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At least 1,747 records · Page 97Linked to original sources
PubMed2000

Globotriaosyl ceramide (CD77/Gb3) in the glycolipid-enriched membrane domain participates in B-cell receptor-mediated apoptosis by regulating lyn kinase activity in human B cells.

The role of CD77 expressed on a fraction of germinal center B cells, also known as glycosphyngolipid Gb3, and as a functional receptor for Shiga toxins (Stx) in B-cell receptor (BCR)-mediated apoptosis was investigated. Using Stx1-sensitive Burkitt's lymphoma Ramos cells as an in vitro model of CD77(+) germinal center B cells, intracellular signaling events mediated by either Stx1 or anti-CD77 antibody were examined immunobiochemically and immunocytologically. We observed prompt activation of Lyn and Syk kinases leading to increased binding of these proteins to surface IgM (sIgM) in Ramos cells after Stx1 treatment. We also observed microscopic colocalization of CD77 and sIgM after stimulation with Stx1. Along with the synergism between the cross-linking of CD77 and that of sIgM in their effect on apoptosis induction, it was highly probable that CD77 cross-linking induces activation of the BCR signaling cascade. Analysis using sucrose density gradient centrifugation suggested that Stx1 binding to CD77 induced recruitment and activation of Lyn in the glycolipid-enriched membrane (GEM) fractions. Once activated, however, Lyn seemed to acquire an increased detergent solubility and moved outside of the GEM fractions. This study describes the participation of the GEM domain in BCR-signaling cascade and suggests a possible role of CD77 as a regulator of BCR-induced apoptosis in human B cells.

Apoptosis↗
PubMedDate not supplied

Synthesis, in vitro kinetics, and in vivo studies on protein conjugates of AZT: evaluation as a transport system to increase brain delivery.

Our method to increase the delivery of polar drugs to the central nervous system is via drug-protein conjugates with proteins that interact with and cross brain capillary endothelial cells. As a model for drugs containing a reactive hydroxyl group, AZT was conjugated via a succinate linker to two such protein carriers, the highly cationic histone H1 and an anti-transferrin receptor antibody, OX-26. The protein carriers were selected on the basis of their ability to interact with brain capillary endothelial cells by absorptive or receptor-mediated events, respectively. An in vitro pH profile of the rate of AZT release indicated that the observed hydrolysis proceeds by a specific base-catalysis mechanism. At 37 degrees C, the release of AZT proceeded at a rate approximately 10-fold faster (Kobs approximately 8 x 10(-4) min-1) than expected for a simple ester (AZT succinate; Kobs approximately 1.25 x 10(-4) min-1). Using simple model systems, product analysis revealed that intramolecular cyclization of the succinate linker accounts for the observed rate enhancement. Drug delivery in vivo was assessed using immunohistochemical techniques and quantitative brain uptake measurements with singly and doubly labeled AZT-OX-26 conjugates. Immunohistochemical staining of brain sections showed the colocalization of AZT and OX-26 in the brain vasculature. Therefore, drug can be linked to the antibody without affecting the targeting property of the antibody. Furthermore, an in vivo time course using radiolabeled conjugate showed that AZT is delivered to the brain capillaries but is not transported into the brain parenchyma with the antibody.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2004

The ammonium transporter RhBG: requirement of a tyrosine-based signal and ankyrin-G for basolateral targeting and membrane anchorage in polarized kidney epithelial cells.

RhBG is a nonerythroid member of the Rhesus (Rh) protein family, mainly expressed in the kidney and belonging to the Amt/Mep/Rh superfamily of ammonium transporters. The epithelial expression of renal RhBG is restricted to the basolateral membrane of the connecting tubule and collecting duct cells. We report here that sorting and anchoring of RhBG to the basolateral plasma membrane require a cis-tyrosine-based signal and an association with ankyrin-G, respectively. First, we show by using a model of polarized epithelial Madin-Darby canine kidney cells that the targeting of transfected RhBG depends on a YED motif localized in the cytoplasmic C terminus of the protein. Second, we reveal by yeast two-hybrid analysis a direct interaction between an FLD determinant in the cytoplasmic C-terminal tail of RhBG and the third and fourth repeat domains of ankyrin-G. The biological relevance of this interaction is supported by two observations. (i) RhBG and ankyrin-G were colocalized in vivo in the basolateral domain of epithelial cells from the distal nephron by immunohistochemistry on kidney sections. (ii) The disruption of the FLD-binding motif impaired the membrane expression of RhBG leading to retention on cytoplasmic structures in transfected Madin-Darby canine kidney cells. Mutation of both targeting signal and ankyrin-G-binding site resulted in the same cell surface but nonpolarized expression pattern as observed for the protein mutated on the targeting signal alone, suggesting the existence of a close relationship between sorting and anchoring of RhBG to the basolateral domain of epithelial cells.

