(1/617) Enhanced fatty streak formation in C57BL/6J mice by immunization with heat shock protein-65.
Recent data suggest that the immune system is involved in atherogenesis. Thus, interest has been raised as to the possible antigens that could serve as the initiators of the immune reaction. In the current work, we studied the effects of immunization with recombinant heat shock protein-65 (HSP-65) and HSP-65-rich Mycobacterium tuberculosis (MT) on early atherogenesis in C57BL/6J mice fed either a normal chow diet or a high-cholesterol diet (HCD). A rapid, cellular immune response to HSP-65 was evident in mice immunized with HSP-65 or with MT but not in the animals immunized with phosphate-buffered saline (PBS) alone. Early atherosclerosis was significantly enhanced in HCD-fed mice immunized with HSP-65 (n=10; mean aortic lesion size, 45 417+/-9258 microm2) or MT (n=15; 66 350+/-6850 microm2) compared with PBS-injected (n=10; 10 028+/-3599 microm2) or nonimmunized (n=10; 9500+/-2120 microm2) mice. No fatty streak lesions were observed in mice fed a chow diet regardless of the immunization protocol applied. Immunohistochemical analysis of atherosclerotic lesions from the HSP-65- and MT-immunized mice revealed infiltration of CD4 lymphocytes compared with the relatively lymphocyte-poor lesions in the PBS-treated or nonimmunized mice. Direct immunofluorescence analysis of lesions from HSP-65- and MT-immunized mice fed an HCD exhibited extensive deposits of immunoglobulins compared with the fatty streaks in the other study groups, consistent with the larger and more advanced lesions found in the former 2 groups. This model, which supports the involvement of HSP-65 in atherogenesis, furnishes a valuable tool to study the role of the immune system in atherogenesis. (+info)
(2/617) Erythropoietin modulates intracellular calcium in a human neuroblastoma cell line.
1. Recent investigations have shown that the glycoprotein erythropoietin (Epo) and its specific receptor (EpoR) are present in the mammalian brain including human, monkey and mouse. These findings suggest a local action of Epo in the nervous system. The aim of this study was to elucidate a possible functional interaction of Epo with neuronal cells. 2. To examine the influence of externally applied Epo on Ca2+ homeostasis the human neuroblastoma cell line SK-N-MC was chosen as a suitable in vitro model for undifferentiated neuronal cells. 3. Expression of the EpoR in SK-N-MC cells was detected by reverse transcription-PCR, Western blot and immunofluorescence analysis. 4. Patch-clamp studies of SK-N-MC cells confirmed the expression of T-type Ca2+ channels, whose peak macroscopic current was increased by the addition of recombinant human Epo (rhEpo) to the bathing medium. 5. Confocal laser scanning microscopy analysis of SK-N-MC cells confirmed a transient increase in intracellular free [Ca2+] in response to externally applied rhEpo. 6. The transient response to Epo was dependent on external Ca2+ and remained even after depletion of internal Ca2+ stores by caffeine or thapsigargin. However, after depletion the response to Epo was absent when cells were superfused with the T-type Ca2+ channel blocker flunarizine. 7. This study demonstrates that Epo can interact with neuronal cells by affecting Ca2+ homeostasis through an increase in Ca2+ influx via plasma membrane T-type voltage-dependent Ca2+ channels. (+info)
(3/617) Different prevalences of Renibacterium salmoninarum detected by ELISA in Alaskan chinook salmon Oncorhynchus tshawytscha spawned from freshwater and seawater.
Soluble antigen of Renibacterium salmoninarum (Rs) was detected by a polyclonal enzyme-linked immunosorbent assay (ELISA) at significantly higher prevalences in adult chinook salmon Oncorhynchus tshawytscha that matured in freshwater than in the same cohort of fish spawned after maturation in seawater. The cumulative results were consistent during 4 yr of comparison at the Little Port Walter Hatchery on Baranof Island, Alaska, USA. Possible causes for this difference are discussed. Maturation of chinook salmon broodstock in seawater has become a practical strategy at this hatchery to reduce the prevalence of Rs-positive parent fish and the numbers of culled eggs. (+info)
(4/617) Distinct transient outward potassium current (Ito) phenotypes and distribution of fast-inactivating potassium channel alpha subunits in ferret left ventricular myocytes.
