Search

Background Adjunctive therapies that increase the incidence of normal reperfusion after thrombolysis for acute myocardial infarction (MI) could enhance clinical outcomes. Direct thrombin inhibitors may offer an advantage over standard adjunctive therapies. Methods We randomized 336 patients with acute MI at 33 sites to receive 1 of 5 doses of efegatran sulfate, a direct thrombin inhibitor, or heparin for 72 to 96 hours, both with accelerated alteplase and aspirin. The primary end point was the incidence of thrombolytic failure (death, reinfarction, or TIMI grade 0-2 flow in the infarct artery from 90 minutes to discharge or 30 days, whichever occurred earlier). Results Significantly more patients randomized to efegatran had evidence of heart failure at admission. The lowest-dose efegatran arm was terminated at 15 patients because of unacceptably increased thrombolytic failure. The primary end point occurred in 53.0% of patients treated with heparin, in 53.8% of patients treated with efegatran overall (P =.90), and in 55.4% of patients given intermediate-dose efegatran (P =.74). These findings were unaffected after adjustment was done for baseline differences. Most bleeding was minor; major bleeding and the use of blood transfusions did not differ significantly by treatment. Three patients in the high-dose efegatran group had intracranial hemorrhage, as did 1 patient in the heparin group. Continuous ST monitoring showed a shorter time to recovery for the efegatran group (median 107 minutes) compared with the heparin group (154 minutes; P =.025). Conclusions Efegatran sulfate appeared to offer no clear advantage over heparin as an adjunct to thrombolysis for acute myocardial infarction, although there may be a modest improvement in time to reperfusion.

Objectives. We sought to determine the prognostic value of the admission electrocardiogram (ECG) in patients with unstable angina and non–Q wave myocardial infarction (MI).Background. Although the ECG is the most widely used test for evaluating patients with unstable angina and non–Q wave MI, little prospective information is available on its value in predicting outcome in the current era of aggressive medical and interventional therapy.Methods. ECGs with the qualifying episode of pain were analyzed in patients enrolled in the Thrombolysis in Myocardial Ischemia (TIMI) III Registry, a prospective study of patients admitted to the hospital with unstable angina or non–Q wave MI.Results. New ST segment deviation ≥1 mm was present in 14.3% of 1,416 enrolled patients, isolated T wave inversion in 21.9% and left bundle branch block (LBBB) in 9.0%. By 1-year follow-up, death or MI occurred in 11% of patients with ≥1 mm ST segment deviation compared with 6.8% of patients with new, isolated T wave inversion and 8.2% of those with no ECG changes (p < 0.001 when comparing ST with no ST segment deviation). Two other high risk groups were identified: those with only 0.5-mm ST segment deviation and those with LBBB, whose rates of death or MI by 1 year were 16.3% and 22.9%, respectively. On multivariate analysis, ST segment deviation of either ≥1 mm or ≥0.5 mm remained independent predictors of death or MI by 1 year.Conclusions. The admission ECG is very useful in risk stratifying patients with non–Q wave MI. The new criteria of not only ≥1-mm ST segment deviation but also ≥0.5-mm ST segment deviation or LBBB identify high risk patients, whereas T wave inversion does not add to the clinical history in predicting outcome.(J Am Coll Cardiol 1997;30:133–40)

which 25,997 (38.6%) involved at least one transfer from the index hospitalization. Generally, more young patients and more males were transferred (Table). Transfer rates decreased as age increased in both males and females, and differed significantly between sexes in the older age groups (Table). Overall, patients who were transferred had lower 30-day mortality rates compared to those who were not transferred (3.9% vs. 19.5%, p < 0.0001); these differences in mortality increased with age for both females and males.

The introduction of cyclosporine has resulted in significant improvement in the survival of cardiac allograft recipients due to decreased mortality from infection and rejection. The original oil-based cyclosporine formulation exhibits variable and unpredictable bioavailability that correlates with an increased incidence of acute and chronic rejection in those patients in whom this is most pronounced. The primary objectives of this prospective, multicenter, randomized, double-blind study in cardiac transplant patients were: to compare the efficacy of cyclosporine microemulsion (CsA-NL) with oil-based cyclosporine (CsA-SM) as measured by cardiac allograft and recipient survival and the incidence and severity of acute rejection episodes; and to assess the safety and tolerability of CsA-NL compared with CsA-SM in this population. This report represents the analysis of results 6 months after transplantation.A total of 380 patients undergoing their first cardiac transplant at 24 centers in the United States, Canada, and Europe were enrolled in this double-blind, randomized trial examining the safety and efficacy of CsA-NL versus CsA-SM. Rejection was diagnosed using endomyocardial biopsy and were graded according to standardized criteria of the International Society of Heart and Lung Transplantation (ISHLT). Clinical parameters were monitored during the study. Survival and freedom from were used for analysis as was Fisher's exact test for comparisons between groups.At 6 months after transplantation, allograft and patient survival were the same for both groups. The frequency of ISHLT grade 3A or greater episodes in the two groups was identical. Fewer CsA-NL patients (5.9%) required antilymphocyte antibody (ATG or OKT-3) therapy for rejection compared with the CsA-SM-treated patients (14.1%, P=0.01). Females with ISHLT rejection gradeor = 3A treated with CsA-NL had a 46% lower incidence of rejection compared with the CsA-SM-treated group (31.3% vs. 57.6%, P=0.032). Fewer infections were seen in the CsA-NL. With the exception of baseline and 1 week posttransplant creatinines which were higher in the CsA-NL group, the overall creatinine was not significantly different between the two groups.This multicenter, randomized study of cardiac transplant recipients documented less severe rejection (in particular those requiring antibody therapy) and a lower incidence of infection in CsA-NL-treated patients. Results from the female subgroup analysis suggest that the improved bioavailability of CsA-NL might reduce the frequency of rejection episodes in female patients. The use of CsA-NL was not associated with an increased risk of adverse events.

