Search

In the title molecule, C15H12BrClO2, the two benzene rings form a dihedral angle of 69.30 (3)°. In the crystal structure, weak intermolecular C—H...O hydrogen bonds link molecules into chains propagating along the b axis.

This article is a concise review of our efforts in understanding the biological functions of traction forces, particularly in relation to the detection of rigidity of adhesive materials by fibroblasts.

Actin filaments, microtubules, and intermediate filaments, have all been found to be dynamic structures in living cells. Recent studies have shed important light on the assembly, disassembly, and mobility of these structures. In addition, a growing emphasis has been placed on the regulation of cytoskeletal activities by various signal transduction pathways.

The study was designed to examine the effect of selective alpha1 receptor agonist midodrine hydrochloride in the treatment of children with postural orthostatic tachycardia syndrome.Fifty-five children (23 male, 32 female, age 5 - 19 yrs, mean age 12.3 +/- 3.1 yrs) who came from Peking University First Hospital were included in the study and clinical investigations as well as standing test, basic head-up tilt test and sublingual nitroglycerin-provocated head-up tilt test under quiet circumstance were conducted. They were randomly divided into treatment group (with midodrine hydrochloride and oral rehydration salt treatment) and control group (with oral rehydration salt treatment only). At last, the disease-free rate, improvement rate and effective rate of symptoms, and the rate of HUT from positive to negative response were compared between control group and treatment group. SPSS 10.0 software was used for the statistical analysis of these data.The symptom improvement rate in treatment group was significantly higher than that of control group after three and six weeks of treatment (100.0% vs. 42.4%, P0.001; 100.0% vs. 42.4%, chi2 = 19.352, P0.001). The disease-free rate at follow-up end-point in treatment group was significantly higher than that of control group (77.3% vs. 27.3%, chi2 = 13.239, P0.001). The effective rate at follow-up end-point in treatment group was also significantly higher than that of control group (100.0% vs. 36.4%, chi2 = 22.647, P0.001). The rate of HUT changing from positive to negative response between two groups after three weeks of treatment was not significantly different (31.8% vs. 12.1%, P0.05), but it was significantly different (81.0% vs. 48.5%, P0.05) after six weeks of treatment.Selective alpha1 receptor agonist midodrine hydrochloride is effective in the treatment of children with postural orthostatic tachycardia syndrome.

This paper deals with the experimental study on the insulation parameters characteristics of XLPE cable in the Course of its aging within CuSO4 electrolyte. The aging tests were performed on a section of 10kV transmission XLPE cable by means of water needle method. The insulation characteristic parameters such as insulation resistance, direct current component, capacitance, dielectric loss, mean discharge power, discharge frequency were found to be significantly dependent on the corresponding aging degree of insulation in the course of aging of XLPE cable, and different kind of aging stage has different characteristics, especially during the later period near to breakdown, the amplitude of them has a significantly change. Therefore, according to the changing regularity of the insulation characteristic parameters within XLPE judging the aging degree of XLPE insulation will be an effective method.

Automated segmentation of time-lapse images is a method to facilitate the understanding of the intricate biological progression, e.g., cancer cell migration. To address this problem, we introduce a shape representation enhancement over popular snake models in the context of confident scale-space such that a higher level of interpretation can hopefully be achieved. Our proposed system consists of a hierarchical analytic framework including feedback loops, self-adaptive and demand-adaptive adjustment, incorporating a steerable boundary detail term constraint based on multiscale B-spline interpolation. To minimize the noise interference inherited from microscopy acquisition, the coarse boundary derived from the initial segmentation with refined watershed line is coupled with microscopy compensation using the mean shift filtering. A progressive approximation is applied to achieve represented as a balance between a relief function of watershed algorithm and local minima concerning multi-scale optimality, convergence, and robust constraints. Experimental results show that the proposed method overcomes problems with spurious branches, arbitrary gaps, low contrast boundaries and low signal-to-noise ratio. The proposed system has the potential to serve as an automated data processing tool for cell migration applications.

Syncope is a common pediatric emergency. Based on an epidemiologic survey in the USA, around 15% of children experienced syncopal attack, which strongly influenced the life, study and hurt the children mentally and physiologically. Therefore, exploring the therapeutic regimen has become a hot topic in the field of pediatric cardiology. The aim of this study was to examine the effect of beta receptor blocker in the treatment of children with autonomous nerve mediated syncope.Totally 103 children (43 males, 60 females, age 5 - 19 yrs, median 12.0 +/- 2.6 yrs) with autonomous nerve mediated syncope from Beijing, Hunan, Hubei and Shanghai, were included in this study. Forty-nine of them suffered from vasovagal syncope (VVS) and 54 suffered from postural tachycardia syndrome (POTS). They were randomly divided into treatment group accepting oral metoprolol treatment and control group accepting oral rehydration salt treatment. The frequency of syncopal episodes and the outcome of head-up tilt (HUT) test were observed. SPSS 10.0 software was used for the statistical analysis of these data.The cure rate of children who suffered from VVS and POTS and took oral metoprolol was 60.61% and 68.75%, respectively, but in the control group, the cure rate was only 18.75% and 0.00%, respectively. The rate of improvement of children who suffered from VVS and POTS and were treated with oral metoprolol was 15.15% and 15.63%, respectively, and in the control group, it was 6.25% and 40.91%, respectively. The effective rates for cases of VVS and POTS treated with oral metoprolol were higher than those of cases received oral rehydration salt treatment (P0.01). The percentage of the change from positive HUT to negative for children with VVS and POTS who took oral metoprolol therapy was 60.61% and 68.75%, respectively, but in control group, it was only 18.75% and 9.09%, respectively (P0.01). There was a significant difference in the percentage of the change from positive HUT to negative between children with VVS treated with oral metoprolol and with oral rehydration salt (P0.01). Also, a significant difference was found in the percentage of the change from positive HUT to negative between children with POTS treated with oral metoprolol and with oral rehydration salt (P0.01).beta receptor blocker is effective in the treatment of children with VVS or POTS.

