Plethysmography and impulse oscillometry assessment of tiotropium and ipratropium bromide; a randomized, double-blind, placebo-controlled, cross-over study in healthy subjects.

AIMS: Spirometry, plethysmography and impulse oscillometry (IOS) measure different aspects of lung function. These methods have not been compared for their ability to assess long- and short-acting anticholinergic agents. We therefore performed a double-blind, placebo-controlled, four-way cross-over study in 30 healthy subjects. METHODS: Single doses of tiotropium bromide (Tio) 54 and 18 mcg, ipratropium bromide (IB) 40 mcg and placebo were administered. Specific conductance (sGaw), total lung capacity (TLC), inspiratory capacity (IC) and residual volume (RV) were measured using plethysmography, while IOS measured resistance (R5-25) and reactance (RF and X5). Pulmonary function was measured for 26 h post dose. RESULTS: Tio caused significant improvements in sGaw, forced expiratory voume in 1 s (FEV(1)), maximum mid-expiratory flow (MMEF) and R5-R25 at time points up to 26 h, with no clear differences between doses. IB improved the same parameters, but only up to 8 h. The weighted mean change (0-24 h) caused by Tio 54 mcg compared with placebo for FEV(1) was 240 ml (95% confidence interval 180, 300), while for sGaw the ratio of geometric means (Tio compared with placebo) was 1.35 (1.28, 1.41). Neither drug caused consistent statistically significant changes in RF, forced vital capacity, TLC or IC over 26 h. RV was significantly improved from 8 to 24 h by Tio 54 mcg only. CONCLUSIONS: In addition to spirometry, IOS resistance measurements and sGaw can distinguish between the effects of long- and shortacting anticholinergic effects in healthy subjects.

Pharmacokinetic and glucodynamic variability: assessment of insulin glargine, NPH insulin and insulin ultralente in healthy volunteers using a euglycaemic clamp technique.

AIMS/HYPOTHESIS: This single-dose, double-blind, randomised, parallel-group study evaluated the reproducibility in systemic exposure and glucodynamic effect of insulin glargine, NPH insulin (NPH) and insulin ultralente (ultralente) using the manually adjusted euglycaemic clamp technique. METHODS: In total, 36 healthy volunteers received two consecutive s.c. injections (0.4 IU/kg) of glargine, NPH or ultralente with a wash-out period of 7 days between treatments. RESULTS: In healthy volunteers, glargine presented well-reproduced flat concentration profiles and no pronounced peaks in activity. NPH, by contrast, showed well-defined peaks in concentration and glucose disposal, while ultralente had highly variable profiles. Within-subject variability (ANOVA) for insulin exposure over 24 h was 15% for glargine and 19% for NPH, compared with 67% for ultralente (p<0.05, glargine and NPH vs ultralente). The 49% within-subject variability in total glucose disposal (glucose infusion rate [GIR]-AUC0-24 h) with ultralente was about twice as large as the 22% with NPH (p<0.05), but was intermediate with glargine at 31% (p=NS). By contrast, variability in the diurnal time-action profile (SD of diurnal day-to-day differences in GIR) for glargine was 30% (p<0.05) and 50% (p<0.05) less than with NPH and ultralente, respectively. No serious adverse events were reported. CONCLUSIONS/INTERPRETATION: Although representing insulins of different profiles, glargine and NPH showed a high and similar reproducibility of total absorption and glucodynamic effect, whereas ultralente proved to have poor reproducibility. However, while NPH yields peaks in concentration and activity, glargine shows flat and non-fluctuating profiles resulting in less variation in day-to-day 24-h activity.

Biotransformation of insulin glargine after subcutaneous injection in healthy subjects.

OBJECTIVE: It is important to establish pharmacokinetic or pharmacodynamic differences between novel insulin analogues and human insulin. This study examined the primary metabolic degradation products of insulin glargine (LANTUS) in humans. DESIGN: In this single dose, open-label study, insulin glargine was administered subcutaneously at a dose of 0.6 IU/kg; placebo was administered to one control subject. PATIENTS: Four healthy male subjects, plus one control subject, aged 18-50 years were enrolled in this study. MEASUREMENTS: Following insulin glargine administration, blood glucose levels were clamped at the subjects' fasting concentration for 6 h and the amount of 20% glucose infused to maintain this baseline concentration was recorded. Metabolite profiling was performed in plasma and injection site tissue using HPLC and radioimmunoassay (RIA). Pharmacokinetics were evaluated by RIA of serum and plasma immunoreactive insulin levels. The primary pharmacodynamic measure was the glucose infusion rate (GIR). Safety was evaluated by measuring blood glucose concentrations during the clamp and adverse events were observed by the investigator or reported by the subject. RESULTS: Metabolic profiling revealed a clear pattern: insulin glargine is metabolised by sequential cleavage at the carboxy terminus of the B chain, to yield products M1 and M2, which are both structurally similar to human insulin. These degradation products are present both at the injection site and in plasma. CONCLUSION: Thus, during treatment with a subcutaneous injection of insulin glargine, metabolic degradation is likely to be initiated at the injection site and continued within the circulatory system.

