Biomedical subjects

L Taylor

Publications and source records attributed to L Taylor.

At least 19 recordsLinked to original sources

PubMed2000

Isolation, characterization and expression of a human brain mitochondrial glutaminase cDNA.

Various cDNAs that encode overlapping portions of the full-length human brain glutaminase (GA) cDNA were cloned and sequenced. The overall nucleotide sequence of hGA has a very high degree of identity with that of the rat kidney-type GA cDNA (77.4%) and the known portion of the cDNA that encodes the 5.0-kb porcine GA mRNA (81.1%). The identity is even more remarkable at the amino acid level, particularly in the C-terminal half where the three proteins share a 99.7% sequence identity. The hGA cDNA encodes a 73,427-Da protein that contains an N-terminal mitochondrial targeting signal and retains the primary proteolytic cleavage site characterized for the cytosolic precursor of the rat renal mitochondrial glutaminase. The entire coding region was assembled through the use of unique restriction sites and cloned into a baculovirus. Sf9 cells infected with the recombinant virus express high levels of properly processed and active glutaminase. Thus, expression of the isolated hGA cDNA should provide a means to purify large amounts of the mitochondrial glutaminase, a protein that catalyzes a key reaction in the metabolism of glutamine and the synthesis of important excitatory and inhibitory neurotransmitters.

Amino Acid Sequence↗

PubMed2000

Characterization of an aerosol chamber for human exposures to endotoxin.

The objective of this study was to develop and characterize an exposure chamber in which human subjects could be exposed to low dust concentrations carrying an endotoxin coating. An exposure chamber, dust dispersion method, and endotoxin characterization technique were developed for inhalation exposures. A 6.27 m3 exposure chamber was designed and constructed from cinder block, glass windows, and Plexiglas. Using an acetone adhesion process, Enterobacter agglomerans were adsorbed onto respirable cellulose particles to create the endotoxin aerosol. The size distribution of the endotoxin-treated particles was verified using light microscopy and cascade impactors. A dry powder dust generator was refined to consistently disperse small quantities of the aerosol into the chamber to maintain dust concentrations at approximately 250 micrograms/m3. Dust levels during the chamber exposures were monitored using a portable continuous aerosol monitor (PCAM). During initial exposure runs, PCAM monitoring stations were positioned at different locations within a 0.5-meter matrix to document mixing patterns. Total dust and cascade impactor samples were collected throughout each exposure period to characterize the chamber operating system and insure the mean airborne dust concentration fulfilled target levels. A one-factor analysis of variance at the 95 percent confidence interval illustrated that there was not a statistically significant difference in the mean dust concentration throughout the exposure runs compared to the individual runs. Together the consistency of the total dust filters, endotoxin concentrations, and aerosol-monitoring instrument were adequate to allow use of the chamber for experimental studies involving human volunteers.

Aerosols↗

PubMed1999

SCF ubiquitin protein ligases and phosphorylation-dependent proteolysis.

Many key activators and inhibitors of cell division are targeted for degradation by a recently described family of E3 ubiquitin protein ligases termed Skp1-Cdc53-F-box protein (SCF) complexes. SCF complexes physically link substrate proteins to the E2 ubiquitin-conjugating enzyme Cdc34, which catalyses substrate ubiquitination, leading to subsequent degradation by the 26S proteasome. SCF complexes contain a variable subunit called an F-box protein that confers substrate specificity on an invariant core complex composed of the subunits Cdc34, Skp1 and Cdc53. Here, we review the substrates and pathways regulated by the yeast F-box proteins Cdc4, Grr1 and Met30. The concepts of SCF ubiquitin ligase function are illustrated by analysis of the degradation pathway for the G1 cyclin Cln2. Through mass spectrometric analysis of Cdc53 associated proteins, we have identified three novel F-box proteins that appear to participate in SCF-like complexes. As many F-box proteins can be found in sequence databases, it appears that a host of cellular pathways will be regulated by SCF-dependent proteolysis.

