A role for the cytoskeleton-associated protein palladin in neurite outgrowth.

The outgrowth of neurites is a critical step in neuronal maturation, and it is well established that the actin cytoskeleton is involved in this process. Investigators from our laboratory recently described a novel protein named palladin, which has been shown to play an essential role in organizing the actin cytoskeleton in cultured fibroblasts. We investigated the expression of palladin in the developing rat brain by Western blot and found that the E18 brain contained a unique variant of palladin that is significantly smaller (approximately 85 kDa) than the common form found in other developing tissues (90-92 kDa). Because the expression of a tissue-specific isoform suggests the possibility of a cell type-specific function, we investigated the localization and function of palladin in cultured cortical neurons. Palladin was found preferentially targeted to the developing axon but not the dendrites and was strongly localized to the axonal growth cone. When palladin expression was attenuated by transfection with antisense constructs in both the B35 neuroblastoma cell line and in primary cortical neurons, a reduction in the expression of palladin resulted in a failure of neurite outgrowth. These results implicate palladin as a critical component of the developing nervous system, with an important role in axonal extension.

Palladin is expressed preferentially in excitatory terminals in the rat central nervous system.

Palladin is a recently described intracellular protein associated with the actin cytoskeleton and cell adhesion in fibroblasts. In Western and Northern blot analyses, palladin expression is ubiquitous in embryonic mice, but it is down-regulated dramatically in most adult tissues. Significant amounts of palladin persist in the brain of adult rodents, as assessed by Western blot analysis. With this work, we extend preliminary observations and determine the overall distribution and subcellular location of palladin throughout the rat brain. In sagittal and coronal sections of the central nervous system, immunostain for palladin is present throughout the brain and spinal cord, but not uniformly. The densest regions of immunostain include the olfactory bulb, cerebral and cerebellar cortex, hippocampus, amygdala, superior colliculus, and superficial laminae of the spinal dorsal horn. Because immunostain characteristically is punctate, we performed double staining for palladin and the presynaptic marker synaptophysin. Confocal microscopy showed that palladin-immunopositive puncta are also immunopositive for synaptophysin; the proportion of synaptophysin-immunopositive puncta that also stained for palladin ranged from 100% of mossy fiber terminals in field CA3 of the hippocampus and in the cerebellar cortex to 60--70% of terminals in the cerebral cortex, striatum, and spinal dorsal horn. The presence of palladin in synaptic terminals was confirmed by electron microscopy. Because immunostained terminals commonly establish asymmetric synapses, the selectivity of palladin expression in synaptic terminals was tested by double staining for palladin and gamma-aminobutyric acid. The modest level of colocalization in this material at both the light microscopic and electron microscopic levels suggests a selectivity of palladin for terminals that release excitatory neurotransmitters. As concomitant work in cell cultures has shown that palladin participates in axonal development and migration, the present results suggest that palladin persists at excitatory synapses of the adult nervous system.

Activity of proximal promoter of the human beta(1)-integrin gene was increased in Sézary syndrome.

Changes in beta1-integrin expression have been involved in abnormal cellular interactions between malignant lymphocytes from Sézary (Sz) patients and keratinocytes. In this paper, we compare the activity of both distal and proximal promoters of the beta1-integrin gene in malignant lymphocytes from Sz patients with human normal lymphocytes. Activity of both beta1-integrin promoters was also analysed in human normal keratinocytes. Northern blot analysis shows that beta1-integrin mRNA expression is higher in malignant Sz lymphocytes than in normal lymphocytes. CAT assays show that the activity of proximal beta1-integrin promoter is markedly increased (up to 6-fold) in malignant lymphocytes from Sz patients, in comparison to normal lymphocytes. These results suggest that changes in activity of the proximal promoter of beta1-integrin subunit could be, in part, responsible for the abnormal cellular interactions between malignant lymphocytes and keratinocytes observed in Sz syndrome.

MR imaging of the knee at 0.2 and 1.5 T: correlation with surgery.