In 2005, the Canadian Cardiovascular Society established an ad hoc working group that was charged with writing a commentary to bring together many of the issues that have arisen in the past few years surrounding physician human resources in cardiac sciences. These issues cover the supply side of the workforce (eg, physician shortages, excessive workloads and possible changes to the training of medical specialists) and the recently developed projections of the growth in demand for these services. The present commentary also notes factors that contribute to fundamental shifts in the balance between demand and supply, particularly the potential challenges that our specialists will face with the introduction of wait time targets for key services and procedures. It should come as no surprise to any health care provider that the burden of cardiovascular disease on the Canadian health care system is increasing. Advances in technology, treatment and health care delivery have improved outcomes in patients with cardiovascular illness. As a result, cardiac patients are living longer, often with greater comorbidities. This burden will continue to increase, because approximately 24% of the population will be older than 65 years of age by the year 2030. The pressure is being felt at every level of the health care delivery platform – from the community cardiologist to the subspecialist in a tertiary care centre. Cardiologists, on average, work 57 h a week, more than the average of 52 h for all specialists. In addition, they have an average of 106 h of on-call responsibilities every month (1). The heavy workload is not new; similar workloads were reported in the specialist physician workforce survey conducted by the Canadian Cardiovascular Society in 2001 (2). The continued high workload is taking its toll. In 2004, 31% of cardiologists reported that they were somewhat or very dissatisfied with the balance between personal and work commitments. Female cardiologists (44%) and all cardiologists younger than 45 years of age (37%) were even more likely to be dissatisfied with this balance (1). Despite this work effort, cardiologists cannot keep up with the need for their services. In the same survey, 21% of cardiologists reported that a new patient with a nonurgent condition would wait longer than three months for a first consultation. Urgent patients have more timely access to initial consults (64% of cardiologists reported a wait time of one week or less). In the words of a participant in one of the Canadian Medical Association’s focus groups on access to care, “the quality of health care in Canada is good once you get in” (3). However, for the nonurgent patient, ‘getting in’ is the real challenge, and the risk is that the nonurgent patient will wait and ultimately enter the system as an urgent referral or through the emergency room. With the introduction – and, we hope, the eventual adoption – of wait time benchmarks for key cardiovascular services and procedures (3), the demand for timely care will intensify. Recently published wait time benchmarks for cardiovascular care (4) suggest that elective consults should be seen within six weeks and urgent consults within seven days. Longer working hours are not an option; we need more trained health care providers within cardiovascular medicine. Indications are that the situation is not likely to improve any time soon. Just as the general population is aging, so is the cardiologist workforce. In 2003, 29% of cardiologists were 55 years of age and older, compared with only 22% in 2001 (5). Even if current training programs could replace the number of retiring cardiologists, the demographic mix and subspecialty choice of medical graduates is causing some concern. In 2004, 53.4% of all medical school graduates were female (6). Female physicians prefer, on average, a shorter work week than their male colleagues. This creates two problems for physician human resource planning. First, if a higher number of female physicians were attracted to cardiology, more physicians would be needed to provide the same service capacity. Second, cardiology has not been very successful in recruiting female medical graduates, likely in part due to the workload conditions. Although one-half of all medical school graduates are female, in 2006, only 30% of cardiologists younger than 35 years of age were female, according to the Canadian Medical Association Masterfile (7). It is possible that the high workload and challenges in balancing work and family life in this profession have been, and will continue to be, real barriers to attracting top candidates into the profession. Advances in treatment are also contributing to pressures on the existing workforce. Over the past decade, the rapid adoption of new procedures (such as angioplasty) and technological advances (such as drug-eluting stents), which have made these procedures the first choice for the majority of revascularizations, has increased the demand for interventional cardiologists. Similarly, expanding indications for implantable cardioverter defibrillators, which are now indicated for primary prevention of sudden cardiac death in patients with depressed left ventricular function, will increase the demand for electro-physiologists needed for related consultations, procedures (including device implantations) and follow-up. The epidemic of heart failure and proven impact of disease management programs on outcomes will result in a greater need for physicians with training in heart failure. This subspecialization of our profession is competing with the need to promote the training of more generalist community cardiologists, who are the most appropriate first point of access to cardiovascular care for the majority of patients. In addition to all of these factors, the Canadian population is aging, and elderly Canadians are living longer. We expect the number of people who have had an acute myocardial infarction, or heart attack, in 2001, to almost triple by the year 2021 (J Tepper, personal communication). For the same period, it has been projected that the number of people living with chronic heart failure will double (S Schultz, personal communication). In short, at present we are already understaffed, and the Canadian population is consequently underserviced by cardiovascular specialists; with current trends, this will only get worse. We cannot afford to reduce the number of medical graduates who choose to train in cardiology. However, that is exactly what The Association of Faculties of Medicine of Canada Postgraduate Medical Education (AFMC PGME) Standing Committee proposed in an undated position paper entitled, “The internal medicine R4 match: Time for a change. A position paper of the AFMC PGME Committee”. The AFMC PGME proposed to assign a defined portion of the postgraduate year 4 spots to general medicine to increase the number of generalists and decrease the number of subspecialists. Although we are sympathetic with the shortage of general internists, we must not allow this problem to be corrected at the expense of cardiology trainee positions. Although general practitioners and internal medicine specialists play an important role in the delivery of basic cardiovascular care in some communities, they are not an acceptable substitute for cardiologists. Studies have shown that cardiologists have the greatest propensity to provide cardiovascular disease prevention services (8), higher referral rates for important diagnostic services such as angiography (9) and are more likely to prescribe the Heart Failure Society of America-recommended medications for congestive heart failure patients on admission and at discharge (10). A recent Canadian study of 38,702 heart failure patients (11) confirmed that cardiologist care was associated with higher adjusted rates of invasive interventions and postdischarge prescriptions of heart failure medications. These services translate into improved outcomes. The study also found a lower one-year risk-adjusted mortality for patients attended by cardiologists compared with those who were attended by general internists, family practitioners or other physicians. This difference in outcomes is not specific to the Canadian context (12,13). The current shortage of cardiologists, and the resulting workloads and wait times, cannot be solved over the short term. At a minimum, we need to maintain the number of cardiology training positions, and preferably, expand our ranks to meet current and projected demands for cardiology services. Perhaps it is time to adopt the two-track approach to training in cardiology that the American College of Cardiology is proposing: a combined three-year plus two-year internal medicine and cardiology program for community cardiologists, and a combined two-year plus three-year internal medicine and cardiology program for those planning a career in academic cardiology or cardiology in tertiary centres with cardiology-only call schedules. This approach would double the number of cardiology graduates for the year that the first graduates of the five-year program enter practice in the same year as the last graduates of the current six-year program. After years of lobbying for more cardiology training positions, we still have not solved the long-term problem of too many patients and too few cardiologists to meet the needs of their services and procedures. What we need now are innovative approaches to recruit and train our next generation of cardiologists and cardiovascular health care providers. This is absolutely essential if we are to ensure that Canadians will always have access to quality and timely cardiovascular care if and when they need it.

