Contact guidance persists under myosin inhibition due to the local alignment of adhesions and individual protrusions.

Contact guidance-cell polarization by anisotropic substrate features-is integral to numerous physiological processes; however the complexities of its regulation are only beginning to be discovered. In particular, cells polarize to anisotropic features under non-muscle myosin II (MII) inhibition, despite MII ordinarily being essential for polarized cell migration. Here, we investigate the ability of cells to sense and respond to fiber alignment in the absence of MII activity. We find that contact guidance is determined at the level of individual protrusions, which are individually guided by local fiber orientation, independent of MII. Protrusion stability and persistence are functions of adhesion lifetime, which depends on fiber orientation. Under MII inhibition, adhesion lifetime no longer depends on fiber orientation; however the ability of protrusions to form closely spaced adhesions sequentially without having to skip over gaps in adhesive area, biases protrusion formation along fibers. The co-alignment of multiple protrusions polarizes the entire cell; if the fibers are not aligned, contact guidance of individual protrusions still occurs, but does not produce overall cell polarization. These results describe how aligned features polarize a cell independently of MII and demonstrate how cellular contact guidance is built on the local alignment of adhesions and individual protrusions.

Hyperoxaluria, hyperglycoluria and renal oxalosis in Gilbert's potoroos (Potorous gilbertii).

CASE REPORT: Six Gilbert's potoroos (Potorous gilbertii) in a captive colony, five of which were closely related, died or were euthanased with severe renal disease. Clinical signs were mostly non-specific. Renal calculi were seen on ultrasound of two affected potoroos and oxalate crystalluria was seen in two of three affected potoroos that had urine samples examined. Necropsies revealed extensive severe renal oxalosis in all affected potoroos. These findings and markedly increased concentrations of glycolate in the urine of the four affected potoroos for which it was measured, confirmed a disorder of oxalate metabolism and suggested a condition similar to primary hyperoxaluria type 1 in humans. Liver alanine : glyoxylate aminotransferase activity and intracellular location were assessed as normal in one affected potoroo, which is inconsistent with human primary hyperoxaluria type 1. Although a condition similar to human primary hyperoxaluria type 2 or 3 was not ruled out, other clinicopathological findings were not consistent with those seen in humans with these conditions. A lack of faecal oxalate-degrading activity was observed in two affected potoroos in which it was measured, whereas oxalate-degrading activity was variably present in healthy captive and wild potoroos. CONCLUSION: Although the pathogenesis of renal oxalosis in these cases was not clear, the biochemical findings of elevated urinary oxalate and glycolate excretion indicate an abnormality of oxalate metabolism. The familial pattern of disease suggests it could be an inherited condition.

Breast cancer antiestrogen resistance 3-p130Cas interactions promote adhesion disassembly and invasion in breast cancer cells.

Adhesion turnover is critical for cell motility and invasion. We previously demonstrated that the adaptor molecule breast cancer antiestrogen resistance 3 (BCAR3) promotes adhesion disassembly and breast tumor cell invasion. One of two established binding partners of BCAR3 is the adaptor molecule, p130Cas. In this study, we sought to determine whether signaling through the BCAR3-Cas complex was responsible for the cellular functions of BCAR3. We show that the entire pool of BCAR3 is in complex with Cas in invasive breast tumor cells and that these proteins colocalize in dynamic cellular adhesions. Although accumulation of BCAR3 in adhesions did not require Cas binding, a direct interaction between BCAR3 and Cas was necessary for efficient dissociation of BCAR3 from adhesions. The dissociation rates of Cas and two other adhesion molecules, α-actinin and talin, were also significantly slower in the presence of a Cas-binding mutant of BCAR3, suggesting that turnover of the entire adhesion complex was delayed under these conditions. As was the case for adhesion turnover, BCAR3-Cas interactions were found to be important for BCAR3-mediated breast tumor cell chemotaxis toward serum and invasion in Matrigel. Previous work demonstrated that BCAR3 is a potent activator of Rac1, which in turn is an important regulator of adhesion dynamics and invasion. However, in contrast to wild-type BCAR3, ectopic expression of the Cas-binding mutant of BCAR3 failed to induce Rac1 activity in breast cancer cells. Together, these data show that the ability of BCAR3 to promote adhesion disassembly, tumor cell migration and invasion, and Rac1 activity is dependent on its ability to bind to Cas. The activity of BCAR3-Cas complexes as a functional unit in breast cancer is further supported by the co-expression of these molecules in multiple subtypes of human breast tumors.

