Delayed Decline of Cognitive Function by Antihypertensive Agents: A Cohort Study Linked with Genotype Data.

BACKGROUND: Arterial hypertension is among factors with the potential for increasing the risk of cognitive impairment in elderly subjects. However, studies investigating the effects of antihypertensives on cognitive function have reported mixed results. METHODS: We have used the National Alzheimer's Coordinating Center (NACC) Uniform Data Set (UDS) to investigate the effect of each class of antihypertensives, both as single and combined, in reducing the rate of conversion from normal to mild cognitive impairment (MCI). RESULTS: The use of antihypertensive drugs was associated with 21% (Hazard ratio: 0.79, p<01001) delay in the rate of conversion to MCI. This effect was modulated by age, gender, and genotypic APOE4 allele. Among different antihypertensive subclasses, calcium channel blockers (CCBs) (24%, HR: 0.76, P=0.004), diuretics (21%, HR: 0.79, P=0.006), and α1-adrenergic blockers (α1-ABs) (23%, HR: 0.77, P=0.034) significantly delayed the rate of MCI conversion. A significant effect was observed with the selective L-type voltage-gated CCBs, dihydropyridines, amlodipine (47%, HR=0.53, P<0.001) and nifedipine (49%, HR=0.51, P=0.012), whereas non-DHPs showed insignificant effect. Loop diuretics, potassium sparing diuretics, and thiazides all significantly reduced the rate of MCI conversion. Combination of α1-AB and diuretics led to synergistic effects; combination of vasodilators plus ß-blockers (ßBs), and α1-AB plus ßBs led to additive effect in delaying the rate of MCI conversion, when compared to a single drug. CONCLUSION: Our results could have implications for the more effective treatment of hypertensive elderly adults who are likely to be at high risk of cognitive decline and dementia. The choice of combination of antihypertensive therapy should also consider the combination which would lead to an optimum benefit on cognitive function.

Analyses of multiple single-nucleotide polymorphisms in the SUMO4/IDDM5 region in affected sib-pair families with type I diabetes.

Previous studies suggested that the SUMO4 gene, located in the IDDM5 interval on chromosome 6q25, was associated with type I diabetes (T1D) and several other autoimmune diseases. Subsequent analyses of the SUMO4 variants with T1D suggested that the association was stronger and more consistent in the Asian populations. In addition, considerable heterogeneity has been observed in the Caucasian populations. In this report, a 40-kb genomic interval including the SUMO4 gene was tagged with 15 single-nucleotide polymorphisms. A total of 2317 affected sib-pair families from the Type I Diabetes Genetic Consortium were genotyped using both the Illumina and Sequenom genotyping platforms. In these Caucasian families, we found little evidence supporting an association between SUMO4 and T1D.

Candidate genes and growth curves for adiposity in African- and European-American youth.

OBJECTIVE: Obesity is associated with multiple health problems and often originates in childhood. This study investigated the association of genes with the development of general and central obesity from childhood into adulthood. DESIGN: Individual growth curves for measures of general adiposity were examined in an 11-year (1987-1998) cohort study. Single-nucleotide polymorphisms (SNPs) in 11 candidate genes were genotyped. SUBJECTS: Five hundred and twenty-six subjects classified by race (49% African American (AA)), sex (47% male) and socio-economic status (SES). RESULTS: AA female carriers of the 27Glu allele in the ADRB2 gene had a larger waist circumference (P<0.05). Subjects of high SES with the ApoB 4145Lys allele had a larger mean waist circumference than those without this allele (P<0.05). Only in the presence of an adverse environment (low SES) did carriers of the NOS3 298Asp allele have a larger mean body mass index, waist circumference and sum of skinfolds (P<0.05). CONCLUSION: These results suggest that several polymorphisms are associated with the mean level of adiposity, with the effects depending on other factors such as race, sex and/or SES.

Radiation-induced untargeted germline mutations in Japanese medaka.

