Nature and significance of orbital autoantigens and their corresponding autoantibodies in thyroid-associated ophthalmopathy.

There is now considerable evidence that the pathogenesis of thyroid-associated ophthalmopathy is closely linked to the presence of a shared autoantigen(s) in the thyroid and the eye muscle, against which cytotoxic mechanisms are directed. Although the orbital connective tissue is certainly involved in the orbital inflammatory process, a 64 kDa membrane protein expressed by both the eye muscle and the thyroid and recognized consistently by antibodies in the sera of TAO patients, seems to be the most likely target candidate. While its presence in non ocular skeletal muscle is not as well established, more recent data tend to suggest the existence of a 64 kDa molecule in the three tissues. The availability of a cDNA encoding a 572 amino acid protein corresponding to a MW of 63-64 kDa, which may be the same molecule, will allow us to determine more clearly the structural characteristics of the different molecules proposed as targets. The role of the corresponding autoantibodies in the pathogenesis of the eye disease is far less well defined. Whether they play a role in the induction of the ophthalmopathy or only represent helpful markers remains to be clarified.


A novel congenital haemostatic defect: combined factor VII and factor XI deficiency.

Isolated deficiencies of factors VII and XI are both rare. Not surprisingly, therefore, combined factor VII and XI deficiency has not been reported previously. We report here a kindred with a combined heterozygous deficiency for both factors VII and XI. The proposita is a 28-year-old woman who had both a prolonged prothrombin time (PT) and a prolonged activated partial prothrombin time (APTT) associated with a mild bleeding tendency. Coagulation studies were performed on the six available members of this kindred. The PT and APTT were normal or mildly abnormal in five of these individuals. Factor VII coagulant activity (VII:C) varied from 0.33 to 0.77 units/ml in affected subjects. In contrast, the concentration of factor VII-related antigen for the six individuals ranged from 0.68 to 2.10 units/ml. Comparable factor VII:C levels were obtained when each subject's plasma was tested with either a rabbit or a human thromboplastin reagent. Factor XI coagulant activity was less than 0.5 units/ml in three of the six subjects and normal (approximately 1.0 units/ml) in the other three. The concentrations of thrombin-antithrombin-III and prothrombin fragment 1.2 were within normal limits for all individuals. In addition to being associated with heterozygous factor XI deficiency, the abnormal factor VII molecule in the plasma of affected individuals in this kindred appears to represent a newly described mutation. This is suggested by the pattern of reactivity with thromboplastin from different species, the normal tissue factor binding and the bleeding tendency in heterozygous individuals in this kindred.


Human fibrinogen heterogeneity: the COOH-terminal residues of defective A alpha chains of fibrinogen II.

Fibrinogen fraction I (340 kDa) and fraction II (305 kDa) were isolated by glycine precipitation. The subunit chains of the two fractions were separated, after reduction, by reverse-phase high performance liquid chromatography. The amino acid compositions of the B beta and tau chains of fibrinogen II were identical with those of fibrinogen I. In contrast, the A alpha chains of fibrinogen II were composed of two populations, one comprising homogeneous, intact A alpha chains and the other consisting of heterogeneous, deficient A alpha chains (A alpha' chains) of lengths varying according to the sizes of their COOH-terminal defects. The molar ratio of the A alpha to the A alpha' chains in fibrinogen II was 1.16:1. The amino acid composition and sequence analyses of the TPCK-trypsin peptides derived from the A alpha' chains revealed that the COOH-terminal residues of the A alpha' chains were mainly Asn-269, Gly-297 and Pro-309. These results indicate that the fibrinogen II molecule is asymmetrical and can be represented by the formula (A alpha) (A alpha')(B beta)2(tau)2 and that fibrinogen II cannot be a plasmin degradation product of fibrinogen I.


Behaviour of tissue plasminogen activator, plasminogen activator inhibitor 1 and their complex in various disease states.

Plasma levels of tissue plasminogen activator (t-PA) antigen, plasminogen activator inhibitor 1 (PAI-1) antigen and t-PA/PAI-1 complex were measured in plasmas from 18 healthy subjects and 75 patients with various diseases (28 patients with haematological malignancies, 20 with thrombotic diseases, five with infectious diseases, four with liver diseases, ten with bleeding disorders and eight miscellaneous conditions). In addition, we studied ten patients with bleeding disorders after DDAVP infusion and 18 healthy subjects after venous occlusion. Plasma levels of t-PA antigen, PAI-1 antigen and t-PA/PAI-1 complex were increased in the patients compared with the healthy subjects. t-PA/PAI-1 complex levels correlated well with t-PA antigen levels and molar concentrations of t-PA antigen were similar to those of the t-PA/PAI-1 complex. Venous occlusion induced an increase in both t-PA antigen and PAI-1 antigen and the molar concentration of the t-PA/PAI-1 complex was equivalent to that of t-PA antigen. Following DDAVP infusion, the levels of t-PA antigen and t-PA/PAI-1 complex increased but PAI-1 antigen levels decreased, and the increase of t-PA antigen was greater than that of t-PA/PAI-1 complex. These findings indicate that PAI-1 antigen exceeds t-PA antigen in healthy subjects and in patients with various diseases. We conclude that part of the t-PA/PAI-1 complex is rapidly cleared from the circulation and that free t-PA increases after DDAVP infusion.


Membrane fluidity and platelet aggregation: dibucaine permits ristocetin-induced platelet aggregation with low-molecular-weight von Willebrand multimers.

The effects of brief incubation of platelets with dibucaine were studied. Membrane fluidity was increased after incubation with 1 mM dibucaine for 1 min as determined by fluorescence polarization. Platelet aggregation was increased when ristocetin was employed as modulator and normal plasma used as a source of von Willebrand factor (vWF). In contrast, aggregation was decreased with botrocetin. After cryoprecipitation the supernatant, which contained predominantly low-molecular-weight vWF multimers, was effective with ristocetin only after prior incubation of the platelets with dibucaine. This indicates that low-molecular-weight vWF multimers can also support ristocetin-induced aggregation when the membrane fluidity is increased. This idea was supported by the use of plasma from a patient with von Willebrand disease type IIa. Being a multimeric protein, von Willebrand factor contains multiple binding sites for glycoprotein Ib (GP Ib). GP Ib-dependent aggregation by agents that do not contain multiple binding sites, i.e. wheat germ agglutinin or polyclonal antibodies to GP Ib, was decreased after brief incubation with dibucaine. These observations are discussed in relation to the hypothesis that the increased membrane fluidity allows an increased number of GP Ib molecules to bind to each vWF multimer.


Antiphospholipid antibodies and thrombosis in systemic lupus erythematosus: comparison of three lupus anticoagulant assays and anticardiolipin ELISA in 188 patients.

The presence of antiphospholipid antibodies (aPL) in 188 unselected patients with systemic lupus erythematosus was studied using the recalcification time, kaolin clotting time (KCT), dilute Russell's viper venom time (dRVVT) and anticardiolipin ELISA (aCL) to identify patients with a high or low risk of thrombosis among patients with aPL. aPL were detected by at least one method in 104 (55%) of the patients. Despite heterogeneity, lupus anticoagulant (LA) methods correlated reasonably well with each other (r = 0.736-0.968), but poorly with aCL (r = 0.241-0.549). Positivity in LA assays and immunoglobulin G (IgG)-aCL were associated with patients who experienced thrombosis (P less than 0.001 for all assays). Patients with both LA and aCL had experienced thrombosis more often than those having only one (odds = 6.3, P less than 0.001). When patients with aPL were ranked by relative strength of the finding and divided into tertiles, a history of thrombosis was associated with membership in the strongest tertile of at least one assay (odds = 4.2, P = 0.002). LA and aCL had similar sensitivities for thrombosis (61% and 63%, respectively), but LA was more specific than aCL (79% vs 53%). The best combination of two assays was KCT with dRVVT (61% sensitivity, 87% specificity). Maximal sensitivity (71%) for thrombosis could be achieved by adding IgG-aCL to these two assays, but specificity was lower (73%). In conclusion, a high thrombotic risk among patients with aPL was indicated by the simultaneous presence of both LA and aCL, strongly positive aPL, and, among aCL, IgG-class antibodies.


Molecular study of von Willebrand disease: identification of potential mutations in patients with type IIA and type IIB.

The defective von Willebrand Factor (vWF) in type IIA von Willebrand disease (vWD) has decreased binding affinity for platelet membrane glycoprotein Ib (GPIb) while in type IIB vWD, the abnormal vWF has increased affinity for this receptor. Segments of exon 28 of the vWF gene were amplified by the polymerase chain reaction and sequenced in two patients with type IIA and two patients with type IIB vWD. One type IIB patient showed an arginine to tryptophan substitution at amino acid residue 543 in the mature vWF and the other patient had a valine to methionine change at residue 553. Including these two new cases, substitutions at residues 543 and 553 now account for more than half of the documented mutations in patients with type IIB vWD. One patient with type IIA vWD showed an isoleucine to threonine change at amino acid 865. This substitution has been reported in another patient with type IIA vWD. The other patient showed a novel proline to serine change at residue 885. The C to T nucleotide transition which causes the amino acid change was not found in over 100 normal chromosomes tested by allele specific oligonucleotide hybridization and was linked to type IIA vWD in the family. This potential mutation is more carboxyterminal in the vWF subunit than other reported mutations in type IIA vWD. It is apparent that mutations associated with type IIA vWD are not as tightly grouped as defects in type IIB vWD, supporting the evidence that the type IIA vWD phenotype is generated by diverse mechanisms.


Quantitative assay of the carbohydrate in urokinase-type plasminogen activator by lectin-enzyme immunoassay.

