Prognostic impact of six versus eight cycles of standard regimen in patients with diffuse large B-cell lymphoma: propensity score-matching analysis.

BACKGROUND: R-CHOP-21 has been the standard treatment for diffuse large B-cell lymphoma (DLBCL), but there is a paucity of evidence focusing on the number of cycles of regimens. PATIENTS AND METHODS: We conducted a retrospective study to compare the effectiveness of six cycles of standard regimens versus eight cycles for overall survival (OS) in DLBCL patients using propensity score matching, in consideration of relative dose intensity (RDI). RESULTS: A total of 685 patients with newly diagnosed DLBCL were identified in three institutions from 2007 to 2017. Patients treated using six cycles of standard regimens were matched by propensity scores with those treated using eight cycles. A 1 : 1 propensity score matching yielded 138 patient pairs. Eight cycles did not significantly improve OS in the conventional Cox proportional hazards model (hazard ratio 0.849, 95% confidence interval 0.453-1.588, P = 0.608). Restricted cubic spline Cox models for OS confirmed that the effect of the number of cycles was not modified by total average RDI, the International Prognostic Index, and age. Occurrence of adverse events did not differ between six and eight cycles. CONCLUSION: Even considering the impact of RDI, six cycles of the initial standard regimen for DLBCL is not inferior to eight cycles.

Half-dose vs one-third-dose photodynamic therapy for chronic central serous chorioretinopathy.

PURPOSE: To prospectively compare the effects of half-dose verteporfin (3 mg/m(2)) photodynamic therapy (1/2 PDT) with those of one-third-dose verteporfin (2 mg/m(2)) PDT (1/3 PDT) for chronic central serous chorioretinopathy (CSC). METHODS: Sixteen eyes of 16 consecutive patients with chronic CSC were enrolled and followed up for a 3-month study period. The first 10 patients received 1/2 PDT and the next 6 patients received 1/3 PDT. The resolution rate of subretinal fluid (SRF) was compared between the two groups. The changes in the choroidal thickness inside and outside the PDT-applied area in both groups were also evaluated. RESULTS: SRF disappeared in all eyes (100%) in the 1/2 PDT group and in two eyes (33%) in the 1/3 PDT group. In the 1/2 PDT group, choroidal thickness inside and outside the PDT-applied area reduced significantly from the baseline (inside, from 387 ± 24 to 325 ± 25 µm; outside, from 292 ± 25 to 249 ± 19 µm; both P=0.005). In the 1/3 PDT group, choroidal thickness decreased in two eyes where SRF disappeared (inside, 87.2 and 90.9% of the baseline; outside, 91.4 and 92.6% of the baseline), but did not change in the other four eyes where SRF remained (inside, 104.1, 100.0, 105.1, and 100.5% of the baseline; outside, 98.9, 103.0, 100.0, and 99.0% of the baseline). CONCLUSIONS: 1/2 PDT is more effective than 1/3 PDT in the resolution of SRF for chronic CSC. Decrease in the choroidal thickness after PDT may be related to the resolution of SRF in chronic CSC.

Behaviors of microtubules (MTs) driven by biological motors (dynein c) at collisions against micro-fabricated tracks and mts for potential nano-bio-machines.

In vitro motility assays, where protein motors (attached to a surface) move protein filaments, have been used for investigating protein motors' functions. In recent decades, these assays are extended to explore potential applications of motor proteins as biological motors in nano-bio-machine development. Recent attempts include fabricating micrometer-scale tracks on the surface to confine and guide the flow of bio-filaments as a power transfer medium driven by the motor proteins. Understanding the interaction between bio-filaments and fabricated tracks as well as the mutual interaction between bio-filaments is of importance to the design of potential nano-bio-machines. In this study, we investigate the behaviors of a microtubule driven by axonemal dynein at the collision against another microtubule and micro-fabricated walls, respectively. Based on experimental observations, we propose a model to study possible mechanisms for the microtubule-microtubule and microtubule-wall interactions, which involve bumping force, bending moment and torque generation.