This paper reviewed the progress in researches on antiviral activity of chemical constituents from plants in recent years, the antiviral activity and mechanism of action of flavonoids, alkaloids, terpenoids, coumarins and polysaccharoses were sammarszed, provided new leading compound for antivirus new drugs from the plares in prospect.

We have developed a top-down, rule-based mathematical model to explore the basic principles that coordinate mechanochemical events during animal cell migration, particularly the local-stimulation-global-inhibition model suggested originally for chemotaxis. Cells were modeled as a shape machine that protrudes or retracts in response to a combination of local protrusion and global retraction signals. Using an optimization algorithm to identify parameters that generate specific shapes and migration patterns, we show that the mechanism of local stimulation global inhibition can readily account for the behavior of Dictyostelium under a large collection of conditions. Within this collection, some parameters showed strong correlation, indicating that a normal phenotype may be maintained by complementation among functional modules. In addition, comparison of parameters for control and nocodazole-treated Dictyostelium identified the most prominent effect of microtubules as regulating the rates of retraction and protrusion signal decay, and the extent of global inhibition. Other changes in parameters can lead to profound transformations from amoeboid cells into cells mimicking keratocytes, neurons, or fibroblasts. Thus, a simple circuit of local stimulation-global inhibition can account for a wide range of cell behaviors. A similar top-down approach may be applied to other complex problems and combined with molecular manipulations to define specific protein functions.

Amoeboid movement is believed to involve a pressure gradient along the cell length, with contractions in the posterior region driving cytoplasmic streaming forward. However, a parallel mechanism has yet to be demonstrated in migrating adhesive cells. To probe the distribution of intracellular forces, we microinjected high molecular weight linear polyacrylamide (PAA) as a passive force sensor into migrating NIH3T3 fibroblasts. Injected PAA appeared as amorphous aggregates that underwent shape change and directional movement in response to differential forces exerted by the surrounding environment. PAA injected into the posterior region moved toward the front, whereas PAA in the anterior region never moved to the posterior region. This preferential forward movement was observed only in migrating cells with a defined polarity. Disruption of myosin II activity by blebbistatin inhibited the forward translocation of PAA while cell migration persisted in a disorganized fashion. These results suggest a myosin II-dependent force gradient in migrating cells, possibly as a result of differential cortical contractions between the anterior and posterior regions. This gradient may be responsible for the forward transport of cellular components and for maintaining the directionality during cell migration.

Cytokinesis of animal cells involves the formation of the circumferential actin filament bundle (contractile ring) along the equatorial plane. To analyze the assembly mechanism of the contractile ring, we microinjected a small amount of rhodamine-labeled phalloidin (rh-pha) or rhodamine-labeled actin (rh-actin) into dividing normal rat kidney cells. rh-pha was microinjected during prometaphase or metaphase to label actin filaments that were present at that stage. As mitosis proceeded into anaphase, the labeled filaments became associated with the cortex of the cell. During cytokinesis, rh-pha was depleted from polar regions and became highly concentrated into the equatorial region. The distribution of total actin filaments, as revealed by staining the whole cell with fluorescein phalloidin, showed a much less pronounced difference between the polar and the equatorial regions. The sites of de novo assembly of actin filaments during the formation of the contractile ring were determined by microinjecting rh-actin shortly before cytokinesis, and then extracting and fixing the cell during mid-cytokinesis. Injected rhodamine actin was only slightly concentrated in the contractile ring, as compared to the distribution of total actin filaments. Our results indicate that preexisting actin filaments, probably through movement and reorganization, are used preferentially for the formation of the contractile ring. De novo assembly of filaments, on the other hand, appears to take place preferentially outside the cleavage furrow.

Ratio imaging is playing an increasingly important role in modern cell biology. Combined with ratiometric dyes or fluorescence resonance energy transfer (FRET) biosensors, the approach allows the detection of conformational changes and molecular interactions in living cells. However, the approach is conducted increasingly under limited signal-to-noise ratio (SNR), where noise from multiple images can easily accumulate and lead to substantial uncertainty in ratio values. This study demonstrates that a far more serious concern is systematic errors that generate artificially high ratio values at low SNR. Thus, uneven SNR alone may lead to significant variations in ratios among different regions of a cell. Although correct average ratios may be obtained by applying conventional noise reduction filters, such as a Gaussian filter before calculating the ratio, these filters have a limited performance at low SNR and are prone to artefacts such as generating discrete domains not found in the correct ratio image. Much more reliable restoration may be achieved with multi-resolution denoising filters that take into account the actual noise characteristics of the detector. These filters are also capable of restoring structural details and photometric accuracy, and may serve as a general tool for retrieving reliable information from low-light live cell images.

