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PubMed2006

Cellular and subcellular localization of paralemmin-1, a protein involved in cell shape control, in the rat brain, adrenal gland and kidney.

Paralemmin-1 is a phosphoprotein, lipid-anchored to the cytoplasmic face of membranes and implicated in plasma membrane dynamics and cell process formation. We report an immunoperoxidase histochemical analysis of the cellular and subcellular localization of paralemmin-1 in the rat tissues where its expression is highest: the brain, the adrenal gland and the kidney. Paralemmin-1 is detected throughout the brain, in neuronal perikarya, axons and dendrites including dendritic spines and also in glial processes. In the adrenal gland, paralemmin-1 is highly expressed in the medulla. The kidney displays a pattern of differential paralemmin-1 expression in various structures and cell types, with high concentrations in cells of the parietal epithelium of Bowman's capsule, intermediate tubules, distal tubules and principal cells of outer medullary collecting ducts. Mosaics of paralemmin-positive and paralemmin-negative cells are observed in proximal tubules, the parietal epithelium of Bowman's capsule and the endothelium of many blood vessels. Plasma membrane association in epithelia is often polarized: paralemmin-1 concentrates at the apical membranes of adrenal chromaffin cells, but at the basolateral plasma membranes of proximal and distal tubule cells in the kidney. Paralemmin-1 immunoreactivity exhibits a spotted pattern and can be seen both at plasma membranes and within the cytoplasm, where it is often associated with endomembranes. This discontinuous distribution and the detergent extraction properties of paralemmin-1 suggest an association with lipid microdomains. The findings are consistent with a role for paralemmin-1 in the formation and stabilization of plasma membrane elaborations, in neurons as well as in other cell types.

Adrenal Glands↗

PubMed1990

Cellular oxidation of lignoceric acid is regulated by the subcellular localization of lignoceroyl-CoA ligases.

The acyl-CoA ligases convert free fatty acids to acyl-CoA derivatives, and these enzymes have been shown to be present in mitochondria, peroxisomes, and endoplasmic reticulum. Because their activity is obligatory for fatty acid metabolism, it is important to identify their substrate specificities and subcellular distributions to further understand the cellular regulation of these pathways. To define the role of the enzymes and organelles involved in the metabolism of very long chain (VLC) fatty acids, we studied human genetic cell mutants impaired for the metabolism of these molecules. Fibroblast cell lines were derived from patients with X-linked adrenoleukodystrophy (X-ALD) and Zellweger's cerebro-hepato-renal syndrome (CHRS). While peroxisomes are present and morphologically normal in X-ALD, they are either greatly reduced in number or absent in CHRS. Palmitoyl-CoA ligase is known to be present in mitochondria, peroxisomes, and endoplasmic reticulum (microsomes). We found enzyme-dependent formation of lignoceroyl-CoA in these same organelles (specific activities were 0.32 +/- 0.12, 0.86 +/- 0.12, and 0.78 +/- 0.07 nmol/h per mg protein, respectively). However, lignoceroyl-CoA synthesis was inhibited by an antibody to palmitoyl-CoA ligase in isolated mitochondria while it was not inhibited in peroxisomes or endoplasmic reticulum (ER). This suggests that palmitoyl-CoA ligase and lignoceroyl-CoA are different enzymes and that mitochondria lack lignoceroyl-CoA ligase. This conclusion is further supported by data showing that oxidation of lignoceric acid was found almost exclusively in peroxisomes (0.17 nmol/h per mg protein) but was largely absent from mitochondria and the finding that monolayers of CHRS fibroblasts lacking peroxisomes showed a pronounced deficiency in lignoceric acid oxidation in situ (1.8% of control). In spite of the observation that lignoceroyl-CoA ligase activity is present on the cytoplasmic surface of ER, our data indicate that lignoceroyl-CoA synthesized by ER is not available for oxidation in mitochondria. This organelle plays no physiological role in the beta-oxidation of VLC fatty acids. Furthermore, the normal peroxisomal oxidation of lignoceroyl-CoA but deficient oxidation of lignoceric acid in X-ALD cells indicates that cellular VLC fatty acid oxidation is dependent on peroxisomal lignoceroyl-CoA ligase. These studies allow us to propose a model for the subcellular localization of various acyl-CoA ligases and to describe how these enzymes control cellular fatty acid metabolism.