Amino Acid Motifs↗
PubMed2000

A rat kidney-specific calcium transporter in the distal nephron.

Active absorption of calcium from the intestine and reabsorption of calcium from the kidney are major determinants of whole body calcium homeostasis. Two recently cloned proteins, CaT1 and ECaC, have been postulated to mediate apical calcium uptake by rat intestine and rabbit kidney, respectively. By screening a rat kidney cortex library with a CaT1 probe, we isolated a cDNA encoding a protein (CaT2) with 84.2 and 73.4% amino acid identities to ECaC and CaT1, respectively. Unlike ECaC, CaT2 is kidney-specific in the rat and was not detected in intestine, brain, adrenal gland, heart, skeletal muscle, liver, lung, spleen, thymus, and testis by Northern analysis or reverse transcription polymerase chain reaction. The expression pattern of CaT2 in kidney was similar to that of calbindin D(28K) and the sodium calcium exchanger 1, NCX1, by in situ hybridization of adjacent sections. Furthermore, the mRNAs for CaT2 and calbindin D(28K) were colocalized in the same cells. CaT2 mediated saturable calcium uptake with a Michaelis constant (K(m)) of 0.66 mm when expressed in Xenopus laevis oocytes. Under voltage clamp condition, CaT2 promoted inward currents in X. laevis oocytes upon external application of Ca(2+). Sr(2+) and Ba(2+) but not Mg(2+) also evoked inward currents in CaT2-expressing oocytes. Similar to the alkaline earth metal ions, application of Cd(2+) elicited inward current in CaT2-expressing oocytes with a K(m) of 1.3 mm. Cd(2+), however, also potently inhibited CaT2-mediated Ca(2+) uptake with an IC(50) of 5.4 micrometer. Ca(2+) evoked currents were reduced at low pH and increased at high pH and were only slightly affected by the L-type voltage-dependent calcium channel antagonists, nifedipine, verapamil, diltiazem, and the agonist, Bay K 8644, even at relatively high concentrations. In conclusion, CaT2 may participate in calcium entry into the cells of the distal convoluted tubule and connecting segment of the nephron, where active reabsorption of calcium takes place via the transcellular route. The high sensitivity of CaT2 to Cd(2+) also provides a potential explanation for Cd(2+)-induced hypercalciuria and resultant renal stone formation.

Amino Acid Sequence↗
PubMed2001

Binding of Ikaros to the lambda5 promoter silences transcription through a mechanism that does not require heterochromatin formation.

The Ikaros family of proteins are DNA binding factors required for correct development of B and T lymphocytes. Cytogenetic studies have shown that these proteins form complexes with pericentromeric heterochromatin in B cells, and the colocalization of transcriptionally silent genes with these complexes suggests that Ikaros could silence transcription by recruiting genes to heterochromatin. Here we show that a site in the lambda5 promoter that binds Ikaros and Aiolos is required for silencing of lambda5 expression in activated mature B cells. Analysis of methylation and nuclease accessibility indicates that the silenced lambda5 gene is not heterochromatinized in B cells, despite being associated with pericentromeric heterochromatin clusters. We also found that a promoter mutation, which affects Ikaros-mediated silencing of lambda5 expression, is not rescued in a transgenic line that has the gene integrated into pericentromeric heterochromatin. Our results indicate that the Ikaros proteins initiate silencing of lambda5 expression through a direct effect on the promoter with localization to pericentromeric heterochromatin likely to affect the action of Ikaros on regulatory sequences rather than causing heterochromatinization of the gene.

Animals↗
PubMed2002

Hantaan virus infection causes an acute neurological disease that is fatal in adult laboratory mice.