Matrix microarchitecture and myosin II determine adhesion in 3D matrices.

BACKGROUND: Reports of adhesions in cells growing in 3D vary widely-from nonexistent to very large and elongated-and are often in apparent conflict, due largely to our minimal understanding of the underlying mechanisms that determine 3D cell phenotype. We address this problem directly by systematically identifying mechanisms that determine adhesion in 3D matrices and, from our observations, develop principles widely applicable across 2D and 3D substrates. RESULTS: We demonstrate that nonmuscle myosin II activity guides adhesion phenotype in 3D as it does in 2D; however, in contrast to 2D, decreasing bulk matrix stiffness does not necessarily inhibit the formation of elongated adhesions. Even in soft 3D matrices, cells can form large adhesions in areas with appropriate local matrix fiber alignment. We further show that fiber orientation, apart from influencing local stiffness, modulates the available adhesive area and thereby determines adhesion size. CONCLUSIONS: Thus adhesion in 3D is determined by both myosin activity and the immediate microenvironment of each adhesion, as defined by the local matrix architecture. Important parameters include not only the resistance of the fiber to pulling (i.e., stiffness) but also the orientation and diameter of the fiber itself. These principles not only clarify conflicts in the literature and point to adhesion modulating factors other than stiffness, but also have important implications for tissue engineering and studies of tumor cell invasion.

Cross-sensitization of histamine-independent itch in mouse primary sensory neurons.

Overexpression of pruritogens and their precursors may contribute to the sensitization of histamine-dependent and -independent itch-signaling pathways in chronic itch. We presently investigated self- and cross-sensitization of scratching behavior elicited by various pruritogens, and their effects on primary sensory neurons. The MrgprC11 agonist BAM8-22 exhibited self- and reciprocal cross-sensitization of scratching evoked by the protease-activated receptor-2 (PAR-2) agonist SLIGRL. The MrgprA3 agonist chloroquine unidirectionally cross-sensitized BAM8-22-evoked scratching. Histamine unidirectionally cross-sensitized scratching evoked by chloroquine and BAM8-22. SLIGRL unidirectionally cross-sensitized scratching evoked by chloroquine. Dorsal root ganglion (DRG) cells responded to various combinations of pruritogens and algogens. Neither chloroquine, BAM8-22 nor histamine had any effect on responses of DRG cell responses to subsequently applied pruritogens, implying that their behavioral self- and cross-sensitization effects are mediated indirectly. SLIGRL unilaterally cross-sensitized responses of DRG cells to chloroquine and BAM8-22, consistent with the behavioral data. These results indicate that unidirectional cross-sensitization of histamine-independent itch-signaling pathways might occur at a peripheral site through PAR-2. PAR-2 expressed in pruriceptive nerve endings is a potential target to reduce sensitization associated with chronic itch.

Raster image correlation spectroscopy (RICS) for measuring fast protein dynamics and concentrations with a commercial laser scanning confocal microscope.

Raster image correlation spectroscopy (RICS) is a new and novel technique for measuring molecular dynamics and concentrations from fluorescence confocal images. The RICS technique extracts information about molecular dynamics and concentrations from images of living cells taken on commercial confocal systems. Here we develop guidelines for performing the RICS analysis on an analogue commercial laser scanning confocal microscope. Guidelines for typical instrument settings, image acquisition settings and analogue detector characterization are presented. Using appropriate instrument/acquisition parameters, diffusion coefficients and concentrations can be determined, even for highly dynamic dye molecules in solution. Standard curves presented herein demonstrate the ability to detect protein concentrations as low as approximately 2 nM. Additionally, cellular measurements give accurate values for the diffusion of paxillin-enhanced-green fluorescent protein (EGFP), an adhesion adaptor molecule, in the cytosol of the cell and also show slower paxillin dynamics near adhesions where paxillin interacts with immobile adhesion components. Methods are presented to account for bright immobile structures within the cell that dominate spatial correlation functions; allowing the extraction of fast protein dynamics within and near these structures. A running average algorithm is also presented to address slow cellular movement or movement of cellular features such as adhesions. Finally, methods to determine protein concentration in the presence of immobile structures within the cell are presented. A table is presented giving guidelines for instrument and imaging setting when performing RICS on the Olympus FV300 confocal and these guidelines are a starting point for performing the analysis on other commercial confocal systems.

