Mutational screening of the CD40 ligand (CD40L) gene in patients with X linked hyper-IgM syndrome (XHIM) and determination of carrier status in female relatives.

AIMS: To analyse the gene encoding the CD40 ligand (CD40L) in 11 Australian patients from 10 unrelated families with the X linked hyper-IgM (XHIM) phenotype. METHODS: The CD40L gene was screened for mutations using direct sequencing of exon specific polymerase chain reaction (PCR) products. RESULTS: Ten mutations were identified. Seven of these mutations have been described previously, whereas three new nonsense mutations were identified, namely: E108X (c.322G>T), G167X (c.499G>T), and C218X (c.654C>A). Ten of 15 female family members revealed both a mutated allele and a normal allele, indicating that they were XHIM carriers. CONCLUSION: The 10 mutations (including the three new ones) identified in this study reflect the heterogeneity of the CD40L gene, and indicate the need for accurate and reliable molecular testing of those patients suspected of XHIM.

NMR trial models: experiences with the colicin immunity protein Im7 and the p85alpha C-terminal SH2-peptide complex.

Two cases of successful molecular replacement using NMR trial models are presented. One is the crystal structure of the Escherichia coli colicin immunity protein Im7; the other is a heretofore unreported crystal structure of a specific PDGF receptor-derived peptide complex of the carboxy-terminal SH2 domain from the p85alpha subunit of human phosphatidylinositol 3-OH kinase. In both cases, molecular replacement was non-trivial. Success was achieved using trial models that consisted of an ensemble of NMR structures from which the more flexible portions had been excised. Use of maximum-likelihood refinement proved critical to be able to refine the poor starting models. The challenges typical of the use of NMR trial models in molecular replacement are discussed.

Assessment of male CVID patients for mutations in the Btk gene: how many have been misdiagnosed?

The presentation of hypogammaglobulinaemia in young males without a family history of immunodeficiency can pose a diagnostic problem. In the past, the presence of B-cells has suggested a diagnosis of common variable immunodeficiency (CVID), although genotypic analysis has now clarified that individuals with B cells may have mutations in their Btk gene. In order to address the issue of how many male individuals with a clinical diagnosis of CVID do in fact have mutations in the Btk gene, we analysed a group of 24 male patients. Single-strand conformation polymorphism (SSCP) analysis was used to screen the patient cohort for mutations in the Btk gene. Given the size of the Btk gene, the number of patients in the cohort and the amount of available DNA, multiplex PCR reactions were utilized to span the 19 exons and promoter region of the gene. Where abnormal migration patterns were observed with multiplex PCR reactions, in nine of the 24 patients, the individual Btk gene fragments were re-amplified and analysed again by SSCP. Following this analysis, four patients continued to demonstrate abnormal SSCP migration patterns. However, direct sequencing of the relevant Btk gene fragments for these four CVID patients revealed a mutation in only one patient. The mutation was the previously described polymorphism at position 2031 of Btk gene within exon 18. These results indicate that caution should be taken with the application of SSCP analysis to mutation detection. While it has a role to play in screening large patient cohorts, direct sequencing is a necessary adjunct to such analysis. Finally, the clinical diagnosis of CVID in this cohort successfully excluded males with Btk mutations.

Intermittent claudication: an objective office-based assessment.

OBJECTIVES: We sought to compare standard lower extremity vascular laboratory treadmill exercise with the office-based active pedal plantarflexion technique. BACKGROUND: Intermittent claudication is relatively common in elderly patients and is an important predictor of cardiovascular morbidity and mortality. Noninvasive testing using resting and posttreadmill exercise ankle:brachial systolic blood pressure indices is often required to confirm the diagnosis and objectively assess the severity of lower extremity arterial occlusive disease. This is traditionally performed in a formal vascular laboratory setting. METHODS: Fifty consecutive patients (100 lower extremities) with known or suspected intermittent claudication referred for lower extremity treadmill exercise testing were also tested with active pedal plantarflexion using a prospective, randomized crossover design. Supine ankle:brachial systolic blood pressure indices were measured immediately before and after each form of exercise. RESULTS: There was an excellent correlation (r = 0.95, 95% confidence interval 0.93 to 0.97) between mean postexercise ankle:brachial systolic blood pressure indices for treadmill exercise and active pedal plantarflexion. There was no significant difference in outcome based on the order of testing or the severity of arterial occlusive disease. Symptoms of angina or dyspnea occurred in 11 patients (22%) with treadmill exercise versus zero patients with active pedal plantarflexion. CONCLUSIONS: Active pedal plantarflexion is an office-based test that compares favorably with treadmill exercise for the noninvasive, safe, objective and economical assessment of lower extremity arterial occlusive disease.