Amino Acid Sequence↗

PubMed1999

Development of low cost peptide-based anti-hepatitis C virus screening and confirmatory assays: comparison with commercially available tests.

Screening and confirmatory low cost reagent tests have been developed for detection of anti-hepatitis C virus (HCV). Assays are based on the use of specific synthetic peptides from several structural and non-structural viral proteins. The efficacy of the screening anti-HCV EIA-Spep assay was compared with both Abbott EIA 2.0 (Abbott Laboratories, North Chicago, IL) and Ortho EIA 2.0 (Ortho Diagnostic Systems, Raritan, NJ) anti-HCV detection kits and the confirmatory EIA-Cpep assay was compared with the Abbott Matrix anti-HCV confirmation test. In the EIA-Spep, a pool of 3 peptides was added to each well of a microtiter plate. In EIA-Cpep, each well was separately coated with 1 of 4 peptides and 1 recombinant protein. A total of 867 blood donor samples from Costa Rica tested simultaneously with the 3 screening assays yielded the same specificity and negative predictive values of > or =99.9% and 100%, respectively. A comparative study on voluntary blood donor samples from Honduras, Nicaragua, and El Salvador using the 2 anti-HCV confirmatory assays revealed different patterns that are 46% positive, 24% indeterminate, and 30% negative with the EIA-Cpep assay vs. 31% positive, 48% indeterminate, and 21% negative with the Matrix assay. A study of 71 patient samples from Costa Rica showed a higher correlation between initially reactive samples when analyzed by the Abbott and Ortho kits, than when the assay results were compared between the Abbott and EIA-Spep kits; the latter detected 7 and 15 non-reactive samples, respectively. These results could reflect the use of a similar antigen source for the 2 commercial assays. The presence of HCV RNA in a group of 29 samples analyzed was related to the simultaneous reactivity in all 3 screening assays. None of the discordant samples had detectable levels of HCV RNA. Economic difficulties for health care services in the developing countries of Central America have prevented implementation of routine anti-HCV blood donor screening tests. This is likely to be the primary reason for uncontrolled dissemination of HCV, and the lack of identification of potential high risk groups. Alternative low cost reagents developed locally as described in this article could be a useful tool in the control of HCV spread throughout the developing world.

Amino Acid Sequence↗

PubMed1999

Australia's oldest human remains: age of the Lake Mungo 3 skeleton.

We have carried out a comprehensive ESR and U-series dating study on the Lake Mungo 3 (LM3) human skeleton. The isotopic Th/U and Pa/U ratios indicate that some minor uranium mobilization may have occurred in the past. Taking such effects into account, the best age estimate for the human skeleton is obtained through the combination of U-series and ESR analyses yielding 62,000+/-6000 years. This age is in close agreement with OSL age estimates on the sediment into which the skeleton was buried of 61,000+/-2000 years. Furthermore, we obtained a U-series age of 81,000+/-21,000 years for the calcitic matrix that was precipitated on the bones after burial. All age results are considerably older than the previously assumed age of LM3 and demonstrate the necessity for directly dating hominid remains. We conclude that the Lake Mungo 3 burial documents the earliest known human presence on the Australian continent. The age implies that people who were skeletally within the range of the present Australian indigenous population colonized the continent during or before oxygen isotope stage 4 (57,000-71,000 years).

Animals↗

PubMed1999

Posttranscriptional destabilization of the bradykinin B1 receptor messenger RNA: cloning and functional characterization of the 3'-untranslated region.