Hantaan virus, the etiological agent of Korean hemorrhagic fever, is transmitted to humans from persistently infected mice (Apodemus agrarius), which serve as the primary reservoir. Here we demonstrate that several strains of adult Mus musculus domesticus (C57BL/6, BALB/c, AKR/J, and SJL/J) were susceptible to Hantaan virus infection when infected intraperitoneally. First clinical signs were loss of weight, ruffled fur, and reduced activity, which were followed by neurological symptoms, such as paralyses and convulsions. Within 2 days of disease onset, the animals died of acute encephalitis. PCR analysis indicated a systemic infection with viral RNA present in all major organs. Immunohistochemical and in situ hybridization analyses of postmortem material detected viral antigen and RNA in the central nervous system (predominantly brain), liver, and spleen. In the central nervous system, viral antigen and RNA colocalized with perivascular infiltrations, the predominant pathological finding. To investigate the involvement of the interferon system in Hantaan virus pathogenesis, we infected alpha/beta interferon receptor knockout mice. These animals were more susceptible to Hantaan virus infection, indicating an important role of interferon-induced antiviral defense mechanisms in Hantaan virus pathogenesis. The present model may help to overcome shortcomings in the development of therapeutic and prophylactic measurements against hantavirus infections.

Acute Disease↗
PubMed2003

Microtubule-dependent PKC-alpha localization in A7r5 smooth muscle cells.

Using laser scanning confocal, fluorescence resonance energy transfer (FRET), and atomic force (AFM) microscopy, we investigated association of protein kinase C (PKC)-alpha with microtubules during stimulus-induced relocalization in A7r5 smooth muscle cells. Confocal microscopy with standard immunostaining techniques confirmed earlier observations that colchicine disruption of microtubules blocked PKC-alpha localization in the perinuclear region of the cell caused by phorbol 12,13-dibutyrate (PDBu; 10-6M). Dual immunostaining suggested colocalization of PKC-alpha and beta-tubulin in both unstimulated and PDBu-treated cells. This finding was verified by FRET microscopy, which indicated that association of PKC-alpha was heterogeneous in distribution and confined primarily to microtubules in the perinuclear region. FRET analysis further showed that association between the molecules was not lost during colchicine-induced dissolution of microtubules, suggesting formation of tubulin-PKC-alpha complexes in the cytosol. Confocal imaging indicated that perinuclear microtubular structure was more highly sensitive to colchicine dissolution than other regions of the cell. Topographic imaging of fixed cells by AFM indicated a well-defined elevated structure surrounding the nucleus that was absent in colchicine-treated cells. It was calculated that the volume of the nuclear sleevelike structure of microtubules increased approximately fivefold in PDBu-treated cells, suggesting a probable increase in microtubular mass. In light of PKC-alpha localization, increased colchicine sensitivity, and their volume change in stimulated cells, the results suggest that perinuclear microtubules form a specialized structure that may be more dynamically robust than in other regions of the cell. PKC-alpha could contribute to this dynamic activity. Alternatively, perinuclear microtubules could act as a scaffold for regulatory molecule interaction at the cell center.

Animals↗
PubMed1995

Molecular characterization of renal calcium channel beta-subunit transcripts.

An apical, hormone-regulated, calcium entry channel in the distal convoluted tubule and/or connecting tubule (DCT/CNT) is thought to play an important role in controlling renal calcium excretion. We previously identified a gene transcript encoding the pore-forming alpha 1-subunit of a calcium channel (alpha 1A, or CaCh4) which may be a candidate for such a molecule. The properties of voltage-dependent calcium channels are known to be modulated by their beta-subunits. To identify the accessory beta-subunit of DCT/CNT calcium channels, degenerate primers based on published beta-subunit sequences were used to amplify rat kidney cDNA by the polymerase chain reaction (PCR), and the products were subcloned and sequenced. Alternatively spliced transcripts of three beta-subunit genes (beta 2, beta 3, and beta 4) were identified. Northern blot analysis indicated that beta 4-subunit is preferentially expressed in kidney cortex. Transcripts of all three beta-subunit genes were detected by PCR in microdissected nephron segments, but only beta 4-subunit was found in DCT/CNT. As the beta 4- and alpha 1A-subunits colocalize to the DCT/CNT, we hypothesize that they may be constituent subunits of a renal calcium channel regulated by a hormone(s).

Alternative Splicing↗
PubMed2004

Subnuclear distribution of progesterone receptors A and B in normal and malignant endometrium.