Kinetic model for lamellipodal actin-integrin 'clutch' dynamics.

In migrating cells, with especial prominence in lamellipodial protrusions at the cell front, highly dynamic connections are formed between the actin cytoskeleton and the extracellular matrix through linkages of integrin adhesion receptors to actin filaments via complexes of cytosolic "connector" proteins. Myosin-mediated contractile forces strongly influence the dynamic behavior of these adhesion complexes, apparently in two counter-acting ways: negatively as the cell-generated forces enhance complex dissociation, and at the same time positively as force-induced signaling can lead to strengthening of the linkage complexes. The net balance arising from this dynamic interplay is challenging to ascertain a priori, rendering experimental studies difficult to interpret and molecular manipulations of cell and/or environment difficult to predict. We have constructed a kinetics-based model governing the dynamic behavior of this system. We obtained ranges of parameter value sets yielding behavior consistent with that observed experimentally for 3T3 cells and for CHO cells, respectively. Model simulations are able to produce results for the effects of paxillin mutations on the turnover rate of actin/integrin linkages in CHO cells, which are consistent with recent literature reports. Overall, although this current model is quite simple it provides a useful foundation for more detailed models extending upon it.

Cryptococcosis in Gilbert's and long-nosed potoroo.

Two cases of fatal cryptococcosis are described, one of Cryptococcus neoformans infection in a Gilbert's potoroo (Potorous gilbertii) and one of Cryptococcus gattii infection in a long-nosed potoroo (Potorous tridactylus). The diagnoses were confirmed by culture and specific immunohistochemistry, respectively. The long-nosed potoroo tested positive using the latex cryptococcal antigen test (LCAT), whereas the Gilbert's potoroo had a negative LCAT result despite having advanced disease of some duration. In both cases, the clinical presentation was a progressive neurologic disease associated with a central nervous system infection. Pulmonary infection was also observed in the long-nosed potoroo. Specific treatment with antifungal agents was unsuccessful in the long-nosed potoroo.

Isolated femoral hypoplasia: an intrauterine differential diagnosis to campomelia.

The isolated form of femoral bowing is an important differential diagnosis of campomelia. Therefore, knowledge of isolated anomalies is fundamental for prenatal diagnosis, especially for the differential diagnosis from severe syndromes. Four cases are presented to discuss the differential diagnosis of femoral bowing including a review of the literature. We report four newborn babies with unilateral bowing and shortening of the femur. Three had no further anomaly; one child had additional abnormalities due to coumarin embryopathy. The radiological findings were shortened femora with bowing and varus deformity and cortical thickening on the concave side. All other parts showed normal bone structure. The aetiology of femoral bowing is unknown. Early damage of the cartilaginous model followed by remodelling with thickening on the concave side of the bone similar to the healing of malaligned fractures is suspected. The isolated form of femoral bowing without any other anomalies has to be differentiated from complex and more often severe congenital syndromes such as campomelia. Postpartum radiological examination should be reduced to a single exposure of the affected limb and follow-up should be done by clinical examination.

Spatial mapping of integrin interactions and dynamics during cell migration by image correlation microscopy.

Image correlation microscopy methodology was extended and used to determine retrospectively the density, dynamics and interactions of alpha5-integrin in migrating cells. Alpha5-integrin is present in submicroscopic clusters containing 3-4 integrins before it is discernibly organized. The integrin in nascent adhesions, as identified by the presence of paxillin, is approximately 1.4 times more concentrated, approximately 4.5 times more clustered and much less mobile than in surrounding regions. Thus, while integrins are clustered throughout the cell, they differ in nascent adhesions and appear to initiate adhesion formation, despite their lack of visible organization. In more mature adhesions where the integrin is visibly organized there are approximately 900 integrins microm(-2) (about fivefold higher than surrounding regions). Interestingly, alpha5-integrin and alpha-actinin, but not paxillin, reside in a complex throughout the cell, where they diffuse and flow together, even in regions where they are not organized. During adhesion disassembly some integrins diffuse away slowly, alpha-actinin undergoes a directed movement at speeds similar to actin retrograde flow (0.29 microm min(-1)), while all of the paxillin diffuses away rapidly.

Infidelity, initiation, and the emotional climate of divorce: are there implications for mental health?