Phenomenological simulation of self-organization of microtubule driven by dynein c.

It was recently noticed that in vitro motility assays, driven by random distributed dynein c, microtubules could form self-organized circular patterns, which could be of importance to the design of nanobiomechanical machines. In order to determine key parameters that control the self-organized movement of microtubules, a phenomenological modeling study taking account of the microtubule joining probability distribution and microtubule bias was conducted to investigate the self-organization of microtubules driven by dynein motors.

The coordination of protein motors and the kinetic behavior of microtubule--a computational study.

Utilizing the mechanical energy converted from chemical energy through hydrolysis of ATP, motor proteins drive cytoskeleton filaments to move in various biological systems. Recent technological advance has shown the potential of the motor proteins for powering future nano-bio-mechanical systems. In order to effectively use motor proteins as a biological motor, the interaction between the protein motors and bio-filaments needs to be well clarified, since such interaction is largely influenced by many factors, such as the coordination among the motors, their dynamic behavior, physical properties of microtubules, and the viscosity of solution involved, etc. In this study, a two-dimensional model was proposed to simulate the motion of a microtubule driven by protein motors based on a dissipative particle dynamics (DPD) method with attempt to correlate the microtubule's kinetic behavior to the coordination among protein motors.

The relationship between 24-hour blood pressure readings, subcortical ischemic lesions and vascular dementia.

BACKGROUND: Twenty-four-hour blood pressure (BP) readings have been found to correlate with hypertensive target organ damage. Lacunar infarcts (LI) and white matter lesions (WML) probably represent manifestations of cerebral hypertensive target organ damage. This study was conducted to better delineate the relationships between 24-hour BP measurements, LI/WML and small vessel disease cognitive impairment/vascular dementia (CI/VD). METHODS: Two hundred patients with first-time symptomatic LI were examined with 24-hour BP monitoring. The degree of nocturnal BP dip, (daytime BP - nighttime BP)/daytime BP, was categorized into three groups: dippers (>0.1), nondippers (0-0.1) and reverse dippers (<0). WML were subdivided into periventricular hyperintensities (PVH) and subcortical hyperintensities. RESULTS: The breakdown of patients was: 50% nondippers, 27.5% reverse dippers and 22.5% dippers. Forty-one patients (20.5%) were found to have CI and dementia. Male sex (OR 3.35; 95% CI 1.20-9.34), advanced PVH (OR 14.42; 95% CI 5.62-36.98) and absence of a dipping status (nondipper: OR 12.62; 95% CI 1.37-115.95; reverse dipper: OR 11.95; 95% CI 1.27-112.11) were independently associated with CIVD after multivariate analysis. High nighttime systolic BP (OR 3.93; 95% CI 1.38-11.17), high daytime (OR 2.06; 95% CI 1.03-4.04) and nighttime diastolic BP (OR 2.48; 95% CI 1.13-5.45) and absence of a dipping status (nondipper: OR 2.7; 95% CI 1.03-7.05; reverse dipper: OR 3.78; 95% CI 1.38-10.34) were significantly associated with PVH. CONCLUSIONS: High prevalence of a nondipping status was found in the LI cohort. A nondipping status appears to be directly associated with CIVD independent of PVH. This study indicates the need for further studies to investigate whether or not controlling nighttime BP will help reduce the risk for CI/VD development.

The structure of dynein-c by negative stain electron microscopy.

Dynein ATPases contain six concatenated AAA modules within the motor region of their heavy chains. Additional regions of sequence are required to form a functional ATPase, which a previous study suggested forms seven or eight subdomains arranged in either a ring or hollow sphere. A more recent homology model of the six AAA modules suggests that these form a ring. Therefore both the number and arrangement of subdomains remain uncertain. We show two-dimensional projection images of dynein-c in negative stain which reveal new details of its structure. Initial electron cryomicroscopy shows a similar overall morphology. The molecule consists of three domains: stem, head, and stalk. In the absence of nucleotide the head has seven lobes of density forming an asymmetric ring. An eighth lobe protrudes from one side of this heptameric ring and appears to join the elongated cargo-binding stem. The proximal stem is flexible, as is the stalk, suggesting that they act as compliant elements within the motor. A new analysis of pre- and post-power stroke conformations shows the combined effect of their flexibility on the spatial distribution of the microtubule-binding domain and therefore the potential range of power stroke sizes. We present and compare two alternative models of the structure of dynein.