Regulation of caldesmon activity by Cdc2 kinase plays an important role in maintaining membrane cortex integrity during cell division.

To study the mitosis-specific phosphorylation of caldesmon (CaD), we generated a mutant of the C-terminal fragment (amino acids 244-538) of human fibroblast CaD (CaD39-6F), as well as a mutant of the full-length CaD (CaD-6F), in which all six potential phosphorylation sites for Cdc2 kinase were abolished. The mitotic CaD39-6F-overexpressing cells required more time to progress from anaphase start to 50% cytokinesis, exhibited larger size, and abnormally formed numerous small blebs. In contrast, overexpression of the wild-type C-terminal fragment of CaD (CaD39) did not result in abnormal bleb formation, but led to larger size and prolonged the time requirement between anaphase start and 50% cytokinesis. Similar abnormal blebs were also observed in the CaD-6F-overexpressing cells. CaD-6F-overexpressing cells did not show larger size but required more time to progress from anaphase start to 50% cytokinesis. These results suggest that mitosis-specific phosphorylation of CaD plays a role in inhibiting bleb formation and that the N-terminal fragment of CaD is required for cell size determination.

Equipotency of insulin glargine and regular human insulin on glucose disposal in healthy subjects following intravenous infusion.

The absolute glucose disposal of insulin glargine (Lantus) was compared to that of regular human insulin in healthy subjects (n=20) using the euglycaemic clamp technique in a single-dose, double-blind, randomized, two-way crossover design. Subjects received 30-minute intravenous infusions of insulin glargine (0.1 IU/kg) or human insulin (0.1 IU/kg) and a 20% glucose solution infused at a variable rate to maintain euglycaemia at the subject's baseline glucose level. At equal baseline blood glucose levels (4.42 mmol/l [range, 4.00-5.16 mmol/l] and 4.42 mmol/l [range, 4.01-4.94 mmol/l], respectively), the area under the glucose infusion rate (GIR) time curves from 0-6 hours (AUC(0-6h)) was within the bioequivalence range (insulin glargine, 663.92 mg/kg; human insulin, 734.85 mg/kg). Both the time to maximum GIR and the suppression of serum C-peptide were similar with insulin glargine and human insulin. The resulting maximum serum insulin concentrations (Cmax) were 151.16 microIU/ml and 202.23 microIU/ml, and the time to Cmax (Tmax) was 30 minutes (the duration of the infusion). The observed differences in the Cmax (the mean value for insulin glargine was about 25% lower than that of human insulin) could be explained by lower cross-reactivity of insulin glargine in the human insulin radioimmunoassay. The employed intravenous route, though definitely not the intended clinical use of insulin glargine, provided the clinical evidence in healthy subjects that on a molar basis insulin glargine is equipotent to regular human insulin regarding glucose disposal.

Pharmacokinetics and absolute oral bioavailability of an 800-mg oral dose of telithromycin in healthy young and elderly volunteers.

BACKGROUND: This two-way, randomized, single-dose, crossover study determined the pharmacokinetics and absolute oral bioavailability of telithromycin in young and elderly healthy subjects. METHODS: Twelve young (18-40 years) and 12 elderly (>65 years and

A role for myosin VII in dynamic cell adhesion.

