5), had a substantial degree of resistance of chlorsulfuron (80-fold increased I50), but not to imazaquin (7-fold increased I50).

Chromosomal resistance of gonococci to antibiotics.

Three hundred and twenty nine strains of non-penicillinase-producing Neisseria gonorrhoeae (non-PPNG) isolated from men and women were tested for their susceptibility to a range of antibiotics, and were also auxotyped and serogrouped. Nearly 6% (18) of 312 strains tested were resistant to 1 mg/l or more penicillin (compared with 4.4% of PPNG strains isolated in 1981). Many (198, 64%) strains showed intermediate resistance to penicillin (0.12-0.5 mg/l). Nearly 5% (15) of 312 strains tested were resistant to 0.5 mg/l or more cefuroxime, and there was a high degree of cross resistance between these two antibiotics. High levels of resistance to erythromycin and tetracycline were also found, and there was also appreciable cross resistance between these antibiotics and the beta lactam drugs. Resistance to spectinomycin was rare, and there was no cross resistance between spectinomycin and other agents tested. Levels of resistance between strains isolated from different anatomical sites did not differ, except that resistance to erythromycin was greater in rectal isolates. Four main auxotypes were detected. Strains requiring arginine, hypoxanthine, and uracil (AHU-) were more prevalent from the cervix. We have shown that there is an association between auxotype, serogroup, and level of sensitivity to penicillin, cefuroxime, and tetracycline.

Biochemical and cross-resistance studies with HeLa cell mutants resistant to cardiac glycoside SC4453. Regulation of the resistant form of Na+/K+-ATPase in the mutant cells.

In HeLa cells, stable mutants which are between 25-to about 200-fold resistant to the cardiac glycoside derivative SC4453 (a digoxin analog which contains a pyridazine ring in place of a lactone ring in the C-17 position) have been isolated after a single step selection in the presence of the drug. Based on their cross-resistance pattern towards various cardiac glycosides, the mutants resistant to SC4453 (SCR mutants) appear to be of two different kinds and they differ from the two classes of ouabain-resistant mutants described previously (Gupta, R. S., and Chopra, A. (1985) J. Biol. Chem. 260, 6843-6850). One type of SCR mutants (designated as group C) exhibit a high degree of cross-resistance to all cardiac glycosides and their genins (viz. ouabain, digitoxin, digoxin, digoxigenin, convallatoxin, gitoxin, strophanthidin, and bufalin). In contrast, the second type of SCR mutant (group D) exhibit considerable resistance to only SC4453, digoxin, and digoxigenin, but showed very little or no cross-resistance to the other cardiac glycosides examined. The cross-resistance of the mutants towards cardiac glycosides was highly specific as they exhibited no cross-resistance towards a large number of other structurally and functionally related compounds (viz. ethacrynic acid, sanguinarine nitrate, penicillic acid, methyl quinolizinum bromide, 5,5'-diphenylhydantoin, deoxycorticosterone, vanadium pentoxide, and adriamycin). The cellular uptake of 86Rb in the mutant cells was found to be resistant to specific cardiac glycosides. Studies on the sensitivity of plasma membrane Na+/K+-ATPase to cardiac glycosides show that about 10-15% of the enzymic activity in the mutant cells was highly resistant to inhibition by the specific drugs to which the mutants exhibit increased resistance. Very interestingly, when the mutant cells are grown in cardiac glycoside-containing medium, the resistant form of the enzyme accounts for about 50-60% of the total enzyme. These results show that both classes of SCR mutants are affected in Na+/K+-ATPase and that the amount of the resistant enzyme in the mutant cells is regulated in response to cardiac glycosides.

Chinese hamster ovary cell mutants specifically affected in the phosphorylation of C-purine nucleosides.

In Chinese hamster ovary cells, two different types of mutants resistant to purine nucleoside analogs have been isolated. One type of mutants selected in presence of C-nucleosides formycin A and formycin B (FomR mutants) exhibited a high degree of cross resistance to different C-nucleosides but showed very slight to no cross resistance to various N-nucleosides. In contrast, mutants selected in presence of toyocamycin (Toyr mutants) exhibited a high degree of cross resistance to all C- and N-adenosine analogs. Studies on the cellular uptake and phosphorylation of [3H]adenosine, [3H]tubercidin, and [3H]formycin A show that, unlike the Toyr mutants which show reduced phosphorylation of all three compounds, the FomR mutants show reduced cellular phosphorylation of only [3H]formycin A. It is suggested that the genetic lesion in the FomR mutants affects adenosine kinase in a novel manner that specifically affects the phosphorylation of C-purine nucleosides.

GENETIC AND BIOCHEMICAL CHARACTERISTICS OF THREE DIFFERENT TYPES OF MUTANTS OF CHINESE HAMSTER OVARY CELLS AFFECTED IN ADENOSINE KINASE: 76

In CHO cells, three different types of mutants affected in purine nucleoside salvage pathway enzyme adenosine kinase (AK) have been isolated. One class of mutants obtained at high frequency using various adenosine analogs (Class A) exhibit high degree of cross resistance to various C- as well as N- adenosine analogs. These mutants show greatly reduced phosphorylation of all adenosine analogs and cell extracts from these contain no measurable activity of AK. The second type of mutants (Class B) obtained using formycin A (a C- adenosine analog) exhibit increased cross resistance to various C- nucleosides (viz. 9-deazaade-nosine, pyrazofurin, etc.),but their sensitivity towards various N- adenosine analogs was found to be unaltered. Cell extracts from these mutants also contained no measurable activity of AK. Reduced phosphorylation of C- adenosine analogs but not N- adenosine analogs by Class B mutants provides strong evidence that they contain a novel genetic lesion affecting AK that specifically affects the phosphorylation of C- nucleosides. The third type of mutants (Class C) selected using inosine analog formycin B, exhibit cross resistance and reduced phosphorylation of both C- as well as N- adenosine analogs. However, their degree of resistance was lower in comparison to the Class A mutants. Cell extracts from the Class C mutants contained between 50-100% of AK activity, but its affinity towards adenosine analogs was found to be altered in comparison to the wild-type enzyme.