Ultrastructure of the capillary pericytes and the expression of smooth muscle alpha-actin and desmin in the snake infrared sensory organs

The infrared sensory membranes of pit organs of pit vipers have an extremely rich capillary vasculature that forms many vascular loops, each serving a small number of infrared nerve terminals. We clarified the ultrastructure of capillary pericytes in the pit membranes by scanning and transmission electron microscopy, and examined the immunoreactivity in their cytoplasm to two contractile proteins: smooth muscle alpha-actin (SM alpha-actin) and desmin. The capillary pericytes had two major cytoplasmic processes: thickened primary processes that radiate to embrace the endothelial tube and flattened secondary processes that are distributed widely on the endothelium. Coexpression of SM alpha-actin and desmin was observed in the pericytes of entire capillary segments, and SM alpha-actin was characterized by prominent filament bundles directed mainly at right angles to the capillary long axis. This expression pattern was different from that of capillary pericytes of the scales, where SM alpha-actin was expressed diffusely in the cytoplasm. In a series of electron microscopic sections, we often observed the pericyte processes depressing the endothelial wall. We also observed a close relationship of the pericytes with inter-endothelial cell junctions, and pericyte processes connected with the endothelial cells via gap junctions. From these findings, we surmised that capillary pericytes in the pit membrane have a close functional relationship with the endothelium, and through their contractile and relaxing activity regulate capillary bloodflow to stabilize production of infrared nerve impulses.     

Early stage of human adenovirus type 12-inoculated retinal tissue of F344 newborn rat

To elucidate the pathogenesis of adenovlrus type 12 (Ad12)-induced rat retinal tumor, an experimental animal model of human retinobiastoma (RB), DNA analysis, in situ hybridizatlon and immunohistochemlstry were performed. The adenovirus oncogene EIA was detected in the host genome by Southern blot hybridization. Examined retinal tlssues did not show any histological changes, but the number of retinal cells lmmunoreactive with an antibody to proliferating cell nuclear antigen (PCNA) increased with the course of study. In in situ hybridization, E1A gene expression was recognized at the Inner granular layer of the retina at an early stage arer virus inoculation, and subsequently, N-myc gene expression was recognized at the same region. No alteration was found in the retinoblastoma susceptibility gene ( Rb gene) expression. The product of the virus oncogene integrated into the host genome could induce an Increase in N-myc expression, without any abnormality of the Rb gene itself. Results from the present study could be useful in clarifying the tumorige-nesis of this experimental model.     

Difference in the expression of three tight junction proteins, barmotin, occludin, and ZO-1, in phenotypically different human colon cancer cell lines

Epithelioid disorganization is a hallmark of gastrointestinal cancers and is believed to be associated with malignant phenotypes such as invasiveness and the potentiality for metastasis. Although tight junctions (TJs) are known to be crucial for the maintenance of polarized organization of the gastrointestinal epithelium, changes in the TJ proteins in human cancers have not yet been fully elucidated. In this report, we investigated the expression and localization of three TJ proteins-barmotin (7H6 antigen), occludin, and ZO-1-in three phenotypically different human colon cancer cell lines exhibiting differnt grades of epithelioid organization. All three proteins were localized at the most apical part of the cell border corresponding to the site of TJs in T₈₄ cells, in which epithelioid organization was well preserved. In contrast, in COLO320DM cells, which showed no epithelioid phenotypes, occludin was not detectable at either the protein or mRNA level, although barmotin and ZO-1 were present in the cytoplasm. In the third cell line, DLD-1, which showed an epithelioid phenotype intermediate between T₈₄ and COLO320DM, aberrant expression of occludin was found in the basolateral cell membrane. On the other hand, barmotin was present in the cytoplasm, whereas ZO-1 was localized at the cell border. These observations showed that changes in the expression of TJ proteins occur in close correlation with epithelioid disorganization in human colon cancers.     