BACKGROUND: The initial stages of phagocytosis and cell motility resemble each other. The extension of a pseudopod at the leading edge of a migratory cell and the formation of a phagocytic cup are actin dependent, and each rely on the plasma membrane adhering to a surface during dynamic extension. RESULTS: A myosin VII null mutant exhibited a drastic loss of adhesion to particles, consistent with the extent of an observed decrease in particle uptake. Additionally, cell-cell adhesion and the adhesion of the leading edge to the substratum during cell migration were defective in the myosin VII null cells. GFP-myosin VII rescued the phagocytosis defect of the null mutant and was distributed in the cytosol and recruited to the cortical cytoskeleton, where it appeared to be enriched at the tips of filopods. It was also localized to phagocytic cups, but only during the initial stages of particle engulfment. During migration, GFP-myosin VII is found at the leading edge of the cell. CONCLUSIONS: Myosin VII plays an important role in mediating the initial binding of cells to substrata, a novel role for an unconventional myosin.

Tortoise, a novel mitochondrial protein, is required for directional responses of Dictyostelium in chemotactic gradients.

We have identified a novel gene, Tortoise (TorA), that is required for the efficient chemotaxis of Dictyostelium discoideum cells. Cells lacking TorA sense chemoattractant gradients as indicated by the presence of periodic waves of cell shape changes and the localized translocation of cytosolic PH domains to the membrane. However, they are unable to migrate directionally up spatial gradients of cAMP. Cells lacking Mek1 display a similar phenotype. Overexpression of Mek1 in torA- partially restores chemotaxis, whereas overexpression of TorA in mek1- does not rescue the chemotactic phenotype. Regardless of the genetic background, TorA overexpressing cells stop growing when separated from a substrate. Surprisingly, TorA-green fluorescent protein (GFP) is clustered near one end of mitochondria. Deletion analysis of the TorA protein reveals distinct regions for chemotactic function, mitochondrial localization, and the formation of clusters. TorA is associated with a round structure within the mitochondrion that shows enhanced staining with the mitochondrial dye Mitotracker. Cells overexpressing TorA contain many more of these structures than do wild-type cells. These TorA-containing structures resist extraction with Triton X-100, which dissolves the mitochondria. The characterization of TorA demonstrates an unexpected link between mitochondrial function, the chemotactic response, and the capacity to grow in suspension.

Pharmacokinetics of the new ketolide telithromycin (HMR 3647) administered in ascending single and multiple doses.

Telithromycin (HMR 3647) is a novel ketolide antimicrobial with good activity against both common and atypical respiratory pathogens, including many resistant strains. This randomized, three-period crossover study determined the dose proportionality of telithromycin pharmacokinetics after single and multiple dosing in healthy subjects. In each treatment period, subjects received a single oral dose of 400, 800 or 1,600 mg of telithromycin followed 4 days later by the same dose once daily for 7 days. Blood and urine samples were taken throughout the study for determination of pharmacokinetic parameters for telithromycin and RU 76363, its main metabolite. Telithromycin and RU 76363 achieved steady state within 2 to 3 days of once-daily dosing. A slight accumulation of telithromycin was observed after 7 days of therapy, with values of the area under the concentration-time curve from 0 to 24 h approximately 1.5 times higher than those achieved with the single dose. The pharmacokinetics of telithromycin and RU 76363 deviated moderately from dose proportionality. At a dose of 800 mg/day, telithromycin attained mean maximal and trough plasma concentrations of 2.27 and 0. 070 mg/liter respectively. Elimination was biphasic; initial and terminal half-lives were 2.87 and 9.81 h for the 800-mg dose. Study medication was well tolerated, although adverse events tended to be more frequent at the 1,600-mg dose. This study showed that telithromycin was generally well tolerated and suggests that a once-daily 800-mg oral dose of telithromycin maintains an effective concentration in plasma for the treatment of respiratory tract infections involving the key respiratory pathogens.

Three-dimensional reconstruction and motion analysis of living, crawling cells.

Cell behavior is three-dimensional (3-D), even when it takes place on a flat surface. Migrating cells form pseudopods on and off the substratum, and the cell body undergoes height changes associated with a 1 min behavior cycle. Inside the cell, the nucleus has a 3-D migratory cycle, and vesicles move up and down in the z-axis as a cell locomotes. For these reasons, the two-dimensional (2-D) analysis of cellular and subcellular behavior is, in many cases, inadequate. We have, therefore, developed 3-D motion analysis systems that reconstruct the cell surface, nucleus, pseudopods, and vesicles of living, crawling cells in 3-D at time intervals as short as 1 s, and compute more than 100 parameters of motility and dynamics morphology at 1-s intervals. We are now in the process of developing a multimode reconstruction system that will allow us to reconstruct and analyze fluorescently tagged molecular complexes within the differential interference contrast-imaged subcellular architecture of a crawling cell. These evolving technologies should find wide application for a host of biomedical problems.

