Heat-shock induced nuclear retention and recycling inhibition of importin alpha

Heat-shock induces a strong stress response and modifies all aspects of cellular physiology, which involves dynamic changes in the nucleocytoplasmic distributions of a variety of proteins. Many distinct nucleocytoplasmic transport pathways exist in eukaryotic cells, but how a particular transport pathway is regulated under different cellular conditions remains elusive. The finding of this study indicate that conventional nuclear import, which is mediated by importin alpha/beta, is down-regulated, while the nuclear import of 70 kD heat-shock cognate protein is up-regulated in heat-shock cells. Among the factors involved in the mediation of the conventional nuclear import, significant levels of importin alpha accumulate in the nucleus in response to heat-shock. An analysis of the behaviour of importin alpha with fluorescence recovery after photobleaching and fluorescence loss in photobleaching studies show that nuclear importin alpha becomes less mobile and its nucleocytoplasmic recycling is impaired in heat-shock cells. These data coincided well with biochemical and cytological studies. Our present data show that heat-shock induces the nuclear accumulation, nuclear retention, and recycling inhibition of importin alpha, resulting in the suppression of conventional nuclear import. This suggests a new regulatory mechanism for the adaptation of cells to environmental changes, such as heat-shock.     

Depletion of mitochondrial DNA in HIV-1-infected patients and its amelioration by antiretroviral therapy

Mitochondrial DNA (mtDNA) of peripheral blood mononuclear cells (PBMCs) collected from Human immunodeficiency virus 1 (HIV-1)-infected patients and healthy controls were measured longitudinally using real-time polymerase chain reaction to evaluate the effects of antiretroviral agents on mtDNA synthesis in vivo and to assess the value of monitoring mtDNA in PBMCs to predict adverse events amongst these patients. MtDNA levels in PBMCs were significantly decreased in treatment-naive HIV-1-infected patients compared with healthy people. MtDNA levels were not only significantly correlated with CD4(+) T-cell count, but also inversely correlated with HIV-1 viral load. MtDNA levels in untreated patients and healthy controls were stable during the period of observation. On the other hand, amongst patients treated with regimens containing AZT/3TC or d4T/3TC, mtDNA increased during treatment and recovered to levels comparable to healthy controls. In contrast, mtDNA decreased immediately after the initiation of an AZT/ddC-containing regimen. We did not find a correlation between mtDNA levels and changes in clinical parameters. There was no significant difference in mtDNA levels between patients with and those without lipoatrophy. Furthermore, there was no obvious difference in mtDNA levels amongst those patients exhibiting signs and symptoms of peripheral neuropathy. In conclusion, the decrease in mtDNA levels in PBMCs amongst HIV-1-infected patients and its amelioration by antiretroviral therapy may suggest the influence of direct effects on mitochondria or mtDNA by HIV-1 infection. Further investigations are needed to elucidate the mechanisms contributing to decreased mtDNA and the value of mtDNA measurement in the care of HIV-1-infected individuals.     

Use of an Enrichment Broth Cultivation-PCR Combination Assay for Rapid Diagnosis of Swine Erysipelas

We have previously described the creation by Tn916 mutagenesis of avirulent transposition mutants from a highly virulent strain of Erysipelothrix rhusiopathiae, the causative agent of swine erysipelas. In this study, we cloned a 2.2-kb DNA fragment which flanked the Tn916 insertion in an avirulent mutant (strain 33H6) and evaluated the possibility that this region could be used for the specific detection of E. rhusiopathiae. According to the sequences of this region, oligonucleotide primers were designed to amplify a 937-bp fragment of the E. rhusiopathiae chromosome by PCR. The specificity of the PCR was investigated by analyzing 64 strains of Erysipelothrix species and 27 strains of other genera different from Erysipelothrix. A 937-bp DNA fragment could be amplified from all E. rhusiopathiae strains tested, and no amplification was observed by using DNAs from the other species tested. To make a rapid and definite diagnosis of swine erysipelas in slaughterhouses, we developed an enrichment broth cultivation-PCR combination assay, which used a commercially available DNA extraction kit, to identify E. rhusiopathiae in the specimens from swine with arthritis. After samples were enriched in selective broth culture, detection of E. rhusiopathiae was tested by either conventional methods or the PCR. Of 102 samples tested, 15 samples were positive by conventional methods and 12 of the 15 samples were positive by the PCR. The detection limit of the PCR was 10³ CFU per reaction mixture for the PCR-positive samples. These results indicate that this PCR technique could be used as a first-line screening technique for the specific detection of E. rhusiopathiae in specimens.     

Follicular hyperplasia presenting with a marginal zone pattern in a reactive lymph node lesion