Radiation has been shown to increase mutation frequencies at tandem repeat loci by indirect interactions of radiation with DNA. We studied germline mutations in chronically exposed Japanese medaka (Oryzias latipes) using microsatellite loci. After screening 26 randomly selected loci among unirradiated parents and their 200 offspring, we selected seven highly mutable loci (0.5-1.0 x 10(-2) mutants per locus per gamete) and two bonus loci for further study. To determine if radiation exposure increases mutation frequencies in these loci, medaka were chronically irradiated from subadults through maturation at relatively low dose rates of 68 mGy/d. Total doses for males and females were 10.4 and 3 Gy, respectively. The mean number of mutations for the offspring of exposed families (0.149+/-0.044) was significantly higher (P=0.018) than for control families (0.080+/-0.028), indicating induction of germline mutations from chronic irradiation. This increase in the microsatellite mutation rate is greater than expected from direct interaction of radiation with DNA, suggesting indirect, untargeted mechanism(s) for mutations. This study identified microsatellite loci with a high mutational background in medaka, variation among loci and families as important variables, and demonstrated the usefulness of this fish model for studying radiation-induced germline mutations.

Integrating Disease Thresholds with TOM-CAST for Carrot Foliar Blight Management.

Cercospora carotae and Alternaria dauci cause foliar blight on carrot (Daucus carota subsp. sativus) and are managed with fungicides to prevent yield loss. Sprays are initiated prior to disease symptoms and reapplied frequently, but some of these applications may not be necessary when the threat of disease is low. Delaying the initial fungicide spray until disease symptoms are observed and applying subsequent sprays according to the TOM-CAST disease forecaster may reduce fungicide inputs. The objective of this 2-year field study was to compare preventive, calendar-based application schedules with an integrated management approach that incorporates disease scouting to initiate fungicide application and the TOM-CAST system for timing subsequent fungicide sprays to manage foliar blight on processing, fresh market, and cut-and-peel carrot cultivars in Michigan. Applications of the fungicides chlorothalonil alternated with azoxystrobin were made prior to disease symptoms (0% blight) or when the foliage became blighted at a trace, 5%, or 10% severity level. Fungicides were reapplied every 7 or 10 days or according to TOM-CAST using disease severity value (DSV) thresholds of 15, 20, or 25. Initiating fungicide treatment at a trace level of disease and timing subsequent sprays according to the TOM-CAST 15-DSV forecaster was comparable to the preventive, calendar-based fungicide regime. One to five fewer applications were needed, while fungicide costs were reduced by $21 to $141 per hectare, when spraying at the trace disease threshold and reapplying according to the TOM-CAST 15-DSV program compared with the 7- or 10-day intervals initiated at 0% blight. Fungicide programs initiated at 5 or 10% leaf blight often provided less control than programs initiated at 0% and trace disease. This study highlights the importance of initiating a fungicide program prior to advanced foliar blight and validates the TOM-CAST 15-DSV forecaster for managing Cercospora leaf spot and Alternaria leaf blight in three carrot production systems.

The application of genomic and proteomic technologies in predictive, preventive and personalized medicine.

The long asymptomatic period before the onset of chronic diseases offers good opportunities for disease prevention. Indeed, many chronic diseases may be preventable by avoiding those factors that trigger the disease process (primary prevention) or by use of therapy that modulates the disease process before the onset of clinical symptoms (secondary prevention). Accurate prediction is vital for disease prevention so that therapy can be given to those individuals who are most likely to develop the disease. The utility of predictive markers is dependent on three parameters, which must be carefully assessed: sensitivity, specificity and positive predictive value. Specificity is important if a biomarker is to be used to identify individuals either for counseling or for preventive therapy. However, a reciprocal relationship exists between sensitivity and specificity. Thus, successful biomarkers will be highly specific without sacrificing sensitivity. Unfortunately, biomarkers with ideal specificity and sensitivity are difficult to find for many diseases. One potential solution is to use the combinatorial power of a large number of biomarkers, each of which alone may not offer satisfactory specificity and sensitivity. Recent technological advances in genetics, genomics, proteomics, and bioinformatics offer a great opportunity for biomarker discovery. The newly identified biomarkers have the potential to bring increased accuracy in disease diagnosis and classification, as well as therapeutic monitoring. In this review, we will use type 1 diabetes (T1D) as an example, when appropriate, to discuss pertinent issues related to high throughput biomarker discovery.

Comparing Disease Forecasters for Timing Fungicide Sprays to Control Foliar Blight on Carrot.