Tomatoes have natural anti-thrombotic effects.

The prevention of arterial thrombotic diseases has a high priority in developed countries. An inappropriate diet may be an important risk factor for thrombotic events. The daily intake of an anti-thrombotic diet may offer a convenient and effective way of prevention. The aim of the present study was to test tomato extracts for anti-thrombotic effects and to identify those varieties that have such an effect. A shear-induced platelet-function test (haemostatometry) was used to test anti-thrombotic potential in vitro. Extracts from those tomato varieties that showed a significant anti-thrombotic activity in vitro were further assessed in vivo, using a laser-induced thrombosis test in mice. One tomato variety (KG99-4) showed significant anti-thrombotic activity both in vitro and in vivo. KG99-4 inhibited not only platelet-rich thrombus formation but also had a thrombolytic effect. It is concluded that haemostatometry can detect and classify the anti-thrombotic potential of fruits and vegetables and offers a simple way of screening for such effects.

A new model to evaluate the diet-induced prothrombotic state, using He-Ne laser-induced thrombogenesis in the carotid artery of apolipoprotein E-deficient and low-density lipoprotein receptor-deficient mice.

Epidemiological studies suggest the high fat content of the Western diet to be responsible for atherosclerosis and its thrombotic complications. Despite such a prevailing view, few animal experiments have so far succeeded in demonstrating enhanced thrombogenicity due to a high fat diet. Even a high fat and a very high cholesterol (1%) diet has failed to demonstrate an enhanced thrombotic reaction in rodents and rabbits. The aim of the present study was twofold. First, we wanted to establish a new, sensitive and specific thrombosis model in mice, which can then be used to study the effect of diets. Second, we intended to employ such a thrombosis model in investigations into the effect of high or low fat diets on thrombosis. The technique described uses a laser to induce thrombus formation in the exposed carotid artery of apolipoprotein E-deficient and low-density lipoprotein receptor-deficient mice. Thrombus formation was recorded on video, analysed by computer, and the size of thrombus was calculated by image analysis software. Thrombotic status was evaluated by analysing a total of 61 individual images of the thrombotic reaction, which were taken over 600 s. The severity of atherosclerosis was assessed by image analysis of the stained elastic fibers. Two kinds of diets were used: the Western type, high fat diet contained 20% fat (w/w) and 0.05% cholesterol (w/w); the low fat diet contained 7% fat, without cholesterol. These diets were on the basis of AIN93G and were given to mice for 4 or 8 weeks. The high fat diet significantly enhanced both the thrombotic reaction and the development of atherosclerosis as compared with the low fat diet.

Twenty-four-hour blood pressure and MRI as predictive factors for different outcomes in patients with lacunar infarct.