We showed previously that the inducible bradykinin B1 receptor (BKB1R) gene expression is regulated, in part, through mRNA stabilization. Here we clone the 3'-untranslated region (3'-UTR) of the BKB1R. This region proves to be very short, containing only 14 bases with an alternative polyadenylation signal (AUUAAA) which overlaps with the stop codon. Reverse transcription confirms the presence of this alternative polyadenylation signal. Northern blot shows a single species of BKB1R mRNA of approximately 1.4 kb in agreement with its calculated length. The BKB1R mRNA induced by TNFalpha, phorbol ester, bradykinin, and desArg10-kallidin contain the same 3'-UTR species. To test the role of this region in the regulation of mRNA stability, we generated a chimeric luciferase construct containing the BKB1R 3'-UTR. The mRNA transcribed from the wild-type luciferase gene displayed a half-life of approximately 6 h. The mRNA transcribed from the chimeric construct displayed a half-life of only 1 h. This decrease was also reflected at the level of enzyme activity. Luciferase activity from cells transfected with the chimeric construct was 10 times less than from cells transfected with wild-type luciferase. The data presented provide compelling evidence that the 3'-UTR is participating in the regulation of BKB1R mRNA stability and its ultimate expression.

3' Untranslated Regions↗

PubMed1999

Effect of the G-protein, G alpha(i2), and G alpha(i3) subunit knockdown on bradykinin-induced signal transduction in rat-1 cells.

The bradykinin (BK) B2 receptor (BKB2R) has been shown to interact with the G alpha(q) subunit family. However, it has remained unclear whether this receptor also interacts with the G alpha(i) subunit family. To further resolve this issue, two antisense expression plasmids were generated. In these, the 5'-untranslated regions of rat G alpha(i2) and G alpha(i3) cDNAs were used as specific antisense templates. The plasmids were transfected into Rat-1 cells, which expressed a stably transfected rat BKB2R cDNA and bound BK with a Kd of approximately 3 nM. In these cells, the transfected BKB2R was fully linked to inositol phosphate production, arachidonic acid (ARA) release, and Ca2+ flux. A number of cell lines, each a G alpha(i2) or G alpha(i3) knockdown, were isolated. Of these, two cell lines were chosen for study. One, designated 2-E3, displayed over a 70% decrease in the expression of G alpha(i2) without a change in the expression of G alpha(i3) or G alpha(q). Another, 3-G9, exhibited over a 70% decrease of G alpha(i3) protein without a change in G alpha(i2) or G alpha(q) expression. Knockdown of either G alpha(i2) or G alpha(i3) protein production did not affect the binding of bradykinin. In the G alpha(i2)-depleted 2-E3 cells, BK induced ARA release was reduced by more than 60%. Interestingly, the production of total inositol phosphate in response to BK was also reduced by approximately 35%. However, G alpha(i2) knockdown had no significant effect on BK-induced Ca2+ influx. In the G alpha(i3)-depleted 3-G9 cells, BK-induced ARA release was decreased by over 50%. In this case [Ca2+]i increase in response to BK was reduced by over 50%. This knockdown also resulted in reduced BK-activated total inositol phosphate production. Moreover, cAMP augmented the BK-induced ARA release. Depletions of G alpha(i2) and G alpha(i3) further enhanced this cAMP-dependent BK induction of ARA release. Taken together, these results delineate direct interaction of the BKB2R with both G alpha(i2) and G alpha(i3) subunits in addition to the heretofore described interaction of BKB2R with the G alpha(q) subunit family.