Referral to autopsy: effect of antemortem cardiovascular disease: a population-based study in Olmsted County, Minnesota.

PURPOSE: Autopsy studies can provide insight into disease trends and their determinants, including data on the prevalence of atherosclerosis. However, such studies are subject to autopsy bias, which limits their generalizability to the source population. The impact of this bias on autopsy based estimates of time trends in heart disease prevalence is unknown. To report on the trends over time in autopsy rates in Olmsted County, MN, to examine the association between clinical diagnoses of cardiovascular diseases (CVDs) and referral to autopsy and how this association may have changed over time. METHODS: We examined the trends in autopsy rates between 1979 and 1994 in Olmsted County, and the association between antemortem characteristics including cardiovascular diagnoses and autopsy referral. RESULTS: From 1979 to 1994, a total of 9110 residents died in Olmsted County. The average annual autopsy rate was 30%. Autopsy rates declined from 36% in 1979 to 23% in 1994, corresponding to an average decline of 0.6%/year (p < 0.01). Referral to autopsy was positively associated with younger age, male sex, in-hospital place of death, antemortem diagnoses of myocardial infarction (MI) or peripheral vascular disease (PVD), and earlier calendar period. There was no evidence of an interaction between calendar period and any of these predictor variables. Antemortem diagnosis of heart failure was associated with a decrease in the odds of referral to autopsy over time as compared to persons without such diagnosis. CONCLUSIONS: In Olmsted County, autopsy rates, although declining over time, have remained on average approximately 30%. Antemortem diagnoses of MI or PVD are associated with autopsy referral but this association did not change over time. While the greater decline overtime in the use of autopsy observed among decedents with an antemortem diagnosis of congestive heart failure (CHF) deserves further studies, the present findings reduce the concern for bias of time trends in the prevalence of atherosclerosis by changes in the clinical characteristics of decedents referred to autopsy.

Time trends in the prevalence of atherosclerosis: a population-based autopsy study.

PURPOSE: Mortality from coronary heart disease is declining but little is known about trends in the prevalence of atherosclerosis. Autopsy rates in Olmsted County, Minnesota, are higher than the national average, offering an opportunity to address this matter. In this study, we determined the prevalence of anatomic coronary disease among autopsied Olmsted County residents and examined the generalizability of these findings. SUBJECTS AND METHODS: Reports of the 2,562 autopsies performed between 1979 and 1994 on Olmsted County residents > or =20 years of age were reviewed for the presence of coronary disease. RESULTS: Among autopsied decedents less than 60 years old at death and among coroner's cases, the prevalence of anatomic coronary disease declined with time (P for trend = 0.05); no trend was detected among older persons or noncoroner's cases. By logistic regression analysis, the crude odds ratio ([OR] per 5 years) for the association between time and anatomic coronary disease was 0.94 (95% confidence interval [CI]: 0.86 to 1.03; P = 0.18]. Age, sex, and antemortem diagnosis of heart disease were also strongly related to the presence of disease. After adjustment for sex and antemortem diagnosis of heart disease, the prevalence of anatomic coronary disease decreased more in younger people than in older people (age 40 years: OR 0.43 [95% CI: 0.24 to 0.80]; age 60 years: OR 0.62 [95% CI: 0.45 to 0.87]; age 80 years: OR 0.89 [95% CI: 0.64 to 1.23]). CONCLUSION: The prevalence of anatomic coronary disease at autopsy decreased between 1979 and 1994, particularly among younger people, supporting the notion that the burden of coronary disease has shifted toward the elderly. These results suggest that the decreased incidence of coronary artery disease has contributed to the recent decrease in coronary mortality, particularly among younger people.