Lysosome is a primary organelle in B cell receptor-mediated apoptosis: an indispensable role of Syk in lysosomal function

To investigate the mechanism of B cell receptor (BCR)-mediated apoptosis, we utilized immature B cell lines, DT40 and WEHI-231. In both cell lines, BCR-crosslinking caused the increase in lysosomal pH with early apoptotic changes characterized by chromatin condensation and phosphatidylserine exposure. This increase was detected in c-Abl-deficient DT40 cells but not in Syk-deficient cells, which corresponded to the fact that the former cells but not the latter revealed BCR-induced apoptosis. In contrast, BCR-crosslinking caused no apparent change in mitochondrial transmembrane potential. Therefore, the lysosomal change might be a primary event in BCR-induced apoptosis in DT40 cells. The increased activity of cathepsin B and apoptosis-preventing effect of a cathepsin inhibitor suggested a significant role of lysosomal enzymes in this apoptosis. By microscopic studies, lysosomes of wild-type DT40 cells fused to BCR-carrying endosomes became enlarged and accumulated one another. In contrast, these changes of lysosomal dynamics did not occur in Syk-deficient cells but transfer of wild-type Syk restored the lysosomal changes and apoptosis. These results demonstrated that the lysosomal change accompanied with the activation of lysosomal enzymes is a primary step in BCR-crosslinking-mediated apoptosis and Syk is responsible for this step through the fusion of BCR-carrying endosomes to lysosomes.     

Role of protein-tyrosine kinase syk in oxidative stress signaling in B cells

Oxidative stress induces the activation of multiple signaling pathways related to various cellular responses. In B cells, Syk has a crucial role in intracellular signal transduction induced by oxidative stress as well as antigen receptor engagement. Treatment of B cells with hydrogen peroxide (H(2)O(2)) induces enzymatic activation of Syk. Syk is essential for Ca(2+) release from intracellular pools through phospholipase C-gamma2 and the activation of c-Jun N-terminal kinase, p38 mitogen-activated protein kinase, and phosphatidylinositol 3-kinase-Akt survival pathway following H(2)O(2) stimulation. Oxidative stress-induced cellular responses in B cells follow different patterns, such as necrosis, apoptosis, and mitotic arrest, according to the intensity of H(2)O(2) stimulation. Syk is involved in the protection of cells from apoptosis and induction of G2/M arrest. Syk leads to the activation of the phosphatidylinositol 3-kinase-Akt survival pathway, thereby enhancing cellular resistance to oxidative stress-induced apoptosis. On the other hand, Syk-dependent phospholipase C-gamma2 activation is required for acceleration toward apoptosis following oxidative stress. These findings suggest that oxidative stress-induced Syk activation triggers the activation of several pathways, such as proapoptotic and survival pathways, and the balance among these various pathways is a key factor in determining the fate of a cell exposed to oxidative stress.     

Raft.1, a monoclonal antibody raised against the raft microdomain,recognizes G-protein beta1 and 2, which assemble near nucleus after shiga toxin binding to human renal cell line

SUMMARY: Raft microdomains are glycolipid-enriched microdomain scaffolding molecules involved in signal transduction. The binding of Shiga toxin to globotriaosyl ceramide in raft microdomains of the human renal tubular cell line ACHN causes temporal activation of Src-kinase Yes. To study the downstream signaling mechanism proceeding to the activation of Yes, we raised monoclonal antibodies (MAbs) against raft microdomains. The MAbs were screened on the basis of, first, binding to raft microdomains with dot-blot immunostaining, second, intracellular localization of the epitope by flowcytometry after permeabilization, and third, translocation of the antigen molecules after Stx treatment by immunohistochemical staining. Raft.1 MAb bound to the molecules that accumulated to the particular region near the nucleus after Stx treatment. Two-dimensional Western blotting and matrix-assisted laser desorption/ionization time of flight mass spectrometry analysis revealed that the antigen molecule is GTP binding protein beta subunits 1 and 2 (Gbeta1 and 2). That Raft.1 recognized Gbeta1 and 2 was further confirmed by the reactivity to recombinant Gbeta1 and 2 proteins. To our knowledge, this is the first report of production of a MAb recognizing Gbeta1 and 2. Because Gbeta1 and 2 are highly conserved all through organisms and are deeply involved in signal transduction, Raft.1 is expected to be utilized frequently in research.     