Alternaria dauci and Cercospora carotae cause foliar blight on carrot and can reduce yield in severely blighted fields. Historically, fungicides are applied every 7 to 14 days even though applications may be made when environmental conditions do not favor blight development. The purpose of this study was to compare a calendar-based application schedule with three disease forecasting systems for timing fungicide sprays to limit foliar blight, and included (i) an A. dauci disease forecaster, (ii) TOM-CAST, using a threshold of 15 disease severity values, and (iii) a disease forecaster developed to control C. apii on celery. Chlorothalonil was applied weekly or according to the forecasting systems to blight-susceptible 'Cellobunch' carrot plants in 2001 and 2002. Overall petiole health was poor ≥8.3; 10 = 100% petiole necrosis) when fungicides were not used. Although all disease forecasters maintained petiole health (≤5.3; 1 = healthy and vigorous), the TOM-CAST program had the best petiole health rating each year (≤2.8). TOM-CAST prompted 38 to 54% fewer applications than the weekly application schedule, resulting in a fungicide savings of $105 and $147/ha in 2001 and 2002, respectively, while providing similar blight control. The number of sprays also was reduced when fungicides were applied according to the A. dauci and C. apii forecasters, but acceptable blight control was not always achieved.

Limiting Populations and Spread of Clavibacter michiganensis subsp. michiganensis on Seedling Tomatoes in the Greenhouse.

Symptomless greenhouse tomato transplants may harbor high populations of Clavibacter michiganensis subsp. michiganensis, the causal agent of bacterial canker, leading to yield loss in the field. The objective of this study was to determine whether resistant cultivars, acibenzolar-S-methyl, avirulent strains of C. michiganensis subsp. michiganensis, or standard bactericides reduce pathogen populations and spread among greenhouse tomato seedlings. All treatments limited pathogen populations compared with the untreated inoculated susceptible cultivar in 1996 and 1998, but not in 1997. In 1996, copper hydroxide alone or mixed with mancozeb or streptomycin limited pathogen populations relative to acibenzolar-S-methyl, acibenzolar-S-methyl mixed with copper hydroxide, and avirulent strains. Copper hydroxide mixed with streptomycin limited pathogen populations compared with copper hydroxide mixed with mancozeb. Adding copper hydroxide to acibenzolar-S-methyl limited pathogen populations compared with acibenzolar-S-methyl alone. In 1998, treatments did not differ significantly from each other in limiting pathogen populations. The treatments limited spread of the bacterium only in 1997. Copper hydroxide mixed with mancozeb limited spread compared with copper hydroxide mixed with streptomycin. Pathogen spread was also reduced among resistant cultivars compared with the susceptible cultivar treated with streptomycin. In the field, the untreated inoculated susceptible cultivar produced yields that were 61% (1996) and 93% (1997) of those produced by the uninoculated susceptible cultivar. Fruit spotting occurred regardless of treatment. To prevent severe bacterial canker disease in the field, growers should initiate and sustain bactericide applications to tomato transplants while in the greenhouse to suppress pathogen populations. Cultivar resistance and acibenzolar-S-methyl may be helpful in disease management of bacterial canker on tomato.

Evolution of egg target size: an analysis of selection on correlated characters.

In broadcast-spawning marine organisms, chronic sperm limitation should select for traits that improve chances of sperm-egg contact. One mechanism may involve increasing the size of the physical or chemical target for sperm. However, models of fertilization kinetics predict that increasing egg size can reduce net zygote production due to an associated decline in fecundity. An alternate method for increasing physical target size is through addition of energetically inexpensive external structures, such as the jelly coats typical of eggs in species from several phyla. In selection experiments on eggs of the echinoid Dendraster excentricus, in which sperm was used as the agent of selection, eggs with larger overall targets were favored in fertilization. Actual shifts in target size following selection matched quantitative predictions of a model that assumed fertilization was proportional to target size. Jelly volume and ovum volume, two characters that contribute to target size, were correlated both within and among females. A cross-sectional analysis of selection partitioned the independent effects of these characters on fertilization success and showed that they experience similar direct selection pressures. Coupled with data on relative organic costs of the two materials, these results suggest that, under conditions where fertilization is limited by egg target size, selection should favor investment in low-cost accessory structures and may have a relatively weak effect on the evolution of ovum size.

Optimal Fungicide Management of Purple Spot of Asparagus and Impact on Yield.

