Biomedical subjects

J Brouwer

Publications and source records attributed to J Brouwer.

At least 19 recordsLinked to original sources

PubMed2006

Difference in long-term prognostic value of renal function between ischemic and non-ischemic mild heart failure.

INTRODUCTION: Renal function is one of the strongest prognostic markers in patients with chronic heart failure, but it has been suggested that this might be due to (local, i.e. renal) vascular atherosclerosis. The aim of the present study is to evaluate the prognostic value of renal function in both ischemic and non-ischemic mild chronic heart failure. METHODS: From 161 patients with early chronic heart failure and NYHA class II, who had been enrolled in a multicenter trial, ischemic chronic heart failure was present in 120 patients and non-ischemic chronic heart failure in 41 patients. Estimated glomerular filtration rate was calculated by the Cockcroft-Gault equation (GFRc). RESULTS: Follow-up duration was 13 years (mean 11.7 years). Mean age was 60.5+/-8.0 years, 86% was male and mean left ventricular ejection fraction was 0.29+/-0.08. Baseline characteristics were not statistically different between the groups. Multivariate Cox regression analysis revealed that in non-ischemic chronic heart failure, renal function was an important predictor of all-cause mortality (Relative Risk; 1.65 (1.05-2.58); p=0.029). In contrast, in ischemic chronic heart failure, renal function was not related to all-cause mortality (Relative Risk; 1.07 (0.92-1.23); p=0.40). CONCLUSION: In mild chronic heart failure, renal function is a prognostic risk marker for long-term mortality in non-ischemic chronic heart failure, but not in patients with coronary artery disease. These data suggest that renal vascular abnormalities are not primarily responsible for the prognostic value of renal function in patients with mild chronic heart failure.

Aged↗

PubMed2005

Screening for distant metastases in patients with head and neck cancer: what is the current clinical practice?

OBJECTIVES: The detection of distant metastases during screening influences the choice of treatment in patients with head and neck squamous cell carcinoma (HNSCC). There is no consensus on the diagnostic technique that has to be used nor on the subgroup of HNSCC patients that may benefit from screening. DESIGN: Questionnaire on current practice concerning the diagnostic work-up in HNSCC patients for screening for distant metastases. PARTICIPANTS: Investigators in the 12 otolaryngology / head and neck and seven oromaxillofacial departments treating head and neck cancer in the Netherlands. RESULTS: The response rate was 100%. Indications for screening were cervical lymph node metastases (63%), mutilating surgery (58%), locoregional recurrence (47%), advanced T-stage (32%), second primary tumour (21%). Diagnostic techniques routinely used for screening besides chest X-ray were chest CT (84%), liver ultrasound (53%), liver CT (16%) and bone scintigraphy (42%). Forty-two per cent of the clinicians were not satisfied with the current methods of screening. CONCLUSION: This survey shows a substantial variation in indications and diagnostic techniques used for screening for distant metastases between the major institutions treating head and neck cancer in the Netherlands. There is a need for guidelines for screening for distant metastases in patients with head and neck cancer.

Bone and Bones↗

PubMed2003

Daily induction combination treatment with alpha 2b interferon and ribavirin or standard combination treatment in naive chronic hepatitis C patients. A multicentre randomized controlled trial.

The standard treatment for patients with chronic hepatitis C is a 6-12-month combination therapy with interferon alpha and ribavirin. Induction treatment could result in a faster early decline of the hepatitis C virus (HCV) load and a better response rate. Naive chronically infected HCV patients (n = 454) were randomized into two arms to receive either induction treatment with interferon alpha 2b 5 million units (MU) subcutaneously (s.c.) daily during a period of 8 weeks (arm A); or treatment with interferon alpha 2b 5 MU s.c. three times a week (TIW) for a period of 8 weeks (arm B). After week 8, interferon treatment in both arms was 3 MU s.c. TIW for a total period of 12 months. In both arms, ribavirin (1000-1200 mg orally per day) was added at week 4. Induction treatment resulted in a higher virological response at week 8 of treatment (66%vs 47%; P < 0.01). However, response at the end of treatment and at 6 months follow-up was not different (53%vs 50%, 41%vs 33%). The occurrence of adverse events and the drop-out rate were similar in both arms. Although an early virological response is observed more frequently in the induction treatment, end of treatment response and sustained responses did not differ.

Adolescent↗

PubMed2003

Cardiac failure following group A streptococcal infection with echocardiographically proven pericarditis, still insufficient arguments for acute rheumatic fever: a case report and literature update.