Histologically, the marginal zone pattern of the lymph node is characterized by lymphoid follicles with three distinct layers. The inner layer is composed of follicular center zones, the middle layer of darkly stained mantle zones, and the outer layer of marginal zones. However, the marginal zone pattern is rarely seen in reactive lymph nodes except for mesenteric lymph nodes. We describe the clinicopathologic, immunohistochemical and genotypic findings of six cases of reactive follicular hyperplasia exhibiting the marginal zone pattern. The patients comprised three males and three females (age range 24 to 63 years; medium 56 years). Follow-up data were obtained from five patients. None of them developed malignant lymphomas during the follow-up period of from 5 to 204 months (median 68 months). Histologically, the lesion was characterized by numerous lymphoid follicles and partial distortion of lymph node structure. Varying degrees of progressive transformation of the germinal center (PTGC) were noted in the four cases. The marginal zone pattern was observed in some or most of the lymphoid follicles including PTGC. The marginal zone B cells were small to medium-sized lymphocytes with round or slightly indented nuclei and a broad rim of pale cytoplasm. Some of them had a monocytoid appearance. They were CD20+, CD79a+, sIgM+/-, sIgD-, CD5-, CD10-, CD21-, CD23-, CD43-, CD45RO-, Bcl-6-, cyclin D1-, EMA- and p53-. A portion of them were Bcl-2 positive. Occasional large lymphoid cells with round or indented nuclei and moderate amounts of cytoplasm were observed in the marginal zone in four cases. These large lymphoid cells were usually CD20 positive, but Bcl-6 negative. A small number of them contained polytypic intracytoplasmic immunoglobulins. The polytypic nature of B lymphocytes was demonstrated by immunohistochemistry and polymerase chain reaction. Recognition of unusual marginal zone hyperplasia in reactive lymph node lesions is important to avoid confusion with nodal involvement in various low-grade B cell lymphomas presenting a marginal zone distribution pattern.     

Freeze-fracture studies of muscle plasma membranes in myopathic patients with hypo-and hyperthyroidism

To examine the influence of thyroid hormone on the skeletal muscle plasma membrane, we analyzed the changes in ultrastructural architecture and membrane area complexed with digitonin of muscle plasma membrane in myopathic patients with hypo-and hyperthyroidism by the conventional freeze-fracture (F-F) technique and F-F cytochemistry using the sterol-specific ligand digitonin. The densities of flask-shaped invaginations, which are mainly thought to correspond to caveolae, intramembranous particles, and orthogonal arrays, and the changes of digitoninreacted membrane areas in the muscle plasma membranes in three patients with hypothyroid myopathy and one patient with both myasthenia gravis and hyperthyroidism were compared with those in age-matched controls. In the conventional F-F study, the muscle plasma membrane of hypothyroid patients showed increased invagination density, whereas that of the hyperthyroid patient was normal ultrastructurally. In the F-F cytochemistry study, however, the ratio of digitonin-reacted membrane areas versus fractured membrane areas was not different between hypothyroid patients and controls, whereas that of the hyperthyroid patient was lowered in comparison with that of control. These results suggest that thyroid hormone may alter the biochemical properties and ultrastructural architecture of muscle plasma membrane.This study was presented at the 29th annual meeting of the Clinical Electron Microscopy Society of Japan, Sapporo, October 2–3, 1997     

Role of nectin in organization of tight junctions in epithelial cells

BACKGROUND: In polarized epithelial cells, cell-cell adhesion forms specialized membrane structures comprised of claudin-based tight junctions (TJs) and of E-cadherin-based adherens junctions (AJs). These structures are aligned from the apical to the basal side of the lateral membrane, but the mechanism of this organization remains unknown. Nectin is a Ca2+ independent immunoglobulin-like cell-cell adhesion molecule which localizes at AJs. Nectin is associated with E-cadherin through their respective cytoplasmic tail-binding proteins, afadin and catenins, and involved in the formation of AJs in cooperation with E-cadherin. We show here that nectin is also involved in the formation of TJs. RESULTS: During the formation of the junctional complex consisting of AJs and TJs in Madin-Darby canine kidney (MDCK) cells, claudin and occludin accumulated at the apical sites of the nectin-based cell-cell adhesion sites. This accumulation of claudin and occludin was inhibited by inhibitors acting on the trans interaction of nectin. The barrier function of TJs was also impaired by the nectin inhibitors. It has been shown that a phorbol ester promotes the formation of a TJ-like structure in an E-cadherin-independent manner. This phorbol ester-induced formation of the TJ-like structure was also inhibited by the nectin inhibitors. CONCLUSIONS: These results suggest a role of the nectin-afadin system in the organization of TJs as well as AJs in epithelial cells.     

Cdc42 and Rac small G proteins activated by trans-interactions of nectins are involved in activation of c-Jun N-terminal kinase,but not in association of nectins and cadherin to form adherens junctions,in fibroblasts

BACKGROUND: Nectins are Ca2+-independent immunoglobulin-like cell-cell adhesion molecules which associate with cadherins to form adherens junctions (AJs) in epithelial cells and fibroblasts. Nectin-1 and -3 are members of the nectin family which most strongly trans-interact, causing cell-cell adhesion. The trans-interaction between nectin-1 and -3 induces the activation of both Cdc42 and Rac small G proteins in epithelial cells. We studied the roles of Cdc42 and Rac activated in this way in L fibroblasts stably expressing both nectin-1 and E-cadherin (nectin-1-EL cells). RESULTS: The trans-interaction between nectin-1 and -3 induced the activation of Cdc42 and Rac in nectin-1-EL cells. Cdc42, and presumably Rac, activated in this way, induced the activation of c-Jun N-terminal kinase (JNK), but not p38 mitogen-activated protein (MAP) kinase or extracellular signal-regulated kinase (ERK). Cdc42 or Rac was not essential for the association of nectin-1 and E-cadherin to form AJs. Reorganization of the actin cytoskeleton was not required for the association of nectin-1 and E-cadherin. CONCLUSION: These results indicate that Cdc42 and Rac activated by the trans-interaction of nectins selectively induce the activation of JNK, but are not essential for the association of nectins and cadherin to form AJs in fibroblasts.