BACKGROUND AND PURPOSE: A long-term follow-up study was conducted in patients with lacunar infarct to assess how 24-hour blood pressure monitoring values and MRI findings, in particular lacunar infarcts and diffuse white matter lesions, can predict subsequent development of dementia and vascular events, which include cerebrovascular and cardiovascular events. METHODS: One hundred seventy-seven patients were tracked for a mean of 8.9 years of follow-up. Documented events comprise the development of dementia and the occurrence of vascular events. The predictors for developing dementia and vascular events were separately evaluated by Cox proportional hazards analysis. RESULTS: Twenty-six patients developed dementia (0.17/100 patient-years). Male sex (relative risk [RR], 4.2; 95% CI, 1.2 to 14.7), cognitive impairment (RR, 3.0; 95% CI, 1.0 to 8.5), confluent DWML (moderate: RR, 7.1; 95% CI, 1.6 to 31.5; severe: RR, 35.8; 95% CI, 7.2 to 177.3), and nondipping status (RR, 7.1; 95% CI, 2.2 to 22.0) were independent predictors for dementia. Forty-six patients suffered from vascular events (3.11/100 patient-years). Diabetes mellitus (RR, 5.7; 95% CI, 2.7 to 11.9), multiple lacunae (moderate: RR, 6.4; 95% CI, 2.5 to 15.8; severe: RR, 8.5; 95% CI, 3.1 to 23.3), and high 24-hour systolic blood pressure (>145 mm Hg versus <130 mm Hg) (RR, 10.3; 95% CI, 1.3 to 81.3) were independent predictors for vascular events. CONCLUSIONS: Predictors for developing dementia and vascular events appear to differ. Male sex, confluent diffuse white matter lesions, and nondipping status were independent predictors for subsequent development of dementia, while diabetes mellitus, multiple lacunae, and high 24-hour systolic blood pressure were independent predictors for vascular events.

Mechanical Properties of a Single-Headed Processive Motor, Inner-Arm Dynein Subspecies-c of ChlamydomonasStudied at the Single Molecule Level.

Dynein from inner arms of Chlamydomonasflagella contains sevendistinct subspecies, a through g. Several lines of evidence suggest thesesubspecies play important roles in generating flagellar beating and thatthe different subspecies are functionally diverse. To evaluate theirroles and diversity, the mechanical properties of subspecies-c, which isa single-headed motor, were examined using optical trap nanometry. Apolystyrene bead coated with a small number of subspecies-c moleculeswas captured with the optical trap and brought into contact with amicrotubule fixed to a coverslip. Movements of the bead were measured bya quadrant photodiode sensor with sub-nanometer- and millisecond-resolution.Beads carrying a single active subspecies-c molecule moved processivelyalong the microtubules in 8-nm steps but slipped backwards under highloads. Force-velocity relationships of single subspecies-c molecules werealmost linear and the shapes of the normalized curves at 5 µM and 100µM ATP were similar. These results indicate that dynein subspecies-cfunctions in a very different way from conventional motor proteins, suchas myosin and kinesin, and has properties that could produceself-oscillation in vivo.

Dynamic behavior of microtubules during dynein-dependent nuclear migrations of meiotic prophase in fission yeast.

During meiotic prophase in fission yeast, the nucleus migrates back and forth between the two ends of the cell, led by the spindle pole body (SPB). This nuclear oscillation is dependent on astral microtubules radiating from the SPB and a microtubule motor, cytoplasmic dynein. Here we have examined the dynamic behavior of astral microtubules labeled with the green fluorescent protein during meiotic prophase with the use of optical sectioning microscopy. During nuclear migrations, the SPB mostly follows the microtubules that extend toward the cell cortex. SPB migrations start when these microtubules interact with the cortex and stop when they disappear, suggesting that these microtubules drive nuclear migrations. The microtubules that are followed by the SPB often slide along the cortex and are shortened by disassembly at their ends proximal to the cortex. In dynein-mutant cells, where nuclear oscillations are absent, the SPB never migrates by following microtubules, and microtubule assembly/disassembly dynamics is significantly altered. Based on these observations, together with the frequent accumulation of dynein at a cortical site where the directing microtubules interact, we propose a model in which dynein drives nuclear oscillation by mediating cortical microtubule interactions and regulating the dynamics of microtubule disassembly at the cortex.

Enhanced thrombolysis induced by argatroban or activated protein C in the presence or absence of staphylokinase, measured in an in vivo animal model using mesenteric arterioles.