We recently encountered a 49-year-old female who developed fever due to group A streptococcal (GAS) bacteriaemia spreading to an abscess in the iliac muscle and a bacterial monarthritis of the right knee with a sterile arthritis of her left knee. Treatment was started with a six-week course of intravenous penicillin. She developed a mitral valve insufficiency and pericarditis on the tenth day of admission. In the third week heart failure developed with, on echocardiograph, a high output left ventricular failure without signs of valvulitis or myocarditis. Using a diuretic regimen she was recompensated. Because of the pericarditis with mitral valve insufficiency corticosteroids were given, which had a rapid beneficial effect. A discussion follows on the position of acute rheumatic fever versus post-streptococcal reactive arthritis in this clinical picture and the literature is updated.

Arthritis, Infectious↗

PubMed2002

One-pot two-step enzymatic coupling of pyrimidine bases to 2-deoxy-D-ribose-5-phosphate. A new strategy in the synthesis of stable isotope labeled deoxynucleosides.

The enzymatic synthesis of thymidine from 2-deoxy-D-ribose-5-phosphate is achieved, in a one-pot two-step reaction using phosphoribomutase (PRM) and commercially available thymidine phosphorylase (TP). In the first step the sugar-5-phosphate is enzymatically rearranged to alpha-2-deoxy-D-ribose-1-phosphate. Highly active PRM is easily obtained from genetically modified overproducing E. coli cells (12,000 units/84 mg protein) and is used without further purification. In the second step thymine is coupled to the sugar-1-phosphate. The thermodynamically unfavorable equilibrium is shifted to the product by addition of MnCl(2) to precipitate inorganic phosphate. In this way the overall yield of the beta-anomeric pure nucleoside increases from 14 to 60%. In contrast to uracil, cytosine is not accepted by TP as a substrate. Therefore, 2'-deoxy-cytidine is obtained by functional group transformations of the enzymatically prepared 2'-deoxy-uridine. The method has been demonstrated by the synthesis of [2',5'-(13)C(2)]- and [1',2',5'-(13)C(3)]thymidine as well as [1',2',5'-(13)C(3)]2'-deoxyuridine and [3',4'-(13)C(2)]2'-deoxycytidine. In addition the nucleoside bases thymine and uracil are tetralabeled at the (1,3-(15)N(2),2,4-(13)C(2))-atomic positions. All compounds are prepared without any scrambling or dilution of the labeled material and are thus obtained with a very high isotope enrichment (96-99%). In combination with the methods that have been developed earlier it is concluded that each of the (13)C- and (15)N-positions and combination of positions of the pyrimidine deoxynucleosides can be efficiently labeled starting from commercially available and highly (13)C- or (15)N-enriched formaldehyde, acetaldehyde, acetic acid, potassium cyanide, methylamine hydrochloride, and ammonia.

Catalysis↗

PubMed2002

Prediction of 6 months left ventricular dilatation after myocardial infarction in relation to cardiac morbidity and mortality. Application of a new dilatation model to GISSI-3 data.

AIMS: To predict the long-term left ventricular volume index early after myocardial infarction and to investigate the relationship between long-term left ventricular dilatation risk and clinical outcome. METHODS AND RESULTS: By applying a previously developed dilatation model, we predicted the 6-month left ventricular volume index early after myocardial infarction (median 9 days) in 13,679 GISSI-3 patients, to identify patients at high risk of long-term left ventricular dilatation. The left ventricular systolic and diastolic volume indexes at 6 months were predicted with r=0.72 and r=0.68, respectively, in the subgroup of patients in whom a pre-discharge echo was available (n=7842). Patients predicted to be at risk for long-term left ventricular dilatation had an increased risk of mortality (RR 1.87, 95% CI: 1.48 to 2.36) and heart failure at 6 months (RR 2.59, 95% CI:2.04 to 3.28), but no increased risk of reinfarction at 6 months (RR 1.12, 95% CI: 0.87 to 1.45) or of angina pectoris (RR 1.07, 95% CI: 0.95 to 1.20). CONCLUSION: Our prediction of long-term left ventricular dilatation, obtained by applying our new dilatation model in over 13,000 GISSI-3 patients, correlated well with mortality and heart failure after myocardial infarction. Therefore, our new dilatation model may contribute to more efficient risk stratification early after myocardial infarction.

Dilatation, Pathologic↗

PubMed2001

SKY1 is involved in cisplatin-induced cell kill in Saccharomyces cerevisiae, and inactivation of its human homologue, SRPK1, induces cisplatin resistance in a human ovarian carcinoma cell line.