We developed an enzyme immunoassay (EIA) for the measurement of Asn302-linked carbohydrate in urokinase-type plasminogen activator (u-PA) using peroxidase (HRP)-labelled lectins. u-PA antigen in the sample was immunologically bound to microtitre plate wells by anti-u-PA IgG and the binding of HRP-labelled lectins [Con A (Concanavalin A), WGA (wheat germ agglutinin), PNA (peanut agglutinin), CSA (Scotch broom), GS-I (Groffonia simplicifollia) and SBA (soybean agglutinin)] to the carbohydrate of u-PA was measured. The lectin-EIA was dose-dependent in the range 6-6000 IU/ml of u-PA using Con A and WGA. The assay did not detect the carbohydrate of bovine albumin, ovalbumin, human albumin, plasminogen, tau-globulin and fibrinogen. The binding of HRP-labelled Con A and WGA to the carbohydrate of u-PA was specifically inhibited by alpha-methylmannose and N-acetylglucosamine respectively. Endo F treatment of the carbohydrate of u-PA decreased the binding of Con A and WGA. The carbohydrate of u-PA obtained from chest fluid, ascites and U937 cell culture medium reacted with Con A and WGA by this assay forming a band in the 55 kDa region. These results suggest that the lectin-EIA method is suitable for the assay of the carbohydrate in the B-chain of u-PA.


The present status of tissue factor pathway inhibitor.

Tissue factor pathway inhibitor (TFPI) is the factor Xa-dependent inhibitor of the factor VIIa/tissue factor complex. The plasma concentration of this 276 amino acid, 40 kDa glycoprotein is normally about 100 ng/ml. There are three intravascular pools of TFPI: 50-90% is on the endothelium, 10-50% is in plasma and less than 2.5% is in platelets. The TFPI in plasma is mainly associated with lipoproteins-only about 5% is free TFPI. The lipoprotein-associated TFPI seems to be of less anticoagulant effect than the free TFPI. Both unfractionated heparin, low-molecular-weight heparins and pentosan polysulphate induce release of TFPI after intravenous injection, whereas dermatan sulphate does not. The interactions with TFPI account for a considerable amount of the anticoagulant effect of heparin. Studies have shown increased TFPI levels in plasma from patients with advanced malignancy and in subjects with fatal DIC or septicaemia. The reason for this is unknown. For measuring the anticoagulant activity of TFPI in plasma, end-point or antigen assays may be less useful than the clotting assay with dilute tissue factor. Animal studies indicate that the main physiological role of TFPI is the inhibition of small amounts of tissue factor. TFPI is probably essential for a normal haemostatic balance.


Venous thromboembolism and cancer.

The literature was reviewed in an attempt to determine whether patients with cancer have an increased risk of venous thromboembolism, i.e. deep vein thrombosis and pulmonary embolism. From case reports it was apparent that various thromboembolic or thrombophlebitic manifestations may be found in a small number of patients, although it is possible that not all cases belong to the same clinical or pathogenetic entity. In clinical series it was found that the risk of postoperative venous thromboembolism was increased in cancer patients, but the possibility that this was due to associated risk factors, rather than to the mere presence of a tumour, could not be excluded. Little is known about patients not undergoing surgery. Retrospective postmortem studies have found more thrombi in patients with malignancy, but a prospective study failed to demonstrate an association between malignancy and pulmonary embolism. It is possible that different types of cancer show various degrees of association with venous thromboembolism. We conclude that further studies should be performed to provide a firm clinical and pathoanatomical basis for investigations into the pathogenesis of venous thromboembolism.


Haemostatic effects in vivo after snakebite by the red-necked keelback (Rhabdophis subminiatus).

We describe a patient who developed a severe coagulopathy after being bitten by a red-necked keelback snake (Rhabdophis subminiatus), a species which is generally considered non-venomous. The patient's blood was incoagulable due to complete depletion of fibrinogen. Comprehensive coagulation studies were performed to identify the mechanism(s) by which the snake toxin caused the coagulopathy. It was found to contain a potent prothrombin activator, probably an activator of protein C and possibly also a factor X activating enzyme. The fibrinolysis was secondary to intravascular fibrin formation; there were no indications for a direct fibrinogenolytic activity in the snake toxin. Remarkably, there was virtually no consumption of antithrombin III, despite extensive thrombin formation; this feature appears to be not uncommon after snake bites, but is still unexplained.


Dental surgery in patients with severe factor XI deficiency without plasma replacement.

Bleeding following dental extraction is frequently the first manifestation of severe factor XI deficiency. Safe oral surgery has previously been performed in such patients by using plasma replacement therapy with or without concomitant administration of antifibrinolytic agents. The aim of this study was to determine whether such patients can undergo safe dental extractions using only an antifibrinolytic agent. The study group consisted of 19 patients with severe factor XI deficiency (factor XI:C level less than 14 U/dl) who had previously bled following dental extractions (14 patients) or other trauma (five patients). Tranexamic acid, 1 g q.i.d., was given from 12 h before surgery, until 7 days afterwards. No excessive bleeding was observed following dental extractions. One patient had slight oozing after 3 days which ceased spontaneously. Thus, plasma replacement no longer appears necessary for patients with severe factor XI deficiency requiring dental extractions.


Prothrombin fragment F1 + 2 and thrombin-antithrombin III complex are useful markers of the hypercoagulable state in atrial fibrillation.

It is well known that atrial fibrillation (AF) is one of the most important diseases that predispose patients to thrombosis. We have attempted to identify patients with AF in the hypercoagulable state by measuring molecular markers such as thrombin-antithrombin III complex (TAT) and prothrombin fragment 1 + 2 (PTF) and determining the effect of antithrombotic therapy on these markers; 83 patients with AF were studied. Increased levels of plasma TAT and PTF were more frequently observed in patients with AF and associated mitral stenosis than in patients with AF alone. In cases of AF without mitral stenosis, plasma levels of TAT and PTF were significantly lower in those patients receiving antithrombotic agents (aspirin or warfarin) than in those receiving no antithrombotic agents. Furthermore, plasma levels of PTF were significantly lower in patients given warfarin than in those receiving aspirin. These results suggest that (1) patients with AF and mitral stenosis who are not given warfarin are in an extremely hypercoagulable state and (2) some patients with AF without mitral stenosis who are not given antithrombotic agents are also moderately hypercoagulable. In vivo activation of blood coagulation was more effectively controlled in patients receiving warfarin than in those taking aspirin.


Thrombolysis using consecutive high dose bolus and infusion of alteplase in a patient with acute massive pulmonary embolism.

We describe the successful use of a novel dosage regimen of alteplase in a 77-year-old man with massive acute pulmonary embolism in the presence of a relative contraindication (recent cranial surgery). An intravenous alteplase bolus of 50 mg was administered, followed 40 min later by a 50 mg infusion over 2 h. Pulmonary arteriography showed a considerable increase in intraluminal filling of thrombotically occluded vessels as a result of alteplase administration, and was accompanied by marked clinical improvement. Peak alteplase plasma concentration after the bolus was 5.5 micrograms/ml, and the steady state concentration during infusion was 0.52 micrograms/ml. Detailed monitoring of haemostasis parameters showed elevated endogenous fibrinolytic activity at baseline, very high levels of fibrin degradation products during treatment resulting from lysis of an extensive thrombus mass, moderate fibrinogenolysis compared to fibrinolysis, long plasma half-lives of fibrin(ogen) degradation products (9.2-11.0 h), and increased thrombin generation. There were no bleeding side-effects. The regimen combines the advantages of rapid onset (bolus) and maintained potency (infusion) of thrombolytic effect in pulmonary embolism.


A comparison of the plasminogen activator activity units of urinary-type plasminogen activator (u-PA) and tissue-type plasminogen activator (t-PA).

The earliest tissue plasminogen activator (t-PA) preparations prepared from melanoma cells were expressed in urinary-type plasminogen activator (u-PA) units of activity using the u-PA International Standard (66/46). This report describes a comparison between u-PA and t-PA units by various types of fibrin plate procedure using both human and bovine fibrin. Within the biometric limits of this procedure it was found that the potency ratio of u-PA/t-PA was 3.5 (human fibrin), 5.29 (enriched bovine fibrin) and 4.6 (crude bovine fibrin). Specific activity figures of approximately 100,000 IU/mg for pure t-PA using the urokinase standard (66/46) would convert to approximately 350,000-530,000 IU/mg using the International Standards for t-PA (83/517 and 87/670). This latter figure is compatible with the reported specific activity for purified preparations of recombinant t-PA.


Intra-cardial thrombosis with systemic and pulmonary embolism as main symptoms in a patient with protein S deficiency.

This report describes the unusual occurrence of both left and right atrial thrombosis with peripheral arterial and pulmonary embolism, respectively, as presenting symptoms of congenital protein S deficiency in a 31-year-old man. The coagulation study performed in the coumarin-treated propositus indicated a heterozygous protein S state. The finding of reduced free protein S antigen and protein S activity levels with normal total protein S and C4B-bp levels in five other family members (father, sister, and three relatives on the paternal side) confirmed the inherited nature of the defect. Since there is an increased frequency of arterial thrombosis in patients suffering from protein S deficiency, any case of idiopathic intra-cardial thrombosis requires careful haemostatic screening. In addition, the possibility of intra-cardial thrombosis should be considered in any thromboembolic event seen in inherited protein S deficiency.


Use of thermal imaging to monitor crural thrombolysis.

Intra-arterial thrombolysis is increasingly being used as the treatment of choice in selected cases of critical limb ischaemia. At present angiography is the established method of monitoring the progress of thrombolysis. We report a case in which thermal imaging was used in conjunction with angiography to monitor the effects of thrombolysis.


[Suitability of capacitive sensors for measuring relative humidity in anesthetic gas systems].

A capacitive sensor was tested for its suitability for measuring relative humidity in an anaesthetic gas circuit. The valvo sensor PH1 was tested using various different anaesthetic gas mixtures. Measuring accuracy was influenced neither by such volatile anaesthetics as isoflurane and halothane, nor by oxygen or nitrous oxide. The response time of the sensor depends on its position within the gas, and in the most favourable case is about 3 minutes. The sensor is readily incorporated within an existing gas circuit. The linearity of the characteristic curve must be corrected by external electronic compensation to avoid measuring problems in the lower humidity range.


