Role of the kinesin neck linker and catalytic core in microtubule-based motility.

Kinesin motor proteins execute a variety of intracellular microtubule-based transport functions [1]. Kinesin motor domains contain a catalytic core, which is conserved throughout the kinesin superfamily, followed by a neck region, which is conserved within subfamilies and has been implicated in controlling the direction of motion along a microtubule [2] [3]. Here, we have used mutational analysis to determine the functions of the catalytic core and the approximately 15 amino acid 'neck linker' (a sequence contained within the neck region) of human conventional kinesin. Replacement of the neck linker with a designed random coil resulted in a 200-500-fold decrease in microtubule velocity, although basal and microtubule-stimulated ATPase rates were within threefold of wild-type levels. The catalytic core of kinesin, without any additional kinesin sequence, displayed microtubule-stimulated ATPase activity, nucleotide-dependent microtubule binding, and very slow plus-end-directed motor activity. On the basis of these results, we propose that the catalytic core is sufficient for allosteric regulation of microtubule binding and ATPase activity and that the kinesin neck linker functions as a mechanical amplifier for motion. Given that the neck linker undergoes a nucleotide-dependent conformational change [4], this region might act in an analogous fashion to the myosin converter, which amplifies small conformational changes in the myosin catalytic core [5,6].

Thrombogenic factors and recurrent coronary events.

BACKGROUND: Thrombosis is a pivotal event in the pathogenesis of coronary disease. We hypothesized that the presence of blood factors that reflect enhanced thrombogenic activity would be associated with an increased risk of recurrent coronary events during long-term follow-up of patients who have recovered from myocardial infarction. METHODS AND RESULTS: We prospectively enrolled 1045 patients 2 months after an index myocardial infarction. Baseline thrombogenic blood tests included 6 hemostatic variables (D-dimer, fibrinogen, factor VII, factor VIIa, von Willebrand factor, and plasminogen activator inhibitor-1), 7 lipid factors [cholesterol, triglycerides, HDL cholesterol, LDL cholesterol, lipoprotein(a), apolipoprotein (apo)A-I, and apoB], and insulin. Patients were followed up for an average of 26 months, with the primary end point being coronary death or nonfatal myocardial infarction, whichever occurred first. The hemostatic, lipid, and insulin parameters were dichotomized into their top and the lower 3 risk quartiles and evaluated for entry into a Cox survivorship model. High levels of D-dimer (hazard ratio, 2.43; 95% CI, 1.49, 3.97) and apoB (hazard ratio, 1.82; 95% CI, 1.10, 3.00) and low levels of apoA-I (hazard ratio, 1.84; 95% CI, 1.10, 3.08) were independently associated with recurrent coronary events in the Cox model after adjustment for 6 relevant clinical covariates. CONCLUSIONS: Our findings indicate that a procoagulant state, as reflected in elevated levels of D-dimer, and disordered lipid transport, as indicated by low apoA-1 and high apoB levels, contribute independently to recurrent coronary events in postinfarction patients.

Direction determination in the minus-end-directed kinesin motor ncd.

Motor proteins of the kinesin superfamily transport intracellular cargo along microtubules. Although different kinesin proteins share 30-50% amino-acid identity in their motor catalytic cores, some move to the plus end of microtubules whereas others travel in the opposite direction. Crystal structures of the catalytic cores of conventional kinesin (a plus-end-directed motor involved in organelle transport) and ncd (a minus-end-directed motor involved in chromosome segregation) are nearly identical; therefore, the structural basis for their opposite directions of movement is unknown. Here we show that the ncd 'neck' made up of 13 class-specific residues next to the superfamily-conserved catalytic core, is essential for minus-end-directed motility, as mutagenesis of these neck residues reverses the direction of ncd motion. By solving the 2.5 A structure of a functional ncd dimer, we show that the ncd neck (a coiled-coil) differs from the corresponding region in the kinesin neck (an interrupted beta-strand), although both necks interact with similar elements in the catalytic cores. The distinct neck architectures also confer different symmetries to the ncd and kinesin dimers and position these motors with appropriate directional bias on the microtubule.

The directional preference of kinesin motors is specified by an element outside of the motor catalytic domain.