The nuclear progesterone receptor (PR), which is expressed as two proteins with different functions (PRA and PRB), is expressed in the normal endometrium and endometrial cancer. Our previous work has shown that there is a disruption to the relative PR isoform expression in progression to malignancy in endometrial cancer in which expression of a single isoform is common. A consistent feature in these studies was discrete punctate distribution of PRA and PRB in the nuclei of endometrial cancer cells. In this study PRA and PRB distribution within the nucleus was examined in vivo in the normal endometrium during the menstrual cycle, and in endometrial cancer, by dual immunofluorescence and confocal microscopy using cohorts in which PRA and PRB expression levels have previously been characterized. In the normal endometrium, PR was distributed evenly within the nucleus and also localized in discrete subnuclear foci. In the proliferative phase, even PR distribution was predominant and both PR isoforms were colocated and distributed evenly. In the secretory phase, there was a marked increase in the proportion of nuclei containing PR distributed into discrete foci, and PRB was the predominant isoform in nuclear foci. There was an inverse relationship between even and focal PR distribution in the menstrual cycle, suggesting that hormonal fluctuations were involved in movement of PR into focal nuclear locations. In endometrial cancers colocalization of PRA and PRB was infrequent, and there was no relationship between even and focal PR isoform distribution, unlike the normal endometrium. PRA was predominantly evenly distributed in endometrial cancers, whereas PRB was focal. Even PRB distribution in endometrial cancer was not often noted. Multivariate analysis showed that PRA expression was highly predictive of even nuclear distribution in endometrial cancers and PRB expression of distribution into foci, and these associations were independent of total PRA and PRB levels. Nuclear distribution of PR isoforms was associated with clinical grade, where tumors of high grade had significantly fewer nuclei containing even PRA distribution and focal PRB distribution, compared with tumors of low grade. In the normal endometrium, localization of PR into nuclear foci coincides with high progesterone levels, suggesting that altered intranuclear PR distribution is hormonally regulated. Nuclear distribution may be an important component of gene regulation in target tissues, and disruptions in PR distribution in endometrial cancer could affect the function of PR and contribute to aberrant hormonal responses.

Adult↗
PubMedDate not supplied

Abundant cysteine-rich protein-1 is localized in the stromal compartment of the human prostate.

The cysteine-rich protein-1 (CRP1) is one of the major proteins of the human prostate. Because of the suspected importance of that protein in cell proliferation and differentiation, its expression was investigated in the prostate, prostatic cancer cells, and other organs of the body. At the mRNA level, the highest concentrations of CRP1 were found in the prostate and the colon followed by the brain and the testis. It was virtually absent from the spleen, liver, heart, and kidney. Prostatic cancer cells PC-3, DU-145, and LNCaP also expressed CRP1 mRNA but virtually no protein. CRP1 protein localization in tissues was determined by immunohistochemical analysis using polyclonal antibodies developed against recombinant CRP1 protein. Strong positive cytoplasmic immunoreactions were observed only in the stromal compartment of the prostate and of other smooth muscle-rich tissues without significant staining in any secretory epithelium. These results, along with previously reported data of colocalization of CRP1 with stress fibers and adhesion plaques, suggest that the main function of CRP1 may be structural.

Carrier Proteins↗
PubMed2000

Expression of leptin receptor (Ob-R) in human atherosclerotic lesions: potential role in intimal neovascularization.

Neovascularization of the adventitial vasa vasorum with extension into the intima of atherosclerotic lesions is frequently observed, but its pathophysiological significance is still subject to debate. Recently, leptin, the product of the Ob gene, was identified. Leptin, via activation of the endothelial receptor (Ob-R), generates a growth signal involving a tyrosine kinase-dependent intracellular pathway and promotes angiogenic processes. We hypothesized that a high concentration of leptin within vasa vasorum and plaque itself, may influence inflammatory and vascular neovascularization coupling with functional upregulation of the vascular endothelial growth factor (VEGF). Microscopic computerized tomography was utilized for the spatial distribution of vasa vasorum and intimal neovascularization from atherosclerotic human coronary arteries. Atherosclerotic coronary arteries showed a dense plexus of microvessels in the adventitia and plaque itself. Microscopic analysis from human atherosclerotic aortas revealed an increase in the intimal thickness with neovascularization. The immunoreactivity for Ob-R, VEGF and matrix metalloproteinase (MMP) increased in atherosclerotic plaque, predominantly in the endothelial lining of the intimal neovessel and macrophages/foam cells. Our observation of a prominent colocalization between Ob-R, VEGF and MMP supports this hypothesis and these factors participate in the neovascularization of atherosclerotic lesions. The present study is the first report on vascular tissue and it opens a promising perspective concerning future investigations of leptin-dependent modulation of atherogenesis and vascular neovascularization under pathophysiolgical conditions.