[Automatic tracking system for laser surgery of the human cornea].

An automatic, closed loop, real-time tracking device is described. The purpose of this device is, despite movements of the eye, to keep the laser beam correctly on target while performing corneal surgery. This type of laser surgery, is employed specifically to ablate some of the cornea to correct myopia, hyperopia and astigmatism.


[Morphological and numerical studies of the stress compatibility of patella implants].

Failure of total knee arthroplasty is relatively often caused by problems of the patellofemoral replacement. The purpose of this study was to analyze the distribution of stresses within an anatomical patella and the changes in stress distribution after patellar resurfacing with a Miller-Galante I patellar implant using two- and three dimensional finite element models (FEM). To assess validity, FEM results were compared with morphological findings from contact radiographs and densitographs. Internal orientation of bone trabeculae is in good agreement with the orientation of theoretically calculated principal stresses. Almost unchanged principal tensile stresses after implantation, together with the lack of extreme stress peaks within the cancellous bone ensure stress compatibility of the implant. In the case of a firmly seated implant with good bone ingrowth, increased von Mises stresses are found near the fixation peg/plate junction. Their relevance for improved bone ingrowth near this part of the interface is emphasized. At the same time, material failure at the peg/plate junction can be better understood. An analysis of the early postoperative period assuming nonlinear interface conditions failed to demonstrate an uniform distribution of normal and tangential interface forces.


Linear regression analysis based on Buckley-James estimating equation.

In this note we consider the problem of drawing inference about the regression parameters in a linear model with survival data. A simple procedure based on the Buckley-James (1979, Biometrika 66, 429-436) estimating equation is proposed and illustrated with an example.


Blocked arteries and multivariate regression.

Ultrasound blood flow waveforms may be used in the diagnosis of arterial occlusive disease in human legs. We develop a statistical model to predict disease severity, conditional on the ultrasound data and some training data. It belongs to the class of models known as seemingly unrelated regressions, for which the Bayesian predictive density function cannot be evaluated analytically. Allowing for missing components of response vectors in the training data, we describe a first-order approximation to the predictive density, based on a Bayes estimate of the precision matrix. This approximation is then used to generate cross-validated predictions of disease severity in a set of 31 patients. We conclude with a discussion of the results.


Testing the effect of treatment in experiments with correlated binary outcomes.

This paper considers the problem of testing for treatment effect in a randomized experiment with correlated binary outcomes, representing success or failure for different "parts" of a randomized unit. Attention is restricted to tests that are based on a summary score for each individual randomized, and thus are valid regardless of the precise nature of the correlation among parts. The focus is on the efficiency of such tests under various correlation structures, with special emphasis on the case in which the correlation among parts within an individual differs across treatment groups. A class of summary score statistics is defined, and optimal testing is discussed for some simple situations. Three potential general-purpose tests also are described: (1) the ratio estimate test discussed by Henderson et al. (1988, Controlled Clinical Trials 9, 189-205); (2) a modified ratio estimate test with adjusted weighting based on the within-individual correlation between parts; (3) a test defined by applying the Mantel-Haenszel procedure to the proportion of individuals with at least one failure, stratifying by the number of parts. For these general-purpose tests, numerical calculations of asymptotic efficiency are presented under a wide range of designs and correlation structures. On the basis of these results, some practical recommendations for choosing a test are made.


Multivariate methods for clustered binary data with multiple subclasses, with application to binary longitudinal data.

Clustered binary data occur frequently in biostatistical work. Several approaches have been proposed for the analysis of clustered binary data. In Rosner (1984, Biometrics 40, 1025-1035), a polychotomous logistic regression model was proposed that is a generalization of the beta-binomial distribution and allows for unit- and subunit-specific covariates, while controlling for clustering effects. One assumption of this model is that all pairs of subunits within a cluster are equally correlated. This is appropriate for ophthalmologic work where clusters are generally of size 2, but may be inappropriate for larger cluster sizes. A beta-binomial mixture model is introduced to allow for multiple subclasses within a cluster and to estimate odds ratios relating outcomes for pairs of subunits within a subclass as well as in different subclasses. To include covariates, an extension of the polychotomous logistic regression model is proposed, which allows one to estimate effects of unit-, class-, and subunit-specific covariates, while controlling for clustering using the beta-binomial mixture model. This model is applied to the analysis of respiratory symptom data in children collected over a 14-year period in East Boston, Massachusetts, in relation to maternal and child smoking, where the unit is the child and symptom history is divided into early-adolescent and late-adolescent symptom experience.


A parametric family of correlation structures for the analysis of longitudinal data.

In epidemiological settings, we are often faced with numerous short time series, and a parsimonious parametrization of the correlation structure is desired in order to optimize the efficiency of the estimation procedure. We propose a damped exponential correlation structure for modeling multivariate Gaussian outcomes. The correlation between two observations separated by s units of time is modeled as gamma s theta, where gamma is the correlation between elements separated by one s-unit, and theta is a damping parameter. For (theta = 0), (theta = 1), and theta----infinity), the correlation structures of compound symmetry, first-order autoregressive, and first-order moving average processes are obtained. Although the AR(2) dependency structure, and the combination of random effects and AR(1) errors are not special cases of the proposed parametric family, these structures can be well approximated within the family for short time series. Maximum likelihood methods for parameter estimation and interpretations of intermediate models (0 less than theta less than 1) are discussed in the context of modeling pulmonary function in an adult population in The Netherlands and T-cell subsets in homosexual men infected with human immunodeficiency virus Type I.


Modelling balanced longitudinal data: maximum likelihood estimation and analysis of variance.

For linear models, assuming a within-experimental-units covariance structure that incorporates errors of measurement, serial correlation, and variation between units, results on explicit estimation of regression parameters are used to simplify maximum likelihood estimation of covariance parameters. The use of an analysis of variance table as a simpler alternative to likelihood inference is illustrated with two examples.


Exchangeability in multivariate Markov chain models.

Time-homogeneous Markov chain models with state space [0, 1]k are useful in analysis of binary follow-up data on k individuals that interact. The number of parameters increases exponentially with k so more restrictive models are imperative for statistical inference. The hypothesis that the matrix of transition probabilities is invariant under permutation of individuals is discussed. It is shown that if individuals are exchangeable, then the process counting the number of individuals occupying a given state is a Markov chain. This reduction of data is sufficient if either at most a single individual may change state between two consecutive time points or if a state is absorbing. Similar results are obtained for exchangeability within two subgroups. Inference in the multivariate process reduces to a univariate problem if individuals are independent given the group's previous response. It is shown how conditional independence could be tested assuming exchangeability. The different hypotheses re examined in an analysis of the occurrence of bacteria in milk samples of Danish dairy cattle.


Sequential monitoring for comparison of changes in a response variable in clinical studies.

The spending function approach proposed by Lan and DeMets (1983, Biometrika 70, 659-663) for sequential monitoring of clinical trials is applied to situations where comparison of changes in a continuous response variable between two groups is the primary concern. Death, loss to follow-up, and missed visits could cause follow-up measurements to be right-censored or missing for some participants. Furthermore, the probability of being censored may be dependent on the parameter value of the response variable (informative censoring). We propose to compare treatment effects by comparing areas under the expected response change curves between the two groups. When the response curves are linear as a function of time in both groups, this comparison is equivalent to comparing the rates of change in the response variable. Covariances of the sequential test statistics are derived. Conditions for having independent increments are presented. For studies designed to evaluate long-term treatment effects, spending functions obtained by shifting the usual spending functions (Kim and DeMets, 1987, Biometrika 74, 149-154) to the right and then rescaling to the remaining interval are also proposed. Such a shifted spending function is applied to the monitoring plan for the Lung Health Study (Anthonisen, 1989, American Review of Respiratory Diseases 140, 871-872).


Use of external rates in nested case-control studies with application to the international radiation study of cervical cancer patients.

A method is proposed for analysis of nested case-control studies that combines the matched comparison of covariate values between cases and controls and a comparison of the observed numbers of cases in the nesting cohort with expected numbers based on external rates and average relative risks estimated from the controls. The former comparison is based on the conditional likelihood for matched case-control studies and the latter on the unconditional likelihood for Poisson regression. It is shown that the two likelihoods are orthogonal and that their product is an estimator of the full survival likelihood that would have been obtained on the total cohort, had complete covariate data been available. Parameter estimation and significance tests follow in the usual way by maximizing this product likelihood. The method is illustrated using data on leukemia following irradiation for cervical cancer. In this study, the original cohort study showed a clear excess of leukemia in the first 15 years after exposure, but it was not feasible to obtain dose estimates on the entire cohort. However, the subsequent nested case-control study failed to demonstrate significant differences between alternative dose-response relations and effects of time-related modifiers. The combined analysis allows much clearer discrimination between alternative dose-time-response models.


Semiparametric estimation of random effects using the Cox model based on the EM algorithm.

Consider a survival experiment where individuals within a certain subset of the population share a common, unobservable, random frailty. Such a frailty could be an unobservable genetic or early environmental effect if individuals were in sibling groups or an environmental effect if individuals were grouped by households. Suppose that if the frailty, omega, is known, the Cox proportional hazards model for the observable covariates is valid with the consequence of the random effect being a multiplicative factor on the hazard rate. Assuming tht the random frailties follow a gamma distribution, estimates of the fixed and random effects are obtained by using an EM algorithm based on a profile likelihood construction. The method developed is applied to the Framingham Heart Study to examine the risks of smoking and cholesterol levels, adjusting for potential random effects.


A statistical distribution with an unbounded hazard function and its application to a theory from demography.

We introduce a statistical distribution with hazard function mu(t) proportional to (psi-t)beta-1 for 0 less than or equal to t less than psi and shape parameter beta satisfying 0 less than beta less than 1. This hazard function is suggested by a theory of aging in demography. We discuss properties of this distribution and maximum likelihood estimates of its parameters. Mixture distributions are considered to account for pooling across dissimilar populations. This model is compared with the Gompertz and generalized Pareto distributions and used to estimate a finite limit on human lifespan based on the survival of a group of female centenarians.