Adrenoleukodystrophy↗

PubMed1984

Subcellular localization of the v-erb-B protein, the product of a transforming gene of avian erythroblastosis virus.

Avian erythroblastosis virus (AEV) is an oncogenic retrovirus capable of transforming both fibroblasts and immature erythroid cells. The v-erb-B locus within the AEV genome encodes a glycosylated protein, expression of which is required for oncogenic transformation of either cell type. Subcellular localization of the v-erb-B glycoprotein in AEV-transformed cells is reported here. Results indicate that the v-erb-B protein is synthesized on dense membrane fractions and appears to possess the properties of an integral membrane protein. The bulk of the v-erb-B protein remains with dense membranes after synthesis, although a small quantity may slowly become associated with the plasma membrane. The biogenesis and subcellular location of the v-erb-B protein are thus quite different from those of the transforming proteins that display protein kinase activity. These differences are especially provocative because the amino acid sequences of the v-erb-B protein and the protein kinases are closely related to one another.

Alpharetrovirus↗

PubMed2002

Marek's disease virus VP22: subcellular localization and characterization of carboxyl terminal deletion Mutations.

Marek's disease virus (MDV) is an alphaherpesvirus that causes T cell lymphoma and severe immunosuppression in chickens. The MDV UL49 gene, which encodes the tegument viral protein 22 (VP22), has been expressed as a green fluorescent protein (GFP) fusion protein in chicken embryonic fibroblasts to examine its subcellular localization. As with both human herpesvirus 1 and bovine herpesvirus 1VP22-GFP fusion proteins, the MDV VP22-GFP product binds to microtubules and heterochromatin. In addition, the MDV protein also binds to the centrosomes. During mitosis, VP22-GFP binds to sister chromatids, but dissociates from the centrosomes and the microtubules of the mitotic spindle. A series of VP22 carboxy terminal truncation mutants were constructed to define regions responsible for these binding properties. These mutants identified separable domains or motifs responsible for binding microtubules and heterochromatin.

Amino Acid Sequence↗

PubMed2000

Subcellular localization of cyclic ADP-ribosyl cyclase and cyclic ADP-ribose hydrolase activities in porcine airway smooth muscle.

Recent studies have provided evidence for a role of cyclic ADP-ribose (cADPR) in the regulation of intracellular calcium in smooth muscles of the intestine, blood vessels and airways. We investigated the presence and subcellular localization of ADP-ribosyl cyclase, the enzyme that catalyzes the conversion of beta-NAD(+) to cADPR, and cADPR hydrolase, the enzyme that degrades cADPR to ADPR, in tracheal smooth muscle (TSM). Sucrose density fractionation of TSM crude membranes provided evidence that ADP-ribosyl cyclase and cADPR hydrolase activities were associated with a fraction enriched in 5'-nucleotidase activity, a plasma membrane marker enzyme, but not in a fraction enriched in either sarcoplasmic endoplasmic reticulum calcium ATPase or ryanodine receptor channels, both sarcoplasmic reticulum markers. The ADP-ribosyl cyclase and cADPR hydrolase activities comigrated at a molecular weight of approximately 40 kDa on SDS-PAGE. This comigration was confirmed by gel filtration chromatography. Investigation of kinetics yielded K(m) values of 30.4+/-1.5 and 695. 3+/-171.2 microM and V(max) values of 330.4+/-90 and 102.8+/-17.1 nmol/mg/h for ADP-ribosyl cyclase and cADPR hydrolase, respectively. These results suggest a possible role for cADPR as an endogenous modulator of [Ca(2+)](i) in porcine TSM cells.