Animals↗

PubMed1999

1997 Peritoneal Dialysis-Core Indicators Study: Dialysis Adequacy and Nutritional Indicators Report.

The 1997 Peritoneal Dialysis-Core Indicators Study: Dialysis Adequacy and Nutritional Indicators Report documents the current status of peritoneal dialysis within the United States. A national random sample of adult peritoneal dialysis (PD) patients participating in the United States End Stage Renal Disease (ESRD) program was surveyed. PD subjects were large, with a mean body weight of 76 +/- 18.9 kg (mean +/- 1 SD) and a body mass index (BMI) of 27 +/- 6.4. The dialysis prescriptions documented achieved a mean weekly Kt/Vurea (wKt/V) and weekly creatinine clearance (wCrCl) of 2. 45 +/- 2.29 and 65.5 +/- 35.2 L/wk/1.73m2, respectively. The serum albumin of these patients was 3.5 +/- 0.48 g/dL, their normalized protein equivalent of nitrogen appearance (nPNA) 1.0 +/- 0.63 g/kg/d, and their normalized creatinine appearance rate (nCAR) 14 +/- 6.0 mg/kg/d. Serum albumin correlated meagerly but in a positive fashion with BMI, nPNA, and nCAR, and negatively with wCrCl. Among adult US PD patients, serum albumin values appear to correlate poorly with alternative measures of nutritional status and are inversely related to the intensity of renal replacement therapy. The presumptive dietary protein intake (nPNA) and creatinine appearance rate (nCAR) derived for PD patients do correlate in a positive fashion with dialysis delivery, at least up to a wCrCl of 60 to 80 L/wk/1.73 m2 and wKt/V = 2.1, but their values suggest that 30% to 50% of PD patients have marginal nutritional status.

Adolescent↗

PubMed1999

Mental health in schools and system restructuring.

Because health is not the primary business of schools, a school's response to mental health and psychosocial concerns usually is limited to targeted problems seen as direct barriers to learning. And because resources are sparse, priority is given to problems defined in legislative mandates. As a result, school-based mental health services are available only to a small proportion of the many students who require assistance, and interventions generally are narrowly focused and short-term. To better meet the needs of those served and to serve greater numbers, emerging trends are pushing for restructuring of school-owned services and greater linkage with community resources to develop multifaceted, comprehensive, integrated approaches. This review (a) provides an overview of what schools currently do related to mental health and psychosocial concerns, (b) clarifies key emerging trends, and (c) explores implications for major systemic changes.

Adolescent↗

PubMed1999

Effect of immunization against melatonin on prolactin concentrations and the timing of reproductive transitions in ewes.

The objective of this experiment was to develop a procedure for immunizing ewes against melatonin that would alter the effects of changing photoperiod on seasonal reproduction and prolactin secretion. Ewes were immunized against human serum albumin (HSA) as controls (n = 9) or a melatonin-human serum albumin conjugate (0.25 mg; n = 10) on December 14th (Day 0) and boosted 9 times. They were maintained on natural photoperiod and then transferred indoors and exposed to long days for 35 d, followed by short days for 146 d, long days for 93 d, and short days for a further 123 d. Antibody titers to melatonin (at a serum dilution of 1:1,250) were significantly higher in immunized ewes (27.3 +/- 6.6%) than controls (0.7 +/- 0.1%; P < 0.001). At the end of the experiment, antibody titers in immunized ewes (at dilution of 1:50) were higher in blood (43.7 +/- 8.2%) than in cerebrospinal fluid (10.8 +/- 3.9%; P < 0.05), and highly correlated (r2 = 0.746). Onset of the breeding season was advanced slightly after the second transfer from long to short days in immunized ewes (April 12 +/- 3 d) compared with controls (April 25 +/- 3 d; P < 0.05). Mean serum prolactin concentrations were lower (P < 0.05) in melatonin-immunized ewes compared with controls on natural photoperiod, after transfer from long to short days, during long days, and after the second transfer from long to short days. In conclusion, despite melatonin-immunization increasing antibody titers in blood and cerebrospinal fluid, and decreasing prolactin concentrations over much of the experiment, minimal effects on the timing of reproductive transitions in the ewes were evident. This discrepancy between the response of the prolactin and reproductive axes to melatonin immunization supports the hypothesis of a dual site of action of melatonin, with melatonin acting in the pituitary gland to mediate the effects of photoperiod on prolactin secretion and in the mediobasal hypothalamus to affect reproductive responses.

Animals↗

PubMed1999

Cognitive reactivity and depressotypic information processing in children of depressed mothers.

Although high-risk research suggests that children of depressed mothers are at increased risk for psychological disorders, the mechanisms of this risk are not well understood. In the current study, the information processing of children of depressed mothers was compared with that of children whose mothers were not depressed. Half of each group received a priming induction designed to activate cognitive schemas prior to assessment. All children then completed a self-referent processing task that examined the recall of negative and positive information. Results indicate that when primed, at-risk children showed a less positive self-concept and more negative information processing than did the children in the other groups. These data may offer potential clues into the mechanisms of cognitive vulnerability in at-risk children.