Purple spot disease of asparagus, caused by the fungus Stemphylium vesicarium, results in lesions on spears and ferns and defoliation of ferns. In two newly established commercial asparagus fields (cvs. Jersey Giant and Jersey Knight), chlorothalonil or mancozeb was applied every 7, 10, or 14 days or according to Tom-Cast with a threshold of 15 disease severity values, and not applied to the control. Tom-Cast prompted four sprays, resulting in a 60% reduction in the number of fungicide applications when compared with the 7-day-interval treatment. When disease pressure was severe, lesions on ferns were significantly less for both cultivars when fungicides were applied according to Tom-Cast or every 7 days compared with spray intervals of 10 or 14 days. Applying fungicides according to Tom-Cast or every 7 days resulted in an increased Jersey Giant fern stand compared with applying fungicides every 10 or 14 days. Unsprayed control plots yielded 77 to 83% (depending on cultivar) of those plots treated according to Tom-Cast using chlorothalonil. Significantly higher yields of Jersey Knight were obtained for chlorothalonil versus mancozeb. When mancozeb was used, Jersey Knight yield was significantly increased with a 7-day versus Tom-Cast application regime. Using chlorothalonil in a Tom-Cast program provided a benefit per hectare (BPH) of $1,005.24 (Jersey Knight) to $2,057.69 (Jersey Giant). In comparison, using mancozeb in a Tom-Cast program provided a BPH of -$484.27 (Jersey Knight) to $1,030.55 (Jersey Giant) over a 2-year period.

Effect of Bactericides on Population Sizes and Spread of Clavibacter michiganensis subsp. michiganensis on Tomatoes in the Greenhouse and on Disease Development and Crop Yield in the Field.

ABSTRACT Chemical applications, with the exception of mancozeb, reduced population sizes and spread of Clavibacter michiganensis subsp. michiganensis among tomato seedlings in the greenhouse and impacted subsequent plant development and yield in the field. While applications of copper hydroxide, copper hydroxide/mancozeb, copper hydroxide/mancozeb (premixed 12 h before spraying), streptomycin, and streptomycin/copper hydroxide to seedlings in the greenhouse did not differ significantly from the inoculated control, the trend was for these treatments to increase the survival of inoculated transplants in the field in comparison to the inoculated control. In the field, inoculated controls produced yields that were 63% (1995) and 51% (1996) of those produced by uninoculated controls. In both years, with the exception of mancozeb in 1995, all treatments resulted in yields similar to those obtained with the uninoculated control. Plant survival and yield in the field were severely affected when transplants had a pathogen population of >/= x 10(8) CFU/g of tissue. All treatments, with the exception of mancozeb, limited C. michiganensis subsp. michiganensis populations to <5.0 x 10(5). None of the treatments significantly reduced the incidence of fruit spotting compared with that of the inoculated control.

Isometric tension and mutant myosin heavy chain content in single skeletal myofibers from hypertrophic cardiomyopathy patients.

Several mutations in the beta-myosin heavy chain (beta-MHC) gene have been linked to hypertrophic cardiomyopathy (HCM). Because this gene is also expressed in slow-twitch fibers of skeletal muscle, we have been able to study the mutant beta-myosin content and mechanical properties associated with these myosin mutations in single skinned skeletal muscle fibers obtained from HCM patients. We found that in patients carrying the 403Arg-->Gln mutation, the mutant beta-MHC comprises 47.3 +/- 9.1% of the total beta-MHC present in single slow-twitch fibers. Therefore, both alleles of the beta-MHC gene are on average equally expressed. Isometric tension was decreased by 18% in slow fibers from HCM patients with the 403Arg-->Gln mutation, but was unchanged in slow fibers from patients with two other beta-MHC gene mutations. Taken together with the previous demonstration of reduced velocities generated by these myosins in an in vitro assay, our results suggest that the mutant beta-myosins are functional molecular motors that are able to generate tension and movement, but with abnormal kinetics.

Adhesion of activated platelets to venous endothelial cells is mediated via GPIIb/IIIa.