Proportional hazards model with covariates subject to measurement error.

When covariates of a proportional hazards model are subject to measurement error, the maximum likelihood estimates of regression coefficients based on the partial likelihood are asymptotically biased. Prentice (1982, Biometrika 69, 331-342) presents an example of such bias and suggests a modified partial likelihood. This paper applies the corrected score function method (Nakamura, 1990, Biometrika 77, 127-137) to the proportional hazards model when measurement errors are additive and normally distributed. The result allows a simple correction to the ordinary partial likelihood that yields asymptotically unbiased estimates; the validity of the correction is confirmed via a limited simulation study.


Estimating the probability of toxicity at the recommended dose following a phase I clinical trial in cancer.

The problem of point and interval estimation following a Phase I trial, carried out according to the scheme outlined by O'Quigley, Pepe, and Fisher (1990, Biometrics 46, 33-48), is investigated. A reparametrization of the model suggested in this earlier work can be seen to be advantageous in some circumstances. Maximum likelihood estimators, Bayesian estimators, and one-step estimators are considered. The continual reassessment method imposes restrictions on the sample space such that it is not possible for confidence intervals to achieve exact coverage properties, however large a sample is taken. Nonetheless, our simulations, based on a small finite sample of 20, not atypical in studies of this type, indicate that the calculated intervals are useful in most practical cases and achieve coverage very close to nominal levels in a very wide range of situations. The relative merits of the different estimators and their associated confidence intervals, viewed from a frequentist perspective, are discussed.


The efficiency of a weighed log-rank test under a percent error misspecification model for the log hazard ratio.

For comparison of two survival distributions, it is natural to use a weighted log-rank test with weight function given by the log hazard ratio function that is anticipated a priori. This paper investigates the efficiency of this test when the a priori estimate of the log hazard ratio is subject to a specified percentage error. The test is shown to be the maximum efficiency robust test over the class of alternatives in question and a simple expression for the maximum efficiency is established.


Starting D-optimal designs for batch kinetics studies of enzyme-catalyzed reactions in the presence of enzyme deactivation.

This paper describes a strategy for the starting experimental design of experiments required by general research in the field of biochemical kinetics. The type of experiments that qualify for this analysis involve batch reactions catalyzed by soluble enzymes where the activity of the enzyme decays with time. Assuming that the catalytic action of the enzyme obeys a Michaelis-Menten rate expression and that the deactivation of the enzyme follows a first-order decay, the present analysis employs the dimensionless, integrated form of the overall rate expression to obtain a criterion (based on the maximization of the determinant of the derivative matrix) that relates the a priori estimates of the parameters with the times at which samples should be withdrawn from the reacting mixture. The analysis indicates that the initial concentration of substrate should be as large as possible, and that the samples should be taken at times corresponding to substrate concentrations of approximately 2/3, 1/4, and I/epsilon of the initial concentration (where epsilon should be as large as possible).


Marginal models for correlated binary responses with multiple classes and multiple levels of nesting.

A model for correlated binary data is presented. Marginal probabilities and odds ratios are allowed to have general regression structures that include multiple classes and multiple levels of nesting. Estimation is done through the generalized estimating equations approach of Liang and Zeger (1986, Biometrika 73, 13-22). They are contrasted with conditional models and recommendations for choosing between the two are given. Examples from genetic epidemiology are presented.


Maximum likelihood estimation for interval-censored data using a Weibull-based accelerated failure time model.

The accelerated failure time regression model is most commonly used with right-censored survival data. This report studies the use of a Weibull-based accelerated failure time regression model when left- and interval-censored data are also observed. Two alternative methods of analysis are considered. First, the maximum likelihood estimates (MLEs) for the observed censoring pattern are computed. These are compared with estimates where midpoints are substituted for left- and interval-censored data (midpoint estimator, or MDE). Simulation studies indicate that for relatively large samples there are many instances when the MLE is superior to the MDE. For samples where the hazard rate is flat or nearly so, or where the percentage of interval-censored data is small, the MDE is adequate. An example using Framingham Heart Study data is discussed.


On estimating standardized risk differences from odds ratios.

An estimator proposed by Greenland and Holland (1991, Biometrics 47, 319-322) for a standardized risk difference parameter is shown to be a maximum likelihood estimator if the consistent estimator of the common odds ratio is appropriately chosen. The statistical problem under consideration is reparameterized. Likelihood equations are derived.


Carotenoids, retinol, and vitamin E and risk of proliferative benign breast disease and breast cancer.

We investigated the relationship between serum levels of retinol, beta-carotene, alpha-carotene, lycopene, alpha-tocopherol, and gamma-tocopherol as well as intakes of retinol, carotene, and vitamin E and the risks of breast cancer and proliferative benign breast disease (BBD) in a case-control study of postmenopausal women in the Boston, MA (United States) area. Serum nutrient data were available for 377 women with newly diagnosed stage I or II breast cancer and 173 women with proliferative BBD. Controls were 403 women who were evaluated at the same institutions but did not require a breast biopsy or whose biopsy revealed nonproliferative BBD. We observed no significant associations between serum levels of these micronutrients and risk of proliferative BBD or breast cancer. The risk of breast cancer was decreased among women in the highest quintile of intake of vitamin E from food sources only (odds ratio [OR] for the highest quintile = 0.4, 95 percent confidence interval [CI] = 0.2-0.9; P, trend across quintiles = 0.02) but less so for total vitamin E intake including supplements (OR = 0.7, CI = 0.4-1.3; P, trend = 0.07).


A theory of site distribution of melanomas: Queensland, Australia.

Although sun exposure is believed to be associated causally with cutaneous melanoma, the high incidence on less sun-exposed areas such as the back, as well as on chronically exposed sites such as the face, suggests that the association with sunlight is less straightforward than for other skin cancers. To explain this enigmatic site distribution, a theory of site-dependent susceptibility of melanocytes to malignant transformation is proposed. As possible evidence, all melanomas diagnosed in the state of Queensland, Australia, over a one-year period were surveyed for histologic evidence of benign melanocytic nevus cells adjacent to the melanoma, and analyzed according to anatomic distribution. Results showed a regional variation in the proportion of melanomas with adjacent nevi not explicable by regional variation in nevus density, which suggests that there is a varying susceptibility of nevi to malignant change. Given that nevus cells are equivalent to melanocytes, this finding would support the hypothesis that melanocytes at-large have a differential response to the mitogenic stimulus of sunlight according to anatomic site.


Atypical hyperplasia and breast cancer risk: a critique.

The purpose of this paper is to examine critically the evidence that atypical hyperplasia (AH) is a risk factor for breast cancer. First, we appraised studies that have examined the association between AH and breast cancer risk for their adherence to widely accepted standards for the conduct of research. Second, we examined the available evidence to determine the plausibility of an association between AH and breast cancer risk using the guidelines proposed by Bradford Hill. A total of 18 studies (11 cohort studies, two case-control studies, and five cross-sectional studies) were found that were published in the English language from January 1960 to March 1992 that examined the association of AH as a distinct entity and breast cancer risk. A systematic approach was adopted to examine the collected studies for their adherence to methodologic standards, which showed wide variation among studies. A meta-analysis was carried out, based on a total sample size of 182,980 women. Of 16 studies that gave point estimates of risk, 14 exceeded unity and 12 were significantly different from unity. The pooled estimate from all studies of the association between AH and breast cancer, gave an overall odds ratio (OR) of 3.67 (95 percent confidence interval = 3.16-4.26). The test of the hypothesis of homogeneous association was rejected (chi 2 = 151.6, df = 14, P < 0.0001), indicating significant variability among the ORs of individual studies. The conclusions from the application of the Bradford Hill criteria indicated strongly that AH is a risk factor for breast cancer.


Childhood cancer among Swedish twins.

The risk of childhood cancer was evaluated among 35,582 twins born in Sweden between 1952 and 1967. Cancers were identified through linkage with national cancer and mortality registries. Overall, 59 childhood cancers and 41 deaths from cancer occurred before the age of 16. For both sexes combined, the cancer incidence was similar to that in the general population of primarily single-born children (number observed/number expected [O/E] = 1.0, 95 percent confidence interval [CI] = 0.7-1.2). For males under age five, cancer incidence was reduced significantly (O/E = 0.3, CI = 0.1-0.7). There was a substantial increase in all-cause mortality among twins (O/E = 3.7), attributable to a high mortality during the first year of life. Excluding this first year, the O/E for death (all causes) was 1.1 (CI = 1.0-1.3). For cancer mortality of both sexes, the O/E was 0.9 (CI = 0.6-1.2), with no significant reduction of risk in any sex-age group. We conclude that with the possible exception of males aged 0-4 years, the childhood cancer risk of twins appears similar to that of singletons.


Urban-rural differences in the management of breast cancer.

We conducted a population-based study with medical-record review of breast cancer patients as part of a larger study of an oncology outreach program to improve cancer management for rural patients within their community hospital. This analysis compares the breast cancer care received by the rural population before the oncology outreach program with that of the contiguous urban population. All 1986-87 cases in selected Illinois (United States) counties were identified using the Illinois State Cancer Registry. Data were obtained by hospital record audit and physician survey. Case follow-back was 98 percent complete. Rural cases were evaluated separately when they were diagnosed in an urban facility. State-of-the-art management was defined by the 1986-87 Physician Data Query and included diagnostic evaluation, prognostic evaluation, and stage-specific treatment. A summary variable incorporated whether or not the most appropriate management was offered. Compared with urban cases, rural cases diagnosed in rural hospitals were less likely to have staged tumors and more likely to have node dissections. Rural cases traveling to urban centers were less likely to have limited surgery, hormone therapy, and a biopsy as a first-step surgical procedure, and more likely to have node dissection.


Accuracy of death certificates: a population-based, complete-coverage, one-year autopsy study in East Germany.