Validation of death certificate diagnosis of out-of-hospital coronary heart disease deaths in Olmsted County, Minnesota.

OBJECTIVE: To evaluate the validity of death certificate diagnosis of out-of-hospital (OOH) coronary heart disease (CHD) and sudden cardiac death (SCD) in Olmsted County, Minnesota, between 1981 and 1994. METHODS: In this review of the medical records, autopsy reports, and coroner's files, OOH deaths with heart disease as the underlying cause of death on the death certificate were classified into CHD (International Classification of Diseases, 9th Revision, Clinical Modification [ICD-9-CM] codes 410-414) and non-CHD (other ICD-9-CM heart disease codes) deaths. A 10% random sample (n = 174) of these death certificates was reviewed by physicians, and published validation criteria were applied to classify these deaths into validated CHD or non-CHD categories. Sudden cardiac death was defined as validated CHD that occurred at an OOH location with less than 24 hours between symptom onset and death. RESULTS: The death certificate definition of OOH CHD death (ICD-9-CM codes 410-414) had high sensitivity and positive predictive value of 91% and 96%, respectively. The specificity and the negative predictive value were slightly lower at 86% and 72%, respectively. The sensitivity of death certificate diagnosis of CHD for validated SCD was 89%, and the positive predictive value was 77%. Using a more restrictive definition of SCD, that is, less than 1 hour between the onset of symptoms and death, the positive predictive value of CHD codes for SCD was lower at 52%. CONCLUSIONS: In Olmsted County, the positive predictive values of death certificate diagnosis for OOH CHD and SCD are high. Relying on death certificate diagnoses results in about 5% underestimation of the true CHD rates, whereas their use as a surrogate for SCD yields a 16% overestimation of the true SCD rates.

Prognostic value of treadmill exercise testing in elderly persons.

BACKGROUND: Recent exercise testing guidelines recognized a gap in knowledge about the prognostic value of treadmill exercise testing in elderly persons. OBJECTIVE: To test the hypothesis that treadmill exercise testing has equal prognostic value among elderly (> or =65 years of age) and younger (<65 years of age) persons and to examine the incremental value of this testing over clinical data. DESIGN: Inception cohort with a median follow-up of 6 years. SETTING: Olmsted County, Minnesota. PATIENTS: All elderly (n = 514) and younger (n = 2593) residents of Olmsted County who underwent treadmill exercise testing between 1987 and 1989. MEASUREMENTS: Overall mortality and cardiac events (cardiac death, nonfatal myocardial infarction, and congestive heart failure). RESULTS: Compared with younger patients, elderly patients had more comorbid conditions, achieved a lower workload (6.0 and 10.7 metabolic equivalents; P < 0.001), and had a greater likelihood of a positive exercise electrocardiogram (28% and 9%; P < 0.001). With median follow-up of 6 years, overall survival (63% and 92%; P < 0.001) and cardiac event-free survival (66% and 95%; P < 0.001) were worse among elderly persons than among younger persons. Workload was the only treadmill exercise testing variable associated with all-cause mortality in both age groups, and the strength of association was similar. Workload and angina with exercise testing were associated with cardiac events in both age groups, whereas a positive exercise electrocardiogram was associated with cardiac events only in younger persons (P < 0.05 for all comparisons). After adjustment for clinical variables, workload was the only additional treadmill exercise testing variable that was predictive of death (P < 0.001) and cardiac events (P < 0.05); the strength of the association was similar in both age groups. Each 1-metabolic equivalent increase in exercise capacity was associated with a 14% and 18% reduction in cardiac events among younger and elderly persons, respectively. CONCLUSIONS: In elderly persons, treadmill exercise testing provided prognostic information that is incremental to clinical data. After adjustment for clinical factors, work-load was the only treadmill exercise testing variable that was strongly associated with outcome, and its prognostic effect was of the same magnitude in elderly and younger persons.