The internal phosphodiesterase RegA is essential for the suppression of lateral pseudopods during Dictyostelium chemotaxis.

Dictyostelium strains in which the gene encoding the cytoplasmic cAMP phosphodiesterase RegA is inactivated form small aggregates. This defect was corrected by introducing copies of the wild-type regA gene, indicating that the defect was solely the consequence of the loss of the phosphodiesterase. Using a computer-assisted motion analysis system, regA(-) mutant cells were found to show little sense of direction during aggregation. When labeled wild-type cells were followed in a field of aggregating regA(-) cells, they also failed to move in an orderly direction, indicating that signaling was impaired in mutant cell cultures. However, when labeled regA(-) cells were followed in a field of aggregating wild-type cells, they again failed to move in an orderly manner, primarily in the deduced fronts of waves, indicating that the chemotactic response was also impaired. Since wild-type cells must assess both the increasing spatial gradient and the increasing temporal gradient of cAMP in the front of a natural wave, the behavior of regA(-) cells was motion analyzed first in simulated temporal waves in the absence of spatial gradients and then was analyzed in spatial gradients in the absence of temporal waves. Our results demonstrate that RegA is involved neither in assessing the direction of a spatial gradient of cAMP nor in distinguishing between increasing and decreasing temporal gradients of cAMP. However, RegA is essential for specifically suppressing lateral pseudopod formation during the response to an increasing temporal gradient of cAMP, a necessary component of natural chemotaxis. We discuss the possibility that RegA functions in a network that regulates myosin phosphorylation by controlling internal cAMP levels, and, in support of that hypothesis, we demonstrate that myosin II does not localize in a normal manner to the cortex of regA(-) cells in an increasing temporal gradient of cAMP.

Forced expression of a dominant-negative chimeric tropomyosin causes abnormal motile behavior during cell division.

Forced expression of the chimeric human fibroblast tropomyosin 5/3 (hTM5/3) in CHO cell was previously shown to affect cytokinesis [Warren et al., 1995: J. Cell Biol. 129:697-708]. To further investigate the phenotypic consequences of misexpression, we have compared mitotic spindle organization and dynamic 2D and 3D shape changes during mitosis in normal cells and in a hTM5/3 misexpressing (mutant) cell line. Immunofluorescence microscopy of wild type and mutant cells stained with monoclonal anti-tubulin antibody revealed that the overall structures of mitotic spindles were not significantly different. However, the axis of the mitotic spindle in mutant cells was more frequently misaligned with the long axis of the cell than that of wild type cells. To assess behavioral differences during mitosis, wild type and mutant cells were reconstructed in 2D and 3D and motion analyzed with the computer-assisted 2D and 3D Dynamic Image Analysis Systems (2D-DIAS, 3D-DIAS). Mutant cells abnormally formed large numbers of blebs during the later stages of mitosis and took longer to proceed from the start of anaphase to the start of cytokinesis. Furthermore, each mutant cell undergoing mitosis exhibited greater shape complexity than wild type cells, and in every case lifted one of the two evolving daughter cells off the substratum and abnormally twisted. These results demonstrate that misexpression of hTM5/3 in CHO cells leads to morphological instability during mitosis. Misexpression of hTM5/3 interferes with normal tropomyosin function, suggesting in turn that tropomyosin plays a role through its interaction with actin microfilaments in the regulation of the contractile ring, in the localized suppression of blebbing, in the maintenance of polarity and spatial symmetry during cytokinesis, and in cell spreading after cytokinesis is complete.

Clathrin plays a novel role in the regulation of cell polarity, pseudopod formation, uropod stability and motility in Dictyostelium.