The biophysical characteristics and alpha subunits underlying calcium-independent transient outward potassium current (Ito) phenotypes expressed in ferret left ventricular epicardial (LV epi) and endocardial (LV endo) myocytes were analyzed using patch clamp, fluorescent in situ hybridization (FISH), and immunofluorescent (IF) techniques. Two distinct Ito phenotypes were measured (21-22 degrees C) in the majority of LV epi and LV endo myocytes studied. The two Ito phenotypes displayed marked differences in peak current densities, activation thresholds, inactivation characteristics, and recovery kinetics. Ito,epi recovered rapidly [taurec, -70 mV = 51 +/- 3 ms] with minimal cumulative inactivation, while Ito,endo recovered slowly [taurec, -70 mV = 3,002 +/- 447 ms] with marked cumulative inactivation. Heteropoda toxin 2 (150 nM) blocked Ito,epi in a voltage-dependent manner, but had no effect on Ito,endo. Parallel FISH and IF measurements conducted on isolated LV epi and LV endo myocytes demonstrated that Kv1.4, Kv4.2, and Kv4.3 alpha subunit expression in LV myocyte types was quite heterogenous: (a) Kv4.2 and Kv4.3 were more predominantly expressed in LV epi than LV endo myocytes, and (b) Kv1.4 was expressed in the majority of LV endo myocytes but was essentially absent in LV epi myocytes. In combination with previous measurements on recovery kinetics (Kv1.4, slow; Kv4.2/4.3, relatively rapid) and Heteropoda toxin block (Kv1.4, insensitive; Kv4.2, sensitive), our results strongly support the hypothesis that, in ferret heart, Kv4.2/Kv4.3 and Kv1.4 alpha subunits, respectively, are the molecular substrates underlying the Ito,epi and Ito,endo phenotypes. FISH and IF measurements were also conducted on ferret ventricular tissue sections. The three Ito alpha subunits again showed distinct patterns of distribution: (a) Kv1.4 was localized primarily to the apical portion of the LV septum, LV endocardium, and approximate inner 75% of the LV free wall; (b) Kv4. 2 was localized primarily to the right ventricular free wall, epicardial layers of the LV, and base of the heart; and (c) Kv4.3 was localized primarily to epicardial layers of the LV apex and diffusely distributed in the LV free wall and septum. Therefore, in intact ventricular tissue, a heterogeneous distribution of candidate Ito alpha subunits not only exists from LV epicardium to endocardium but also from apex to base. (+info)
(5/617) In situ hybridisation and direct fluorescence antibodies for the detection of Chlamydia trachomatis in synovial tissue from patients with reactive arthritis.
BACKGROUND: Chlamydia trachomatis is associated with Reiter's syndrome and reactive arthritis but the form in which the organism survives in synovial cells is unclear. AIM: To compare in situ hybridisation with direct fluorescence in the detection of inapparent chlamydial infection in synovial tissue. METHODS: Synovial tissue from four patients with reactive arthritis patients was examined using biotin labelled probes for chlamydial DNA and fluorescein isothiocyanate (FITC) labelled monoclonal antibodies against the major outer membrane protein. RESULTS: In two of the four patients, evidence of chlamydial infections was detected by in situ hybridisation in parallel sections but not with FITC labelled monoclonal antibodies. CONCLUSIONS: Detection of chlamydial DNA by in situ DNA hybridisation may be a better way to identify chlamydial infection in synovial tissue than phenotype targeting with FITC conjugated antibodies, which is used as a standard procedure for screening clinical specimens for chlamydia. (+info)
(6/617) Evidence of hypoxic areas within the arterial wall in vivo.
The anoxemia theory of atherosclerosis states that an imbalance between the demand and supply of oxygen in the arterial wall is a key factor for the development of atherosclerotic lesions. Direct in vitro and in situ measurements have shown that PO2 is decreased in the more deeply situated parts of the media, but the degree of hypoxia in vivo or the distribution of hypoxia along the arterial tree is not known. For this reason, we have developed a method for the detection of hypoxia in the arterial wall in vivo by using a hypoxia marker, 7-(4'-(2-nitroimidazol-1-yl)-butyl)-theophylline, that may be visualized by immunofluorescence. In the present study, we have used this method in rabbits with experimentally induced atherosclerosis. Our results indicate that zones of hypoxia occur at depth in the atherosclerotic plaque. The mechanism was probably an impaired oxygen diffusion capacity due to the thickness of the lesion, together with high oxygen consumption by the foam cells. Thus, we have for the first time demonstrated that hypoxia actually does exist in the arterial wall in vivo, lending support to the anoxemia theory of atherosclerosis. (+info)
(7/617) A new ER trafficking signal regulates the subunit stoichiometry of plasma membrane K(ATP) channels.
Proper ion channel function often requires specific combinations of pore-forming alpha and regulatory beta subunits, but little is known about the mechanisms that regulate the surface expression of different channel combinations. Our studies of ATP-sensitive K+ channel (K(ATP)) trafficking reveal an essential quality control function for a trafficking motif present in each of the alpha (Kir6.1/2) and beta (SUR1) subunits of the K(ATP) complex. We show that this novel motif for endoplasmic reticulum (ER) retention/retrieval is required at multiple stages of K(ATP) assembly to restrict surface expression to fully assembled and correctly regulated octameric channels. We conclude that exposure of a three amino acid motif (RKR) can explain how assembly of an ion channel complex is coupled to intracellular trafficking. (+info)
(8/617) Caspase-8 is required for cell death induced by expanded polyglutamine repeats.
We show here that caspase-8 is required for the death of primary rat neurons induced by an expanded polyglutamine repeat (Q79). Expression of Q79 recruited and activated caspase-8. Inhibition of caspase-8 blocked polyglutamine-induced cell death. Coexpression of Q79 with the caspase inhibitor CrmA, a dominant-negative mutant of FADD (FADD DN), Bcl-2, or Bcl-xL, but not an N-terminally tagged Bcl-xL, prevented the recruitment of caspase-8 and inhibited polyglutamine-induced cell death. Furthermore, Western blot analysis revealed the presence of activated caspase-8 in the insoluble fraction of affected brain regions from Huntington's disease (HD) patients but not in those from neurologically unremarkable controls, suggesting the relocation and activation of caspase-8 during the pathogenesis of HD. These results suggest an essential role of caspase-8 in HD-related neural degenerative diseases. (+info)