We sought to determine the prognostic value of the admission electrocardiogram (ECG) in patients with unstable angina and non-Q wave myocardial infarction (MI).Although the ECG is the most widely used test for evaluating patients with unstable angina and non-Q wave MI, little prospective information is available on its value in predicting outcome in the current era of aggressive medical and interventional therapy.ECGs with the qualifying episode of pain were analyzed in patients enrolled in the Thrombolysis in Myocardial Ischemia (TIMI) III Registry, a prospective study of patients admitted to the hospital with unstable angina or non-Q wave MI.New ST segment deviationor = 1 mm was present in 14.3% of 1,416 enrolled patients, isolated T wave inversion in 21.9% and left bundle branch block (LBBB) in 9.0%. By 1-year follow-up, death or MI occurred in 11% of patients withor = 1 mm ST segment deviation compared with 6.8% of patients with new, isolated T wave inversion and 8.2% of those with no ECG changes (p0.001 when comparing ST with no ST segment deviation). Two other high risk groups were identified: those with only 0.5-mm ST segment deviation and those with LBBB, whose rates of death or MI by 1 year were 16.3% and 22.9%, respectively. On multivariate analysis, ST segment deviation of eitheror = 1 mm oror = 0.5 mm remained independent predictors of death or MI by 1 year.The admission ECG is very useful in risk stratifying patients with non-Q wave MI. The new criteria of not onlyor = 1-mm ST segment deviation but alsoor = 0.5-mm ST segment deviation or LBBB identify high risk patients, whereas T wave inversion does not add to the clinical history in predicting outcome.

Unstable angina is generally considered to encompass a spectrum of symptomatic manifestations of ischemic heart disease, intermediate between stable angina and acute myocardial infarction. Approximately 75,000 Canadians are hospitalized yearly with unstable angina. The pathophysiology of unstable angina is still imperfectly understood, but is related to the same pathophysiological factors underlying myocardial infarction and sudden cardiac death. In March 1995 a group of Canadian cardiologists met to review the current understanding of unstable angina and to define a Canadian approach to this common problem. Important issues and questions regarding the diagnosis and management of unstable angina were defined. The objective was to outline approaches to the management of unstable angina that would be appropriate in Canada. Topics discussed included definition, incidence, clinical presentations, pathophysiology, initial diagnostic and risk stratification approaches, acute medical management, role of invasive interventions and long term management.