Although the traditional role of clathrin has been in vesicle trafficking and the internalization of receptors, a novel role in cytokinesis was recently revealed in an analysis of a clathrin-minus Dictyostelium mutant (chc(-)). chc(-) cells grown in suspension were demonstrated to be defective in assembling myosin II into a normal contractile ring. To test whether this defect reflected a more general one of cytoskeletal dysfunction, chc(-) cells were analyzed for cell polarity, pseudopod formation, uropod stability, cell locomotion, chemotaxis, cytoskeletal organization and vesicle movement. chc(-) cells crawled, chemotaxed, localized F-actin in pseudopods, organized their microtubule cytoskeleton in a relatively normal fashion and exhibited normal vesicle dynamics. Although chc(-) cells extended pseudopods from the anterior half of the cell with the same frequency as normal chc(+) cells, they extended pseudopods at twice the normal frequency from the posterior half of the cell. The uropods of chc(-) cells also exhibited spatial instability. These defects resulted in an increase in roundness, a reduction in polarity, a reduction in velocity, a dramatic increase in turning, a high frequency of 180 degrees direction reversals and a decrease in the efficiency of chemotaxis. All defects were reversed in a rescued strain. These results are the first to suggest a novel role for clathrin in cell polarity, pseudopod formation, uropod stability and locomotion. It is hypothesized that clathrin functions to suppress pseudopod formation and to stabilize the uropod in the posterior half of a crawling cell, two behavioral characteristics that are essential for the maintenance of cellular polarity, efficient locomotion and efficient chemotaxis.

Dihydrofluorescein diacetate is superior for detecting intracellular oxidants: comparison with 2',7'-dichlorodihydrofluorescein diacetate, 5(and 6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate, and dihydrorhodamine 123.

To detect intracellular oxidant formation during reoxygenation of anoxic endothelium, the oxidant-sensing fluorescent probes, 2',7'-dichlorodihydrofluorescein diacetate, dihydrorhodamine 123, or 5(and 6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate were added to human umbilical vein endothelial cells during reoxygenation. None of these fluorescent probes were able to differentiate the controls from the reoxygenated cells in the confocal microscope. However, dihydrofluorescein diacetate demonstrated fluorescence of linear structures, consistent with mitochondria, in reoxygenated endothelium. This work tests the hypothesis that dihydrofluorescein diacetate is a better fluorescent probe for detecting intracellular oxidants because it is more reactive toward specific oxidizing species. To investigate this, dihydrofluorescein diacetate was exposed to various oxidizing species (hydrogen peroxide, superoxide [KO2], peroxynitrite, nitric oxide, horseradish peroxidase, ferric iron, xanthine oxidase, cytochrome c, and lipoxygenase) and compared with the three other popular probes. Though oxidized dihydrofluorescein has higher molar fluorescence, comparison of the reactions of dihydrofluorescein with these other three probes in a cell-free system indicates that dihydrofluorescein is sometimes less fluorescent than the other probes. In addition, we find that the reactivity of all of the probes is very complex. Based on the results reported here, it is no longer appropriate to think of these probes as detecting a specific oxidizing species in cells, such as H2O2, but rather as detectors of a broad range of oxidizing reactions that may be increased during intracellular oxidant stress. Cell-loading studies indicate that dihydrofluorescein achieves higher intracellular concentrations than the second brightest intracellular probe, 2',7'-dichlorodihydrofluorescein. This fact and its higher molar fluorescence may account for the superior brightness of dihydrofluorescein diacetate. Dihydrofluorescein diacetate may be a superior fluorescent probe for many cell-based studies.

The photic sneeze reflex and ocular anesthesia.

BACKGROUND AND PURPOSE: Intravenous sedation to minimize discomfort from local anesthetic injection has many potential complications including severe involuntary sneezing (i.s.). This prospective study evaluates the occurrence of i.s. and a history of photic sneezing (p.s.). METHODS AND MATERIALS: All patients receiving local anesthesia (retrobulbar or periocular injections) after intravenous thiopentone for eye surgery during eight months were asked about p.s. and observed for i.s. RESULTS: The 557 patients (40% males) had a mean age of 69.9 years and 14% recalled p.s. (29.5% males). I.s. developed in 5.2% of the 557. Only 7.6% of those with p.s. developed i.s. After periocular injections 23.8% developed as compared to 4.5% after retrobulbar injections. (P < 0.001). There was no relationship between p.s. and i.s. (p = 0.43). CONCLUSION: I.s. is not linked to p.s., with males and females at equal risk for either. I.s. is more common after periocular injections.

A computer-assisted system for reconstructing and interpreting the dynamic three-dimensional relationships of the outer surface, nucleus and pseudopods of crawling cells.