The Goerlitz Autopsy Study is a population-based autopsy study, conducted in 1987 in the municipality of Goerlitz, population 78,484, in former East Germany. It is unique in that 1,023 (96.5 percent) of the 1,060 subjects who died in the municipality over a period of one year were investigated by full autopsy. An underlying cause was assigned to every death, using the procedures recommended by the International Classification of Diseases, Injuries and Causes of Death, ninth revision, German edition. Assignment of cause of death was done independently by the attending clinician and the pathology team who performed the autopsy. The data were analyzed through cross-classification of deaths by death certificate diagnosis and autopsy-based diagnosis. Sensitivity and positive predictive values were calculated for the death certificate diagnoses, assuming that the autopsy findings represent the correct reference set. Overall, 47 percent of diagnoses on death certificates differed from those based on autopsy and, for 30 percent of the subjects, the difference crossed a major disease category. The proportion of disagreement was higher for deaths occurring in nursing homes and among the very old, but was unrelated to gender. In the death certificates, diseases of the circulatory system and endocrine and metabolic disorders were over-represented, whereas infectious diseases, neoplasms, and respiratory, digestive, and genitourinary diseases were under-represented. Neoplasms in the death certificates are characterized by relatively high sensitivity and predictive values, even though these were still unsatisfactory, given the chronicity of the clinical course and the opportunities available for correct diagnosis.(ABSTRACT TRUNCATED AT 250 WORDS)


Oral contraceptive use and risk of cutaneous malignant melanoma.

The relation between cutaneous malignant melanoma (MM) and the use of oral contraceptives (OC) was investigated in a case-control study carried out from 1979 to 1991 among patients in hospitals and clinics in the Philadelphia (PA) and New York City (NY) metropolitan areas (United States). Cases were 615 women under age 70 who recently had been diagnosed with invasive melanoma; controls were 2,107 women of the same ages who had been treated for other conditions unrelated either to OC use or to skin diseases. The cases were categorized as severe or nonsevere based on the depth of invasion of the tumor or the presence or absence of metastases. Among the severe cases, OC use was not associated with MM: the relative risk (RR) estimate for ever-use was 1.1 (95 percent confidence interval [CI] = 0.8-1.5) and the estimate for 10 or more years of use was 1.1 (CI = 0.6-2.1). Nor was risk associated with recent use, long latency, or young age at first use. Among the nonsevere cases, ever-use of oral contraceptives was associated positively with MM (RR = 1.5, CI = 1.1-2.4) but there was no trend with increased duration of use. The findings provide evidence against the hypothesis that OC use increases the risk of malignant melanoma. The elevated estimates among the nonsevere cases most likely reflect selection bias rather than a causal relation.


Occupational risk factors for multiple myeloma among Danish men.

A large population-based case-control study evaluated occupational exposures in 1,098 Danish males diagnosed with multiple myeloma from 1970 to 1984 and in 4,169 age- and gender-matched controls alive at the time of case-diagnosis. Industrial histories were obtained from the Supplementary Pension Fund which, since 1964, has recorded employments of adult Danes; occupation came from subjects' most recent tax records. Four industrial hygienists created a job-exposure matrix for 47 substances based on 15,000 unique industry/occupation combinations in subjects' histories. Risk of myeloma was significantly elevated among road and railroad workers, precision metalworkers, and workers in the transportation and communication industries. Risk increased significantly with duration of employment in: production of synthetic yarns, plastic packaging, and miscellaneous chemical compounds; fabricating structural metal and stationary tanks; body factories; electrical plants; and retail sale of paint and wallpaper. Risks of myeloma were elevated, though statistically nonsignificantly, in all categories of exposure to gasoline and engine exhausts. Risks rose with likelihood and duration of exposure to phthalates, and were statistically significant and nearly fivefold with probable exposure to vinyl chloride for five or more years. After adjusting for multiple exposures and disregarding exposures within 10 years of diagnosis (or selection as a control), probable exposure to vinyl chloride was associated with increased risk of myeloma, which rose to fivefold with longer exposure. Associations with gasoline, engine exhausts, and phthalates persisted, but were inconsistent with duration and probability of exposure. Previously reported associations with agriculture were not confirmed by these data.


Computer modeling of fibrin polymerization kinetics correlated with electron microscope and turbidity observations: clot structure and assembly are kinetically controlled.

Although much is known about fibrin polymerization, because it is complex, the effects of various modifications are not intuitively obvious and many experimental observations remain unexplained. A kinetic model presented here that is based on information about mechanisms of assembly accounts for most experimental observations and allows hypotheses about the effects of various factors to be tested. Differential equations describing the kinetics of polymerization were written and then solved numerically. The results have been related to turbidity profiles and electron microscope observations. The concentrations of intermediates in fibrin polymerization, and fiber diameters, fiber and protofibril lengths have been calculated from these models. The simplest model considered has three steps; fibrinopeptide A cleavage, protofibril formation, and lateral aggregation of protofibrils to form fibers. The average number of protofibrils per fiber, which is directly related to turbidity, can be calculated and plotted as a function of time. The lag period observed in turbidity profiles cannot be accurately simulated by such a model, but can be simulated by modifying the model such that oligomers must reach a minimum length before they aggregate. Many observations, reported here and elsewhere, can be accounted for by this model; the basic model may be modified to account for other experimental observations. Modeling predicts effects of changes in the rate of fibrinopeptide cleavage consistent with electron microscope and turbidity observations. Changes only in the rate constants for initiation of fiber growth or for addition of protofibrils to fibers are sufficient to account for a wide variety of other observations, e.g., the effects of ionic strength or fibrinopeptide B removal or thrombospondin. The effects of lateral aggregation of fibers has also been modeled: such behavior has been observed in turbidity curves and electron micrographs of clots formed in the presence of platelet factor 4. Thus, many aspects of clot structure and factors that influence structure are directly related to the rates of these steps of polymerization, even though these effects are often not obvious. Thus, to a large extent, clot structure is kinetically determined.


Brain stimulation using electromagnetic sources: theoretical aspects.

We prove that, at the frequencies generally proposed for extracranial stimulation of the brain, it is not possible, using any superposition of external current sources, to produce a three-dimensional local maximum of the electric field strength inside the brain. The maximum always occurs on a boundary where the conductivity jumps in value. Nevertheless, it may be possible to achieve greater two-dimensional focusing and shaping of the electric field than is currently available. Towards this goal we have used the reciprocity theorem to present a uniform treatment of the electric field inside a conducting medium produced by a variety of sources: an external magnetic dipole (current loop), an external electric dipole (linear antenna), and surface and depth electrodes. This formulation makes use of the lead fields from magneto- and electroencephalography. For the special case of a system with spherically symmetric conductivity, we derive a simple analytic formula for the electric field due to an external magnetic dipole. This formula is independent of the conductivity profile and therefore embraces spherical models with any number of shells. This explains the "insensitivity" to the skull's conductivity that has been described in numerical studies. We also present analytic formulas for the electric field due to an electric dipole, and also surface and depth electrodes, for the case of a sphere of constant conductivity.


Gating of mammalian cardiac gap junction channels by transjunctional voltage.

Numerous two-cell voltage-clamp studies have concluded that the electrical conductance of mammalian cardiac gap junctions is not modulated by the transjunctional voltage (Vj) profile, although gap junction channels between low conductance pairs of neonatal rat ventricular myocytes are reported to exhibit Vj-dependent behavior. In this study, the dependence of macroscopic gap junctional conductance (gj) on transjunctional voltage was quantitatively examined in paired 3-d neonatal hamster ventricular myocytes using the double whole-cell patch-clamp technique. Immunolocalization with a site-specific antiserum directed against amino acids 252-271 of rat connexin43, a 43-kD gap junction protein as predicted from its cDNA sequence, specifically stained zones of contact between cultured myocytes. Instantaneous current-voltage (Ij-Vj) relationships of neonatal hamster myocyte pairs were linear over the entire voltage range examined (0 less than or equal to Vj less than or equal to +/- 100 mV). However, the steady-state Ij-Vj relationship was nonlinear for Vj greater than +/- 50 mV. Both inactivation and recovery processes followed single exponential time courses (tau inactivation = 100-1,000 ms, tau recovery approximately equal to 300 ms). However, Ij recovered rapidly upon polarity reversal. The normalized steady-state junctional conductance-voltage relationship (Gss-Vj) was a bell-shaped curve that could be adequately described by a two-state Boltzmann equation with a minimum Gj of 0.32-0.34, a half-inactivation voltage of -69 and +61 mV and an effective valence of 2.4-2.8. Recordings of gap junction channel currents (ij) yielded linear ij-Vj relationships with slope conductances of approximately 20-30 and 45-50 pS. A kinetic model, based on the Boltzmann relationship and the polarity reversal data, suggests that the opening (alpha) and closing (beta) rate constants have nearly identical voltage sensitivities with a Vo of +/- 62 mV. The data presented in this study are not consistent with the contingent gating scheme (for two identical gates in series) proposed for other more Vj-dependent gap junctions and alternatively suggest that each gate responds to the applied Vj independently of the state (open or closed) of the other gate.


A quasi-elastic light scattering study of smooth muscle myosin in the presence of ATP.

We have investigated the hydrodynamic properties of turkey gizzard smooth muscle myosin in solution using quasi-elastic light scattering (QELS). The effects of ionic strength (0.05-0.5 M KCl) and light chain phosphorylation on the conformational transition of myosin were examined in the presence of ATP at 20 degrees C. Cumulant analysis and light scattering models were used to describe the myosin system in solution. A nonlinear least squares fitting procedure was used to determine the model that best fits the data. The conformational transition of the myosin monomer from a folded form to an extended form was clearly demonstrated in a salt concentration range of 0.15-0.3 M KCl. Light chain phosphorylation regulates the transition and promotes unfolding of the myosin. These results agree with the findings obtained using sedimentation velocity and electron microscopy (Onishi and Wakabayashi, 1982; Trybus et al., 1982; Trybus and Lowey, 1984). In addition, we present evidence for polymeric myosin coexisting with the two monomeric myosin species over a salt concentration range from 0.05 to 0.5 M KCl. The size of the polymeric myosin varied with salt concentration. This observation supports the hypothesis that, in solution, a dynamic equilibrium exists between the two conformations of myosin monomer and filaments.