To investigate the value of hemodynamic changes during head-up tilt (HUT) test and predictive value thereof in evaluating the efficacy of metoprolol therapy in children with vasovagal syncope (VSS).Twenty-six consecutive children with history of VSS diagnosed by head-up tilt (HUT) or sublingual nitroglycerin potentiated head-up tilt (SNHUT), who were treated with metoprolol for 6 approximately 12 months and followed up for (18 +/- 9) months (12 approximately 36 months), were divided into two groups according to effect of metoprolol: effective treatment group (n = 16, aged 12 +/- 2) without VSS recurrence during treatment and fellow-up, and futile treatment group (n = 10, aged 12 +/- 3). The heart rate changes during HUT or SNHUT were evaluated between the two groups.There were no significant differences between these 2 groups with regard to the demographic and clinical characteristics including age, gender, history, syncope spells, follow-up time and heart rate, mean blood pressure in supine position and during positive response. For example, the baseline heart rate of the effective treatment group was 81 +/- 12 beats/min, not significantly different from that of the futile treatment group (78 +/- 8 beats/min, P = 0.804). However, during tilt test 16 of the 26 patients in the effective treatment group showed tachycardia before positive response, with the mean heart rate of (123 +/- 15) beats/min, whereas all 10 patients in the futile group did not have tachycardia before positive response, with the mean heart rate of (96 +/- 17) beats/min. If an increase of 30 beats/min was taken as a borderline in heart rate during positive response in HUT compared with that of baseline value, in respect of predicting the metoprolol efficacy in the treatment of VVS, the sensitivity was 81% (13/16), the specificity was 80% (8/10), and the diagnostic value was 81% (21/26).A marked increase in heart rate in HUT or SNHUT is a better predictor of metoprolol efficacy in the treatment of children with VVS.

In conjunction with surface chemistry, the mechanical properties of cell culture substrates provide important biological cues that affect cell behavior including growth, differentiation, spreading, and migration. The phenomenon has led to the increased use of biological and synthetic polymer-based flexible substrates in cell culture studies. However, widely used methods for measuring the Young's modulus have proven difficult in the characterization of these materials, as they tend to be relatively thin, soft, hydrated, and tethered to glass substrates. Here we describe three methods that have been applied successfully to probe the flexibility of soft culture substrates.

When cultured on two-dimensional surfaces, most adherent cells show profound differences from those in their native habitats. In addition to chemical factors, it is likely that both physical parameters, such as substrate rigidity, and topographical factors, such as the asymmetry in integrin anchorage, play a major role in the differences. We have designed a simple culture system that provides flexible, adhesive substrates for both dorsal and ventral cell surfaces. Fibroblasts in this system show the spindle or stellate morphology found in native tissues. The ease of preparation, versatility, and optical quality of this model system should greatly facilitate the understanding of cellular behavior and functions in vivo.

When cultured on two-dimensional surfaces, most adherent cells show profound differences from those in their native habitats. In addition to chemical factors, it is likely that both physical parameters, such as substrate rigidity, and topographical factors, such as the asymmetry in integrin anchorage, play a major role in the differences. We have designed a simple culture system that provides flexible, adhesive substrates for both dorsal and ventral cell surfaces. Fibroblasts in this system show the spindle or stellate morphology found in native tissues. The ease of preparation, versatility, and optical quality of this model system should greatly facilitate the understanding of cellular behavior and functions in vivo.

Focal adhesions provide physical linkages between the interior of an adhesive cell and the extracellular matrix (ECM). They may be involved in mediating such functions as cell migration, anchorage, mechanical interactions with the ECM, and the detection of physical cues in the environment. Cell biologists have long struggled to piece together the complex array of components found at focal adhesions, and the equally complex network of interactions among these components, into a "machine" that performs these putative functions. Two recent studies, however, indicate that focal adhesions may be more amorphous and dynamic than previously envisioned. These studies found different degrees of correlated retrograde movement of various focal adhesion proteins with actin filaments, while integrins remained largely stationary. Such differential movements appear inconsistent with a precisely engineered machine and may reflect a slippage clutch for transmitting a variable amount of contractile force to the substrate for migration, a gauge of physical interactions at adhesive sites, or a mechanism for releasing signals from the adhesive sites to the interior of the cell.

Detecting the atrioventricular annular velocity along the long axis of ventricle by tissue Doppler imaging (TDI) is a useful modality to quantitatively assess global myocardial function. The present study was designed to quantitatively assess ventricular function in healthy children by TDI and to evaluate the clinical effect of growth and echocardiographic parameters on TDI velocities during childhood.The study enrolled 242 healthy children aged 3 days to 17 years and they were divided into 8 groups:1 month of age group (37 cases), 1 month-of age group (28 cases), 7 months-of age group (21 cases), 1 year-of age group (36 cases), 4 years-of age group (40 cases), 7 years-of age group (26 cases), 10 years-of age group (28 cases) andor = 13 years of age group (26 cases). Pulsed wave TDI velocities were obtained at the lateral mitral annulus (MA-L), basal septum (MA-S) and lateral tricuspid annulus (TA) during ventricular systole (Sa), early diastole (Ea) and late diastole (Aa), and Ea/Aa and E/Ea were obtained. Conventional echocardiography performed done and the parameters of left ventricular end-diastolic dimension (LVEDD), left ventricular ejection fraction (LVEF), the transmitral and transtricuspid flow E wave and A wave velocities and E/A ratio were obtained. TDI parameters were compared with demographic and echocardiographic variables.Sa, Ea and Ea/Aa were the lowest in children1 month of age [MA-L: Sa (4.8 +/- 0.7) cm/s, Ea (6.6 +/- 1.1) cm/s; MA-S: Sa (4.1 +/- 0.6) cm/s, Ea (5.0 +/- 0.8) cm/s; TA: Sa (6.2 +/- 1.2) cm/s, Ea (6.4 +/- 1.0) cm/s], and increased with age. The increase was significant from 1 month- to 1 year-of age group 1 year-of age group: MA-L: Sa (8.5 +/- 2.0) cm/s, Ea (16.3 +/- 2.6) cm/s; MA-S: Sa (7.2 +/- 0.8) cm/s, Ea (12.2 +/- 1.6) cm/s; TA: Sa (12.6 +/- 2.3) cm/s, Ea (14.7 +/- 2.6) cm/s. Ea and Sa of TA reached the older children's value earlier than those of the mitral annulus did. Aa increased in the 1 month-of age group compared to1 month of age group and remained stable beyond 1 year-of age group. Mitral annulus E/Ea ratio was high among neonates to 7-months-old children (MA-L: 9.2 +/- 2.1, MA-S: 12.1 +/- 2.9), and decreased with age, and there was a significant decrease in 1 year-of age group (MA-L: 5.9 +/- 1.2, MA-S: 7.8 +/- 1.3). In these healthy children, all the above TDI parameters except Aa were influenced by age, body surface area (BSA), LVEDD and heart rate. The influence of age and BSA showed a logarithm model. LVEDD was the main factor that influenced Sa and Ea of MA-L and MA-S, and it was the only single factor that influenced E/Ea ratio at mitral annulus.This study demonstrated that the left and right ventricular function developed with age in childhood, and it developed most rapidly during infancy and toddler period. The right ventricular function matured earlier than that of the left ventricle. Cardiac growth, age, and heart rate had important clinical effects on TDI velocities during childhood, and LVEDD had the most important influence on left ventricular systolic and diastolic function.