Adult↗

PubMed1999

Searching the right database. A comparison of four databases for psychiatry journals.

Journal coverage is one factor that affects the retrieval of relevant information from bibliographic databases. The aims of this study were to investigate the coverage of databases for psychiatry journals, and to assess the overlap between databases. Psychiatry journals were identified using Ulrich's International Periodicals Directory and then analysed to see which bibliographic databases indexed the most journals. A total of 213 abstracting and indexing services were listed as indexing at least one of the 977 psychiatry journals identified. The four most frequently cited databases (PSYCLIT, EMBASE, BIOSIS and MEDLINE) indexed 506 (52%) of the psychiatry journals. Of these 506 journals, PSYCLIT indexed 367 (73%), EMBASE 337 (67%), BIOSIS 243 (48%) and MEDLINE 236 (47%). Combining the databases with the highest yields (PSYCLIT and EMBASE) increased the number of journals indexed to 461 (91%). The four databases combined accounted for 90% of all psychiatry journals found to be indexed by at least one abstracting and indexing service. More than 400 journals were not indexed at all. Variations in the overlap between PSYCLIT, EMBASE, BIOSIS and MEDLINE, and the high proportion (35%) of journals indexed in only one of these four databases emphasize the importance of searching more than one or even two databases to ensure optimal coverage of the literature.

Abstracting and Indexing↗

PubMed1999

Psychometric validation of a constipation symptom assessment questionnaire.

BACKGROUND: Clinical management of constipation is complicated by the lack of a gold standard for evaluation of symptoms. A constipation symptom assessment instrument, the PAC-SYM, was developed to address the patient perspective on the disorder. Instrument content was based on literature review and results of focus groups. METHODS: Two hundred and sixteen patients at nine sites participated in a 6-week psychometric evaluation of the PAC-SYM. The final instrument contained 12 items assigned to 3 subscales: stool symptoms, rectal symptoms, and abdominal symptoms. The psychometric properties of this final instrument were assessed. RESULTS: Internal consistency and test-retest reliability of the final instrument was high (Cronbach's alpha = 0.89; intraclass correlation = 0.75). Concurrent validity was supported by the correlation with both subject and investigator constipation severity ratings (r= 0.68 and 0.72, respectively; P < 0.0001). Scores were moderately correlated with instruments measuring quality of life. Comparison of treatment responders with nonresponders showed the ability of the instrument to differentiate between groups on the basis of clinical severity (t = -6.12, P < 0.0001 ). Scores changed significantly over time among responders, indicating instrument responsiveness. CONCLUSIONS: The PAC-SYM is internally consistent, reproducible under stable conditions, valid, and responsive to change and provides a comprehensive means to assess the effectiveness of treatment for constipation.

Adolescent↗

PubMed1999

Trends in births at and beyond term: evidence of a change?

OBJECTIVE: To examine trends in the distribution of births at and beyond term in New South Wales and in particular, to determine whether any changes are associated with changes in the obstetric practices of induction and elective caesarean section. DESIGN: Cross-sectional analytic study. SETTING: New South Wales, Australia. POPULATION: All 540,162 women delivering a singleton cephalic-presenting infant of gestational age > or = 37 weeks from 1 January 1990 to 31 December 1996. METHODS: Data were obtained from the New South Wales midwives data collection, a population-based surveillance system covering all births in New South Wales. The data were analysed to examine changes over time and associations between gestational age, maternal factors and onset of labour. MAIN OUTCOME MEASURES: Induction of labour and elective caesarean section rates. RESULTS: From 1990 to 1996 there was a significant decrease in births reported as 40 weeks of gestation, from 35,670 (46.3%) to 30,651 (40.3%). These declines were offset by significant increases in births at 38 and 39 weeks. Births > or = 42 weeks declined from 3321 (4.6%) to 2132 (2.8%). The decline in prolonged pregnancies was associated with increasing induction rates at 41 weeks. The re-distribution of some births from 40 to 38-39 weeks was associated with increasing rates of elective caesarean sections and induction at 38 and 39 weeks, and increasing maternal age. CONCLUSIONS: Clinicians appear to be implementing the recommendations of randomised controlled trials to offer induction after 41 weeks of gestation. However the trend of performing elective caesarean sections at earlier gestational ages may be unnecessarily putting some infants at increased risk of respiratory morbidity.