Adult↗
PubMed1988

Structural integrity of the glycoprotein IIb and IIIa genes in Glanzmann thrombasthenia patients from Israel.

Glanzmann thrombasthenia is an autosomal recessive disorder of the platelet glycoproteins (GP) IIb and IIIa. These glycoproteins normally serve as receptors for other adhesive glycoproteins, including fibrinogen, von Willebrand factor, and fibronectin. Most patients affected by Glanzmann thrombasthenia have low levels of GPIIb and GPIIIa; however, the separate mechanisms responsible for the deficiency in each remain to be determined. cDNA clones coding for the GPIIb and GPIIIa have been recently isolated, and their corresponding genomic sequences have been colocalized to the long arm of chromosome 17. Since a deletional event involving one or both of these structural genes could explain the disease phenotype, we have studied the DNA of two previously well-characterized cohorts of Glanzmann thrombasthenia patients from Israel. We performed Southern analysis with near full-length cDNA probes on genomic DNA obtained from 20 individuals. Four restriction enzyme digests were completed on each DNA sample. The similarity of banding patterns among probands, family members, and controls indicated that there were no major insertions or deletions in either the GPIIb or GPIIIa genes. Thus, the genetic defect in these patients with Glanzmann thrombasthenia is most likely due to either a small change in the nucleotide sequence of the coding region or a defect in the regulatory region of one or both genes.

Blood Platelet Disorders↗
PubMed2004

MM-TRAG (MGC4175), a novel intracellular mitochondrial protein, is associated with the taxol- and doxorubicin-resistant phenotype in human cancer cell lines.

In the search for novel genes involved in the taxol resistance phenotype, prior studies of gene expression in taxol-resistant cell lines and their paired drug-sensitive parental lines using high-density Affymetrix microarrays identified MGC4175 as an overexpressed transcript. In this report, we characterize MGC4175 and demonstrate that seven of eight taxol- and doxorubicin-resistant cell lines overexpressed MGC4175 as compared to their chemotherapy naive parent lines. Sequence analyses of MGC4175 cDNA and the predicted amino acid sequence did not show significant homology to any known sequence or protein domain, with an open reading frame of 356 bp that is predicted to encode a protein product of 118 amino acids. Both the MGC4175 and MDR1 genes are located at chromosome position 7q21. Southern blot analysis demonstrated that a single copy of MGC4175 is present in the human genome, and MGC4175 overexpression is not caused by genomic amplification or gene arrangement. Human MGC4175 fused to the carboxy terminus of enhanced green fluorescent protein (EGFP) and expressed in U-2OS cells localized the protein to the perinuclear region with further studies colocalizing this protein to the mitochondria. The Cancer Profiling Arrays and the Cancer Cell Line Profiling Array demonstrated that MGC4175 is broadly expressed in various tissues with no significant difference of MGC4175 expression between chemotherapy naive tumor cells and normal cells. However, MGC4175 is overexpressed 1.2- to 12.3-fold after 48 h of taxol induction and 0.65- to 6.5-fold after doxorubicin induction in various human cancer cell lines. In light of the overexpression of MGC4175 in association with taxol exposure, drug resistance, the coexpression of MDR1 and the mitochondrial localization of its protein, we propose to name this transcript MDR1 and Mitochondrial Taxol Resistance Associated Gene (MM-TRAG) and suggest that MM-TRAG may play a role in the development of taxol resistance in human cancer.

Blotting, Northern↗
PubMed1996

Glutamate immunoreactivity in the tiger salamander retina differentiates between GABA-immunoreactive and glycine-immunoreactive amacrine cells.