Successful administration of thrombolytic agents is associated with vessel reocclusion in a high proportion of cases. We have previously established an animal model to investigate platelet-rich thrombolytic mechanisms in vivo and demonstrated that recombinant staphylokinase (rSAK)-induced thrombolysis was enhanced by the concomitant administration of the direct thrombin inhibitor argatroban. The present study expanded the use of this model by comparing arterial and venous thrombolysis using advanced image analysis software. Mural thrombi were formed by Helium-Neon laser irradiation in mesenteric arterioles and were shown to be lysed, dose-dependently, by smaller amounts of rSAK than those required in venules. Activated protein C (APC), as well as argatroban, enhanced the rSAK-induced thrombolysis. APC or argatroban also induced thrombolysis in the absence of rSAK, and the effect was inhibited by tranexamic acid. The enhanced thrombolysis induced by APC or argatroban in the presence or absence of rSAK may be due to increased endogenous thrombolysis mediated by the inhibition of thrombin activity or delayed thrombin generation.

Controlling the direction of kinesin-driven microtubule movements along microlithographic tracks.

Motor proteins are able to move protein filaments in vitro. However, useful work cannot be extracted from the existing in vitro systems because filament motions are in random directions on two-dimensional surfaces. We succeeded in restricting kinesin-driven movements of microtubules along linear tracks by using micrometer-scaled grooves lithographically fabricated on glass surfaces. We also accomplished the extraction of unidirectional movement from the bidirectional movements along the linear tracks by adding arrowhead patterns on the tracks. These "rectifiers" enabled us to construct microminiturized circulators in which populations of microtubules rotated in one direction, and to actively transport microtubules between two pools connected by arrowheaded tracks in the fields of micrometer scales.

An in vitro assay reveals essential protein components for the "catch" state of invertebrate smooth muscle.

"Catch," a state where some invertebrate muscles sustain high tension over long periods of time with little energy expenditure (low ATP hydrolysis rate) is similar to the "latch" state of vertebrate smooth muscles. Its induction and release involve Ca(2+)-dependent phosphatase and cAMP-dependent protein kinase, respectively. Molecular mechanisms for catch remain obscure. Here, we describe a quantitative microscopic in vitro assay reconstituting the catch state with proteins isolated from catch muscles. Thick filaments attached to glass coverslips and pretreated with approximately 10(-4) M free Ca(2+) and soluble muscle proteins bound fluorescently labeled native thin filaments tightly in catch at approximately 10(-8) M free Ca(2+) in the presence of MgATP. At approximately 10(-4) M free Ca(2+), the thin filaments moved at approximately 4 microm/s. Addition of cAMP and cAMP-dependent protein kinase at approximately 10(-8) M free Ca(2+) caused their release. Rabbit skeletal muscle F-actin filaments completely reproduced the results obtained with native thin filaments. Binding forces >500 pN/microm between thick and F-actin filaments were measured by glass microneedles, and were sufficient to explain catch tension in vivo. Synthetic filaments of purified myosin and twitchin bound F-actin in catch, showing that other components of native thick filaments such as paramyosin and catchin are not essential. The binding between synthetic thick filaments and F-actin filaments depended on phosphorylation of twitchin but not of myosin. Cosedimentation experiments showed that twitchin did not bind directly to F-actin in catch. These results show that catch is a direct actomyosin interaction regulated by twitchin phosphorylation.

Twenty-four-hour blood pressure changes in the course of lacunar disease.