Syncope is common in children and adolescents, with 15% estimated to have had at least one syncopal episode by age 18. In recent years, an increasing number of children, especially girls at their school age, have developed unexplained syncope. The mechanism of an unexplained syncope exhibited by children is incompletely studied; the association between different hemodynamic patterns and clinical features is also not clear. The aim of the study was to investigate the hemodynamic patterns of children with unexplained syncope and to examine the clinical relevance.Two hundred and eight children [87 boys, 121 girls, aged 3 - 19 years, mean (11.66 +/- 2.72) years] were selectively recruited from May 2000 to April 2006 when they presented syncope as their main complaint at the Multi-center Network for Childhood Syncope in Beijing, Hunan Province, Hubei Province, and Shanghai of China. All of the patients underwent head-up tilt tests; data were analyzed using SPSS version 10.0 for Windows. Continuous variables were expressed as the mean +/- standard deviation. Dichotomous variables were compared through a chi(2) test. A value of P0.05 (two sided) was regarded as statistically significant.The age distribution of children with syncope was approximately normal. Head-up tilt tests was positive in 155 children, and the incidence of positive response of the baseline head-up tilt test for diagnosing unexplained syncope was 50.48%. The sensitivity value and diagnostic value of sublingual nitroglycerin head-up tilt test were both 74.52%. The hemodynamic pattern was normal in 53 children. The 155 children, who were positive in head-up tilt tests, showed signs of postural orthostatic tachycardia syndrome (60, 28.8%), the vasoinhibitory pattern (72, 34.6%), the cardioinhibitory pattern (5, 2.4%), and the mixed pattern (18, 8.7%). The gender distribution between the two age groups (age12 years vs ageor = 12 years) was not different (P0.05). The distribution of hemodynamic patterns between the children of the two age groups (age12 years vs ageor = 12 years), and the children with different complaints (dizziness vs syncope) was significantly different (P0.05), while the distribution between the children of different sexes and different lasting time of syncope (or = 5 minutes vs5 minutes) was not significantly different (P0.05). Different hemodynamic patterns were differentiated by differing syncope inducements, presymptoms, and complicated symptoms during and after syncope.The tested girls were more prone when compared with the boys to have unexplained syncope, and the peak age was around twelve years old. The incidence of positive response of head-up tilt tests was also relatively higher for the girls. The distribution of hemodynamic patterns for different ages was different. For children with unexplained syncope, we should use head-up tilttests to distinguish the hemodynamic patterns in order to adopt rational therapeutic measures.

Publisher Summary This chapter introduces to the basic concept of several computational image restoration techniques that are particularly useful for processing fluorescence images. An optimal computational noise filter should aggressively remove random fluctuations in the image while preserving as much as possible the non random features. Filters that apply a uniform algorithm across the image, such as low-pass convolution and median filters, usually lead to the degradation of image resolution or amplification of artifacts. Better performance may be obtained using filters that adapt to the local characteristics of the image. In anisotropic diffusion, local intensity fluctuations are reduced through a simulated division process by diffusing from pixels of high intensity toward neighboring pixels of low intensity. Mathematical reversal of systemic defects of the microscope is known as image “Deconvolution.” This is because the degradation effects of the microscope can be mathematically described as the convolution of input signals by the pointspread function of the optical system. In many cases, the fluorescence image contains specific characteristics, which may be used as powerful preconditions for image restoration. As optical microscopy continues to push for maximal speed and minimal irradiation, overcoming the limitation in signal-to-noise ratio will likely remain a major challenge for both the retrieval of information from unprocessed images and the application of various restoration algorithms. In addition, as many biological structures contain characteristic features, the application of feature recognition and extraction will play an increasingly important role in both light and electron microscopy.