Sex differences in left ventricular geometry in aortic stenosis: impact on outcome.

OBJECTIVE: This study of surgical aortic stenosis characterized sex differences in left ventricular (LV) geometry and outcome. MATERIALS AND METHODS: We examined 92 women and 82 men who underwent echocardiography before valve replacement for aortic stenosis. RESULTS: Women had a smaller cavity size (LV end-diastolic diameter 48.2 +/- 7 mm in women vs 53.6 +/- 7.6 mm in men; p = 0.0001) and higher ejection fraction (59% in women vs 54% in men; p = 0.02). LV mass was greater in men than women (300.4 +/- 88 g in men vs 250.6 +/- 85.8 g in women; p = 0.0055) but when corrected for body surface area, the difference was not significant. The prevalence of LV hypertrophy was similar in both sexes (51% in women vs 49% in men; p = 0.62). The 5-year survival was 82% in women and 79% in men (p = 0. 9). CONCLUSION: Several descriptors of LV geometry differed between men and women. These differences were largely eliminated after normalizing for body surface area. No differences in surgical mortality or long-term outcome were noted.

Sex differences in evaluation and outcome of unstable angina.

CONTEXT: The existence of sex bias in the delivery of cardiac care is controversial, and little is known about the association between sex and delivery of care and outcomes at an early point in the diagnostic sequence, such as when patients present for the evaluation of chest pain. OBJECTIVE: To test the hypothesis that female sex is negatively associated with care delivered to and outcomes of persons diagnosed as having unstable angina. DESIGN: Inception population-based cohort study with an average of 6 years of follow-up. SETTING: Emergency departments (EDs) in Olmsted County, Minnesota. PATIENTS: A total of 2271 Olmsted County residents (1306 men and 965 women) who presented to the ED for the first time with symptoms meeting criteria for unstable angina between 1985 and 1992. MAIN OUTCOME MEASURES: Use of cardiac procedures within 90 days of ED visit, overall mortality, and cardiac events (cardiac death, nonfatal myocardial infarction, nonfatal cardiac arrest, and congestive heart failure), compared by sex and Agency for Health Care Policy and Research cardiovascular risk category (low, intermediate, or high). RESULTS: Women were older (P<.001), more likely to have a history of hypertension (P = .001), and less likely to present with typical angina (P = .004) than men. Men were more likely than women to undergo noninvasive cardiac tests (relative risk [RR], 1.27; 95% confidence interval [CI], 1.14-1.40) as well as invasive cardiac procedures (RR, 1.72; 95% CI, 1.51-1.97). After adjustment, male sex was associated with a 24% increase in the use of cardiac procedures. Survival of both men and women in the high and intermediate risk categories was significantly lower than expected per the general population (P<.001). Women had a worse outcome than men, but after multivariate adjustment, male sex was associated with a trend toward an increase in the risk of death (RR, 1.23; 95% CI, 0.99-1.54) and significantly associated with increased risk of cardiac events (RR, 1.21; 95% CI, 1.03-1.42). CONCLUSIONS: Our population-based data indicate that after an ED visit for symptoms of unstable angina, the use of cardiac procedures was lower in women, but after taking into account baseline characteristics, men experienced worse outcomes.

Sex differences in the epidemiology and outcomes of heart disease: population-based trends.

We examined trends in heart disease (HD) mortality and the delivery of cardiac in Olmsted County, MN. Between 1979 and 1994, women experienced 51% of the total number of HD (ICD9 codes 390-398,402,404-429) deaths (3095). Age-adjusted HD mortality rate declined from 123 per 100,000 (95%CI 102, 144) in 1979 to 81 (67,95) in 1994. The risk ratio (RR) of HD death in 1994 compared to 1979 was 0.69 for women vs 0.53 for men (P = 0.06). This equates to a decline in HD mortality of 2.5%/y in women and 4.2%/y in men. The decline in HD mortality was less pronounced in older age groups (P < 0.001), reflecting a shift of the burden of HD towards women and the elderly. Compared to men, there was less use of stress tests among women, of cardiology visits after stress testing, and of cardiac procedures among women presenting to the emergency room with unstable angina. Further studies are needed to examine causal links between these trends.