BACKGROUND: Changes in blood pressure (BP) over time have not been considered in investigations on the relationship between BP and cerebrovascular disease (CVD). OBJECTIVE: To investigate BP changes throughout a 24-hour period in lacunar infarct patients with different outcomes. METHODS: Twelve control subjects (group 1) and 56 patients with symptomatic lacunar infarcts were studied. The infarct patients were divided into three groups: group 2, 25 patients with a fair outcome without any cerebrovascular attack or progressive dementia (mean follow-up period: 4.4 years); group 3, 14 patients with worsening of clinical dementia rating and silent lesions, which included lacunae and diffuse white matter lesions (4.5 years), and group 4, 17 patients who developed symptomatic infarcts (1.7 years). MRIs and ambulatory BP monitoring were performed for each patient on two separate occasions. No patient was treated with antihypertensive agents during the course of the study. RESULTS: In group 2, the second measurements were significantly higher than the first for 24-hour systolic BP (SBP), daytime SBP, 24-hour diastolic BP (DBP), daytime DBP (p < 0.01, for all) and nighttime DBP (p < 0.05). In group 3, the second measurements were significantly lower than the first for 24-hour SBP, daytime SBP, 24-hour DBP, and daytime DBP (p < 0.01, for all). In group 4, the second measurements were significantly lower than the first for 24-hour SBP and daytime SBP (p < 0.01). The correlation between BP and pulse rate became positive for group 2 in second measurements, but was not positive for groups 3 and 4. CONCLUSIONS: BP tended to elevate over time in patients with a fair outcome. In contrast, BP tended to decrease in those who developed dementia and symptomatic infarct. Autonomic functions including sympathetic activity might play a role in changes in BP in lacunar infarct patients during the course of disease.

X-ray diffraction evidence for the lack of stereospecific protein interactions in highly activated actomyosin complex.

The structure of actomyosin complex while hydrolyzing ATP was investigated by recording X-ray diffraction patterns from rabbit skeletal muscle fibers, in which exogenously introduced rabbit skeletal subfragment-1 (S1) was covalently cross-linked to the endogenous actin filaments in rigor by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). Approximately two-thirds of the introduced S1 was cross-linked. The cross-linking procedure did not affect the profile of the S1-induced enhancement of the actin-based layer line reflections in rigor, indicating that the acto-S1 interactions remained highly stereospecific. In the presence of ATP, the MgATPase of the S1 was highly activated regardless of calcium levels, presumably because the availability of the stereospecific binding sites for both proteins was maximized by the cross-linking. However, the diffraction pattern in the presence of ATP was striking in that the intensity profile of the strong 1/5.9 nm(-1) layer lines was indistinguishable from that from bare actin filaments, despite the fact that the majority of the S1 was still associated with actin. The change of the intensity profiles upon addition of ATP was completely reversible. Model calculations showed that this result can be explained if the S1 is not only swinging around its pivoting point, but the pivoting point itself is also moving on the actin surface in a range of a few nanometers. The results suggest that the stereospecific binding sites, which have been considered important for actomyosin cycling, are paradoxically left unoccupied for most of the time in this highly activated actomyosin complex.

[Mechanisms involved in large subcortical infarcts].

Large subcortical infarcts(maximum diameter of infarct > or = 20 mm) result from various stroke patterns, including striatocapsular infarcts (SCI), corona radiata infarcts, centrum semiovale infarcts, and internal borderzone infarcts. A systematic investigation of stroke pathogenesis involved in large subcortical infarcts, however, has not been performed. This study attempted to clarify the stroke mechanisms involved in large subcortical infarcts, by examining 50 patients with large subcortical infarcts out of 430 ischemic stroke patients consecutively registered in our department. The subjects were divided into two groups according to the vascular territories involved on the MRI: 1) the lenticulostriate arteries group for 39 patients whose infarcts were restricted to within the vicinity of the lenticulostriate arteries; 2) the internal borderzone group for 11 patients whose infarcts mainly involved the internal borderzone (the upper part of the corona radiata and the centrum semiovale) between the territories of the deep perforating branches from the basal cerebral arteries and the medullary branches from the superficial pial arteries. Stroke pathogenesis were classified into the following 6 categories: A) cardiogenic embolism, 9 patients; B) artery-to-artery embolism, 6 patients; C) cryptogenic embolism, 2 patients; D) thrombotic MCA (M1) occlusion, 9 patients; E) thrombotic ICA occlusion, 10 patients; F) undetermined cause, 14 patients. The lenticulostriate arteries group consisted of 9 patients with cardiogenic embolism, 6 with artery-to-artery embolism, 2 with cryptogenic embolism, 8 with thrombotic M1 occlusion, and 14 with undetermined cause. The internal borderzone group consisted of 10 patients with thrombotic ICA occlusion and 1 patient with thrombotic M1 occlusion. The stroke pathogenesis of the undetermined cause is considered to be thrombotic occlusion at the orifice of the lateral lenticulostriate artery, a so-called "branch atheromatous disease (BAD)". The patients in this group experienced a gradual onset, and did not have a cardiac source of the embolism or proximal large artery disease. Among the patients reported as having SCI, BAD may play a role in some cases, especially in those whose the cause was classified as "undetermined". In conclusion, the lenticulostriate arteries group exhibited a higher frequency of cerebral embolisms (cardiogenic embolism, artery-to-artery embolism, and cryptogenic embolism) and thrombotic M1 occlusion, whereas the internal borderzone group had a higher frequency of thrombotic ICA occlusion.