ADP-ribosyl Cyclase↗

PubMed2005

Subcellular localization and functional expression of the glycerol uptake protein 1 (GUP1) of Saccharomyces cerevisiae tagged with green fluorescent protein.

GFP (green fluorescent protein) from Aequorea victoria was used as an in vivo reporter protein when fused to the N- and C-termini of the glycerol uptake protein 1 (Gup1p) of Saccharomyces cerevisiae. The subcellular localization and functional expression of biologically active Gup1-GFP chimaeras was monitored by confocal laser scanning and electron microscopy, thus supplying the first study of GUP1 dynamics in live yeast cells. The Gup1p tagged with GFP is a functional glycerol transporter localized at the plasma membrane and endoplasmic reticulum levels of induced cells. The factors involved in proper localization and turnover of Gup1p were revealed by expression of the Gup1p-GFP fusion protein in a set of strains bearing mutations in specific steps of the secretory and endocytic pathways. The chimaerical protein was targeted to the plasma membrane through a Sec6-dependent process; on treatment with glucose, it was endocytosed through END3 and targeted for degradation in the vacuole. Gup1p belongs to the list of yeast proteins rapidly down-regulated by changing the carbon source in the culture medium, in agreement with the concept that post-translational modifications triggered by glucose affect proteins of peripheral functions. The immunoelectron microscopy assays of cells expressing either Gup1-GFP or GFP-Gup1 fusions suggested the Gup1p membrane topology: the N-terminus lies in the periplasmic space, whereas its C-terminal tail has an intracellular location. An extra cytosolic location of the N-terminal tail is not generally predicted or determined in yeast membrane transporters.

Carrier Proteins↗

PubMed2006

Association between Pak1 expression and subcellular localization and tamoxifen resistance in breast cancer patients.

BACKGROUND: p21-activated kinase 1 (Pak1) phosphorylates many proteins in both normal and transformed cells. Its ability to phosphorylate and thereby activate the estrogen receptor alpha (ERalpha) potentially limits the effectiveness of antiestrogen treatment in breast cancer. Here we studied associations between Pak1 expression and subcellular localization in tumor cells and tamoxifen resistance. METHODS: Pak1 protein expression was evaluated in 403 primary breast tumors from premenopausal patients who had been randomly assigned to 2 years of adjuvant tamoxifen or no treatment. Tamoxifen response was evaluated by comparing recurrence-free survival in relation to Pak1 and ERalpha expression in untreated versus tamoxifen-treated patients. Tamoxifen responsiveness of human MCF-7 breast cancer cells that inducibly expressed constitutively active Pak1 or that transiently overexpressed wild-type Pak1 (Wt-Pak1) or Pak1 that lacked functional nuclear localization signals (Pak1DeltaNLS) was evaluated by analyzing cyclin D1 promoter activation and protein levels as markers for ERalpha activation. The response to tamoxifen in relation to Pak1 expression was analyzed in naturally tamoxifen-resistant Ishikawa human endometrial cancer cells. All statistical tests were two-sided. RESULTS: Among patients who had ERalpha-positive tumors with low Pak1 expression, those treated with tamoxifen had better recurrence-free survival than those who received no treatment (hazard ratio [HR] = 0.502, 95% confidence interval [CI] = 0.331 to 0.762; P = .001) whereas there was no difference in recurrence-free survival between treatment groups for patients whose tumors had high cytoplasmic (HR = 0.893, 95% CI = 0.420 to 1.901; P = .769) or any nuclear Pak1 expression (HR = 0.955, 95% CI = 0.405 to 2.250; P = .916). In MCF-7 cells, overexpression of Wt-Pak1, but not of Pak1DeltaNLS, compromised tamoxifen response by stimulating cyclin D1 expression. Treatment of Ishikawa cells with tamoxifen led to an increase in the amount of nuclear Pak1 and Pak1 kinase activity, suggesting that tamoxifen, to some extent, regulates Pak1 expression. CONCLUSIONS: Our data support a role for Pak1, particular Pak1 localized to the nucleus, in ERalpha signaling and in tamoxifen resistance.