Trends in heart disease deaths in Olmsted County, Minnesota, 1979-1994.

BACKGROUND: Although age-adjusted heart disease mortality has declined since the 1960s, this decline may not have applied equally to all subgroups. OBJECTIVE: To examine recent trends in heart disease mortality, specifically in women and in the elderly. METHODS: Age- and sex-specific heart disease mortality (International Classification of Diseases, Ninth Edition, Clinical Modification [ICD-9-CM] codes 390-398, 402, 404-429) in Olmsted County, Minnesota, between 1979 and 1994 were studied. RESULTS: The total number of heart disease deaths was 3095; 1578 (51%) occurred in women and 1984 (64%) in persons aged 75 years or older. Most heart disease deaths (77%) were coronary disease deaths (ICD-9-CM codes 410-414). Age-adjusted heart disease mortality rates declined from 123 per 100,000 (95% confidence interval [CI], 102-144/100,000) in 1979 to 81 per 100,000 (95% CI, 67-95/100,000) in 1994. Poisson regression analyses indicated that the trends differed according to sex and age. For women, the relative risk (RR) of heart disease death in 1994 compared with 1979 was 0.69 vs 0.53 for men (P = .06). This equates to a decline in heart disease mortality of 2.5% per year in women or 32% over the period and 4.2% per year in men or 47% over the period. The decline was less pronounced as age increased (P < .001). For 60-year-old women, the RR for 1994 compared with 1979 was 0.59, whereas for 80-year-old women, the RR for 1994 compared with 1979 was 0.76. For men, the RR for 1994 compared with 1979 was 0.60 for 80-year-old men vs 0.46 for 60-year-old men. CONCLUSIONS: Between 1979 and 1994, in Olmsted County, the decline in heart disease mortality was of lesser magnitude in women and in the elderly, emphasizing the importance of age- and sex-specific trends to characterize time patterns in heart disease deaths to target preventive measures.

Crystal structure of the anti-fungal target N-myristoyl transferase.

N-myristoyl transferase (NMT) catalyzes the transfer of the fatty acid myristate from myristoyl-CoA to the N-terminal glycine of substrate proteins, and is found only in eukaryotic cells. The enzyme in this study is the 451 amino acid protein produced by Candida albicans, a yeast responsible for the majority of systemic infections in immuno-compromised humans. NMT activity is essential for vegetative growth, and the structure was determined in order to assist in the discovery of a selective inhibitor of NMT which could be developed as an anti-fungal drug. NMT has no sequence homology with other protein sequences and has a novel alpha/beta fold which shows internal two-fold symmetry, which may be a result of gene duplication. On one face of the protein there is a long, curved, relatively uncharged groove, at the center of which is a deep pocket. The pocket floor is negatively charged due to the vicinity of the C-terminal carboxylate and a nearby conserved glutamic acid residue, which separates the pocket from a cavity. These observations, considered alongside the positions of residues whose mutation affects substrate binding and activity, suggest that the groove and pocket are the sites of substrate binding and the floor of the pocket is the catalytic center.

The entropic penalty of ordered water accounts for weaker binding of the antibiotic novobiocin to a resistant mutant of DNA gyrase: a thermodynamic and crystallographic study.