Background Ligand binding to the platelet membrane receptor glycoprotein (GP) IIb/IIIa, the final and obligatory step to platelet aggregation, can now be inhibited by pharmacological agents. This study was designed to evaluate the potential of lamifiban, a novel nonpeptide antagonist of GP IIb/IIIa, for the management of unstable angina. Methods and Results In a prospective, dose-ranging, double-blind study, 365 patients with unstable angina were randomized to an infusion of 1, 2, 4, or 5 μg/min of lamifiban or of placebo. Treatment was administered for 72 to 120 hours. Outcome events were measured during the infusion period and after 1 month. Concomitant aspirin was administered to all patients and heparin to 28% of patients. Lamifiban, all doses combined, reduced the risk of death, nonfatal myocardial infarction, or the need for an urgent revascularization during the infusion period from 8.1% to 3.3% ( P =.04). The rates were 2.5%, 4.9%, 3.3%, and 2.4% with increasing doses. At 1 month, death or nonfatal infarction occurred in 8.1% of patients with placebo and in 2.5% of patients with the two high doses ( P =.03). The highest dose of lamifiban additionally prevented the need for an urgent intervention. Lamifiban dose-dependently inhibited platelet aggregation. Bleeding times were significantly prolonged with platelet inhibition of >80%. Major (but neither life-threatening nor intracranial) bleedings occurred in 0.8% of patients with placebo and 2.9% with lamifiban. Conclusions The nonpeptide GP IIb/IIIa antagonist lamifiban protected patients with unstable angina from severe ischemic events during a 3- to 5-day infusion and reduced the incidence of death and infarction at 1 month, suggesting considerable promise for this new therapeutic approach.

To investigate the natural history and response to treatment of patients with unstable angina or non-Q-wave myocardial infarction (MI).Inception cohort.Patients in general community, primary care, or referral hospitals.All patients with an episode of unstable exertional chest pain or chest pain at rest presumed to be ischemic in origin lasting 5 minutes or more but without persisting ST-segment elevation greater than 30 minutes or the development of Q-waves were identified and enumerated in 18 participating hospitals. A subset of enumerated patients was selected to be followed prospectively using specific sampling strategies that would provide adequate numbers of black, women, and elderly (agedor = 75 years) patients for comparison with their respective counterparts.The primary analysis compared the incidence of death or MI at 42 days after entry into the prospective study according to race, sex, and age. Other outcomes considered were recurrent ischemia and the combined outcomes of death, MI, or recurrent ischemia by 42 days after entry.A total of 8676 admissions with unstable angina or non-Q-wave MI were enumerated and, of these, 3318 patients were selected for the prospective study. The direct adjusted mean age of 3318 patients was 63.8 years. There were 943 blacks and 2375 nonblacks. Compared with nonblacks, blacks were less likely to be treated with intensive anti-ischemic therapy for their qualifying anginal episode and less likely to undergo invasive procedures (risk ratio [RR], 0.65%; 95% confidence interval [CI], 0.58 to 0.72; P.001). However, of those who underwent angiography (45% of blacks and 61% of nonblacks), blacks had less extensive and severe coronary stenoses than nonblacks. The incidence of death and MI was similar for blacks and nonblacks, but blacks had a lower incidence of recurrent ischemia. There were 1678 men and 1640 women. Women were less likely than men to receive intensive anti-ischemic therapy and less likely to undergo coronary angiography (RR, 0.71; 95% CI, 0.65 to 0.78; P.001). Women had less severe and extensive coronary disease and were less likely to undergo revascularization, yet had a similar risk of experiencing an adverse cardiac event by 6 weeks. There were 2490 patients aged 75 years or less and 828 patients aged more than 75 years. Elderly patients received less aggressive anti-ischemic therapy and were less likely to undergo coronary angiography than their younger counterparts. Elderly patients had more severe and extensive coronary disease but fewer revascularization procedures than younger patients and experienced a much higher incidence of adverse cardiac events both in hospital and by 6 weeks.Among patients presenting with acute ischemic chest pain without persistent ST-segment elevation, blacks appeared to have less severe coronary disease, received revascularization less frequently, and had less recurrent ischemia compared with nonblacks. Women were also found to have less severe coronary disease and were treated less intensely than men, but experienced similar outcomes. Elderly patients had more severe coronary disease than younger patients on coronary angiography, but were more likely to be treated medically, and they experienced far more adverse outcomes. These data suggest that more aggressive strategies should be directed to those patients with the greatest likelihood of adverse outcomes.