A higher proportion of retinal amacrine cells, largely thought to be inhibitory, in tiger salamander retina are immunoreactive (+) for the excitatory amino acid glutamate than for either of the inhibitory amino acids, GABA and glycine. The colocalizations of glutamate immunoreactivity, GABA immunoreactivity and glycine immunoreactivity were studied in the proximal retina of tiger salamander using postembedding immunocytochemical methods in consecutive semithin sections. A considerable degree of colocalization was observed between glutamate immunoreactivity and GABA immunoreactivity and to a lesser extent with glycine immunoreactivity. No glutamate-immunoreactive cells were single labelled in INL3 (proximal inner nuclear), 12% were single labelled in INL2 (mid INL; these are bipolar cells), 62% were singled labelled in the ganglion cell layer (GCL) and likely to be ganglion cells. Single-labelled glycine-immunoreactive cells comprised 16% in INL3, 2%; INL2 and GCL, 0%). Glutamate immunoreactivity co-localized four times as often with GABA immunoreactivity than glycine immunoreactivity in INL3 (77% versus 19%), comparably in INL2 (37% and 32%) and six times as often in the GCL (29% versus 5%). A computerized image analysis of the relative intensities of glutamate immunoreactivity in GABA-immunoreactive/glutamate-immunoreactive and glycine-immunoreactive/glutamate-immunoreactive double labelled amacrine cells in INL3 revealed that all glycine-immunoreactive cells contained low intensities of glutamate immunoreactivity; whereas 35% of GABA-positive cells had medium to high intensities of glutamate immunoreactivity. There was a significant correlation (r = 0.44; p < 0.01) between the intensities of glutamate immunoreactivity and GABA immunoreactivity in GABA-immunoreactive/glutamate-immunoreactive amacrine cells. The common colocalization of glutamate immunoreactivity with GABA immunoreactivity and the positive correlation of their relative intensities suggests that glutamate is likely to serve as a precursor for GABA synthesis. However, intense glutamate immunoreactivity in 5% of the amacrine cells suggests a transmitter function. Glycine-immunoreactive amacrine cells contained little or no glutamate immunoreactivity, suggesting a minimal role for glutamate as a neurotransmitter in these cells. The major distribution of intense glutamate immunoreactivity is in bipolar cells and ganglion cells, consistent with data suggesting that glutamate is the transmitter in the retinal through pathway.

Animals↗
PubMed2002

GABA-, glycine-, and glutamate-immunoreactive bouton profiles in apposition to neurons of the central cervical nucleus in the rat.

The neurons of the central cervical nucleus (CCN) convey information about the position and movements of the head, and receive excitatory input from dorsal neck muscles and the labyrinth. Both of these afferent sources form glutamatergic synaptic contacts with CCN neurons. However, these sensory afferent sources can also inhibit CCN neurons. To further elucidate the synaptic organization, we made an electron microscopic investigation, identifying and evaluating the relative frequency of bouton profiles containing the inhibitory transmitters GABA and glycine in apposition to identified CCN neurons. In addition, labeling for glutamate was performed. The identification of the CCN neurons was made possible by injections of retrograde tracer substances into the cerebellum. These substances were made visible by preembedding immunocytochemistry or postembedding immunogold staining. Such staining was also used to detect the three amino acids that were found in boutons apposed to the identified neurons (cf. Ornung et al., J. Comp. Neurol. 1996;365:413-426; Lindå et al., J. Comp. Neurol. 2000;425:10-23). Due to the relatively poor transport of the tracer substances into dendrites of the CCN neurons, the analysis was restricted to the cell body and included bouton profiles in direct apposition to the soma membrane. Data from 10 CCN neurons revealed that about 50% of the apposing bouton profiles were immunoreactive for GABA, and about 34% for glycine. In four neurons, the degree of colocalization of GABA and glycine was determined to be close to 30%. Thus, the vast majority of glycine-labeled profiles also contained GABA, while a considerable fraction of the profiles were immunoreactive for only GABA. The values for glycine immunoreactive bouton profiles presented here may represent somewhat low estimates, depending on the method used. Data from four neurons showed that about 18% of the profiles were labeled for glutamate. The large fraction of purely GABA immunoreactive profiles, or at least a substantial group of them, is suggestive of their derivation from axons descending from the brainstem.

Animals↗
PubMed2002

Synergistic induction of HSP40 and HSC70 in the mouse hippocampal neurons after cerebral ischemia and ischemic tolerance in gerbil hippocampus.