In conjunction with surface chemistry, the mechanical properties of cell culture substrates provide important biological cues that affect cell behavior including growth, differentiation, spreading, and migration. The phenomenon has led to the increased use of biological and synthetic polymer-based flexible substrates in cell culture studies. However, widely used methods for measuring the Young's modulus have proven difficult in the characterization of these materials, as they tend to be relatively thin, soft, hydrated, and tethered to glass substrates. Here we describe three methods that have been applied successfully to probe the flexibility of soft culture substrates.

Remodeling of cell membranes takes place during motile processes such as cell migration and cell division. Defects of proteins involved in membrane dynamics, including clathrin and dynamin, disrupt cytokinesis. To understand the function of clathrin-containing structures (CCS) in cytokinesis, we have expressed a green fluorescent protein (GFP) fusion protein of clathrin light chain a (GFP-clathrin) in NRK epithelial cells and recorded images of dividing cells near the ventral surface with a spinning disk confocal microscope. Punctate GFP-CCS underwent dynamic appearance and disappearance throughout the ventral surface. Following anaphase onset, GFP-CCS between separated chromosomes migrated toward the equator and subsequently disappeared in the equatorial region. Movements outside separating chromosomes were mostly random, similar to what was observed in interphase cells. Directional movements toward the furrow were dependent on both actin filaments and microtubules, while the appearance/disappearance of CCS was dependent on actin filaments but not on microtubules. These results suggest that CCS are involved in remodeling the plasma membrane along the equator during cytokinesis. Clathrin-containing structures may also play a role in transporting signaling or structural components into the cleavage furrow.

Publisher Summary This chapter introduces fluorescence imaging of live cells, defining its various features. An inverted microscope generally allows more flexibility and workspace for the culture and manipulation of live cells than an upright microscope. All lenses for fluorescence imaging should be checked upon delivery for the quality of point-spread function, using fluorescent beads as the sample. The system should include both a mercury arc lamp and a 100-W quartz-halogen lamp for epi-illumination, coupled through a selection mirror to the microscope. Contrary to common practice, the most suitable lamp for fluorescence imaging of live cells is often a 100-W quartz-halogen lamp. The mercury arc lamp should consist of a well shielded housing, power supply, and power supply cable to minimize the potentially damaging electromagnetic wave during ignition. Cooled CCD cameras are used in most fluorescent imaging applications. In choosing a camera, important parameters include quantum efficiency, noise level, pixel size and full-well capacity, and scanning frequency.

Two new compounds, taraxafolide (1) and (+)-taraxafolin-B (2) together with eighteen known compounds, which include one sesquiterpene, thirteen benzenoids, two indole alkaloids, one pyridine derivative and steroid mixtures were isolated and characterized from the fresh roots of Taraxacum formosanum. Structures of new compounds were determined by spectral analysis. (+)-Taraxafolin-B had the bioactive caffeic acid moiety, but its activity was weaker than alpha-tocopherol in DPPH radicals scavenging activity assay.

To explore the effect of sodium butyrate (SB) on the expression of costimulatory molecules in acute leukemia cells and its mechanism.The expression of CD86 and CD80 was examined on the surfaces of NB4, HL-60, Kasumi-1, U937 and Jurkat cells by flow cytometric analysis after treated by SB or not. Allogeneic mixed lymphocyte reaction was used to evaluate the immunomodulatory effects of cells treated by SB. Activated NF-kappaB was measured with an NF-kappaB assay kit.Up-regulation of CD86 and CD80 at various levels was observed on these leukemia cells treated by SB. The ratio of CD86 expressing cell in NB4 cells treated by 0.5 mmol/L SB was 36.8 times higher than that in control. Up-regulation of NF-kappaB was similar to that of CD86. Allogeneic lymphocyte proliferation was strongly stimulated by the SB treated cells.SB can improve the expression of CD86 in acute leukemia cells. NF-kappaB was an important transcription factor involved in the up-regulation of CD86.

Microinjection of messenger RNA (mRNA) represents a convenient, effective approach to probe protein functions during specific period of the cell cycle. Keywords: protein expression; mitosis; mRNA; microinjection

SummaryThe involvement of myosin II in cytokinesis has been demonstrated with microinjection, genetic, and pharmacological approaches; however, the exact role of myosin II in cell division remains poorly understood. To address this question, we treated dividing normal rat kidney (NRK) cells with blebbistatin, a potent inhibitor of the nonmuscle myosin II ATPase. Blebbistatin caused a strong inhibition of cytokinesis but no detectable effect on the equatorial localization of actin or myosin. However, whereas these filaments dissociated from the equator in control cells during late cytokinesis, they persisted in blebbistatin-treated cells over an extended period of time. The accumulation of equatorial actin was caused by the inhibition of actin filament turnover, as suggested by a 2-fold increase in recovery half-time after fluorescence photobleaching. Local release of blebbistatin at the equator caused localized accumulation of equatorial actin and inhibition of cytokinesis, consistent with the function of myosin II along the furrow. However, treatment of the polar region also caused a high frequency of abnormal cytokinesis, suggesting that myosin II may play a second, global role. Our observations indicate that myosin II ATPase is not required for the assembly of equatorial cortex during cytokinesis but is essential for its subsequent turnover and remodeling.