Adult↗

PubMed1999

The treatment and prevention of coronary heart disease in Canada: do older patients receive efficacious therapies? The Clinical Quality Improvement Network (CQIN) Investigators.

OBJECTIVES: To review the evidence for clinical efficacy and cost-effectiveness of proven medications in the treatment and prevention of myocardial infarction (MI) in older patients; to summarize Canadian data on treatment patterns and clinical outcomes for younger and older patients with coronary heart disease; to explore the reasons for gaps between best care, based on the evidence of efficacy from trials, and usual care, based on the population effectiveness audits; and to explore potential approaches to closing the care gaps. DESIGN: Review of the recent clinical trial literature on the management of MI, highlighting results in older patients. Review of medication utilization and outcomes data from a series of large, consecutively enrolled patient cohorts with acute MI (N = 7070) in a variety of cardiac care settings (10 centers in five Canadian provinces, including university-based teaching hospitals, community hospitals, cardiologist and family physician out-patient clinics) from 1987 to 1996. RESULTS: There is no qualitative interaction of cardiac therapies: thrombolytics, beta-blockers, acetylsalicylic acid (ASA), and statins are efficacious in all clinically relevant patient subgroups, including older people. However, there are consistent gaps between usual care and best care, particularly among older patients (in whom there is also a concomitantly higher mortality risk). Repeated multivariate analyses confirm older age to be an independent contributor to increased risk. Use of efficacious medications is, in contrast, consistently associated with increased survival. Analysis of temporal trends suggests beneficial changes in practice patterns and outcomes are possible to achieve. However, "best care" has not been rapidly or completely achieved. Review of strategies to close these care gaps suggests that audit and feedback, critical pathways, and multifactorial interventions involving patients and other members of the healthcare team as well as physicians may be the most efficacious strategies for change. CONCLUSIONS: Despite equal or enhanced efficacy, there is consistently less prescription of proven drugs among older cardiac patients. These care patterns may contribute to their enhanced risk. The causes underlying these practice patterns are complex, and their population impact may be undervalued by clinicians and managers. Improvement of these patterns is difficult, but ultimately it would be beneficial for this presently disadvantaged, readily identified, high risk patient population.

Aged↗

PubMed1999

HHV-8 (KSHV) is not associated with bacillary angiomatosis.

AIMS: Bacillary angiomatosis is a rare pseudoneoplastic angioproliferative lesion occurring in patients with AIDS. This condition has been associated with Bartonella henselae and Bartonella quintana infections. Human herpesvirus 8 (HHV-8) is thought to be the causative agent of Kaposi's sarcoma, a vasoproliferative neoplasm, also commonly found in patients with AIDS. The presence of HHV-8 in a cohort of patients with bacillary angiomatosis was investigated. METHODS: Eight cutaneous cases of biopsy confirmed bacillary angiomatosis were assessed for HHV-8 using standard solution phase polymerase chain reaction (PCR). RESULTS: No case of bacillary angiomatosis harboured HHV-8 DNA. CONCLUSIONS: HHV-8 was not demonstrated in the lesions of bacillary angiomatosis and therefore does not appear to play a role in the pathogenesis of this pseudoneoplastic angioproliferative disorder. This finding might be useful in the distinction of bacillary angiomatosis from Kaposi's sarcoma, because lesions from patients with Kaposi's sarcoma almost always contains HHV-8 DNA.