Adult↗

PubMed1998

Activation of the proprotein transcription factor pro-sigmaE is associated with its progression through three patterns of subcellular localization during sporulation in Bacillus subtilis.

The activity of the sporulation transcription factor sigmaE in Bacillus subtilis is governed by an intercellular signal transduction pathway that controls the conversion of the inactive proprotein pro-sigmaE to the mature and active form of the factor. Here I use immunofluorescence microscopy to show that the activation of the proprotein is associated with its progression through three patterns of subcellular localization. In the predivisional sporangium, pro-sigmaE was found to be associated with the cytoplasmic membrane. Next, at the stage of asymmetric division, pro-sigmaE accumulated at the sporulation septum. Finally, after processing, mature sigmaE was found to be distributed throughout the mother cell cytoplasm. The results of subcellular fractionation and sedimentation in density gradients of extracts prepared from postdivisional sporangia confirmed that pro-sigmaE was chiefly present in the membrane fraction and that sigmaE was predominantly cytoplasmic, findings that suggest that the pro-amino acid sequence is responsible for the sequestration of pro-sigmaE to the membrane. The results of chemical cross-linking experiments showed that pro-sigmaE was present in a complex with its putative processing protein, SpoIIGA, or with a protein that depended on SpoIIGA. The membrane association of pro-sigmaE was, however, independent of SpoIIGA and other proteins specific to B. subtilis. Likewise, accumulation of pro-sigmaE at the septum did not depend on its interaction with SpoIIGA. Sequestration of pro-sigmaE to the membrane might serve to facilitate its interaction with SpoIIGA and may be important for preventing its premature association with core RNA polymerase. The implications of these findings for the compartmentalization of sigmaE are discussed.

Bacillus subtilis↗

PubMed2006

Mutual regulation of c-Jun and ATF2 by transcriptional activation and subcellular localization.

ATF2 and c-Jun are key components of activating protein-1 and function as homodimers or heterodimers. c-Jun-ATF2 heterodimers activate the expression of many target genes, including c-jun, in response to a variety of cellular and environmental signals. Although it has been believed that c-Jun and ATF2 are constitutively localized in the nucleus, where they are phosphorylated and activated by mitogen-activated protein kinases, the molecular mechanisms underlying the regulation of their transcriptional activities remain to be defined. Here we show that ATF2 possesses a nuclear export signal in its leucine zipper region and two nuclear localization signals in its basic region, resulting in continuous shuttling between the cytoplasm and the nucleus. Dimerization with c-Jun in the nucleus prevents the export of ATF2 and is essential for the transcriptional activation of the c-jun promoter. Importantly, c-Jun-dependent nuclear localization of ATF2 occurs during retinoic acid-induced differentiation and UV-induced cell death in F9 cells. Together, these findings demonstrate that ATF2 and c-Jun mutually regulate each other by altering the dynamics of subcellular localization and by positively impacting transcriptional activity.

Activating Transcription Factor 2↗

PubMed1994

Rapid effects of aldosterone on free intracellular calcium in vascular smooth muscle and endothelial cells: subcellular localization of calcium elevations by single cell imaging.

Rapid in vitro effects of aldosterone on the intracellular concentrations of sodium, potassium and calcium, cell volume and the sodium-proton-antiport have been described in human mononuclear leukocytes and vascular smooth muscle cells (VSMC). These nongenomic effects are signalled through membrane receptors with a high affinity for aldosterone, but not for cortisol, and through the phosphoinositide pathway. In the present study, we demonstrate that free intracellular calcium is increased rapidly by aldosterone in VSMC and endothelial cells (EC) as determined by single cell imaging of Fura2-fluorescence. In VSMC, calcium elevation is localized to the perinuclear region whereas in EC, a predominant increase of subplasmalemmal calcium is seen. In VSMC, effects are half maximal at 0.1 nM aldosterone; cortisol is inactive up to 0.1 microM. These data show that intracellular signalling for aldosterone also involves calcium, but the subcellular localization of this signal varies between cell types.