Normal circulating platelets do not adhere to intact, undisturbed endothelium. Studies have shown, however, that platelets will adhere to virally infected or thrombin-stimulated human umbilical vein endothelial cells. Using a novel platelet/endothelial cell adhesion assay we studied the interaction of thrombin-activated platelets to human saphenous vein endothelial cells (HSVEC), and its mechanism(s). Biotinylated platelets were exposed to Hepes-Tyrode buffer, 10E5 or PAC-1 [monoclonal antibodies (Mabs) blocking GPIIb-IIIa], AK4 (Mab blocking P-selectin, 6D1 (Mab blocking vWf binding to GPIb), RGDS (small peptide blocking the fibrinogen binding site), or EDTA (dissociates GPIIb-IIIa complex) and then activated with thrombin. The platelets were subsequently exposed to thrombin-stimulated monolayer HSVEC. Phycoerythrin-streptavidin was added to the wells to fluorescently label the platelets, followed by formaldehyde fixation and washing to remove nonadherent platelets. Adhesion of platelets to HSVEC was assessed using a fluorescent multiwell plate reader. Antibodies which blocked the GPIIb-IIIa receptor and agents which competitively bound the receptor all significantly inhibited activated platelet adhesion to the activated HSVEC. We have found that thrombin significantly increases platelet/HSVEC adhesion, and this event is mediated via the integrin GPIIb-IIIa (fibrinogen receptor). These GPIIb-IIIa receptor blocking Mabs and RGDS may be useful adjuncts for improving patency following angiographic intervention and/or vein grafting in patients with high risk of thrombosis. The assay we have developed is a valuable and relatively simple method for assessing platelet/endothelial cell adhesion and activation.

Population structure of morphological traits in Clarkia dudleyana. I. Comparison of FST between allozymes and morphological traits.

Studies of genetic variation at allozyme loci, assumed to be selectively neutral, have provided valuable insights into the genetic structure of numerous populations. The degree to which population structure of allozyme variation reflects that of quantitative traits, however, is not well resolved. Here, we compare estimates of population differentiation (FST) of 11 populations for allozymes with those for nine discrete and nine continuous morphological traits. Overall, the allozymes have the lowest FST estimates, indicating relatively little population differentiation. Excepting two traits, petal width and long internode length, the continuous morphological traits have estimates similar to those from allozymes. The discrete morphological traits tend to have the highest estimates. On a single trait basis, estimates of FST for four discrete and two continuous traits are higher than those for allozymes. A more detailed (narrow-sense quantitative) genetic study of two populations suggests that these estimates of FST may underestimate the true value because of dominance. Clustering analyses show that the pattern of differentiation for the discrete morphological traits strongly reflects the geographical distribution of the populations, whereas the patterns for the continuous traits and allozymes do not. These results suggest that selection has been occurring on the discrete morphological traits, selecting toward a common optimum within each geographic group, and optima differing among geographic groups.

Quantitative genetics of response to competitors in Nemophila menziesii: a field experiment.

Recent investigations of evolution in heterogeneous environments have begun to accommodate genetic and environmental complexity typical of natural populations. Theoretical studies demonstrate that evolution of polygenic characters depends heavily on the genetic interdependence of the expression of traits in the different environments in which selection occurs, but information concerning this issue is scarce. We conducted a field experiment to assess the genetic variability of the annual plant Nemophila menziesii in five biotic regimes differing in plant density and composition. Significant, though modest, additive genetic variance in plant size was expressed in particular treatments. Evidence of additive genetic tradeoffs between interspecific and intraspecific competitive performance was found, but this result was not consistent throughout the experiment. Two aspects of experimental design may tend to obscure genetically based tradeoffs across environments in many previously published experiments: (1) inability to isolate additive genetic from other sources of variation and (2) use of novel (e.g., laboratory) environments.

Capsaicin-induced gastric hyperemia and protection are NO- dependent.

Topical treatment of gastric mucosa with capsaicin (cap) increases gastric mucosal blood flow (GMBF) and protects the mucosa from injury by acidified bile salts. The purpose of this study was to test the hypothesis that this hyperemia related "cytoprotection" is mediated by nitric oxide. Male Sprague-Dawley rats were anesthetized and the glandular stomach (blood supply intact) was chambered between two plastic rings. Animals were divided into four groups. All groups received a 5-min topical saline exposure. Groups 1 and 2 received iv saline or nitro-L-arginine methyl ester (L-NAME, 25 mg/kg iv), a specific nitric oxide inhibitor, 5 min prior to baseline treatment, followed by a 15-min preinjury period of saline and a 15-min injury period of 10 mM acidified taurocholate (ATC, pH 1.2). Groups 3 and 4 were treated as above except topical cap (160 microM) was used during the preinjury period. GMBF was measured with a laser Doppler flowmeter (ml/min/100 g tissue). Injury was assessed grossly (grade 0-3), histologically (grade 0-3), and by measuring DNA content of a 5-min N-acetylcysteine wash (DNAE). Baseline GMBF of 30 +/- 1.5 significantly decreased to 15 +/- 1.2 in group 1 versus group 2 (P < 0.05). When topical ATC was used GMBF increased to 59 +/- 4.9 and 25 +/- 2.8, respectively. Injury by grade and DNAE was not significantly different between these groups. GMBF during cap exposure was 42 +/- 4 and 22 +/- 2 in groups 3 and 4, respectively. Graded histologic and gross injuries were significantly worse in group 4 compared to group 3 (P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Temperature and water viscosity: physiological versus mechanical effects on suspension feeding.