Members of the kinesin superfamily share a similar motor catalytic domain yet move either toward the plus end (e.g., conventional kinesin) or the minus end (e.g., Ncd) of microtubules. The structural features that determine the polarity of movement have remained enigmatic. Here, we show that kinesin's catalytic domain (316 residues) in a dimeric construct (560 residues) can be replaced with the catalytic domain of Ncd and that the resultant motor moves in the kinesin direction. We also demonstrate that this chimera does not move processively over many tubulin subunits, which is similar to Ncd but differs from the highly processive motion of conventional kinesin. These findings reveal that the catalytic domain contributes to motor processivity but does not control the polarity of movement. We propose that a region adjacent to the catalytic domain serves as a mechanical transducer that determines directionality.

Angina pectoris after recovery from an acute coronary event--the role of psychological factors in Japanese vs North American patients.

To determine whether specific psychological characteristics are associated with angina pectoris in clinically stable patients 1 to 6 months after recovery from an acute coronary event, a battery of tests was administered to 92 Japanese and 646 North American participants (22% females) in the Multicenter Study of Myocardial Ischemia. Of these 738 patients, 541 had originally suffered acute myocardial infarction, 188 had unstable angina, and 9 were admitted for other acute ischemic events. At the time of enrollment, an average of 2.7 months after the index event, 205 patients reported having had anginal symptoms during the preceding months. Compared to those who did not report angina, these patients scored higher on a modified Autonomic Perception Questionnaire (p = 0.04) and lower on the Internal Health Locus of Control Scale (p = 0.004). These differences were generalized across the Japanese and North American cohorts. These results indicate that in these patients, angina pectoris was associated with an increased awareness of a wide range of physical symptoms and a decreased sense of personal control over one's own health and prognosis.

Molecular cloning of Drosophila mus308, a gene involved in DNA cross-link repair with homology to prokaryotic DNA polymerase I genes.

Mutations in the Drosophila mus308 gene confer specific hypersensitivity to DNA-cross-linking agents as a consequence of defects in DNA repair. The mus308 gene is shown here to encode a 229-kDa protein in which the amino-terminal domain contains the seven conserved motifs characteristic of DNA and RNA helicases and the carboxy-terminal domain shares over 55% sequence similarity with the polymerase domains of prokaryotic DNA polymerase I-like enzymes. This is the first reported member of this family of DNA polymerases in a eukaryotic organism, as well as the first example of a single polypeptide with homology to both DNA polymerase and helicase motifs. Identification of a closely related gene in the genome of Caenorhabditis elegans suggests that this novel polypeptide may play an evolutionarily conserved role in the repair of DNA damage in eukaryotic organisms.

Psychological determinants of anginal pain perception during exercise testing of stable patients after recovery from acute myocardial infarction or unstable angina pectoris.

The objective of this study was to test the hypothesis that psychological factors are determinants of anginal symptoms during positive exercise tests. The sample consisted of clinically stable patients who were enrolled in the Multicenter Study of Myocardial Ischemia 1 to 6 months after admission to a coronary care unit. Among 186 post-myocardial infarction patients, 151 developed ischemia (i.e., a stress-induced myocardial perfusion defect) without symptoms (silent ischemia) and 35 developed angina with ischemia (symptomatic ischemia) during a thallium exercise test; among 39 patients who had been hospitalized for unstable angina, 24 developed silent ischemia and 15 developed symptomatic ischemia. Two sets of psychometric tests were administered: set 1, factors that influence awareness of physical symptoms, and set 2, factors associated with biases toward or against reporting perceived symptoms. Two hundred eleven patients produced complete data in each set. Analysis of set 1 factor scores revealed significant effects of symptom status (p = 0.006) and index event (p = 0.02), but no interaction. No effects were found in set 2. Patients who are clinically stable after recovery from an acute coronary event and who experience angina during exercise testing are more aware of physical symptoms in general than are comparable patients with silent ischemia. Psychological biases toward or against reporting perceived symptoms do not differentiate these groups. Thus, it appears that silent ischemia is probably "silent" in the sense of being truly asymptomatic rather than of stoic endurance or denial of perceived symptoms.

Change of intradisc pressure versus volume change.