A large literature has examined the role of "secondary" stressors, such as problems with finances, social support, residential mobility, and children, in producing the well-documented association between divorce and a variety of psychopathological conditions. Much less attention, however, has been paid to variation in the "primary" disruption experience. We address this omission using data from the National Survey of Families and Households to investigate the interrelationships among depression, initiator status, and spousal infidelity. While we find little evidence of direct effects of initiator status or spousal infidelity on post-divorce depression, the importance of these characteristics emerges when they are considered in an interactive context. Specifically, while divorce initiation is associated with reduced depression among individuals with unfaithful spouses, initiation is associated with increased depression in the absence of spousal infidelity. Taken together, our findings suggest that characteristics of the divorce experience may interact in complex ways to produce variation in mental health outcomes.

The LD4 motif of paxillin regulates cell spreading and motility through an interaction with paxillin kinase linker (PKL).

The small GTPases of the Rho family are intimately involved in integrin-mediated changes in the actin cytoskeleton that accompany cell spreading and motility. The exact means by which the Rho family members elicit these changes is unclear. Here, we demonstrate that the interaction of paxillin via its LD4 motif with the putative ARF-GAP paxillin kinase linker (PKL) (Turner et al., 1999), is critically involved in the regulation of Rac-dependent changes in the actin cytoskeleton that accompany cell spreading and motility. Overexpression of a paxillin LD4 deletion mutant (paxillinDeltaLD4) in CHO.K1 fibroblasts caused the generation of multiple broad lamellipodia. These morphological changes were accompanied by an increase in cell protrusiveness and random motility, which correlated with prolonged activation of Rac. In contrast, directional motility was inhibited. These alterations in morphology and motility were dependent on a paxillin-PKL interaction. In cells overexpressing paxillinDeltaLD4 mutants, PKL localization to focal contacts was disrupted, whereas that of focal adhesion kinase (FAK) and vinculin was not. In addition, FAK activity during spreading was not compromised by deletion of the paxillin LD4 motif. Furthermore, overexpression of PKL mutants lacking the paxillin-binding site (PKLDeltaPBS2) induced phenotypic changes reminiscent of paxillinDeltaLD4 mutant cells. These data suggest that the paxillin association with PKL is essential for normal integrin-mediated cell spreading, and locomotion and that this interaction is necessary for the regulation of Rac activity during these events.

The impact of childhood abuse and neglect on adult mental health: a prospective study.

This paper examines the impact of three types of victimization in childhood--sexual abuse, physical abuse, and neglect--on lifetime measures of mental health among adults. In contrast to research that relies on retrospective recall of childhood victimization, this work uses a prospective sample gathered from records of documented court cases of childhood abuse and neglect in a midwestern city around 1970. These subjects were interviewed about twenty years later. In addition, this research compares outcomes of the 641 members of the abuse and neglect group with a matched control group of 510 persons who did not have documented cases of abuse or neglect. The results indicate that men who were abused and neglected as children have more dysthymia and antisocial personality disorder as adults than matched controls, but they did not have more alcohol problems. Abused and neglected women report more symptoms of dysthymia, antisocial personality disorder, and alcohol problems than controls. After controlling for stressful life events, however, childhood victimization had little direct impact on any lifetime mental health outcome. This research indicates the importance of adopting an approach that places childhood victimization in the context of other life stressors and of prospective changes over the life course.

Negative and supportive social interactions and quality of life among persons diagnosed with severe mental illness.

This study examined the relative importance of negative and supportive social interactions in predicting different aspects of quality of life (QOL) in a sample of persons diagnosed with severe mental illness (n = 104). Controlling for other variables that might explain such a relationship, we found that negative social interactions were significantly related to lower QOL in three subjective domains, while supportive social interactions were related to higher QOL in four objective domains and one subjective domain. We found negative social interactions that are stigmatizing to be particularly important in predicting QOL; additional analyses suggested that perceived stigma partially mediated the relationship between negative social interactions and QOL. We discuss the implications of the present findings for the study of the link between social relationships and QOL among persons with mental illness.

Differential dynamics of alpha 5 integrin, paxillin, and alpha-actinin during formation and disassembly of adhesions in migrating cells.