Objective. —To investigate the natural history and response to treatment of patients with unstable angina or non—Q-wave myocardial infarction (MI). Design. —Inception cohort. Setting. —Patients in general community, primary care, or referral hospitals. Patients. —All patients with an episode of unstable exertional chest pain or chest pain at rest presumed to be ischemic in origin lasting 5 minutes or more but without persisting ST-segment elevation greater than 30 minutes or the development of Q-waves were identified and enumerated in 18 participating hospitals. A subset of enumerated patients was selected to be followed prospectively using specific sampling strategies that would provide adequate numbers of black, women, and elderly (aged ≥75 years) patients for comparison with their respective counterparts. Main Outcome Measures.—The primary analysis compared the incidence of death or Ml at 42 days after entry into the prospective study according to race, sex, and age. Other outcomes considered were recurrent ischemia and the combined outcomes of death, Ml, or recurrent ischemia by 42 days after entry. Results. —A total of 8676 admissions with unstable angina or non—Q-wave Ml were enumerated and, of these, 3318 patients were selected for the prospective study. The direct adjusted mean age of the 3318 patients was 63.8 years. There were 943 blacks and 2375 nonblacks. Compared with nonblacks, blacks were less likely to be treated with intensive anti-ischemic therapy for their qualifying anginal episode and less likely to undergo invasive procedures (risk ratio [RR], 0.65; 95% confidence interval [CI], 0.58 to 0.72;P Conclusions. —Among patients presenting with acute ischemic chest pain without persistent ST-segment elevation, blacks appeared to have less severe coronary disease, received revascularization less frequently, and had less recurrent ischemia compared with nonblacks. Women also were found to have less severe coronary disease and were treated less intensely than men, but experienced similar outcomes. Elderly patients had more severe coronary disease than younger patients on coronary angiography, but were more likely to be treated medically, and they experienced far more adverse outcomes. These data suggest that more aggressive strategies should be directed to those patients with the greatest likelihood of adverse outcomes. (JAMA. 1996;275:1104-1112)

The digestion of a human IgG1 K myeloma protein with pepsin in the presence of 8M-urea was observed to produce a fragment, designated Fb′2, which differed from the products of aqueous peptic digestion and from other characteristic immunoglobulin digestion products. 2. Fragment Fb′s was also found when two other IgG1/K proteins were treated similarly. 3. Sedimentation-equilibrium studies showed the mol.wt. of fragment Fb′2 to be 56800. 4. On reduction, two equivalents of each of three peptides were released from fragment Fb′s; these were characterized by N- and C-terminal determinations and by amino acid sequencing. 5. Fragment Fb′2 was shown to consist of the constant regions of both light chains, from residue Ile-117 to the C-terminus, and the CH1 domains and hinge region of the heavy chains, from residue Val-113 to residue Met-252, with a gap of five residues within the intrachain disulphide loop, between residues Leu-174 and Tyr-180.

C1t is a 9.5 S α1 glycoprotein that has been shown to be a fourth subcomponent of the first component of complement. The m.w. of C1t was found to be 233,000 by sedimentation equilibrium in the ultracentrifuge. A subunit m.w. of 23,000 was obtained by sedimentation equilibrium in 5.95 M guanidinium chloride. No change in either m.w. was produced by prior reduction and alkylation. In the electron microscope characteristic pentagonal figures of 85 Å diameter were observed together with rod-like figures which appear to be stacked assemblies of the pentagonal figures. These observations lead us to propose that C1t is a noncovalent, decameric protein with the subunits disposed at the vertices of two regular pentagons joined at one of their faces. A possible relationship between C1t and the P-component of amyloid is discussed.

1. The "oxygenated" form of cytochrome oxidase has been generated by treatment of the enzyme with ascorbic acid.2. "Oxygenated oxidase" so generated is stable over long periods (24 h).3. Sedimentation velocity experiments have shown the "oxygenated oxidase to be a less compact molecule than the oxidized.

A wheat germ protein (Ec), the dominant site of cysteine incorporation during early (E) germination of isolated wheat embryos, has been partially sequenced by automated Edman degradation. The sequence of residues 1–59 is not significantly similar to the amino acid sequence known for any other protein, including wheat germ agglutinin which, like Ec, is very rich in half-cystine and glycine. The partial sequence for Ec contains an almost identical pattern of half-cystine residues in segments 6–20 and 35–48, a duplication which includes 10 of the 12 half-cystine residues in the sequence. The partial sequence of Ec may include a large part of the complete sequence, but this remains uncertain because it has not been possible to arrive at a definitve estimate of molecular weight using different physical techniques. Protein Ec can be prepared from a reticulocyte lysate in which cell-free synthesis is programmed by bulk wheat germ mRNA. Determination of the distribution of half-cystine moieties between residues 1 and 20 by Edman degradation of the [35S]cysteine-labeled product of cell-free synthesis shows that it is devoid of an N-terminal extension. Unlike wheat germ agglutinin, Ec does not seem to arise by processing of a conspicuously larger precursor protein. Unlike Ec, another wheat germ protein, Em, the most conspicuous methionine-labeled protein when cell-free protein synthesis is directed by wheat germ mRNA, is refractory to direct sequence analysis by Edman degradation. However, again unlike Ec, uncertainty about the molecular weight of Em, based on its mobility in different sodium dodecyl sulphate – polyacrylamide gel systems, has been resolved by virtue of Em being ideally suited to study by the Yphantis high-speed sedimentation equilibrium method.