An ischemia-induced gene was screened using a differential display technique in mouse transient forebrain ischemia. One of the ischemia-responsive clones was found to encode mouse hsp40. HSP40 has a critical regulatory function in the HSC70 ATPase activity. Expression of hsp40 mRNA was low in the nonischemic mouse hippocampus, but it was significantly upregulated 4 hr after ischemia by Northern blot analysis. In situ hybridization analysis revealed hsp40 mRNA induction in the neuron. HSP40 protein expression was also enhanced in the pyramidal and dentate granular neurons from 2 to 4 days after ischemia. The temporal expression and distribution profile of HSC70 protein was similar to that of HSP40, and both proteins were colocalized in ischemic hippocampal neurons. In the gerbil transient forebrain ischemia model, both HSP40 and HSC70 proteins were expressed strongly in ischemia-resistant CA3 neurons and dentate granule cells 1 day after 5 min ischemia, but were not expressed in vulnerable CA1 neurons. However, both proteins were in parallel expressed in the tolerance-acquired CA1 neurons. Based on the current observation that both HSP40 and HSC70 proteins were synergistically expressed in the ischemia-resistant and tolerance-acquired neurons, cochaperone HSP40 may play a significant role against postischemic neuronal response and lead to cell survival through interaction with simultaneously induced HSC70.

Animals↗
PubMed1995

Well-differentiated endocrine tumours of the middle ear and of the hindgut have immunocytochemical and ultrastructural features in common.

The immunocytochemical analysis of two cases of well-differentiated endocrine tumours (carcinoids) of the middle ear revealed predominant cell populations producing pancreatic polypeptide (PP)-related peptides, glucagon-related peptides, and serotonin (the latter only in one case). In consecutive sections PP- and glucagon-related immunoreactivities mainly colocalized in the same tumour cells. Ultrastructurally tumour cells were characterized by medium-sized to large granules of moderate to high density, on which PP and glicentin were localized by the immunogold technique. No amphicrine cells were found. These features are consistent with those of similar tumours in the rectal mucosa that are mainly composed of L cells coexpressing both PP-related and glucagon-related peptides. Additional tumour antigens of hindgut type detected immunohistochemically were prostatic acid phosphatase and CAR-5 mucin. Expression of the CAR-5 antigen was also found in samples of normal middle ear mucosa, in which endocrine cells have not been identified. In case 1 peritumoral mucosal invaginations showed a proliferation of endocrine cells identical immunophenotypically to tumour cells, possibly representing a precursor lesion. It is concluded that well-differentiated endocrine tumours of the middle ear are a distinct pathological entity characterized by multiple hormone production, typically involving three classes of hormones (pancreatic polypeptide-related peptides, glucagon-related peptides, and serotonin) of the hindgut endocrine system.

Adult↗
PubMed1999

Immuno-localization of sulphonylurea receptor 1 in rat pancreas.

AIMS/HYPOTHESIS: A sulphonylurea receptor, SUR1, and an inward rectifier potassium channel, Kir6.2, reconstitute the ATP-sensitive K(+) channel that mediates glucose-induced insulin secretion in pancreatic beta cells. We reported previously that Kir6.2 were localized at insulin-, glucagon-, and somatostatin-producing cells. In this new study we aimed to determine the distribution of SUR1 in rat pancreatic islets and to suggest the location of the ATP-sensitive K(+) channels in the islet. METHODS: Western blot analysis was carried out using two anti-SUR1 antibodies, which had been raised against different portions of rat SUR1. SUR1, Kir 6.2, and islet hormones were then localized by indirect immunofluorescence staining of the cryosections of rat pancreas. RESULTS: In Western blot analysis, each of the anti-SUR1 antibodies detected a band at 140 kDa, which is close to the predicted molecular weight of SUR1, in the homogenate of isolated pancreatic islets. Double immunofluorescence staining of cryosections showed that SUR1 occurred all over the islets, and that SUR1 colocalized with insulin, glucagon, somatostatin, and pancreatic polypeptide. Kir6.2 was also shown to be present in pancreatic polypeptide cells. CONCLUSION/INTERPRETATION: Together with our previously reported data, the above findings indicate that K(ATP) channels comprising SUR1 and Kir6.2 occur not only in beta cells but also in the alpha, delta, and pancreatic polypeptide cells of the pancreatic islets, suggesting that therapeutic sulphonylureas could act on these cells directly. [Diabetologia (1999) 42: 1204-1211]

ATP-Binding Cassette Transporters↗