AIDS-Related Opportunistic Infections↗

PubMed1999

bcl-2 immunoreactivity, human papillomavirus DNA, and cervical intraepithelial neoplasia.

The aim of this study was to identify the role of bcl-2 protein expression in precancerous lesions of the cervix in patients from Johannesburg, South Africa and to correlate this expression with human papillomavirus (HPV) status. Archival cervical biopsy specimens (n = 107) of normal squamous epithelia (n = 18), pure HPV squamous epithelial lesions (n = 15), cervical intraepithelial neoplasia (CIN) I lesions (n = 17), CIN II lesions (n = 26), and CIN III lesions (n = 31) underwent bcl-2 immunohistochemical analysis with use of the streptavidin-biotin complex/horseradish peroxidase system and nonisotopic in situ hybridization for the detection of HPV DNA. Although 45 (61%) of the 74 CIN lesions demonstrated bcl-2 protein expression in the epithelia, most seemed to be in a patchy basal cell distribution, with a 1+ to 2+ intensity. Furthermore, comparison of bcl-2 immunoreaction between the low and high grades of the CIN lesions did not reveal significant differences. In addition, there was no apparent link between the presence of HPV DNA and bcl-2 expression in the CIN lesions. In contrast to previous studies that showed an increase in bcl-2 immunostaining intensity with increasing severity of CIN, only 4 (5.4%) of our 74 CIN specimens satisfied this pattern. Hence, we suggest that bcl-2 protein expression might not play a significant role in the majority of CIN lesions in this population group and that it might not correlate with HPV status.

DNA, Viral↗

PubMed1998

Motif mutation of bradykinin B2 receptor second intracellular loop and proximal C terminus is critical for signal transduction, internalization, and resensitization.

In the search for the structural elements participating in signal transduction, internalization, and resensitization of the bradykinin B2 receptor, we identified two critical motifs, one in the second intracellular loop (IC2), the other in the proximal C terminus. We previously described the contribution of tyrosines within each of the two motifs (Tyr131 and Tyr322) to signal transduction and receptor internalization (Prado, G. N., Taylor, L., and Polgar, P. (1997) J. Biol. Chem. 272, 14638-14642). Here, we investigate the effect of exchanging both tyrosine residues simultaneously for alanine, phenylalanine, or serine, termed YAYA (Y131A/Y322A), YFYF (Y131F/Y322F), and YSYS (Y131S/Y322S) receptors, respectively. All of these mutants bound bradykinin (BK) normally, with a Kd of approximately 1.1 nM. However, although phosphoinositide (PI) turnover in response to BK by Y131A and Y131S proved negligible, the YAYA mutant returned BK-activated PI turnover to wild type (WT). In contrast, PI turnover with YSYS remained unresponsive to BK. Importantly, the pattern of BK-activated arachidonate release differed markedly in the mutant receptors. For example, whereas Y131S ablated BK-activated arachidonic acid release, conversion of this mutant to YSYS returned the BK-activated receptor function to a level above that of WT. However, YAYA showed only a partial recovery from the poor BK response of Y131A. These and additional results suggest that Tyr131 and Tyr322 interact cooperatively in conjunction with at least two separate signaling functions. Given these results, a molecular model of the receptor was generated with the IC2 and the proximal C terminus in close spatial proximity. Conformations were identified to provide structural explanation for these observations. The conserved Thr137 in the IC2 was next substituted with proline (T137P) to prevent phosphorylation at this position or with aspartate (T137D) to emulate phosphorylation. The T137P mutant demonstrated no change from WT with respect to either BK-activated PI turnover or arachidonic acid release. However, the mutant exhibited a markedly reduced capacity to internalize. It also resensitized poorly. The T137D mutant lacked both BK responsive activities. However, it internalized and resensitized normally, as did WT. These final results suggest that Thr137 is functioning as a switch in termination of signal transduction and the initiation of internalization.

Amino Acid Sequence↗