We would like to express our concerns about the current publication system for basic biomedical research and to propose an improved system that takes better advantage of Web technology. In the present system, journal Web sites serve primarily as “mirrors” of paper journals and therefore can publish only a limited number of accepted manuscripts. However, as the output of research increases, existing journals no longer provide sufficient space for the volume of information. The problem is compounded by the culture of “publish in high-profile journals or perish,” which makes it difficult for authors who publish in specialized journals to be credited favorably for grant competition, job applications, or promotion ([1][1]). There are a number of serious consequences to this problem. The direction of research is dictated more and more by publishability in high-profile journals, instead of by strict scientific considerations, while fields not deemed fashionable are facing an increasing shortage of young researchers. Furthermore, because of the unpredictability of the publication process, scientists become increasingly apprehensive about sharing their preliminary findings and interacting with one another openly. The fierce competition for publication in high-profile journals may encourage aggressive behavior and discourage others from staying in academic research. We believe that modifying the current system may alleviate the above problems. Because of the striking differences in volume and cost, paper journals should be used to complement, rather than duplicate, what is published on the Web. Thus, the new system we propose would consist of a high-capacity Web site for posting peer-reviewed papers. A paper version of the journal would be reserved for the fraction of the Web-published papers that have made the strongest impact. Editors would continue to solicit reviews and enforce an objective scientific standard. However, in this system, a paper would not be rejected on the basis of judgments of “novelty,” “priority,” or “descriptiveness.” Reports that confirm or challenge previous studies or describe significant negative findings would all be considered for publication. This approach should provide cross-examination of results and help fight against misconduct. The scientific editors would also select from the Web-published papers the outstanding papers for republication in the paper version ([2][2]). With more time and objective parameters ([3][3]), the decision will be influenced less by haggling and more by quality. In addition, the reviewers' comments and the authors' responses to those comments would be published on the Web, thereby encouraging constructive reviews and providing additional insights for readers. Authors of thoughtful, named reviews may be credited accordingly, as part of their productivity and evidence of stature. The new system may also correct the bias against specialized journals: With a high acceptance rate for Web journals but no guarantee for selection in the paper version, scientists will likely choose journals more rationally during submission and develop a balanced view of the value of different journals. The problems of publishing in basic biomedical research have already exerted serious negative impacts on scientific interactions and education. It would be highly irresponsible for scientific publishers to fail to correct these problems, and equally irresponsible for the scientific community to allow the situation to deteriorate. [1]: #fn-1 [2]: #fn-2 [3]: #fn-3

Although myosin II is known to play an important role in cell migration, little is known about its specific functions. We have addressed the function of one of the isoforms of myosin II, myosin IIB, by analyzing the movement and mechanical characteristics of fibroblasts where this protein has been ablated by gene disruption. Myosin IIB null cells displayed multiple unstable and disorganized protrusions, although they were still able to generate a large fraction of traction forces when cultured on flexible polyacrylamide substrates. However, the traction forces were highly disorganized relative to the direction of cell migration. Analysis of cell migration patterns indicated an increase in speed and decrease in persistence, which were likely responsible for the defects in directional movements as demonstrated with Boyden chambers. In addition, unlike control cells, mutant cells failed to respond to mechanical signals such as compressing forces and changes in substrate rigidity. Immunofluorescence staining indicated that myosin IIB was localized preferentially along stress fibers in the interior region of the cell. Our results suggest that myosin IIB is involved not in propelling but in directing the cell movement, by coordinating protrusive activities and stabilizing the cell polarity.

The optical flow algorithm presented here is a robust method that rapidly yields a high-density field of substrate displacement vectors based on two optical images. We found that one of the limiting factors, at least for inexperienced experimentalists, is the consistency of focusing or the drift in microscope focus. However, with properly collected images the standard error of the measurement was estimated to be on the order of +/- 0.10 pixels. Finally, although the discussion has been focused on the displacement of flexible substrata, a similar method should be applicable for detecting movements on other types of images, as long as the movement involves a certain degree of local coordination.

Cell motility is driven by the sum of asymmetric traction forces exerted on the substrate through adhesion foci that interface with the actin cytoskeleton. Establishment of this asymmetry involves microtubules, which exert a destabilising effect on adhesion foci via targeting events. Here, we demonstrate the existence of a mechano-sensing mechanism that signals microtubule polymerisation and guidance of the microtubules towards adhesion sites under increased stress. Stress was applied either by manipulating the body of cells moving on glass with a microneedle or by stretching a flexible substrate that cells were migrating on. We propose a model for this mechano-sensing phenomenon whereby microtubule polymerisation is stimulated and guided through the interaction of a microtubule tip complex with actin filaments under tension.

Phagocytosis is an actin-based process used by macrophages to clear particles greater than 0.5 μm in diameter. In addition to its role in immunological responses, phagocytosis is also necessary for tissue remodeling and repair. To prevent catastrophic autoimmune reactions, phagocytosis must be tightly regulated. It is commonly assumed that the recognition/selection of phagocytic targets is based solely upon receptor-ligand binding. Here we report an important new criterion, that mechanical parameters of the target can dramatically affect the efficiency of phagocytosis. When presented with particles of identical chemical properties but different rigidity, macrophages showed a strong preference to engulf rigid objects. Furthermore, phagocytosis of soft particles can be stimulated with the microinjection of constitutively active Rac1 but not RhoA, and with lysophosphatidic acid, an agent known to activate the small GTP-binding proteins of the Rho family. These data suggest a Rac1-dependent mechanosensory mechanism for phagocytosis, which probably plays an important role in a number of physiological and pathological processes from embryonic development to autoimmune diseases.

By modulating adhesion signaling and cytoskeletal organization, mechanical forces play an important role in various cellular functions, from propelling cell migration to mediating communication between cells. Recent developments have resulted in several new approaches for the detection, analysis and visualization of mechanical forces generated by cultured cells. Combining these methods with other approaches, such as green-fluorescent protein (GFP) imaging and gene manipulation, proves to be particularly powerful for analyzing the interplay between extracellular physical forces and intracellular chemical events.