Aldosterone↗

PubMed2001

Dynamic changes in subcellular localization of mineralocorticoid receptor in living cells: in comparison with glucocorticoid receptor using dual-color labeling with green fluorescent protein spectral variants.

Mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) are ligand-dependent transcription factors. Although it is generally accepted that GR is translocated into the nucleus from the cytoplasm only after ligand binding, the subcellular localization of MR is still quite controversial. We examined the intracellular trafficking of MR in living neurons and nonneural cells using a fusion protein of green fluorescent protein (GFP) and rat MR (GFP-MR). Corticosterone (CORT) induced a rapid nuclear accumulation of GFP-MR, whereas in the absence of ligand, GFP-MR was distributed in both cytoplasm and nucleus in the majority of transfected cells. Given the differential action of MR and GR in the central nervous system, it is important to elucidate how the trafficking of these receptors between cytoplasm and nucleus is regulated by ligand. To examine the simultaneous trafficking of MR and GR within single living cells, we use different spectral variants of GFP, yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP), linked to MR and GR, respectively. In COS-1 cells, expressing no endogenous corticosteroid receptors, the YFP-MR chimera was accumulated in the nucleus faster than the CFP-GR chimera in the presence of 10(-9) M CORT, while there was no significant difference in the nuclear accumulation rates in the presence of 10(-6) M CORT. On the other hand, in primary cultured hippocampal neurons expressing endogenous receptors, the nuclear accumulation rates of the YFP-MR chimera and CFP-GR chimera were nearly the same in the presence of both concentrations of CORT. These results suggest that CORT-induced nuclear translocation of MR and GR exhibits differential patterns depending on ligand concentrations or cell types.

Animals↗

PubMed1997

Endogenous fibronectin of blood polymorphonuclear leukocytes: immunochemical characterization and subcellular localization.

Fibronectin, a large dimeric glycoprotein synthesized and secreted by several cell types, mediates cell adherence to surfaces. In infections and inflammatory responses, blood polymorphonuclear leukocytes (PMNs) adhere to cells and matrix proteins during extravasation and accumulation at inflammatory sites. The presence of fibronectin in blood PMNs has been poorly studied, and the characteristics and subcellular localization of this endogenous adhesive molecule are practically unknown. By immunofluorescence flow cytometry, purified rabbit antibodies and a monoclonal antibody to plasma fibronectin reacted with isolated blood PMNs, only after permeabilization of the cells. By Western blot analysis, the antibodies recognized, under reducing conditions, a protein with an apparent molecular mass of 230 kDa in the cell lysate. Eleven monoclonal antibodies to common frame fibronectin epitopes, including the RGD-containing cell-binding domain, also reacted with PMN fibronectin by Western blotting. In contrast, two antibodies to ED-A, the alternatively spliced region characteristic of "cellular" fibronectin, were unreactive, but recognized platelet fibronectin. On average, 1 million PMNs contained 6.8 ng +/- 1.4 (SD) of fibronectin, as measured by sandwich ELISA. Immunogold labeling and electron microscopy studies indicated localization of most fibronectin in PMN granules. Moreover, double-immunofluorescence and digital image analysis demonstrated colocalization of fibronectin with lactoferrin, a marker of specific (secondary) granules. The results indicate that blood PMNs contain approximately 8000 molecules per cell of intact ED-A-negative fibronectin localized mainly in their specific granules.

Antibodies, Monoclonal↗

PubMed2004

Distribution and subcellular localization of a water-soluble hematoporphyrin-platinum(II) complex in human bladder cancer cells.