To gain further insight into the biomechanics of the intervertebral disc and determine a potential mechanism for causation and relief of symptoms related to a herniated disc, the pressure-volume relationship was determined within the nucleus pulposus (NP). In 17 intact human cadaver lumbar discs, pressure was measured continuously within the NP by means of a miniature strain gauge at the tip of a size 4 French (1.3 mm) catheter inserted into the NP. The volume of the NP was increased at the slow, continuous rate of 0.034 ml/min by the pump-regulated infusion of saline colored with methylene blue. In 12 unloaded discs, NP pressure rose in a linear fashion (linear r2 = 0.96) from an initial mean pressure of 174 +/- 81 kPa. The mean rate of pressure rise was 327 +/- 109 kPa/ml of volume increase. The peak pressure reached was limited to 550 kPa by the capacity of the strain gauge. Similar linear relationships were obtained during saline infusion into 5 vertically loaded discs. The data define the pressure-volume relationship within the disc and show that the NP, surrounded by the relatively inelastic annulus and the solid vertebral end-plates, has the properties of a tight hydraulic space in which a large pressure rise will regularly result from a small increase in volume. Presumably, the opposite is also true. The data may provide a biomechanical basis for variation in symptoms related to physiological changes in disc volume, and for any benefit obtained from interventions designed to remove disc tissue.

Living alone after myocardial infarction. Impact on prognosis.

OBJECTIVE: To determine if the presence of a disrupted marriage or living alone would be an independent prognostic risk factor for a subsequent major cardiac event following an initial myocardial infarction. DESIGN: Prospective evaluation in the placebo wing of a randomized, double-blind drug trial in patients with an enzyme-documented acute myocardial infarction who were admitted to a coronary care facility. Data for living alone and/or a marital disruption were entered into a Cox proportional hazards model constructed from important physiologic and nonphysiologic factors in the same database. SETTING: Multicenter trial in a mixture of community and academic hospitals in the United States and Canada. PATIENTS: All consenting patients who were 25 to 75 years of age and without other serious diseases were enrolled (placebo, N = 1234) within 3 to 15 days of the index infarction and followed for a period of 1 to 4 years (mean, 2.1 years). Nine hundred sixty-seven patients were followed for 1.1 years and 530 for 2.2 years. PRIMARY OUTCOME MEASURE: Recurrent major cardiac event (either recurrent nonfatal infarction or cardiac death). RESULTS: Living alone was an independent risk factor, with a hazard ratio of 1.54 (95% confidence interval, 1.04 to 2.29; P less than .03). Using the Kaplan-Meier statistical method for calculation, the recurrent cardiac event rate at 6 months was 15.8% in the group living alone vs 8.8% in the group not living alone. Risk remained significant throughout the follow-up period (P = .001). A disrupted marriage was not an independent risk factor. CONCLUSION: Living alone but not a disrupted marriage is an independent risk factor for prognosis after myocardial infarction when compared with all other known risk factors.

Laser radiation at various wavelengths for decompression of intervertebral disk. Experimental observations on human autopsy specimens.

The interaction of laser radiation with the nucleus pulposus from autopsy specimens of human intervertebral disks was evaluated at different wavelengths (193 nm, 488 nm & 514 nm, 1064 nm, 1318 nm, 2150 nm, 2940 nm, and 10600 nm). A significant correlation of linear least squares fit of the mass ablated as a function of incident energy was found for all lasers used except the Excimer at 193 nm. The 2940-nm Erbium:YAG laser was most efficient in terms of mass of disk ablated per joule in the limited lower range where this wavelength was observed. At higher energy levels, the CO2 laser in the pulsed mode was most efficient. However, the Nd:YAG 1064-nm and 1318-nm lasers are currently best suited for percutaneous laser disk decompression because of the availability of usable waveguides. Carbonization of tissue with the more penetrating Nd:YAG 1064-nm laser increases the efficiency of tissue ablation and makes it comparable to the Nd:YAG 1318-nm laser.

Experimental Nd:YAG laser disintegration of methylmethacrylate. Analysis of gaseous products.