Goblet-cell mucin (mucin 1) was isolated and purified from human small-intestinal scrapings. After application of mucin 1 to DEAE-Bio-Gel (A) columns, most of the glycoprotein (76–94% of hexoses) was eluted in the first peak (designated mucin 2). Minor amounts of acidic glycoproteins were eluted with 0.2m- and 0.4m-NaCl in later peaks. Analyses of mucin 1 and mucin 2 revealed mucin 2 to be a monodisperse highly glycosylated glycoprotein containing 6.3% by wt. of protein, N-acetylgalactosamine, N-acetylglucosamine, galactose and fucose. Mucin 1 was similar in composition, but was polydisperse and contained more protein (12.3% by wt.) as well as N-acetylneuraminic acid. Analytical CsCl-gradient ultracentrifugation showed both mucin 1 and mucin 2 to have a major component with an average buoyant density of 1.47000g/ml. Mucin 1 also contained a slightly less-dense minor glycoprotein component. After exhaustive reduction and alkylation mucin 1 retained its major component, but partly dissociated into two lighter glycoprotein components. Mucin 2, in contrast, did not change its density distribution after reduction. Band ultracentrifugation in 2H2O-containing iso-osmotic buffers showed that mucin 1 contained a major fast-sedimenting component (so=37±2S), and a minor amount of a slower-sedimenting component. After reduction there was an increased quantity of the latter component, for which an so value of 14.5S was calculated. In contrast, mucin 2 was unaltered by reduction (so=33±2S). These findings indicate that the major component of goblet-cell mucin (mucin 2) does not dissociate after S–S-bond reduction, and thus does not apparently rely for its polymeric structure on the association of subunits through covalent disulphide bonds. However, the effects of reduction on mucin 1 suggest that in the native mucin intramolecular disulphide bonds in the minor glycoproteins may stabilize their structure, permitting secondary non-covalent interactions to develop with the major dense mucin (mucin 2) protein.

A simple, practical calculation procedure has been developed for predicting the changes in molecular weight distribution of a polymer undergoing random crosslinking and/or degradation. Simulations of the random crosslinking and degradation of narrow and broad Poisson-type distributions have been made. The results agree with those calculated from Kimura's analytical solutions to Saito's general equations after a correction has been made for a mathematical error in Kimura's solution. This method can be applied to determining the probabilities of crosslinking and scission for any arbitrary molecular weight distribution expressed in tabular form. The importance of using narrow distribution samples to estimate crosslinking from changes in molecular weight distribution is graphically demonstrated.

The total collision cross section has been measured for argon atoms, with energies between 300 and 1100 electron volts, scattered in room temperature argon. In the region between 700 and 1000 volts, the cross section shows a maximum indicative of inelastic scattering, probably due to ionization by the high energy atoms.The repulsive potential between two argon atoms has been evaluated from elastic collision cross sections (deduced from observed values of the total collision cross section) using a method which takes account of the greater probability of small angle scattering by averaging the cross section over the length of the scattering path. The results may be expressed by V(r)=4.61×10−11/r4.33 ergs or V(r)=3.66×10−8 exp(−6.88r12) ergs for values of r between 1.37 and 1.84A.

Inactivation of C3 by enzymatic cleavage, nucleophilic addition, or slow freezing and thawing resulted in the acquisition of similar end-state conformations as judged by near-UV circular dichroism. Although inactivation by the two nonenzymatic processes involves no peptide bond scission, the inactivated C3 resembled C3b in that it possessed a free sulfhydryl group not present in the native protein and an increased surface hydrophobicity as evidenced by enhanced binding of the fluorophore 8-anilino-1-naphthalensulfonate (ANS). The C3b-like functional properties of modified C3 [Pangburn, M. K., & Muller-Eberhard, H. J. (1980) J. Exp. Med. 152, 1102-1114] may thus be understood in terms of the similarity of its conformation to that of C3b. The rate of the conformational change following proteolytic cleavage was fast and appeared to be limited by the rate of the enzymatic reaction. In contrast, the rate of conformational change following addition of methylamine was slow and rate limited by the conformational rearrangement itself, not by the chemical modification. A kinetic analysis of the changes in circular dichroism and ANS fluorescence enhancement suggested that the nucleophilic addition was spectroscopically undetectable and was followed by a minimally biphasic, spectroscopically demonstrable conformational rearrangement. The appearance of C3b-like functional activity in nucleophile-modified C3 largely parallels the time course of the spectroscopically detectable conformational change but is distinctly slower than the rate at which hemolytic activity is lost. While fully transconformed methylamine-inactivated C3 can bind factor B and is susceptible to cleavage by C3b inactivator and its cofactor beta 1H, this cleavage occurs at a substantially slower rate than the equivalent process in C3b. The implications of these findings in terms of the mechanism through which the alterative pathway of complement is initiated are discussed.

An iterative program, Gaushaus, has been adapted to analyze complex DNA reassociation kinetics. With valid experimental data, rapid convergence to a solution is always achieved by this program even when the starting parameters are grossly in error. The output of the program also includes useful statistical information.

A numerical method is described for the elimination of Rayleigh scattering from protein fluorescence emission spectra. The method is based upon the observation that Rayleigh scattering is symmetrical about a wavelength at or near the wavelength of excitation. It works best when an automated, computer-based approach can be applied to spectra collected on a data-logging device, although manual correction of chart-recorded spectra is also possible.