The therapeutic potential of cisplatin, one of the most active and widely used anticancer drugs, is severely limited by the occurrence of cellular resistance. In this study, using budding yeast Saccharomyces cerevisiae as a model organism to identify novel drug resistance genes, we found that disruption of the yeast gene SKY1 (serine/arginine-rich protein-specific kinase from budding yeast) by either transposon insertion or one-step gene replacement conferred cellular resistance to cisplatin. Heterologous expression of the human SKY1 homologue SRPK1 (serine/arginine-rich protein-specific kinase) in SKY1 deletion mutant yeast cells restored cisplatin sensitivity, suggesting that SRPK1 is a cisplatin sensitivity gene, the inactivation of which could lead to cisplatin resistance. Subsequently, we investigated the role of SRPK1 in cisplatin sensitivity and resistance in human ovarian carcinoma A2780 cells using antisense oligodeoxynucleotides. Treatment of A2780 cells with antisense oligodeoxynucleotides directed against the translation initiation site of SRPK1 led to down-regulation of SRPK1 protein and conferred a 4-fold resistance to cisplatin. The human SRPK1 gene has not been associated with drug resistance before. Our new findings strongly suggest that SRPK1 is involved in cisplatin-induced cell kill and indicate that SRPK1 might potentially be of importance for studying clinical drug resistance.

Antineoplastic Agents↗

PubMed2001

Dinuclear alkyldiamine platinum antitumor compounds: a structure-activity relationship study.

Six related dinuclear trans-platinum complexes, with the formula [[trans-PtCl(2)(NH(3))(L)](2)(mu-H(2)N(CH(2))(n)NH(2))](2+) (L = pyridine, 2-picoline, 4-picoline; n = 4, 6) and chloride or nitrate anions, are compared with known cytotoxic dinuclear compounds (L = NH(3); n = 4, 6) that overcome cisplatin resistance. The cytotoxicity of the compounds was determined in L1210 murine leukemia and L1210/2, a cisplatin-resistant derivative. Unlike the L = NH(3) compounds, the substituted n = 4 compounds are more susceptible toward the resistance mechanisms in L1201/2. The n = 6 compounds, however, have comparable IC(50) values in both cell lines. In general, the substituted compounds are less cytotoxic than their NH(3) counterparts. After incubation with equimolar concentrations, the amount of platinum bound to cellular DNA was determined. The compounds show comparable binding, except for the sterically hindered 2-picoline compounds that bind significantly less. The amounts of platinum bound to DNA do not correlate with the cytotoxicity data. As DNA is considered to be the cellular target of platinum antitumor drugs, structural details of the DNA adducts probably account for the differences in cytotoxic activity.

Animals↗

PubMed2001

Stress, glucocorticoids and the susceptibility to develop autoimmune disorders of the central nervous system.

Alterations of the immunoendocrine circuit along the hypothalamic-pituitary-adrenocortical (HPA) axis in various autoimmune diseases have recently been observed, suggesting a modulatory role of this feedback regulation in the pathogenesis of autoimmune diseases. Susceptibility to experimental autoimmune encephalomyelitis (EAE) may be influenced by variations in the production of endogenous glucocorticoid hormones (GC). The adrenocortical response is central to recovery from EAE in the Lewis rat, as reflected by increased severity of the disease in adrenalectomized animals. The key role of GC in modifying the induction and progression of EAE is also emphasized by a reversal of corticoid-mediated effects through treatment with glucocorticoid receptor (GR) antagonists. We studied the relationship between defective GR function and susceptibility to EAE in transgenic (Tg) mice expressing GR antisense RNA. EAE was induced with the encephalitogenic myelin oligodendrocyte peptide (pMOG 36-50) in wild type (Wt) and transgenic (Tg) female mice bearing GR antisense RNA. pMOG 36-50 induced typical EAE in Wt mice but not in Tg mice. Histological examination of brains and spinal cords of Wt mice showed the presence of inflammation and/or demyelination, whereas in Tg mice neither were present. Although the mechanisms underlying the resistance of Tg mice to EAE induction are not yet clarified, compensatory changes at different levels of the HPA-immune axis in response to the potent immunogenic challenge are likely to participate in the observed effects. This work underlies the plasticity of the HPA-immune axis and suggests that pharmacological manipulation of neuroendocrine-immune networks may be a therapy of multiple sclerosis.