Novobiocin is an antibiotic which binds to a 24 kDa fragment from the B subunit of DNA gyrase. Naturally occurring resistance arises from mutation of Arg-136 which hydrogen bonds to the coumarin ring of novobiocin. We have applied calorimetry to characterize the binding of novobiocin to wild-type and R136H mutant 24 kDa fragments. Upon mutation, the Kd increases from 32 to 1200 nM at 300 K. The enthalpy of binding is more favorable for the mutant (DeltaH degrees shifts from -12.1 to -17.5 kcal/mol), and the entropy of binding is much less favorable (TDeltaS degrees changes from -1.8 to -9.4 kcal/mol). Both of these changes are in the direction opposite to that expected if the loss of the Arg residue reduces hydrogen bonding. The change in heat capacity at constant pressure upon binding (DeltaCp) shifts from -295 to -454 cal mol-1 K-1. We also report the crystal structure, at 2.3 A resolution, of a complex between the R136H 24 kDa fragment and novobiocin. Although the change in DeltaCp often would be interpreted as reflecting increased burial of hydrophobic surface on binding, this structure reveals a small decrease. Furthermore, an ordered water molecule is sequestered into the volume vacated by removal of the guanidinium group. There are large discrepancies when the measured thermodynamic parameters are compared to those estimated from the structural data using empirical relationships. These differences seem to arise from the effects of sequestering ordered water molecules upon complexation. The water-mediated hydrogen bonds linking novobiocin to the mutant protein make a favorable enthalpic contribution, whereas the immobilization of the water leads to an entropic cost and a reduction in the heat capacity of the system. Such a negative contribution to DeltaCp, DeltaH degrees , and TDeltaS degrees appears to be a general property of water molecules that are sequestered when ligands bind to proteins.

Molecular characterisation of integrin-procollagen C-propeptide interactions.

The carboxyl-terminal propeptide of type I procollagen (CPP-I) plays a key role in regulation of collagen fibrillogenesis, and may exert feedback control of collagen biosynthesis. We have previously shown that CPP-I is a ligand for the integrin alpha2beta1 [Weston, S. A., Hulmes, D. J. S., Mould, A. P., Watson, R. B. & Humphries, M. J. (1994) Identification of the integrin alpha2beta1 as a cell surface receptor for the C-propeptide of type I procollagen, J. Biol. Chem. 269, 20982-20986] suggesting that some of the phenotypic effects of C-propeptides may be mediated by adhesion receptors. Here we have extended this work to study the molecular basis of this interaction. We have broadened the ligand range by demonstrating that the C-terminal propeptide of type II procollagen supports alpha2beta1-mediated binding of NHS human fibroblasts in cell attachment assays. Also, we have used function-blocking antibodies in cell attachment and solid-phase binding assays with purified integrin to expand the CPP-I receptor family, showing that integrin alpha1beta1 is also a receptor for CPP-I. Integrin alpha-subunit A-domains are known to be major ligand-binding sites and recombinant alpha1 and alpha2 subunit A-domains were able to bind CPP-I. Finally we have shown that peptides corresponding to potential integrin-binding sequences in CPP-I do not mediate integrin-CPP-I adhesion. Taken together, these studies indicate that the interactions between C-propeptides and integrins are more numerous than previously reported, that C-propeptides are a new class of molecule which bind to A-domains, and that the integrin-C-propeptide interaction does not utilise established peptide motifs.

Effect of beta-mercaptoethanol on the detection of biotinylated proteins.

Biotinylated proteins were visualized by enhanced chemiluminescence (ECL) or conventional autoradiography following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and protein transfer onto nitrocellulose. Soaking polyacrylamide gels run under nonreducing conditions in beta-mercaptoethanol (2-ME) prior to protein transfer onto nitrocellulose resulted in a 2- to 10-fold augmentation of the resultant signal. This enhancement was observed for both disulfide- and nondisulfide-bonded proteins. Furthermore, 2-ME had no effect on either the activity of the extravidin-horse-radish peroxidase conjugate, used to detect biotin moieties, or the net protein transfer onto nitrocellulose. Thus, we propose that amplification of either ECL or gamma emission following 2-ME treatment is due to its ability to modify protein conformation, which in turn provides greater access of avidin to biotin.

X-ray structure of recombinant ricin A-chain at 1.8 A resolution.