Chain-length dependence of lipid bilayer properties near the liquid crystal to gel phase transition.

The temperature dependence of the mean orientational order parameter in the vicinity of the liquid crystal to gel phase transition is obtained from the first moment M1 of deuterium nuclear magnetic resonance spectra for bilayers of chain perdeuterated phosphatidylcholines with acyl chains of 12, 14, 16, and 18 carbons. The data clearly show an increasing temperature dependence of the orientational order parameter in the vicinity of the transition, with the effect becoming more pronounced with decreasing chain length. Assuming a linear relationship between the mean orientational order parameter and the extension of the acyl chain, estimates of the change in area of the membrane at the transition are shown to be consistent with those obtained from other measurements. It is shown that the transition may be modeled in terms of a Landau expansion of the free energy involving a small number of phenomenological parameters. From this it is shown that the behavior of these systems in the temperature range of interest is, in large part, controlled by the close proximity of a spinodal to the transition temperature.


Resonance Raman spectroscopic and kinetic consequences of a nitrogen ... sulphur enzyme-substrate contact in a series of dithioacylpapains.

The resonance Raman (RR) spectroscopic, conformational, and kinetic properties of six dithioacylpapain intermediates have been examined. Five of the intermediates are of the form N-(methyloxycarbonyl)-X-glycine-C(= S)S-papain, where X is L-phenyl-alanine, D-phenylalanine, glycine, L-phenylglycine, or D-phenylglycine. The sixth intermediate is N-phenylacetyl-glycine-C(= S)S-papain. Throughout the series there is an approximately 50-fold variation in kcat, the rate constant for deacylation, and a 1750-fold variation in kcat/KM. Existing RR spectra structure correlations allow us to define the torsional angles in the NH-CH2-C(= S)-S-CH2-CH fragment of the functioning intermediates. The values of these angles for each bound substrate appear to be very similar, with the substrates assuming a B-type conformer such that the nitrogen atom of the P1 glycine residue is cis to the thiol sulphur atom of cysteine-25. For each intermediate, the C(= S)S-CH2CH torsional angle is approximately -90 degrees, whereas for the SCH2-CH torisonal angle the cysteine-25 thiol sulphur (S) and cysteine-25 C alpha hydrogen (H) atoms are approximately trans. The three acyl-enzymes with the lowest catalytic rate constants, viz. N-(methyloxycarbonyl)-glycine-glycine-, N-(methyloxycarbonyl)-L-phenylglycine-glycine-, or N-(phenylacetyl)-glycine-dithioacylpapains, have atypical RR spectra in that they show a feature of medium intensity in the 1,085-cm-1 region. This band is sensitive to NH to ND exchange of the P1 glycine residues' (-NH-) function and, thus, the corresponding mode involves an excursion of the NH hydrogen. It is hypothesized that the high intensity is due to a particularly strong interaction between the P1 glycine nitrogen atom and the thiol sulphur of cysteine-25, which also has the effect of retarding deacylation, because the nitrogen . . . sulphur contact has to be broken in the rate-determining step.


Pulmonary surfactant protein SP-C causes packing rearrangements of dipalmitoylphosphatidylcholine in spread monolayers.

The hydrophobic pulmonary surfactant protein SP-C has been isolated from porcine lung surfactant, and it has been incorporated into monolayers of dipalmitoylphosphatidylcholine (DPPC). The monolayers, which contained 1 mol% of a fluorescently-labeled phosphatidylcholine, were observed under various states of compression in an epifluorescence surface balance. SP-C altered the packing arrangements of DPPC in the monolayer, causing the production of many more, smaller condensed lipid domains in its presence than in its absence.


Monomer diffusion and polymer alignment in domains of sickle hemoglobin.

We have used polarized absorbance to observe the process of monomer accretion and polymer alignment which occurs in domains of sickle hemoglobin that are formed and maintained by laser photolysis. These diffusion and alignment processes have been studied as a function of initial concentration and temperature (initial and final), as well as beam size and domain number. Monomers are found to diffuse into growing polymer domains with a rate that is essentially temperature and concentration independent, but which depends on the size of the final domain boundaries, and the number of domains within a boundary. The final concentrations achieved are very close to those found in packed centrifugation experiments (50-55 g/dl) and are approximately independent of starting temperature and concentration. The influx of monomers is accompanied by polymer alignment, and the amount aligned is proportional to the amount diffused throughout the process. We propose that polymer alignment controls the influx of added monomers into the growing domain.


Binding kinetics of an anti-dinitrophenyl monoclonal Fab on supported phospholipid monolayers measured by total internal reflection with fluorescence photobleaching recovery.

Fluorescence photobleaching recovery with total internal reflection illumination (TIR-FPR) has been used to measure the dissociation kinetics of a fluorescein-labeled anti-dinitrophenyl monoclonal Fab specifically bound to supported monolayers composed of a mixture of dipalmitoylphosphatidylcholine and dinitrophenyl-conjugated dipalmitoylphosphatidylethanolamine. The fluorescence recovery curves were not monoexponential; when analyzed as a sum of two exponentials, the rates and fractional recoveries were approximately 1 s-1 (approximately 50%) and approximately 0.1 s-1 (approximately 30%). The data did not change as a function of the Fab solution concentration, indicating that the fluorescence recovery curves were not influenced by the rate of diffusion in bulk solution. Also, the recovery curves were independent of the size of the illuminated area, indicating that surface diffusion did not significantly contribute to the rate and shape of the fluorescence recovery. The measured off rates and apparent association constant (1.6 x 10(5) M-1) were analyzed with the theoretical formalism for a proposed mechanism that accounts for the nonmonoexponential kinetics.


A membrane model for cytosolic calcium oscillations. A study using Xenopus oocytes.

Cytosolic calcium oscillations occur in a wide variety of cells and are involved in different cellular functions. We describe these calcium oscillations by a mathematical model based on the putative electrophysiological properties of the endoplasmic reticulum (ER) membrane. The salient features of our membrane model are calcium-dependent calcium channels and calcium pumps in the ER membrane, constant entry of calcium into the cytosol, calcium dependent removal from the cytosol, and buffering by cytoplasmic calcium binding proteins. Numerical integration of the model allows us to study the fluctuations in the cytosolic calcium concentration, the ER membrane potential, and the concentration of free calcium binding sites on a calcium binding protein. The model demonstrates the physiological features necessary for calcium oscillations and suggests that the level of calcium flux into the cytosol controls the frequency and amplitude of oscillations. The model also suggests that the level of buffering affects the frequency and amplitude of the oscillations. The model is supported by experiments indirectly measuring cytosolic calcium by calcium-induced chloride currents in Xenopus oocytes as well as cytosolic calcium oscillations observed in other preparations.


Simulations of the temperature dependence of energy transfer in the PSI core antenna.

In order to understand the organization of the PSI core antenna and to interpret results obtained from studies of the temperature and wavelength dependence of energy transfer and trapping in the PSI particles, we have constructed a model for PSI in which spectral heterogeneity is considered via a self-consistent approach based on Forster transport. The temperature dependence of the absorption and emission spectra of the individual Chl molecules in the protein matrix is calculated based on a model Hamiltonian which includes a phonon contribution. Time and wavelength resolved kinetics of PSI at different temperatures are investigated by means of two-dimensional lattice models. We conclude that wavelength-dependent fluorescence decay kinetics result only when two or more bottlenecks exist in the energy transfer and trapping process. A single trap or several pseudo-traps with spectrally identical environments do not lead to wavelength dependent decays. Simple funnel arrangements of the spectral types can be ruled out. At least one pigment with energy lower than the photochemical trap located close to the reaction center is required to produce the trends of the fluorescence lifetimes observed experimentally. The remainder of the core antenna is consistent with a random arrangement of spectral types.


Study of mechanisms of electric field-induced DNA transfection. III. Electric parameters and other conditions for effective transfection.

Electric parameters, osmolality, temperature, and pH of the suspending medium and the growth phase of cells, etc., are known to influence the efficiency of the pulsed electric field (PEF)-induced DNA transfection of cells. PEF-induced transfection of Escherichia coli JM105 by plasmid DNA PUC18, PUC19, PBR322, and PMSG has been used as a model system to establish quantitative relationships between these parameters and transfection efficiency. The main findings are summarized for experiments using unipolar square wave PEF. (a) For a given field strength (up to 6 kV/cm), the transfection efficiency (TE) was linearly dependent on the pulse width (up to 1 ms). (b) When field strength is fixed, Log [TE] correlated with the number of pulses applied. Similarly, when field duration was fixed, Log [TE] correlated with the number of pulses. (c) In the absence of MgCl2, TE showed a maximal value at 50 mM sucrose and was reduced by several fold at lower and higher sucrose concentrations. Cell survival was nearly constant in the range 1-300 mM sucrose. (d) E. coli in the early and mid-exponential growth phases was more susceptible to PEF for DNA transfection than it was in the stationary phase. (e) For a given set of electric parameters, TE was the highest at neutral pH and was greatly reduced at acidic and alkaline pH. (f) Increasing the temperature from 0 to 37 degrees C resulted in the reduction of TE by three orders of magnitude. This could reflect a rapid shrinking of pores at higher temperatures. (g) TE was inversely proportional to the square of the size of the plasmid DNA. By adjusting the above parameters to optimize transfection, a TE of 1010 1microg-1 DNA (PUC18) has been recorded. Further improvement in percent cell transfection may be expected by a more exhaustive search of conditions than the present study has done.


Folding kinetics of designer proteins. Application of the diffusion-collision model to a de novo designed four-helix bundle.

A folding algorithm is described, based on the diffusion-collision model, combining static and dynamic calculational methods. The algorithm is applied to predict the basic structure and schematic folding pathways of an artificial four-helix bundle.


Effect of small release on force during sarcomere-isometric tetani in frog muscle fibers.