OBJECTIVE: The purpose of this study was to compare the diagnostic efficacy of low- and high-field-strength MR imagers in the diagnosis of anterior cruciate ligament tears and meniscus tears. SUBJECTS AND METHODS: In 219 patients with suspected internal derangement of the knee, MR imaging at 0.2 and 1.5 T was performed with similar sequences. Only patients with surgically confirmed diagnosis (n = 90) were included in the statistical analysis. Radiologists were unaware of diagnosis and field strength. Sensitivity, specificity, diagnostic accuracy, and inter- and intraobserver variability were determined. RESULTS: There was excellent correlation between the field strengths in accuracy, sensitivity, and specificity for anterior cruciate ligament and meniscus tears. Accuracy for medial meniscus, lateral meniscus, and anterior cruciate ligament tears was 91-93%, 88-90%, and 93-96%, respectively, at 0.2 T and 91-94%, 91-93%, and 97-98%, respectively, at 1.5 T. Inter- and intraobserver variability values showed excellent correlation (kappa > 0.8). CONCLUSION: The level of diagnostic accuracy in anterior cruciate ligament tears and meniscus tears is comparable for low- and high-field-strength MR imagers.

Integrins of the beta1 family influence keratinocyte-lymphocyte interaction.

Data from the literature indicate that ICAM-1 molecules play an important role in keratinocyte interactions with lymphocytes via the lymphocyte function-associated-1 lymphocyte-adhesion molecule. We examined the role of beta1 integrins in keratinocyte-lymphocyte adhesion under different activation conditions. Among the beta1 integrins expressed on keratinocytes and lymphocytes detected by indirect immunofluorescence microscopy and flow cytofluorometry, primarily the alpha2 and the alpha3 subunits on both cell types were involved in keratinocyte-lymphocyte adhesion. Moreover, the highest adhesion level was observed when both cell types were activated by IFN-gamma for keratinocytes and phorbol 12-myristate 13-acetate for lymphocytes, suggesting that the former involved the protein kinase C pathway. Keratinocyte activation, characterized by the expression of ICAM-1, a decrease of beta1 integrins, and the absence of alpha5beta1 integrin, was required for optimal lymphocyte adhesion. Thus, beta1 integrins remaining at the surface of IFN-gamma-treated keratinocytes could be activated by this cytokine, and could synergize with ICAM-1 and lymphocyte function-associated-1 molecules to consolidate keratinocyte-lymphocyte adhesion.

T lymphocytes from Sézary syndrome patients express beta1 integrins whose beta(1-6)-branched N-linked oligosaccharides reflect their adhesive capacity.

Sézary syndrome (Sz), characterized by slowly progressing clonal proliferation of CD4+, CD45 RO+ T cells, has several forms that are distinguished according to the epidermotropic properties of the pathological cells. In a recent paper (Derappe C, Haentjens G, Lemaire S, Feugeas JP, Lebbe C, Pasqualetto V, Bussel A, Aubery M, Néel D. Leukemia 1996;10:138), we observed that T lymphocytes from most of the Sézary patients [Szbeta(1-6)+] expressed high levels of beta(1-6)-GlcNAc-branched N-linked oligosaccharides while T lymphocytes from other patients [Szbeta(1-6)-] did not. Because this observation suggests the possibility of two forms of Sz, distinguished according to the expression rate of these glycans, we looked for a possible relationship between this expression rate and T-cell adhesiveness. Using an original protocol (Braut-Boucher F, Pichon J, Rat P, Adolphe M, Aubery M, Font J. J Immunol Methods 1995;178:41), we observed that T lymphocytes obtained from the Szbeta(1-6)+ patients adhered less to normal keratinocyte monolayers than T lymphocytes from Szbeta(1-6)- patients and normal donors. As assessed by FACS analysis, all the integrin-subunits studied were more expressed on Szbeta(1-6)-, especially alpha4, alpha5, beta1 and beta2, than on Szbeta(1-6)+ and normal lymphocytes. Although these results suggest that beta1- and beta2-integrin expression is involved in the adhesive properties of these T-cells, other factors, such as glycosylation, may also contribute. To demonstrate this possibility, we sought the presence of beta(1-6)-GlcNAc-branched N-linked oligosaccharides on beta1 integrins expressed by T lymphocytes from Sz patients. Immunoblot experiments, performed using the specific lectin from Phaseolus vulgaris (Leukoagglutinin form), showed that only the beta1 integrin subunit expressed by T lymphocytes from Szbeta(1-6)+ patients carried these glycans, supporting the concept of the involvement of T-cell glycosylation in the evolution of Sz.