Water viscosity is inversely related to temperature. This simple physical relation couples two potential influences on organism performance. Seawater viscosity was manipulated, with and without temperature, to distinguish the physiological and mechanical effects of temperature on suspension feeding by ciliated echinoderm larvae. Change in viscosity alone accounted for half of the decline in the feeding rate at lower temperature. High viscosity shifted ingestion toward larger particles, which suggests that viscosity affects particle capture as well as rates of water processing. Temperature-induced change in viscosity, therefore, impacts suspension feeding independently of physiology and has implications for many small-scale biological processes.

Successful preservation of an infected common femoral vein to common femoral vein crossover prosthetic graft.

We used selective graft preservation to treat an infected anastomosis of a functioning common femoral vein to common femoral vein crossover PTFE bypass performed for iliac vein occlusion. The graft was successfully salvaged by operative debridement and placement of a rectus abdominis muscle flap. With the growing interest in venous reconstructive surgery, bypass infections may be seen more frequently. This case illustrates that selective preservation of infected prosthetic grafts anastomosed to a peripheral vein may be a simple and improved method to treat these complications.

Effects of topical isoproterenol on bile acid-induced gastric mucosal injury.

Topical isoproterenol protects the gastric mucosa from the severe necrosis induced by 100 per cent ethanol. Its effect on gastric mucosal blood flow is unknown. The purpose of this study was to determine the effect of topical isoproterenol on gastric mucosal blood flow and on the less severe gastric mucosal injury caused by dilute bile acid. Prior to injury with topical 5 mM acidified taurocholate (pH 1.2), stomachs were pretreated with either saline or isoproterenol (low dose = 50 micrograms/kg; high dose = 500 micrograms/kg). Mucosal injury was assessed by measuring net transmucosal ion fluxes (H, K) and the appearance of DNA into the gastric lumen (DNAE). Gastric mucosal blood flow was determined by using laser doppler. Pretreatment with isoproterenol significantly decreased bile acid-induced net transmucosal ion fluxes and luminal accumulation of DNA, suggesting mucosal protection. Furthermore, this effect was dose-dependent on H and DNAE but not K. Pretreatment with topical high dose isoproterenol had no significant effect on gastric mucosal blood flow. Thus, topical pretreatment with isoproterenol dose dependently protects the gastric mucosa from the superficial injury caused by the application of dilute bile acid. This protective effect appears to be mediated by a mechanism other than augmentation of gastric mucosal blood flow.

Crossbridge rotation in EDC-crosslinked striated muscle fibers.

The rotatability of the strong- and weak-binding myosin heads was tested by stretching glycerinated rabbit psoas fibers after crosslinking the heads to actin by using a carbodiimide EDC. The equatorial 1,1 reflection intensity (I1,1) decreased by approximately 10% upon 1% stretch in the presence of various ligands (ATP, ATP-gamma-S, pyrophosphate and AMPPNP). As the action of ligands to dissociate actomyosin increased, the relaxation of tension response to stretch and the I1,1 decrease were accelerated. This result is best explained if the ligand converts the crosslinked head to a weak-binding state, in which the head is rotatable because of its acquired elasticity. Conversely, the weak-to-strong transition was induced in the crosslinked system by removing a ligand (ATP-gamma-S) from myosin. Force was produced upon weak-to-strong transition and was accounted for by the increased stiffness of each crosslinked myosin head. However, the comparison of stress-strain curves for the weak- and strong-binding myosin showed that the equilibrium angle of myosin attachment was unchanged, making it unlikely that the weak-to-strong transition is the sole mechanism for active contraction. The calcium-activated force of the same crosslinked fibers showed several features in marked contrast to the force produced by the weak-to-strong transition. This leads to a possibility that the active force is supported by a third class of intermediate which is distinct not only from the weak-binding but also from the strong-binding intermediates in a classical sense.