We investigated the effect of small shortening imposed on frog muscle fibers during sarcomere-isometric tetani. Sarcomere length was initially kept constant, then slightly shortened (1%-5% of initial length) and clamped again for the remainder of the tetanus. Force level after the shortening was higher than the force level preceding the release. The size of the increase was larger than that predicted by the descending limb of the linear force-length relation. The difference between measured and predicted force levels increased with sarcomere length. At a sarcomere length of 3.2 microns, the force level after the shortening was higher by 50% than the force level expected from the linear descending limb. Dispersion of sarcomere-length within the sampled region was measured by two independent methods: striation imaging and analysis of the intensity profile of the first diffraction order. Sarcomere-length inhomogeneity in the sampled region was too small (standard deviation from the average sarcomere-length was +/- 0.03 microns) to account for the size of the increase in force. We studied the dependence of increase in tetanic force level after small sarcomere-length release on the size, velocity and timing of the release, as well as on initial sarcomere-length. Release size was the major determinant of the amount of increase in force. Release of 20 nm per half sarcomere was sufficient to produce an almost full force increase. Larger releases increased the force only moderately. Over the range studied, release velocity and timing had little or no effect.


Transport methods for probing the barrier domain of lipid bilayer membranes.

Two experimental techniques have been utilized to explore the barrier properties of lecithin/decane bilayer membranes with the aim of determining the contributions of various domains within the bilayer to the overall barrier. The thickness of lecithin/decane bilayers was systematically varied by modulating the chemical potential of decane in the annulus surrounding the bilayer using different mole fractions of squalene in decane. The dependence of permeability of a model permeant (acetamide) on the thickness of the solvent-filled region of the bilayer was assessed in these bilayers to determine the contribution of this region to the overall barrier. The flux of acetamide was found to vary linearly with bilayer area with Pm = (2.9 +/- 0.3) x 10(-4) cm s-1, after correcting for diffusion through unstirred water layers. The ratio between the overall membrane permeability coefficient and that calculated for diffusion through the hydrocarbon core in membranes having maximum thickness was 0.24, suggesting that the solvent domain contributes only slightly to the overall barrier properties. Consistent with these results, the permeability of acetamide was found to be independent of bilayer thickness. The relative contributions of the bilayer interface and ordered hydrocarbon regions to the transport barrier may be evaluated qualitatively by exploring the effective chemical nature of the barrier microenvironment. This may be probed by comparing functional group contributions to transport with those obtained for partitioning between water and various model bulk solvents ranging in polarity or hydrogen-bonding potential. A novel approach is described for obtaining group contributions to transport using ionizable permeants and pH adjustment. Using this approach, bilayer permeability coefficients of p-toluic acid and p-hydroxymethyl benzoic acid were determined to be 1.1 +/- 0.2 cm s-1 and (1.6 +/- 0.4) x 10(-3) cm s-1, respectively. From these values, the -OH group contribution to bilayer transport [delta(delta G0-OH)] was found to be 3.9 kcal/mol. This result suggests that the barrier region of the bilayer does not resemble the hydrogen-bonding environment found in octanol, but is somewhat less selective (more polar) than a hydrocarbon solvent.


Indo-1 binding to protein in permeabilized ventricular myocytes alters its spectral and Ca binding properties.

We have examined the binding of the fluorescent Ca indicator indo-1 to cellular protein in permeabilized ventricular myocytes and also to soluble and particulate myocyte protein. Using either a filtration technique or equilibrium dialysis, and conditions similar to those in a cardiac myocyte patch clamped with 100 microM indo-1 in the patch pipette, we found that 72% of the total indo-1 was bound to myocyte protein at a protein concentration of 100 mg/ml. This corresponds to a binding of 3.8 +/- 0.5 nmol indo-1/mg protein. Separation of the myocyte protein into a soluble and a particulate fraction showed that 63% of the bound indo-1 was bound to soluble protein, corresponding to a binding of 3.22 +/- 0.99 nmol/mg, whereas 37% of the bound indo-1 was bound to particulate protein (0.85 +/- 0.14 nmol/mg) at a low [Ca] (pCa approximately 9). Binding of indo-1 in permeabilized myocytes was approximately 60% higher at a saturating Ca concentration (pCa = 3), than under Ca free conditions (1 mM EGTA). Simultaneous measurements of free [Ca] with a Ca selective electrode and indo-1 fluorescence showed that, the dissociation constant (Kd) for Ca was increased 4-5 fold in the presence of permeabilized myocytes as compared to the value obtained in vitro. In agreement with the binding experiments we estimate that the true Kd and the apparent Kd (using ratiometric measurements) for Ca binding to indo-1 are increased approximately four fold, at a myocyte protein concentration of 100 mg/ml.


Implications of epidermal growth factor (EGF) induced egf receptor aggregation.

To investigate the role of receptor aggregation in EGF binding, we construct a mathematical model describing receptor dimerization (and higher levels of aggregation) that permits an analysis of the influence of receptor aggregation on ligand binding. We answer two questions: (a) Can Scatchard plots of EGF binding data be analyzed productively in terms of two noninteracting receptor populations with different affinities if EGF induced receptor aggregation occurs? No. If two affinities characterize aggregated and monomeric EGF receptors, we show that the Scatchard plot should have curvature characteristic of positively cooperative binding, the opposite of that observed. Thus, the interpretation that the high affinity population represents aggregated receptors and the low affinity population nonaggregated receptors is wrong. If the two populations are interpreted without reference to receptor aggregation, an important determinant of Scatchard plot shape is ignored. (b) Can a model for EGF receptor aggregation and EGF binding be consistent with the "negative curvature" (i.e., curvature characteristic of negatively cooperative binding) observed in most Scatchard plots of EGF binding data? Yes. In addition, the restrictions on the model parameters required to obtain negatively curved Scatchard plots provide new information about binding and aggregation. In particular, EGF binding to aggregated receptors must be negatively cooperative, i.e., binding to a receptor in a dimer (or higher oligomer) having one receptor already bound occurs with lower affinity than the initial binding event. A third question we consider is whether the model we present can be used to detect the presence of mechanisms other than receptor aggregation that are contributing to Scatchard plot curvature. For the membrane and cell binding data we analyzed, the best least squares fits of the model to each of the four data sets deviate systematically from the data, indicating that additional factors are also important in shaping the binding curves. Because we have controlled experimentally for many sources of receptor heterogeneity, we have limited the potential explanations for residual Scatchard plot curvature.


Atomic force microscopy of three-dimensional membrane protein crystals. Ca-ATPase of sarcoplasmic reticulum.

We have observed three-dimensional crystals of the calcium pump from sarcoplasmic reticulum by atomic force microscopy (AFM). From AFM images of dried crystals, both on graphite and mica, we measured steps in the crystal thickness, corresponding to the unit cell spacing normal to the substrate. It is known from transmission electron microscopy that crystal periodicity in the plane of the substrate is destroyed by drying, and it was therefore not surprising that we were unable to observe this periodicity by AFM. Thus, we were motivated to use the AFM on hydrated crystals. In this case, crystal adsorption appeared to be a limiting factor, and our studies indicate that adsorption is controlled by the composition of the medium and by the physical-chemical properties of the substrate. We used scanning electron microscopy to determine the conditions yielding the highest adsorption of crystals, and, under these conditions, we have obtained AFM images of hydrated crystals with a resolution similar to that observed with dried samples (i.e., relatively poor). In the same preparations, we have observed lipid bilayers with a significantly better resolution, indicating that the poor quality of crystal images was not due to instrumental limitations. Rather, we attribute poor images to the intrinsic flexibility of these multilamellar crystals, which apparently allow movement of one layer relative to another in response to shear forces from the AFM tip. We therefore suggest some general guidelines for future studies of membrane proteins with AFM.


Nonmonotonic alterations in the fluorescence anisotropy of polar head group labeled fluorophores during the lamellar to hexagonal phase transition of phospholipids.

The temperature dependence of the fluorescence anisotropy of polar head group labeled fluorophores (i.e., N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)dipalmitoyl-L- alpha-phosphatidylethanolamine or N-(lissamine rhodamine B sulfonyl)dipalmitoyl-L-alpha-phosphatidylethanol- amine) incorporated into multiple phosphatidylethanolamine molecular species was parabolic, possessing minima (dr/dT = 0) that precisely correlated with the respective lamellar (L alpha) to hexagonal (HII) phase transition temperature of each species. The parabolic alterations in the thermotropic behavior of these fluorophores were due to increased motional constraints in the polar head group region during heating (dr/dT greater than 0), because significant alterations in the fluorescence lifetimes of these probes during the phase transition did not occur. The sensitivity inherent in identification of peak minima was exploited to determine the lamellar to hexagonal phase transition temperatures of several homogeneous molecular species of plasmenylethanolamine (e.g., the transition temperature of 1-O-(Z)-hexadec-1'-enyl-2-octadec-9'- enoyl-sn-glycero-3-phosphoethanolamine was 28 degrees C). Experiments using ethanolamine glycerophospholipids containing either an ester or a vinyl ether linkage at the sn-1 position demonstrated that introduction of the vinyl ether constituent increased the propensity of these species to assume the hexagonal phase. Collectively, these results identify and substantiate a new technique for the characterization of the lamellar to hexagonal phase transition in phospholipids that requires only small amounts of phospholipids present in dilute membrane suspensions.


Optical and physical properties of wet-spun films of Na-hyaluronate. Evidence of a phase transition.

The refractive indices, water content, and volume of wet-spun films of Na-hyaluronate have been measured as a function of relative humidity (rh). These data are used with the Lorentz-Lorenz formula to determine the optical polarizabilities of Na-hyaluronate parallel and perpendicular to the helical axis. The analysis reveals a drop in the optical polarizabilities of approximately 20% between 80 and 88% rh, indicating a phase transition.


Formation of a new stable phase of phosphatidylglycerols.