The difficulty of removing intramedullary methylmethacrylate (MM) in revision of hip prostheses is considerable. To facilitate its removal from bone, Nd:YAG laser energy was delivered to a bovine anatomic specimen of the femur with the medullary canal filled with MM. Through an endoscope the laser was observed to soften and ablate the methyl methacrylate. The vapors generated by this process were analyzed by mass spectrometry. The two chief components were hydrogen and carbon monoxide, followed by carbon dioxide, methane, acetylene, and C3 unsaturated hydrocarbons. A high capacity smoke evacuator must be used in conjunction with laser vaporization of methylmethacrylate. The method should be evaluated under in vivo conditions in dogs before it is attempted in patients.

Association between transient pulmonary congestion during acute myocardial infarction and high incidence of death in six months.

In a prospective multicenter study of 866 patients after acute myocardial infarction (AMI), an increased or excessive mortality rate (13%) was confined to the first 6 months after AMI. In the subsequent 18 months of follow-up, the mortality rate (4%) was similar to that in coronary patients in chronic stable condition. Analysis of patients who died in the first 6 months revealed that 55% had had pulmonary congestion at the time of the index AMI. Neither these patients nor the others who died in the early period were found to have more severe ventricular dysfunction, more malignant arrhythmias or more severe ischemia than patients who died after 6 months. The reason for the high and early mortality in patients with pulmonary congestion is not clear, particularly because 30% had reasonable ventricular function, with an ejection fraction of more than 40%. However, given the poor prognosis of these patients, early and aggressive diagnostic efforts should be undertaken to exclude jeopardized regions remote from the initial AMI.

Plasma lactate and ventilation thresholds in trained and untrained cyclists.

Six trained male cyclists and six untrained sedentary men were studied to determine whether the plasma lactate threshold (PLT) and ventilation threshold (VT) occur at the same work rate in both fit and unfit populations. The PLT was determined from a marked increase in plasma lactate concentration ([La]) and VT from a nonlinear increase in expired minute ventilation (VE) during incremental leg-cycling tests; work rate was increased 30 W every 2 min until volitional exhaustion. The trained subjects' mean VO2 max (63.8 ml O2 X kg-1 X min-1) and VT (65.8% VO2 max) were significantly higher (P less than 0.05) than the untrained subjects' mean VO2max (35.5 ml O2 X kg-1 X min-1) and VT (51.4% VO2 max). The trained subjects' mean PLT (68.8% VO2 max) and VT did not differ significantly, but the untrained subjects' mean PLT (61.6% VO2 max) was significantly higher than their VT. The trained subjects' mean peak [La] (10.5 mmol X l-1) did not differ significantly from the untrained subjects' mean peak [La] (11.5 mmol X l-1). However, the time of appearance of the peak [La] during passive recovery was inversely related to VO2 max. These results suggest that variance in lactate diffusion and/or removal processes between the trained and untrained subjects may account in part for the different relationships between the VT and PLT in each population.

Type A behavior and survival after acute myocardial infarction.

To ascertain the influence of personality factors on the course of coronary artery disease, we measured Type A behavior in 516 patients within two weeks after an acute myocardial infarction, using the Jenkins Activity Survey questionnaire. Over a follow-up period of one to three years, there was no relation between the Type A score and total mortality, cardiac mortality, time to death for nonsurvivors, left ventricular ejection fraction, or duration of the stay in the coronary care unit. These negative findings were not changed by restricting the analyses to men below 61 years of age or by comparing extreme score categories. The contributions of behavioral, demographic, and cardiac physiologic factors to postinfarction mortality were also evaluated by multivariate survivorship analyses. The physiologic factors were the only ones that contributed a significant and independent mortality risk; the Type A score did not enter the survivorship model (relative risk, 0.8; 95 per cent confidence interval, 0.5 to 1.5). Thus, we found no relation between Type A behavior and the long-term outcome of acute myocardial infarction.

Vascular recanalization with the argon laser: the role of blood in the transmission of laser energy.

Successful revascularization of stenosed arteries with the argon laser optical fiber catheter requires delivery of high-power-density energy to the lesion. Because of the high energy absorption by hemoglobin at the argon laser wavelengths, which would attenuate transmission of laser energy to the obstruction, we measured argon laser transmission through blood at different concentrations. We found that no transmission occurred through 1 mm of whole blood with hematocrit of 42 and that blood must be diluted to a hematocrit below 1 for laser energy to be delivered. Successful use of the intravascular argon laser is dependent on the creation of a transmission medium by displacement of blood by saline or other non-absorbing substance.