To investigate the mechanisms by which adhesions form and disperse in migrating cells, we expressed alpha 5 integrin, alpha-actinin, and paxillin as green fluorescent protein (GFP) fusions. All localized with their endogenous counterparts and did not perturb migration when expressed at moderate levels. alpha 5-GFP also rescued the adhesive defects in CHO B2 cells, which are alpha 5 integrin deficient. In ruffling cells, alpha 5-GFP and alpha-actinin--GFP localized prominently at the leading edge in membrane protrusions. Of the three GFP fusion proteins that we examined, paxillin was the first component to appear visibly organized in protrusive regions of the cell. When a new protrusion formed, the paxillin appeared to remodel from older to newer adhesions at the leading edge. alpha-Actinin subsequently entered adhesions, which translocated toward the cell center, and inhibited paxillin turnover. The new adhesions formed from small foci of alpha-actinin--GFP and paxillin-GFP, which grew in size. Subsequently, alpha 5 integrin entered the adhesions to form visible complexes, which served to stabilize the adhesions. alpha 5-GFP also resided in endocytic vesicles that emanated from the leading edge of protrusions. Integrin vesicles at the cell rear moved toward the cell body. As cells migrated, alpha 5 vesicles also moved from a perinuclear region to the base of the lamellipodium. The alpha 5 vesicles colocalized with transferrin receptor and FM 4-64 dye. After adhesions broke down in the rear, alpha 5-GFP was found in fibrous structures behind the cell, whereas alpha-actinin--GFP and paxillin-GFP moved up the lateral edge of retracting cells as organized structures and then dissipated.

The effect of dexamethasone on respirator-dependent very-low-birth-weight infants is best predicted by chest X-ray.

BACKGROUND: Chronic lung disease (CLD) in premature infants shows a variable clinical course with different radiological manifestations. OBJECTIVE: To evaluate the correlation between parameters of trans-membrane permeability [albumin/secretory component (SC)] and oxidative stress [malondialdehyde (MDA)/SC] in tracheal aspirate fluid (TAF) and radiological findings with the effect of a 5-day course of dexamethasone (0.5 mg/kg per day). MATERIALS AND METHODS: Fifty ventilator-dependent premature infants with birth weights < 1,500 g (gestational ages 23-31 weeks) and radiological signs of early chronic lung disease (CLD) were treated with dexamethasone at day of life 5-27 (median 10 days) because of respiratory deterioration. TAF was collected serially. Chest X-rays taken before and 8-10 days after dexamethasone were scored for changes of opacification, consolidation and hyperinflation/emphysema, and classified into three groups. RESULTS: Twenty-four infants had a positive response to dexamethasone, defined as a reduction of the ventilation index FiO2 x mean airway pressure > 40% at day 5, compared to pretreatment values. About 80% of the responders showed homogeneous lung opacification on chest X-ray, reflecting leaky lung syndrome. In contrast, seven of eight infants with predominantly emphysema on radiology were non-responders; 80% of infants with a mixed radiological picture characterized by predominance of consolidations alternating with regions of emphysema were also non-responders. Ratios of albumin/SC and MDA/SC in TAF decreased significantly within 3 days after the onset of dexamethasone. However, MDA/SC was persistently higher in non-responders compared to responders. Opaque lungs were largely improved by dexamethasone, in contrast to streaky or patchy consolidations and emphysema. In a logistic regression model, radiographic classification was the most important factor influencing the response to dexamethasone with a positive predictive value of 86%, followed by albumin/SC ratio. CONCLUSIONS: The optimum timing of dexamethasone treatment may be determined by the stage of developing CLD and radiological findings rather than by the age of the premature infant.

Stably transfected ABCA1 antisense cell line has decreased ABCA1 mRNA and cAMP-induced cholesterol efflux to apolipoprotein AI and HDL.

Using a sensitive real time fluorescent PCR assay, ABCA1 mRNA levels were induced by approximately 50-70-fold following 8Br-cAMP treatment of the RAW264 murine macrophage cell line, concomitant with the induction of cholesterol efflux to apoAI and HDL. A stably transfected ABCA1 antisense cDNA cell line was created, which led to approximately 50-70% reductions in ABCA1 mRNA levels in basal and 8Br-cAMP-treated cells, and diminished to the same extent the 8Br-cAMP-mediated efflux of cholesterol to apolipoprotein AI and HDL. These data demonstrate that ABCA1 is necessary for the cAMP-induced lipid efflux to both apoAI and HDL.