Animals↗

PubMed2001

Autonomic dysfunction in patients with mild heart failure and coronary artery disease and the effects of add-on beta-blockade.

AIM: Autonomic impairment is related to the incidence of sudden death in chronic heart failure (CHF). Our objective was to study autonomic profiles in patients with mild CHF due to coronary artery disease, and to investigate the value of add-on beta-blockade. METHODS AND RESULTS: Measures of autonomic function (plasma norepinephrine, heart rate [HR] variability, autonomic function testing), and exercise capacity, were compared between 24 patients with mild CHF, and 24 healthy controls. In this mechanistic study, we assessed the effect of 26 weeks metoprolol treatment in a double-blind, randomized, placebo-controlled design. All patients received metoprolol sustained release (200 mg; n=12) or placebo (n=12). Assessments were made at baseline and after 10 and 26 weeks' treatment. At baseline, norepinephrine levels were elevated, while HR variability parameters were decreased in patients vs. controls (both P<0.05). Autonomic function testing showed only small differences, although significant alterations were observed with deep breathing and head up tilting (both P<0.05). After 26 weeks', metoprolol did not affect exercise capacity or norepinephrine concentrations. In contrast, HR variability was markedly improved in metoprolol-treated patients vs. placebo-treated patients (P<0.05). In particular, a shift toward normal in the sympathovagal balance was observed (P<0.05). Autonomic function testing showed only small, and generally non-significant trends after metoprolol. CONCLUSIONS: Marked autonomic abnormalities are already present in mild CHF, which may be (partially) reversed by metoprolol. These observations support the reported reduction of sudden death by beta-blockade in patients with CHF.

Adrenergic beta-Antagonists↗

PubMed2001

Possible bradycardic mode of death and successful pacemaker treatment in a large family with features of long QT syndrome type 3 and Brugada syndrome.

INTRODUCTION: We recently identified a novel mutation of SCN5A (1795insD) in a large family with features of both long QT syndrome type 3 and the Brugada syndrome. The purpose of this study was to detail the clinical features and efficacy of pacemaker therapy in preventing sudden death in this family. METHODS AND RESULTS: The study group consisted of 116 adult family members: 60 carriers (29 males) and 56 noncarriers (28 males) of the mutant gene. Investigations included 24-hour Holter monitoring, ergometry, and electrophysiologic studies. Mean, lowest, and highest heart rate were lower in the carriers, but heart rate variability was comparable. In carriers, disproportional QT prolongation was present during bradycardia. No complex ventricular ectopy was recorded, and there were fewer isolated premature beats (both ventricular and atrial) in carriers. All patients were asymptomatic, except for two individuals who experienced syncope; in one of these patients, asystolic episodes (up to 9 sec) were repeatedly recorded. Prolonged HV intervals were present in 5 of 6 patients. Thirty carriers received a prophylactic backup pacemaker. During median follow-up of 4.5 years (range 0.0 to 22.6), their survival rate was 100%. There were five sudden deaths among the remaining 30 carriers without a pacemaker (P = 0.019). CONCLUSION: This family with a high incidence of nocturnal sudden death is characterized by bradycardia-dependent QT prolongation, intrinsic sinus node dysfunction, and generalized conduction abnormalities. There is a striking absence of complex ventricular ectopy, and pacemaker implantation was effective in preventing sudden death. These findings raise the possibility of a bradycardic rather than tachycardic mode of death.

Adult↗

PubMed2000

Spt4 modulates Rad26 requirement in transcription-coupled nucleotide excision repair.

The nucleotide excision repair machinery can be targeted preferentially to lesions in transcribed sequences. This mode of DNA repair is referred to as transcription-coupled repair (TCR). In yeast, the Rad26 protein, which is the counterpart of the human Cockayne syndrome B protein, is implicated specifically in TCR. In a yeast strain genetically deprived of global genome repair, a deletion of RAD26 renders cells UV sensitive and displays a defect in TCR. Using a genome-wide mutagenesis approach, we found that deletion of the SPT4 gene suppresses the rad26 defect. We show that suppression by the absence of Spt4 is specific for a rad26 defect and is caused by reactivation of TCR in a Rad26-independent manner. Spt4 is involved in the regulation of transcription elongation. The absence of this regulation leads to transcription that is intrinsically competent for TCR. Our findings suggest that Rad26 acts as an elongation factor rendering transcription TCR competent and that its requirement can be modulated by Spt4.