The water-soluble porphyrin-platinum complex diammine[7,12-bis[1-(polyethyleneglycol-750-monomethylether-1-yl)ethyl]-3,8,13,17-tetramethylporphyrin-2,18-dipropionato]platinum(II) (PEG-HPPt) was studied with respect to cellular accumulation, subcellular localization, behavior in 3D-cell aggregates and degree of DNA platination on the low-differentiated J82 cells, a model of invasive bladder cancer, and UROtsa, a normal urothelial cell line. Accumulation studies with 2D and spheroid cell cultures revealed that the concentration of PEG-HPPt was 1.7-times higher in J82 cancer cells than in UROtsa cells. Despite its high molecular weight, penetration of PEG-HPPt was not restricted to the peripheral cells of the spheroids. Fluorescence microscopic analysis showed that PEG-HPPt was localized in essential cellular targets of photodynamic therapy. DNA platination in J82 and UROtsa cells was higher by PEG-HPPt than by cisplatin, whereas there was no significant difference between the two cell lines.

Antineoplastic Agents↗

PubMed2001

Two subcellular localizations of eIF3 p170 and its interaction with membrane-bound microfilaments: implications for alternative functions of p170.

We previously identified a 170-kDa protein (p170) highly expressed in lung cancers as the major subunit of the eukaryotic translation initiation factor 3 (eIF3). p170 was recently cloned and little is known concerning its characteristics and subcellular localization. In this paper, we report our surprising findings that about 20% of p170 is associated with membranes while the remaining portion is located in the cytoplasm presumably in the eIF3 complex. We also find that p170 interacts with both endoplasmic reticulum and plasma membranes. The binding of p170 to membranes is through actin filaments, consistent with the fact that p170 contains a spectrin repeat motif that may be involved in actin binding. Furthermore, the cytoplasmic p170 is phosphorylated at serine and threonine residues and the phosphorylation is stimulated by serum. However, the membrane-actin-bound p170 is not phosphorylated. The results obtained in this study suggest that p170 may have other functions in addition to participating in translation initiation. Phosphorylation may play an important regulatory role in the function of p170 in translation initiation and other alternative functions.

ATP Binding Cassette Transporter, Subfamily B↗

PubMed2006

Cellular and subcellular localization of neurokinin-1 and neurokinin-3 receptors in primate globus pallidus.

The primate globus pallidus receives massive innervations from GABAergic striatal neurons that co-release the neuropeptide substance P (SP). To expand our knowledge regarding SP interaction at pallidal level, we used single and double antigen retrieval methods to study the cellular and subcellular localization of SP and its high-affinity receptors neurokinin-1 (NK-1R) and neurokinin-3 (NK-3R) in the globus pallidus of the squirrel monkey (Saimiri sciureus). At the light microscopic level, a large number of neurons and fibers located in both the external (GPe) and internal (GPi) segments of the globus pallidus expressed NK-1R or NK-3R immunoreactivity. At the electron microscopic level, both NK-1R and NK-3R were mainly associated with intracellular sites or located at extrasynaptic positions on the plasma membrane. Presynaptic axon terminals forming symmetric and asymmetric synapses occasionally contained NK-1R and NK-3R. Neurokinin receptors were also observed in a proportion of SP-immunoreactive axon terminals, but these terminals preferentially expressed NK-3R. The pattern of distribution of NK-1R and NK-3R in GPe and GPi indicates that SP effects at pallidal level are mediated through postsynaptic receptor as well as presynaptic autoreceptors and heteroreceptors. These morphological data suggest that, either alone or in conjunction with GABA, SP could have a wide range of effects at pallidal level. This neuroactive peptide may influence in a significant manner the integration and treatment of neural information that flows through the basal ganglia.

Animals↗

PubMed2002

Active nuclear import and export pathways regulate E2F-5 subcellular localization.