Absence of distal emboli during in vivo laser recanalization.

We used an argon laser to recanalize occluded arteries in vivo and to determine the extent of any particulate matter resulting from the procedure that might cause embolization. Thrombosis was achieved by balloon de-endothelialization and thrombin injection in 12 canine femoral or carotid arteries in six dogs. The resulting totally occlusive thrombi (2.5-8.5 cm in length) were laser treated in situ 2-29 days after formation, using laser tip power outputs of 0.8-3.4 W. The treated arterial segment was perfused before, during, and after the recanalization procedure with 1,000 ml of heparinized saline. All saline passing through the artery was collected and studied by filtration through 20-micron pore filter paper; 50-ml unfiltered aliquots were studied by automated, calibrated cell counting and microscopically after centrifugation and staining. All vessels except one were completely recanalized. Filtration yielded a thin reddish, dust-like residue. No large fragments were found in 11 of 12 effluents. The residue after centrifugation was almost entirely composed of erythrocytes plus one to four 20-30-micron strands of amorphous cellular material per high-power field. Cell counting showed that 99.1% of the material in the effluents was smaller than 9.37 micron. These results demonstrate the presence of some filterable debris following laser recanalization of intraarterial thrombi, but it probably lacks any physiologic significance. Further study of this effect of laser recanalization is needed.

Complete patency in thrombus-occluded arteries two weeks after laser recanalization.

The potential problem of rethrombosis after laser recanalization was studied in 16 thrombus-occluded canine femoral arteries. Balloon de-endothelialization and thrombin-human blood injection produced adherent, completely occlusive thrombi 4.13 +/- 1.54 cm in length; laser exposure of the thrombi occurred at 18.35 +/- 22.1 hours. The argon laser catheter was introduced via a proximal arteriotomy and a power of 3.83 +/- 0.58 W delivered for 411.3 +/- 296.87 seconds. Follow-up period was 14 days. All arteries were patent immediately after and 14 days following lasing, as demonstrated by angiography. There was no vessel perforation. Seven of the dogs were maintained on aspirin and dipyridamole 4 days before and throughout the study, but there were no differences in thrombus length, laser power, or duration of laser exposure between these dogs and those receiving no anti-platelet therapy. Control thrombosed arteries (without laser energy application) showed no autolysis within 14 days in all dogs and up to 95 days in three dogs followed for this period of time. These data show that rethrombosis of totally occluded, thrombosed arteries is not present up to 2 weeks later after laser recanalization, with or without the aid of anti-platelet therapy.

A new thermal sensor for surface-based measurement of local myocardial perfusion.

The transient response of a new thermal sensor for continuous surface-based measurement of local tissue blood flow was evaluated in the beating dog heart. The sensor is 2.5 mm square, tests on the heart, and responds to flow changes only within a region of tissue 3-4 mm below its location on the epicardial surface. Initial studies demonstrate excellent sensitivity, good frequency response with a time constant of the order of 10-11 s, and an ability to continuously monitor changes in local myocardial blood flow during a variety of well-understood interventions, including coronary artery occlusion, reactive hyperemia, and intravenous administration of nitroglycerin and dipyridamole.

Steady-state analysis and evaluation of a new thermal sensor for surface measurements of tissue perfusion.

The steady-state response and operating characteristics of a new thermal sensor for surface measurements of local tissue perfusion have been analyzed theoretically and evaluated in vivo. The flow measurement system incorporates an electrically isolated thin-film thermal sensor, which is maintained at a fixed temperature by high frequency response electronic circuitry. The sensor rests on the tissue surface, and the power required to maintain a fixed probe to tissue temperature elevation is measured and related to tissue blood flow. A theoretical analysis of the steady-state probe response to flow changes was carried out employing the bio-heat-transfer equation and a solution based on Fourier series to describe the temperature distribution within the tissue domain. A comparison of steady-state theory to results obtained from initial experimental tests on the surface of the dog heart, over a perfusion range 0.51 to 2,00 ml/min/g, shows close agreement. The probe demonstrates good sensitivity to flow changes, provides stable and continuous measurements, and appears promising for both research and clinical applications.