Antineoplastic Agents, Alkylating↗

PubMed2000

Cloning of a human homolog of the yeast nucleotide excision repair gene MMS19 and interaction with transcription repair factor TFIIH via the XPB and XPD helicases.

Nucleotide excision repair (NER) removes UV-induced photoproducts and numerous other DNA lesions in a highly conserved 'cut-and-paste' reaction that involves approximately 25 core components. In addition, several other proteins have been identified which are dispensable for NER in vitro but have an undefined role in vivo and may act at the interface of NER and other cellular processes. An intriguing example is the Saccharomyces cerevisiae Mms19 protein that has an unknown dual function in NER and RNA polymerase II transcription. Here we report the cloning and characterization of a human homolog, designated hMMS19, that encodes a 1030 amino acid protein with 26% identity and 51% similarity to S.cerevisiae Mms19p and with a strikingly similar size. The expression profile and nuclear location are consistent with a repair function. Co-immunoprecipitation experiments revealed that hMMS19 directly interacts with the XPB and XPD subunits of NER-transcription factor TFIIH. These findings extend the conservation of the NER apparatus and the link between NER and basal transcription and suggest that hMMS19 exerts its function in repair and transcription by interacting with the XPB and XPD helicases.

Amino Acid Sequence↗

PubMed2000

Effect of mibefradil on heart rate variability in patients with chronic heart failure.

BACKGROUND: Mibefradil was recently withdrawn from the market because of an unfavorable clinical profile in patients with chronic heart failure. Although drug interactions appear to play a role, other mechanisms such as proarrhythmia and autonomic deterioration could also be relevant. Chronic heart failure is accompanied by autonomic impairment and analysis of heart rate variability can be used to examine autonomic modulation of heart rate. METHODS: We studied 18 heart failure patients (age 63.2+/-10.1 years (mean+/-S.D. ), ejection fraction 0.21+/-0.07) treated with mibefradil or placebo, who participated in the MACH-I (Mortality Assessment in Chronic Heart failure) trial in our center, and compared them with 18 healthy matched controls. Heart rate variability analysis was performed at baseline and after 7 months of treatment. RESULTS: At baseline, heart rate variability parameters were impaired in patients with heart failure compared to healthy controls (P<0.05). After 7 months of treatment a reduction in (24-h) heart rate was observed (P=0.02, versus placebo). Apart from the effect on mean NN, no significant differences were observed for the remaining heart rate variability parameters. CONCLUSIONS: Mibefradil does not impair autonomic balance and in fact reduces heart rate in patients with heart failure. These findings suggest that autonomic activation did not contribute to the adverse effects of mibefradil.

Autonomic Nervous System↗

PubMed2000

A genome-wide screening in Saccharomyces cerevisiae for genes that confer resistance to the anticancer agent cisplatin.

Cisplatin is a potent DNA-damaging agent that has demonstrated anticancer activities against several tumors. However, manifestation of cellular resistance is a major obstacle in anticancer therapy that severely limits the curative potential of cisplatin. Therefore, understanding the molecular basis of cisplatin resistance could significantly improve the clinical efficacy of this anticancer agent. Here, we employed Saccharomyces cerevisiae as a model organism to study cisplatin resistance mechanisms and describe a one-step cisplatin selection to identify and characterize novel cisplatin resistance genes. Screening a multicopy yeast genomic library enabled us to isolate several yeast clones for which we could confirm that the cisplatin resistance phenotype was linked to the introduced fragment. In a first attempt, a number of open reading frames could be identified. Among these genes, PDE2 and ZDS2 were repeatedly identified as genes whose overexpression confers cellular resistance to cisplatin. PDE2, encoding cAMP-phosphodiesterase 2, is of particular interest because the overexpression of this yeast gene is known to induce cisplatin resistance in mammalian cells as well, providing proof of the principle of our experimental approach. In addition, the identification of PDE2 shows that our yeast screening system can directly be informative for drug resistance in mammalian cells.

Chromosome Mapping↗

PubMed2000

Identification and characterization of the rhp23(+) DNA repair gene in Schizosaccharomyces pombe.

We have identified rhp23(+), the ortholog of the Saccharomyces cerevisiae RAD23 and human HHR23A and HHR23B genes, in Schizosaccharomyces pombe and examined its role in cell survival and DNA repair. In S. pombe two repair mechanisms are operative on UV-induced photoproducts, i.e., UV damage repair (UVDR) and nucleotide excision repair (NER). Here we show that Rhp23 is solely involved in NER and study its role in DNA repair in the absence of the UVDR pathway. S. pombe rhp23-deficient cells are sensitive toward UV irradiation, although not as sensitive as complete NER-deficient cells. Furthermore we demonstrate that the residual survival observed in rhp23-deficient cells is NER dependent. Despite this NER-dependent survival, uvde rhp23 double mutants are unable to repair cyclobutane pyrimidine dimers. The inability to remove these photolesions from both DNA strands clearly demonstrates that rhp23(+) is involved in transcription coupled repair as well as global genome repair.