We have described a powerful tool for the study of mechanical interactions between cells and their physical environment. Although the approach has already been used in a variety of ways to measure traction forces and to characterize active and passive responses of cultured cells to mechanical stimulation, it can be extended easily and combined with other microscopic approaches, including fluorescent analog imaging (Beningo et al., 2001), photobleaching, calcium imaging, micromanipulation, and electrophysiology. This method will be particularly useful for studying the functions of various components at focal adhesions, and the effects of mechanical forces on focal adhesion-mediated signal transduction. In addition, the method can be extended to a 3D setting, e.g., by sandwiching cultured cells between two layers of polyacrylamide to create an environment mimicking that in the tissue of a multicellular organism. Whereas chemical interactions between cells and the environment have been investigated extensively, many important questions remain as to the role of physical forces in cellular functions and the interplay between chemical and physical mechanisms of communication. The present approach, as well as other approaches capable of probing physical interactions, should fill in this important gap in the near future.

Interference fringes are generally found in the photoluminescence (PL) spectrum of heterostructures with large refractive index differences between layers and with a smooth interface. To eliminate the interference effects in an air/GaN/InGaN/GaN/Al2O3 structure, the measured interference fringe wavelengths of the PL spectrum are used to deduce the frequency dependent interference function of the system. This interference function is then used to numerically remove interference fringes from the as-measured PL spectrum. This versatile semiempirical method allows the derivation of the true PL spectrum from the measured data, including angular dependent spectra, without complicated calculations or additional measurements.

Cell migration involves complex physical and chemical interactions with the substrate. To probe the mechanical interactions under different regions of migrating 3T3 fibroblasts, we have disrupted cell-substrate adhesions by local application of the GRGDTP peptide, while imaging stress distribution on the substrate with traction force microscopy. Both spontaneous and GRGDTP-induced detachment of the trailing edge caused extensive cell shortening, without changing the overall level of traction forces or the direction of migration. In contrast, disruption of frontal adhesions caused dramatic, global loss of traction forces before any significant shortening of the cell. Although traction forces and cell migration recovered within 10–20 min of transient frontal treatment, persistent treatment with GRGDTP caused the cell to develop traction forces elsewhere and reorient toward a new direction. We conclude that contractile forces of a fibroblast are transmitted to the substrate through two distinct types of adhesions. Leading edge adhesions are unique in their ability to transmit active propulsive forces. Their functions cannot be transferred directly to existing adhesions upon detachment. Trailing end adhesions create passive resistance during cell migration and readily redistribute their loads upon detachment. Our results indicate the distinct nature of mechanical interactions at the leading versus trailing edges, which together generate the mechanical interactions for fibroblast migration.

Focal adhesion kinase (FAK) is a non-receptor protein tyrosine kinase localized at focal adhesions and is believed to mediate adhesion-stimulated effects. Although ablation of FAK impairs cell movement, it is not clear whether FAK might be involved in the guidance of cell migration, a role consistent with its putative regulatory function. We have transfected FAK-null fibroblasts with FAK gene under the control of the tetracycline repression system. Cells were cultured on flexible polyacrylamide substrates for the detection of traction forces and the application of mechanical stimulation. Compared with control cells expressing wild-type FAK, FAK-null cells showed a decrease in migration speed and directional persistence. In addition, whereas FAK-expressing cells responded to exerted forces by reorienting their movements and forming prominent focal adhesions, FAK-null cells failed to show such responses. Furthermore, FAK-null cells showed impaired responses to decreases in substrate flexibility, which causes control cells to generate weaker traction forces and migrate away from soft substrates. Cells expressing Y397F FAK, which cannot be phosphorylated at a key tyrosine site, showed similar defects in migration pattern and force-induced reorientation as did FAK-null cells. However, other aspects of F397-FAK cells, including the responses to substrate flexibility and the amplification of focal adhesions upon mechanical stimulation, were similar to that of control cells. Our results suggest that FAK plays an important role in the response of migrating cells to mechanical input. In addition, phosphorylation at Tyr-397 is required for some, but not all, of the functions of FAK in cell migration.

portant interactions that modulate intracellular signaling pathways, as well as various cellular events from gene expression to cell locomotion (Juliano and Haskill, 1993). The full response to adhesion seems to involve not only the cross-linking of integrins but also mechanical input through these receptors (Craig and Johnson, 1996; Wang et al., 1993; Ingber, 1993; Chrzanowska-Wodnika and Burridge, 1996; Choquet et al., 1997). To explore this possibility, we have examined the motility and cytoskeletal organization of NRK epithelial cells and 3T3 fibroblasts cultured on substrates having varying mechanical properties (Pelham and Wang, 1997). Flexible, optically clear substrates were prepared by covalently linking type I collagen to polyacrylamide sheets. The flexibility of the substrate was manipulated by maintaining the acrylamide concentration at 10% while varying the bis-acrylamide contents between 0.03% and 0.26%. In this manner, we were able to maintain a constant chemical environment regardless of substrate flexibility. The Young's Modulus of the substrate, determined by measuring the extent of stretching in response to known applied forces, showed a 13-fold difference between sheets of 0.26% and 0.03% bis-acrylamide. When probed microscopically with a calibrated microneedle, the substrates showed 16-fold difference in compliance (-7.3 x 10-7 newtons/tm versus ~4.6 X 10-8 newtons/ym). On more rigid substrates, both NRK epithelial cells and 3T3 fibroblasts were well spread and appeared indistinguishable from those cultured on glass or plastic sur

Over the past 100 years, many models have been proposed and tested for cytokinesis [1]. There is strong evidence that the equator represents a unique region that receives cleavage signals from the mitotic spindle [2, 3]. The nature of such a signal and the mechanism of cleavage, however, remain poorly understood. To probe the contribution of different cortical regions in the cleavage of cultured epithelial cells, we applied cytochalasin D (CD), a known inhibitor of cytokinesis [4], in a highly localized manner to different regions of dividing NRK cells. Surprisingly, equatorial application of CD not only allowed cytokinesis to complete but also appeared to facilitate the process. Conversely, local application of CD near the polar region caused inhibition of cytokinesis. Our results suggest a mechanism that involves global coordination of cortical activities, including controlled cortical disassembly along the equator and possibly global cortical contraction.