Dilauroyl and dimyristoylphosphatidylglycerol (DMPG) form a more stable gel state when aqueous suspensions are incubated several days at low temperature (0-2 degrees C), pH 7.4 with 0.15 M NaCl. This gel state is characterized by a higher transition temperature and a higher transition enthalpy. The geometry of this gel state is distinguishable from the metastable gel state that forms rapidly upon hydration on the basis of its x-ray diffraction pattern. Infrared spectra in the CH2 scissoring region indicate that the stable gel phase of DMPG is also characterized by reduced reorientational fluctuations of acyl chains and increased interchain interactions. Analysis of vibrational bands due to ester carbonyl groups of DMPG suggests that the transition to a new gel phase is initiated by changes in the interfacial and/or headgroup region of the bilayer, most likely via formation of interlipid hydrogen bonds. The melting of the stable gel phase of DMPG is accompanied by a gross morphological change resulting in vesiculation.


Techniques for obtaining analytical solutions to the multicylinder somatic shunt cable model for passive neurones.

The somatic shunt cable model for neurones is extended to the case in which several equivalent cylinders, not necessarily of the same electrotonic length, emanate from the cell soma. The cable equation is assumed to hold in each cylinder and is solved with sealed end conditions and a lumped soma boundary condition at a common origin. A Green's function (G) is defined, corresponding to the voltage response to an instantaneous current pulse at an arbitrary point along one of the cylinders. An eigenfunction expansion for G is obtained where the coefficients are determined using the calculus of residues and compared with an alternative method of derivation using a modified orthogonality condition. This expansion converges quickly for large time, but, for small time, a more convenient alternative expansion is obtained by Laplace transforms. The voltage response to arbitrary currents injected at arbitrary sites in the dendritic tree (including the soma) may then be expressed as a convolution integral involving G. Illustrative examples are presented for a point charge input.


Long tail kinetics in biophysics?

Long tail kinetics describe a variety of data from complex, disordered materials that cannot be described by conventional kinetics. It is suggested that the kinetics of diffusive motion in complex biological media, such as cytoplasm or biomembranes, might also have long tails. The effects of long tail kinetics are investigated for two standard biophysical measurements, fluorescence recovery after photobleaching (FRAP), and dynamic light scattering (DLS). It is shown that long tail kinetic data would yield significantly distorted and misleading results when analyzed assuming conventional kinetics.


Analysis of electric field stimulation of single cardiac muscle cells.

Electrical stimulation of cardiac cells by imposed extracellular electric fields results in a transmembrane potential which is highly nonuniform, with one end of the cell depolarized and the other end hyperpolarized along the field direction. To date, the implications of the close proximity of oppositely polarized membranes on excitability have not been explored. In this work we compare the biophysical basis for field stimulation of cells at rest with that for intracellular current injection, using three Luo-Rudy type membrane patches coupled together as a lumped model to represent the cell membrane. Our model shows that cell excitation is a function of the temporal and spatial distribution of ionic currents and transmembrane potential. The extracellular and intracellular forms of stimulation were compared in greater detail for monophasic and symmetric biphasic rectangular pulses, with duration ranging from 0.5 to 10 ms. Strength-duration curves derived for field stimulation show that over a wide range of pulse durations, biphasic waveforms can recruit and activate membrane patches about as effectively as can monophasic waveforms having the same total pulse duration. We find that excitation with biphasic stimulation results from a synergistic, temporal summation of inward currents through the sodium channel in membrane patches at opposite ends of the cell. Furthermore, with both waveform types, a net inward current through the inwardly rectifying potassium channel contributes to initial membrane depolarization. In contrast, models of stimulation by intracellular current injection do not account for the nonuniformity of transmembrane potential and produce substantially different (even contradictory) results for the case of stimulation from rest.


Evidence for structurally different attached states of myosin cross-bridges on actin during contraction of fish muscle.

Using data from fast time-resolved x-ray diffraction experiments on the synchrotrons at Daresbury and (Deutsches Elektronen Synchrotron [DESY]), it is shown that during contraction of fish muscle there are at least two distinct configurations of myosin cross-bridges on actin, that they appear to have different tension producing properties and that they probably differ in the axial tilt of the cross-bridges on actin. Evidence is presented for newly observed myosin-based layer lines in patterns from active fish muscle, together with intensity changes of the actin layer lines. On the equator, the 110 reflection changes much faster (time for 50% change t1/2 = 21 +/- 4 ms after activation) than the 100 reflection (t1/2 = 35 +/- 8 ms) and tension (t1/2 = 41 +/- 3 ms) during the rising phase of tetanic contractions. These and higher order reflections have been used to show the time course of mass attachment at actin during this rising phase. Mass arrival (t1/2 = 25 ms) precedes tension by approximately 15 ms. Analysis has been carried out to evaluate the effects of changes in sarcomere length during the tetanus. It is shown that any such effects are very small. Difference "equatorial" electron density maps between active muscle at a time when mass arrival at actin is just complete, but the tension is still rising, and at a later time well into the tension plateau, show that the structural difference between the lower and higher force states corresponds to mass movement consistent with axial swinging of heads from a nonstereospecific actin attached state (low force) to a more stereospecific (high force) state.


Virtual intermediates in photosynthetic electron transfer.

We explore the possibility of virtual transfer in the primary charge separation of photosynthetic bacteria within the context of several types of experimental data. We show that the peak that might be expected in the virtual rate as electric fields vary the intermediate state energy is severely broadened by coupling to high-frequency modes. The Stark absorption kinetics data are thus consistent with virtual transfer in the primary charge separation. High-frequency coupling also makes the temperature dependence weak over a wide range of parameters. We demonstrate that Stark fluorescence anisotropy data, usually taken as evidence of virtual transfer, can in fact be consistent with two-step transfer. We suggest a two-pulse excitation experiment to quantify the contributions from two-step and virtual transfer. We show that virtual absorption into a charge transfer state can make a substantial contribution to the Stark absorption spectrum in a way that is not related to any derivative of the absorption spectrum.


Formation of a square-planar Co(I) B12 intermediate. Implications for enzyme catalysis.

X-ray edge and extended x-ray absorption fine structure (EXAFS) techniques provide powerful tools for analysis of local molecular structure of complexes in solution. We present EXAFS results for Co(I) B12 that demonstrate a four-coordinate (distorted) square-planar configuration. Comparison of EXAFS solutions for Co(I) and Co(II) B12 (collected previously; Sagi et al. 1990. J. Am. Chem. Soc. 112:8639-8644) suggest that modulation of the Co-N bond to the axial 5,6-dimethylbenzimidazole (DMB), in the absence of changes in Co-N (equatorial) bond distances, may be a key mechanism in promoting homolytic versus heterolytic cleavage. As Co-C bond homolysis occurs, the Co-N (DMB) bond becomes stronger. However, for heterolytic cleavage to occur, earlier electrochemical studies (D. Lexa and J. M. Saveant. 1976. J. Am. Chem. Soc. 98:2652-2658) and recent studies of methylcobalamin-dependent Clostridium thermoaceticum (Ragsdale et al. 1987. J. Biol. Chem. 262:14289-14297) suggest that removal of the DMB ligand (before Co-C bond cleavage) favors formation of the four-coordinate square-planar Co(I) species while inhibiting formation of the five-coordinate Co(II) B12 complex. This paper presents the first direct evidence that formation of the Co(I) B12 intermediate must involve breaking of the Co-N (DMB) bond.


Influence of the membrane surface on glycolipid conformation and dynamics. An interpretation of NMR results using conformational energy calculations.

Glycolipids constitute an important class of biomolecules that are involved in biomolecular recognition. The importance of carbohydrate head group conformation in such processes is well recognized. Glycolipids typically occur as minor components of the complex heterogeneous matrix of a biological membrane. As a result, the membrane surface may not only influence head group conformation but also serves as a spatial frame in which the glycolipid is oriented and recognized. In this study, conformational energy calculations have been used to assess the conformational space available to the glucose head group of 1,2-di-O-tetradecyl-3-O-(beta-D-glucopyranosyl)-sn-glycerol (beta-DTGL) in a liquid-crystalline membrane matrix. 2H NMR quadrupolar splittings are calculated and compared with those observed experimentally. This study demonstrates the importance of including surface interactions when considering the conformational space accessible to cell surface carbohydrates. The empirical approach taken here provides considerable insight at the molecular level, and offers the possibility of exploring even more complex systems.


Intermediates and kinetics of membrane fusion.

Recently, it has become clear that the influenza virus fusion protein, hemagglutinin (HA), produces membrane destabilization and fusion by a multistep process, which involves the aggregation of the HAs to form a fusion site. While the details of this process are under debate, it is important to recognize that proposing any sequence of "microscopic" fusion intermediates encumbers general "macroscopic" kinetic consequences, i.e., with respect to membrane mixing rates. Using a kinetic scheme which incorporates the essential elements of several recently proposed models, some of these measurable properties have been elucidated. First, a rigorous mathematical relationship between fusion intermediates and the fusion event itself is defined. Second, it is shown that what is measured as the macroscopic "fusion rate constant" is a simple function of all of the rate constants governing the transitions between intermediates, whether or not one of the microscopic steps is rate limiting. Third, while this kinetic scheme predicts a delay (or lag) time for fusion, as has been observed, it will be very difficult to extract reliable microscopic information from these data. Furthermore, it is predicted that the delay time can depend upon HA surface density even when the HA aggregation step is very rapid compared with fusion, i.e., the delay time need not be due to HA aggregation. Fourth, the inactivation process observed for influenza virions at low pH can be described within this kinetic scheme simply, yet rigorously, via the loss of the fusion intermediates. Fifth, predicted Arrhenius plots of fusion rates can be linear for this multistep scheme, even though there is no single rate-determining step and even when a branched step is introduced, i.e., where one pathway predominates at low temperature and the other pathway predominates at high temperature. Furthermore, the apparent activation energies obtained from these plots bear little or no quantitative resemblance to the microscopic activation energies used to simulate the data. Overall, these results clearly show that the intermediates of protein mediated fusion can be studied only by using assays sensitive to the formation of each proposed intermediate.