Newly developed software additions to the three-dimensional dynamic image analysis system, 3D-DIAS, are described for simultaneously reconstructing and motion analyzing in three dimensions the outer surface, nucleus and pseudopods of living, crawling cells. This new system is then used to describe for the first time a nuclear behavior cycle in translocating Dictyostelium discoideum amoebae and to investigate the role of pseudopod extension in this process. The nuclear behavior cycle is tuned to the two phases of the general cell behavior cycle [Wessels et al., 1994], and includes nuclear migration both in the z- and in the x,y-axes from the proximal border of the prior anterior pseudopod to the proximal border of a newly expanding anterior pseudopod. Nuclear migration is cued by pseudopod-substratum contact, achieves velocities in excess of 50 microm/min, and is accompanied by characteristic changes in nuclear shape. The rules and characteristics of nuclear behavior are demonstrated to be intact in two mutants affecting pseudopod formation, a myosin IB null mutant (myoB-) and a myosin II heavy chain phosphorylation mutant (3XALA). The rules and characteristics of nuclear migration, however, are disrupted upon dissolution of microtubules by colcemid. Together the above results demonstrate that the newly developed 3D-DIAS system can be used to gain new insights into the dynamic changes in the intracellular 3D architecture associated with cellular translocation.

Hook and loop fastener on loose prisms assists in measuring ocular deviation.

It is difficult to hold a pair of loose prisms in one hand. Doing so can lead to inaccuracy, which worsens with large-angle exotropia or vertical deviations. The authors applied a self-adhesive hooked Velcro (Velcro Sticky Back Tape, Velcro USA Inc., Manchester, NH) strip to the base and the top of loose prisms. They applied the complementary looped Velcro to a wooden bar. As a result, a pair of prisms could be suspended horizontally and/or vertically while being held in one hand. Forty consecutive patients undergoing strabismus surgery without adjustable sutures were retrospectively studied to evaluate the accuracy of this method. Clinical use confirmed its ease and convenience. Of 19 patients with exotropia and 15 patients with esotropia (mean ages 41.9 and 15.7 years, respectively; mean deviations 44.7 and 49.8 D, respectively), 4 patients with abducens paralysis, and 2 patients with trochlear palsies, 1 surgery achieved less than 10 D of residual deviation in all but 2 (5.9%). This simple, inexpensive system can assist with the clinical evaluation of ocular deviation.

Salvaged viscoelastic reduces irrigation frequency during cataract surgery.

A more viscous solution may decrease the need for frequent irrigations to maintain a clear cornea during cataract surgery. Fifty-four consecutive cataract patients were prospectively randomized to receive a drop of viscoelastic or saline on the cornea at the start of surgery. The surgical scrub nurse was instructed to irrigate only when the cornea appeared hazy or at the surgeon's request. The number of irrigations, ultrasound time and energy, and surgery times were compared using the Student's t test. Only one ampoule of viscoelastic was used for each case. When the patients who received viscoelastic were compared with the patients who received saline, the mean numbers of irrigations were 2 and 18, whereas the mean numbers of irrigations per minute were 0.17 and 1.21, highly significant (P < .0001) differences. The duration of surgery, ultrasound time, and ultrasound energy were less with viscoelastic, but were not statistically significant. Viscoelastic on the cornea reduces the frequency of irrigation without increasing cost.

Phosphorylation of the Dictyostelium myosin II heavy chain is necessary for maintaining cellular polarity and suppressing turning during chemotaxis.

Conversion of the three mapped threonine phosphorylation sites in the myosin II heavy chain tail to alanines results in a mutant (3XALA) in Dictyostelium discoideum, which displays constitutive myosin overassembly in the cytoskeleton and increased cortical tension. To assess the importance of myosin phosphorylation in cellular translocation and chemotaxis, 3XALA mutant cells have been analyzed by 2D and 3D computer-assisted methods in buffer, in a spatial gradient of cAMP, and after the rapid addition of cAMP. 3XALA cells crawling in buffer exhibit distinct abnormalities in cellular shape, the maintenance of polarity and the complexity of the pseudopod perimeter. 3XALA cells crawling in buffer also exhibit a decrease in directionality. In a spatial gradient of cAMP, the behavioral defects are accentuated. In a spatial gradient, 3XALA cells exhibit a repeating 1- to 2-min behavior cycle in which the shape of each cell changes abnormally from elongate to extremely wide with lateral, opposing pseudopods. At the end of each cycle, 3XALA cells turn 90 degrees into the left or right lateral pseudopod, resulting in a dramatic depression in chemotactic efficiency, even though 3XALA cells are chemotactically responsive to cAMP. These results demonstrate that the phosphorylation of myosin II heavy chain plays a critical role in the maintenance of cell shape and in persistent translocation in a spatial gradient of chemoattractant.