Directional cell locomotion is critical in many physiological processes, including morphogenesis, the immune response, and wound healing. It is well known that in these processes cell movements can be guided by gradients of various chemical signals. In this study, we demonstrate that cell movement can also be guided by purely physical interactions at the cell-substrate interface. We cultured National Institutes of Health 3T3 fibroblasts on flexible polyacrylamide sheets coated with type I collagen. A transition in rigidity was introduced in the central region of the sheet by a discontinuity in the concentration of the bis-acrylamide cross-linker. Cells approaching the transition region from the soft side could easily migrate across the boundary, with a concurrent increase in spreading area and traction forces. In contrast, cells migrating from the stiff side turned around or retracted as they reached the boundary. We call this apparent preference for a stiff substrate “durotaxis.” In addition to substrate rigidity, we discovered that cell movement could also be guided by manipulating the flexible substrate to produce mechanical strains in the front or rear of a polarized cell. We conclude that changes in tissue rigidity and strain could play an important controlling role in a number of normal and pathological processes involving cell locomotion.

Publisher Summary The performance of optical microscopes is limited both by the aperture of the lens that causes light from a point source to spread (or diffract) over a finite volume, and by the cross-contamination of light that originates from out of focus planes. To overcome these limitations, approaches have been developed both to improve the microscope design, as exemplified by confocal scanning microscopy, and to reverse mathematically the degrading effects of the conventional microscope. This latter approach is commonly referred to as “deconvolution,” because the degradation effects of the microscope can be described mathematically as the convolution of input signals by the point spread function of the optical system. This chapter introduces the basic rationale of these two methods in languages easily understood by biologists. It also provides details for the implementation of nearest- neighbor deconvolution using readily available hardware and software. A number of deconvolution algorithms have been tested for restoring fluorescence images. The most straightforward approach—three-dimensional (3-D) inverse filtering (Agard et al ., Holmes and Liu) attempts to reverse the effects of image degradation through direct calculations. The two methods currently in wide use are constrained iterative deconvolution and nearest-neighbor deconvolution.

The technique of fluorescence immunolocalization has evolved steadily since its first application in the mid-1960s, incorporating innovations in probe chemistry, microscopy, and image detection. This chapter provides an overview of the current status of indirect immunofluorescence for those starting to use the method. It includes both general considerations from cell culture to image detection and several protocols that should serve as an entry point for this technique.

Responses of cells to mechanical properties of the adhesion substrate were examined by culturing normal rat kidney epithelial and 3T3 fibroblastic cells on a collagen-coated polyacrylamide substrate that allows the flexibility to be varied while maintaining a constant chemical environment. Compared with cells on rigid substrates, those on flexible substrates showed reduced spreading and increased rates of motility or lamellipodial activity. Microinjection of fluorescent vinculin indicated that focal adhesions on flexible substrates were irregularly shaped and highly dynamic whereas those on firm substrates had a normal morphology and were much more stable. Cells on flexible substrates also contained a reduced amount of phosphotyrosine at adhesion sites. Treatment of these cells with phenylarsine oxide, a tyrosine phosphatase inhibitor, induced the formation of normal, stable focal adhesions similar to those on firm substrates. Conversely, treatment of cells on firm substrates with myosin inhibitors 2,3-butanedione monoxime or KT5926 caused the reduction of both vinculin and phosphotyrosine at adhesion sites. These results demonstrate the ability of cells to survey the mechanical properties of their surrounding environment and suggest the possible involvement of both protein tyrosine phosphorylation and myosin-generated cortical forces in this process. Such response to physical parameters likely represents an important mechanism of cellular interaction with the surrounding environment within a complex organism.

Although non-muscle tropomyosins (TM) have been implicated in various cellular functions, such as stabilization of actin filaments and possibly regulation of organelle transport, their physiological role is still poorly understood. We have probed the role of a high molecular mass isoform of human fibroblast TM, hTM3, in regulating organelle transport by microinjecting an excess amount of bacterially-expressed protein into normal rat kidney (NRK) epithelial cells. The microinjection induced the dramatic retrograde translocation of organelles into the perinuclear area. Microinjection of hTM5, a low molecular mass isoform had no effect on organelle distribution. Fluorescent staining indicated that hTM3 injection stimulated the retrograde movement of both mitochondria and lysosomes. Moreover, both myosin I and cytoplasmic dynein were found to redistribute with the translocated organelles to the perinuclear area, indicating that these organelles were able to move along both microtubules and actin filaments. The involvement of microtubules was further suggested by the partial inhibition of hTM3-induced organelle movement by the microtubule-depolymerizing drug nocodazole. Our results, along with previous genetic and antibody microinjection studies, suggest that hTM3 may be involved in the regulation of organelle transport.