Comparative single-molecule and ensemble myosin enzymology: sulfoindocyanine ATP and ADP derivatives.

Single-molecule and macroscopic reactions of fluorescent nucleotides with myosin have been compared. The single-molecule studies serve as paradigms for enzyme-catalyzed reactions and ligand-receptor interactions analyzed as individual stochastic processes. Fluorescent nucleotides, called Cy3-EDA-ATP and Cy5-EDA-ATP, were derived by coupling the dyes Cy3.29.OH and Cy5.29.OH (compounds XI and XIV, respectively, in, Bioconjug. Chem. 4:105-111)) with 2'(3')-O-[N-(2-aminoethyl)carbamoyl]ATP (EDA-ATP). The ATP(ADP) analogs were separated into their respective 2'- and 3'-O-isomers, the interconversion rate of which was 30[OH(-)] s(-1) (0.016 h(-1) at pH 7.1) at 22 degrees C. Macroscopic studies showed that 2'(3')-O-substituted nucleotides had properties similar to those of ATP and ADP in their interactions with myosin, actomyosin, and muscle fibers, although the ATP analogs did not relax muscle as well as ATP did. Significant differences in the fluorescence intensity of Cy3-nucleotide 2'- and 3'-O-isomers in free solution and when they interacted with myosin were evident. Single-molecule studies using total internal reflection fluorescence microscopy showed that reciprocal mean lifetimes of the nucleotide analogs interacting with myosin filaments were one- to severalfold greater than predicted from macroscopic data. Kinetic and equilibrium data of nucleotide-(acto)myosin interactions derived from single-molecule microscopy now have a biochemical and physiological framework. This is important for single-molecule mechanical studies of motor proteins.

Catchin, a novel protein in molluscan catch muscles, is produced by alternative splicing from the myosin heavy chain gene.

Molluscan catch muscles contain polypeptides of 110-120 kDa in size which have the same partial amino acid sequences as those of the myosin heavy chain (MHC). Here we provide evidence that these polypeptides are major components only of the catch-type muscles (their estimated molar ratio to MHC is approximately 1:1) and they are alternative products of the MHC gene. Northern blot analysis of total RNA from Mytilus galloprovincialis catch muscles was carried out with fragments from the 3'-end of the MHC cDNA as probes. We detected two bands of 6.5 kb and 3.5 kb. The former corresponds to the MHC mRNA, and the latter is an mRNA coding for catchin, a novel myosin rod-like protein. By using a 5'-rapid amplification of cDNA ends (RACE) PCR method, the full-length cDNA of Mytilus catchin was cloned. It codes for a protein with a unique N-terminal domain of 156 residues (rich in serine, threonine, and proline), which includes a phosphorylatable peptide sequence. The rest of the sequence is identical with the C-terminal 830 residues of the MHC. We also analyzed Mytilus and scallop (Argopecten irradians) genomic DNAs and found that the 5'-end of the cDNA sequence was located in a large intron of the MHC gene in both species. Since catchin is abundantly expressed only in catch muscles and it is phosphorylatable, we suggest that it may play an important role in the catch contraction of molluscan smooth muscles.