Epidermal keratinocyte differentiation is accompanied by differential regulation of E2F genes, including up-regulation of E2F-5 and its concomitant association with the retinoblastoma family protein p130. This complex appears to play a role in irreversible withdrawal from the cell cycle in differentiating keratinocytes. We now report that keratinocyte differentiation is also accompanied by changes in E2F-5 subcellular localization, from the cytoplasm to the nucleus. To define the molecular determinants of E2F-5 nuclear import, we tested its ability to enter the nucleus in import assays in vitro using digitonin-permeabilized cells. We found that E2F-5 enters the nucleus through mediated transport processes that involve formation of nuclear pore complexes. It has been proposed that E2F-4 and E2F-5, which lack defined nuclear localization signal (NLS) consensus sequences, enter the nucleus in association with NLS-containing DP-2 or pRB family proteins. However, we show that nuclear import of E2F-5 only requires the first N-terminal 56 amino acid residues and is not dependent on interaction with DP or pRB family proteins. Because E2F-5 is predominantly cytoplasmic in undifferentiated keratinocytes and in other intact cells, we also examined whether this protein is subjected to active nuclear export. Indeed, E2F-5 is exported from the nucleus through leptomycin B-sensitive, CRM1-mediated transport, through a region corresponding to amino acid residues 130-154. This region excludes the DNA- and the p130-binding domains. Thus, the subcellular distribution of E2F-5 is tightly regulated in intact cells, through multiple functional domains that direct nucleocytoplasmic shuttling of this protein.

Active Transport, Cell Nucleus↗

PubMed1988

Dipeptidyl peptidase IV--subcellular localization, activity and kinetics in lymphocytes from control subjects, immunodeficient patients and cord blood.

Dipeptidyl peptidase IV (DPIV), a T cell enzyme, has been implicated in the regulation of lymphocyte proliferation in response to lectins and allogeneic cells. A sensitive fluorimetric assay has been established for the enzyme and used to investigate DPIV activity, kinetics and the subcellular localization in lymphocytes from control subjects, cord blood and patients with common variable hypogammaglobulinaemia (CVH) and chronic lymphatic leukaemia (CLL). Using sucrose density gradient centrifugation and organelle marker enzyme assays, in conjunction with digitonin as a selective plasma membrane perturbant and diazotized sulphanilic acid as a non-permeant enzyme inhibitor, DPIV was shown to be a plasma membrane ecto-enzyme. A significant decrease in lymphocyte DPIV activity was observed in cord blood and in patients with CVH and CLL compared to controls. Kinetic analysis showed a marked decrease in the Vmax of lymphocyte DPIV from cord blood and patients with CVH and CLL compared to controls. The apparent Km for the substrate was unaffected in cord blood and patients with CLL. However, in patients with CVH the Km was significantly reduced. Various enzyme inhibitors showed no differences between control subjects and CVH lymphocyte activities. The decreased Km for DPIV provides further evidence for a stem cell defect rather than cell immaturity in CVH.

Agammaglobulinemia↗

PubMedDate not supplied

Subcellular localization of neuronal nitric oxide synthase in turtle retina: electron immunocytochemistry.

Recent studies imaging nitric oxide (NO) production in the retina have indicated a much wider distribution of NO production than would be suggested by previous light-microscopic localizations of neuronal nitric oxide synthase (nNOS). To help resolve this discrepancy, the present study analyzed the ultrastructural localization of nNOS-like immunoreactivity (-LI) in all layers of the retina. In the ellipsoids of rod photoreceptors and the accessory elements of double cones, nNOS-LI was associated with some atypical mitochondria. In the outer plexiform layer, nNOS-LI was in some postsynaptic horizontal and bipolar cell processes at photoreceptor ribbon synapses. In some amacrine and ganglion cell somata, nNOS-LI was diffusely localized in the cytoplasm and associated with the endoplasmic reticulum. In the inner plexiform layer, nNOS-LI diffusely filled some amacrine cell processes, while in other amacrine cells nNOS-LI was selectively localized at the presynaptic specializations of conventional synapses. Neuronal NOS-LI was also found at membrane specializations in bipolar cell terminals that were distinct from their normal ribbon synapses. Finally, some nNOS-LI was found in mitochondria in Muller cells. The diverse subcellular localizations of nNOS-LI indicates that NO may play distinct functional roles in many retinal cells, which correlates well with the widespread NO production found in previous NO imaging studies.

Amacrine Cells↗