Ricin is a potent plant toxin which acts by removing a specific adenine residue from the ribosome. The X-ray crystal structure of a new, tetragonal crystal form of the recombinant ricin A-chain diffracting to 1.8 A resolution has been determined via molecular replacement methods and refined to a crystallographic R-factor of 18.6%. The higher resolution electron density allowed improvements to be made upon previously published models, resulting in an increase in the assigned secondary structure of the protein. The enzyme adopts the same global conformation in this crystal form with differences in detail due only partly to crystal packing. The active site superimposes closely with those of previously published models but the locations of the active-site water molecules differ in this structure. To address the current mechanistic model, an additional two structures are presented: recombinant ricin A-chain complexed with the substrate analogue formycin monophosphate as well as with adenosine monophosphate, which is cleaved by the crystalline enzyme. The formycin monophosphate displaces a putative catalytic water molecule. This supports the notion that the analogue does not bind in a transition state conformation and that contacts from other elements of the 28 S RNA natural substrate are required to achieve full reactivity. The structure of the adenosine monophosphate complex suggests a mechanism for the release of the adenine product via of the side-chain Tyr80. The structures suggest that Glu177 is better positioned for the activation of the catalytic water molecule than Arg180.

Identification of integrin alpha 2 beta 1 as cell surface receptor for the carboxyl-terminal propeptide of type I procollagen.

The carboxyl-terminal propeptide of procollagen type I (CPP-I) plays a key role in the regulation of collagen fibrillogenesis. In addition, it has been reported that, after cleavage from procollagen, CPP-I exerts feedback control of collagen biosynthesis. To further elucidate the mechanisms involved in each of these processes, we have investigated the nature of cell surface receptors for CPP-I. CPP-I affinity chromatography, using detergent extracts of iodinated HT1080 cells and EDTA elution, resulted in the isolation of two polypeptides of molecular mass 160 and 110 kDa. Since the migratory behavior of these polypeptides under nonreducing and reducing conditions was characteristic of a subset of integrin receptors, their reactivity with anti-integrin monoclonal antibodies was tested. Antibodies directed against the alpha 2 and beta 1 subunits specifically immunoprecipitated both CPP-I-binding polypeptides, indicating that the CPP-I receptor is the integrin alpha 2 beta 1. CPP-I was found to support the attachment and spreading of HT1080 cells, demonstrating that it can function as an adhesion protein. Two other approaches supported the identification of alpha 2 beta 1 as the CPP-I receptor. First, anti-functional anti-integrin monoclonal antibodies directed against the alpha 2 and beta 1 subunits completely abrogated the adhesive activity of CPP-I and, second, highly purified CPP-I bound specifically to alpha 2 beta 1-containing integrin preparations in a solid-phase receptor-ligand binding assay. These findings have important implications for the function of fibrillar collagen carboxyl-terminal propeptides and for the role played by integrins in the regulation of cellular phenotype.

Integrins: a review of their structure and mechanisms of ligand binding.

Adhesive interactions between cells and between cells and extracellular matrices play key roles in determining spatiotemporal positioning, influencing site-specific gene expression, and dictating proliferation rate. In addition, aberrant adhesion contributes to various aspects of disease pathology. These phenotypic effects of adhesion are mediated initially by the recognition of adhesive components of the extracellular matrix by membrane-intercalated receptor molecules and ultimately by the transduction of chemical and physical signals to the cell interior. Cell-cell and cell-matrix interactions are highly complex, since they involve the interfacing of surface membrane structures with each other or with three-dimensional aggregates of glycoproteins and proteoglycans, and it is this complexity that provides the necessary versatility for cells to react appropriately to either gross or subtle changes in their environment. Reagents with the ability to modulate adhesion could have many types of use: They could be employed to dissect the role of cell migration in development, provide insight into how adhesion might regulate gene expression and cell phenotype, and they could have widespread therapeutic applications in the treatment of thrombosis, inflammation and cancer. The quest to develop such reagents has necessitated the elucidation of the mechanisms of cell adhesion, and in particular the identification of the molecules involved and their modes of interaction. This article reviews the state of this quest; in particular, the molecular basis of ligand binding by integrin receptors.