Assessment of Baroreceptor Reflex Function During Mental Stress and Relaxation

This paper describes a method of measuring baroreceptor cardiac reflex sensitivity noninvasively from spontaneous patterns of blood pressure and interbeat interval, and the application of this technique in psychophysiology. Baroreflex function was assessed in 24 female volunteers during relaxation and performance of the cold pressor test and a non-verbal mental arithmetic task. Blood pressure and interbeat interval were monitored continuously from the finger using the vascular unloading technique. Sequences of three or more cardiac cycles were identified over which systolic blood pressure increased progressively in conjunction with lengthening interbeat interval, or systolic blood pressure decreased as interbeat interval was reduced. The regression between systolic blood pressure and interbeat interval was computed as an index of baroreflex sensitivity. Relaxation was associated with a small prolongation of interbeat interval, whereas baroreflex sensitivity increased from 17.1 to 19.8 ms/mmHg. Baroreflex sensitivity was reduced significantly during mental arithmetic (mean 14.2 ms/mmHg) but not during the cold pressor test (mean 17.4 ms/mmHg). The difference between mental arithmetic and the cold pressor test may be related to the relative intensity of cardiac and vascular responses in the two situations. The implications of these results for the understanding of behavioural influences on haemodynamic function are discussed and the advantages of noninvasive methods are considered.     

The architecture of variant surface glycoprotein gene expression sites in Trypanosoma brucei

Trypanosoma brucei evades the immune system by switching between Variant Surface Glycoprotein (VSG) genes. The active VSG gene is transcribed in one of approximately 20 telomeric expression sites (ESs). It has been postulated that ES polymorphism plays a role in host adaptation. To gain more insight into ES architecture, we have determined the complete sequence of Bacterial Artificial Chromosomes (BACs) containing DNA from three ESs and their flanking regions. There was variation in the order and number of ES-associated genes (ESAGs). ESAGs 6 and 7, encoding transferrin receptor subunits, are the only ESAGs with functional copies in every ES that has been sequenced until now. A BAC clone containing the VO2 ES sequences comprised approximately half of a 330 kb 'intermediate' chromosome. The extensive similarity between this intermediate chromosome and the left telomere of T. brucei 927 chromosome I, suggests that this previously uncharacterised intermediate size class of chromosomes could have arisen from breakage of megabase chromosomes. Unexpected conservation of sequences, including pseudogenes, indicates that the multiple ESs could have arisen through a relatively recent amplification of a single ES.     

Influences of pressure-injected cyclic AMP on the membrane current and characteristics of an identified neuron of Aplysia kurodai

The ionic mechanism of the effect of intracellularly injected adenosine 3',5'-cyclic monophosphate (cAMP) on the membrane of identified neuron L5 of Aplysia kurodai was investigated with conventional voltage-clamp and ion-substitution techniques. The intracellular elevation of cAMP caused an inward current (IcAMP), which was not accompanied by a significant change in membrane conductance at potentials more hyperpolarized than -60 mV. The current increased over the voltage range (-50 to -30 mV) associated with a conductance decrease and decreased at potentials more hyperpolarized than -60 mV. Elevated intracellular cAMP was found to enhance a region of negative slope resistance in steady-state I-V relations. Duration of the IcAMP was greatly prolonged by bath-applied isobutylmethylxanthine (50 microM), but imidazole (10 mM) had an opposite effect on the IcAMP. Tolbutamide (5 mM), a protein kinase inhibitor, reduced the IcAMP. The current was not affected by the presence of bath-applied TTX (50 microM), ouabain (50 microM), or triaminopyrimidine (5 mM). Reduction of [Na+]0 reversibly decreased the IcAMP. Li+ could largely substitute for Na+. Alterations of [K+]0, and bath application of 4-AP (5 mM) and TEA (30 mM) did not affect the IcAMP. In the presence of Na+, Cl-, and divalent cations such as Ca2+ and Ba2+ inhibited the IcAMP. These results suggest that fast elevation of intracellular cAMP induces a TTX-resistant slow Na+ inward current, and the current might be due to activation of cAMP-dependent protein kinase.