The fluorescent probe, 5-iodoacetamidofluorescein (5-IAF), was specifically attached to the COOH-terminal cysteine residue of the L chains of two monoclonal human IgKκ proteins. The induced circular dichroism and fluorescence properties of the bound 5-IAF probe were used to study changes in its microenvironment upon reassociation of autologous or heterologous L chains and their domains with Fd' fragments. Recombination of 5-IAF-L with Fd' at pH 5.4 was accompanied by a red-shifted visible difference spectrum, an increase in the magnitude of the induced optical activity and an enhancement of fluorescence. These results were compatible with the transfer of the 5-IAF moeity to a less polar and more asymmetric environment. Equilibrium and kinetic data obtained from difference spectroscopy and fluorescence studies indicated that 5-IAF-L bound with high affinity to Fd' in a 1:1 ratio with a second-order rate constant of 1618 M −1 sec −1 at 25°C. Using the same approach, it was shown that the labelled constant region fragment (5-IAF-C κ ) bound to Fd' with an association constant of 5 × 10 6 M −1 and a forward rate constant of 30 M −1 sec −1 . When 5-IAF-C κ was recombined with a preformed Fd'V κ complex, however, the intensity of the visible difference spectrum, the fluorescence emission, the binding affinity and the forward rate constant of the reaction, were significantly increased. In contrast, no spectroscopic changes were observed when V κ was recombined with a preformed Fd'-5-IAF-C κ complex. These data suggest that: (1) the high affinity interaction between Fd' and L results from the summation of relatively weak interactions between paired domains; (2) the binding of Vκ to Fd' modulates the reactivity of Cγl toward Cκ, probably through conformation changes transmitted via V H -Cγl contacts; and (3) conversely, once 5-IAF-Cκ is bound to Fd', the addition of the complementary Vκ domains does not induce any detectable change in the vicinity of the fluorescent probe.

Circular dichroism and ultraviolet absorption difference spectroscopy have been used to study the binding of a series of divalent and trivalent cations to porcine intestinal calcium-binding protein (CaBP). When calcium is bound to the single high-affinity site on CaBP, the aromatic optical activity is greatly increased. Analysis of the circular dichroic spectra, obtained in the presence and absence of calcium, suggested that although changes in the optical activity of the single tyrosyl residue accounted for much of the overall change observed upon binding calcium, one or more of the five phenylalanyl residues was also perturbed. All the cations tested, with the exception of lead which gave rise to unique spectral effects, caused the same changes in optical activity between 300 and 250 nm. In the peptide absorption region, CaBP exhibited optical activity typical of an α-helical protein and no significant changes were observed in the presence of any of the cations tested. Cation-binding curves obtained from the circular dichroic data for the cations bound with high affinity (i.e., calcium, strontium, and the trivalent lanthanide ions) showed that the apparent number of binding sites was inversely related to the protein concentration. This phenomenon was accounted for by the concentration-dependent aggregation of CaBP observed in earlier studies. The binding data, obtained using circular dichroism, clearly indicated that the affinity of CaBP for the various cations was related to their ionic radius. Absorption difference spectra were observed when calcium was bound to CaBP. The features of these spectra confirmed that phenylalanyl as well as tyrosyl transitions were perturbed upon calcium binding. The extent to which the tyrosyl side chain was exposed to solvent was determined by solvent perturbation difference spectroscopy using perturbing agents of differing molecular radius. The apparent degree of exposure increased as the perturbant size decreased suggesting that the side chain was located in a cleft. Bound calcium did not change the degree of exposure. These data, together with complementary data obtained with bovine CaBP, were discussed in terms of the geometry of the cation-binding site.

Spectral properties of pig intestinal Ca2+-binding protein (CaBP) and its apoprotein have been examined by fluorescence, absorption and c.d. Direct fluorescence from some of the five phenylalanine residues is observed and excitation spectra show that there is also energy transfer from some phenylalanine residues to the tyrosine. Absorption and c.d. spectra show that the tyrosine hydroxy group does not ionize significantly below pH 12. Tyrosine fluorescence is reversibly quenched by a lysine residue with a pK of 10.05 in the Ca2+ form. At low pH the tyrosine fluorescence is enhanced with transitions with pK values of approx. 4.2. The c.d. spectrum of the Ca2+ form shows a decrease of the ellipticity band at 276nm with a transition similar to that of the fluorescence titration. The apoprotein, however, shows an additional transition with a pK of about 6. The results are interpreted in terms of the recently published structure of the cow intestinal CaBP [Szebenyi, Obendorf & Moffat (1981) Nature (London) 294, 327-332]. The single tyrosine has a very high pK, although it apparently lies on the surface of the protein molecule.

A lambda light chain, isolated from an immunoglobulin G molecule, was found to reversibly precipitate at low temperatures. This cryoprecipitation was a function of pH, ionic strength, protein concentration, and time as well as temperature. The lambda chain underwent a cooperative conformational change as the temperature was lowered from 26 to 0 degrees C as judged by ultraviolet difference spectroscopy and circular dichroism. Normal lambda chains showed no conformational change. By difference spectroscopy it was possible to calculate the equilibrium constant governing the conformational change. The change was strongly exothermic (delta H approximately -80 kcal mol-1) and accompanied by a large decrease in entropy (delta S approximately -280 eu). The midpoint of the transition was dependent on the initial protein concentration, suggesting that only the noncovalent dimer of the lambda chain exhibited the conformational change. The existence of a monomer-dimer eqiulibrium (KA approximately 4 X 10(5) M-1) was confirmed by sedimentation velocity. No conformational change was observed by circular dichroism at concentrations where greater than 95% of lambda chain was in the form of a monomer. Although high ionic strength inhibited cryoprecipitation, it had no effect on the conformational change. Stabilization of the dimer by forming an interchain disulfide bond between two monomers abolished both the conformational change and cryoprecipitation. A fragment corresponding to the constant region was isolated from both peptic and tryptic digests of the lambda chain. This fragment neither cryoprecipitated nor showed temperature dependence conformational changes. It proved impossible to isolate a fragment corresponding to the variable region. Both qualitative and quantitative models are presented to account for the behavior of the lambda chain at low temperatures.