Amino Acid Sequence↗

PubMed2000

Effect of very early angiotensin-converting enzyme inhibition on left ventricular dilation after myocardial infarction in patients receiving thrombolysis: results of a meta-analysis of 845 patients. FAMIS, CAPTIN and CATS Investigators.

OBJECTIVES: We sought to investigate the effect of angiotensin-converting enzyme (ACE) inhibition <9 h after myocardial infarction (MI) on left ventricular (LV) dilation in patients receiving thrombolysis. BACKGROUND: The ACE inhibitors reduce mortality after MI. Attenuation of LV dilation has been suggested as an important mechanism. METHODS: The data of 845 patients with three-month echocardiographic follow-up after MI were combined from three randomized, double-blind, placebo-controlled studies. The criteria for these studies included: 1) thrombolytic therapy; 2) ACE inhibition within 6 to 9 h; and 3) evaluation of LV dilation as the primary objective. RESULTS: The ACE inhibitor was started 3.2+/-1.7 h after the patients' first (mainly, 85%) anterior MI. After three months, LV dilation was not significantly attenuated by very early treatment with an ACE inhibitor. The diastolic volume index was attenuated by 0.5 ml/m2 (95% confidence interval [CI] -1.5 to 2.5, p = 0.61), and the systolic volume index by 0.5 ml/m2 (95% CI -1.0 to 1.9, p = 0.50). Subgroup analysis demonstrated that LV dilation was significantly attenuated by ACE inhibitor treatment for patients in whom reperfusion failed. In contrast, LV dilation was almost unaffected by ACE inhibitor treatment in successfully reperfused patients. CONCLUSIONS: We could not demonstrate attenuation of LV dilation in patients receiving thrombolysis by ACE inhibitor treatment within 6 to 9 h after MI. We speculate that very early treatment with an ACE inhibitor has a beneficial effect on LV remodeling only in patients in whom reperfusion failed. Other mechanisms may be responsible for the beneficial effects of ACE inhibitors in successfully reperfused patients after MI.

Angiotensin-Converting Enzyme Inhibitors↗

PubMed2000

Why are A(2B) receptors low-affinity adenosine receptors? Mutation of Asn273 to Tyr increases affinity of human A(2B) receptor for 2-(1-Hexynyl)adenosine.

Adenosine A(2B) receptors are known as low-affinity receptors due to their modest-to-negligible affinity for adenosine and prototypic agonists. Despite numerous synthetic efforts, 5'-N-ethylcarboxamidoadenosine (NECA) still is the reference agonist, albeit nonselective for this receptor. In our search for higher affinity agonists, we developed decision schemes to select amino acids for mutation to the corresponding residues in the most homologous, higher affinity, human A(2A) receptor. One scheme exploited knowledge on sequence alignments and modeling data and yielded three residues, V11, L58, and F59, mutation of which did not affect agonist affinity. The second scheme combined knowledge on sequence alignments and mutation data and pointed to Ala12 and Asn273. Mutation of Ala12 to threonine did not affect the affinity for NECA, (R)-N(6)-(phenylisopropyl)adenosine (R-PIA), and 2Cl Ado. The affinity of the N273Y mutant for NECA and R-PIA and for the antagonists xanthine amine congener (XAC), ZM241385, and SCH58261 was also unaltered. However, this mutant had a slightly increased affinity for a 2-substituted adenosine derivative, CGS21680. This prompted us to investigate other 2-substituted adenosines, with selectivity and high affinity for A(2A) receptors. All four compounds tested had improved affinity for the N273Y receptor. Of these, 2-(1-hexynyl)adenosine had submicromolar affinity for the N273Y receptor, 0.18 +/- 0.10 microM, with a 61-fold affinity gain over the wt receptor. In addition, the non-NECA analog (S)-PHP adenosine had an affinity of 1.7 +/- 0.5 microM for the wt receptor. The high affinity of (S)-PHP adenosine for the wt receptor suggests that further modifications at the 2-position may yield agonists with even higher affinity for A(2B) receptors.

Adenosine↗