Previous studies have shown that the forward rate constants governing the non-covalent association of heavy and light chains of different IgG myeloma proteins vary considerably. In the present study rate constants, absorption difference spectra enthalpies associated with recombination have been determined using kappa chain of known variable-region subgroup specificity. The basic experimental approach was to allow a single species by γ chain to react reparately with several kappa chains belonging either to the κIII subgroup. The rate constant varied by two orders of magnitude but there was no evidence that this variability was related to subgroup specificity. There was as much variation within a subgroup as between subgroups. A correlation was noted, however, between the rate constants and the species of heavy chain used in the recombination reactions. The shape and magnitude of the difference spectra generated upon subunit association were also variable but again this could not be correlated with subgroup specificity. Variations in enthalpies of association were less striking but there were some indications that values for δH depended on the heavy chain used. Only for one of the heavy chains used was there evidence to indicate that the preferential recombination of autologous subunits observed in equilibrium systems arises because autologous subunits associate more rapidly than heterologous chains. The data presented indicate that sequence variations in either the hypervariable regions, or the framework within a subgroup, play an important role in subunit association.

A procedure is outlined by which sedimentation analyses of small quantities of mucin glycoproteins can be performed. Rat intestinal goblet cell mucin was stained with periodic acid-Schiff reagent to permit detection by light absorption at 555 nm. PAS treatment resulted in chemical modification of sialic acid and 55% of fucose residues in the mucin. No other chemical or physical alterations were detected. The stained mucus was subjected to band ultracentrifugation using D2O-containing solvents. Sedimentation was monitored by scanning at 555 nm. Results compared favorably with those reported earlier for conventional boundary ultracentrifugation of intact goblet cell mucin. Because of the low concentrations of mucin used in band ultracentrifugation (0.2–1.5 μg protein/ml), S20,w values are comparable to sedimentation coefficients at zero concentration (So values), determined by conventional means.

Single component guinea pig insulin was found by sedimentation equilibrium centrifugation to exist in neutral solution as a monomeric species only, even in the presence of zinc ion. This insulin, in contrast to bovine insulin, did not bind zinc ion at pH 7.4. The biological activity of guinea pig insulin, estimated by mouse convulsion assay, was 2.14 I. U. per mg.

We have recently isolated a 9.5S α1 glycoprotein from human serum and shown it to be a Ca++-dependent subcomponent of the first component of complement and which we have named C1t. It is believed that C1t serves as a bridge between C1q and C1s enhancing and amplifying the ability of C1q to bind C1s and present it to C1r for activation. The molecular weight of C1t was determined by sedimentation equilibrium analysis by using the meniscus depletion method. In neutral solvents, 0.1 mM Na3HEDTA, a molecular weight of 233,000 was found. In 5.95 M guanidinium chloride, a molecular weight of 23,000 was obtained. This value was unaffected by prior reduction and alkylation with 0.1 M mercaptoethanol and 0.22 M iodoacetamide. The molecule appears to be composed of 10 similar subunits each composed of a single polypeptide chain.

A statistical analysis is performed of various low‐order truncations of the two‐dimensional inviscid Navier–Stokes equations. The behavior of the time‐averaged enstrophy and time‐correlation function of each Fourier mode is analyzed and compared to the behavior expected in a random dynamical system. Empirical evidence is presented for the randomness of all isotropically truncated models for which the wavenumber cutoff is large enough to insure that every mode is involved in nontrivial interactions. This evidence is contrasted with the apparent nonrandomness found in other special truncations. The results strengthen the evidence for the validity of statistical descriptions of two‐dimensional turbulence.

Direct numerical solutions of the three-dimensional time-dependent Navier-Stokes equations are presented for the evolution of three-dimensional finite-amplitude disturbances of plane Poiseuille and plane Couette flows. Spectral methods using Fourier series and Chebyshev polynomial series are used. It is found that plane Poiseuille flow can sustain neutrally stable two-dimensional finite-amplitude disturbances at Reynolds numbers larger than about 2800. No neutrally stable two-dimensional finite-amplitude disturbances of plane Couette flow were found.Three-dimensional disturbances are shown to have a strongly destabilizing effect. It is shown that finite-amplitude disturbances can drive transition to turbulence in both plane Poiseuille flow and plane Couette flow at Reynolds numbers of order 1000. Details of the resulting flow fields are presented. It is also shown that plane Poiseuille flow cannot sustain turbulence at Reynolds numbers below about 500.