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Immunocytochemical evidence suggests that taurine is colocalized with GABA in the Purkinje cell terminals, but that the stellate cell terminals predominantly contain GABA: a light- and electronmicroscopic study of the rat cerebellum.

The distributions of taurine-like and GABA-like immunoreactivities in the rat cerebellum were compared by analysis of consecutive semithin and ultrathin sections, postembedding labeled with the peroxidase-antiperoxidase technique or with an indirect immunogold procedure, respectively. Taurine-like immunoreactivity was selectively enriched in Purkinje cell bodies, dendrites and spines, and boutons in the cerebellar nuclei exhibiting ultrastructural features typical of Purkinje cell terminals. The stellate and basket cell bodies and terminals were very weakly labeled. A computer assisted quantitative assessment of the net immunogold labeling revealed that the mean gold particle density in the Purkinje cell terminals was about 70% higher than that in the Purkinje cell dendrites, and about 14 times higher than that in the stellate/basket cell terminals in the molecular layer. Stellate, basket and Purkinje cell terminals emerged as intensely immunoreactive in adjacent sections processed with an antiserum against conjugated GABA. These findings indicate, contrary to recent electrophysiological data, that GABA is a more likely transmitter candidate than taurine in the stellate cells. The apparent colocalization of GABA and taurine in the terminals of Purkinje cells raises the possibility that these terminals are capable of releasing two different inhibitory amino acids.

Animals

Spinal cord NADPH-diaphorase histochemical staining but not nitric oxide synthase immunoreactivity increases following carrageenan-produced hindpaw inflammation in the rat.

Recent reports suggest that NADPH-diaphorase (NADPH-d) may be a histochemical marker for neuronal nitric oxide synthase (nNOS) in the central nervous system. Carrageenan-produced unilateral hindpaw inflammation in the rat results in a bilateral increase in NADPH-d in spinal cord neurons. This suggests there would be a bilateral increase in NO, which mediates thermal hyperalgesia. However, carrageenan-produced unilateral hindpaw inflammation results in hyperalgesia of the inflamed hindpaw only. This study determined (1) if neurons that labeled for NADPH-d following carrageenan-produced unilateral hindpaw inflammation colocalized nNOS, and (2) whether there was an increase in nNOS-ir neurons following inflammation. Following unilateral hindpaw inflammation, double labeling of tissue sections and single labeling of alternate serial sections revealed a lack of colocalization or mismatch between NADPH-d histochemical activity and nNOS-like immunoreactivity in neurons in lamina I, the dorsolateral funiculus and lamina X. Quantitative analysis showed no difference in the number of nNOS-ir neurons and NADPH-d labeled neurons in the superficial laminae of the spinal cord in non-inflamed animals. Following unilateral hindpaw inflammation, there was a 34% increase in the number of NADPH-d labeled neurons but no increase in the number of nNOS-ir neurons. These results indicate that nNOS-immunoreactive neurons and NADPH-diaphorase stained neurons are not identical and that nNOS does not increase as a result of hindpaw inflammation, leaving the source of NO involved in thermal hyperalgesia following injury in question.

Amino Acid Oxidoreductases

Monoclonal antibodies specific for human platelet membrane glycoproteins bind to monocytes by focal absorption of platelet membrane fragments: an ultrastructural immunogold study.

The membrane labeling of monocytes by monoclonal antibodies directed against platelet glycoproteins Ib (AN51), IIb (Tab), IIIa (C17), IIb-IIIa complex (J15) and to antigens common to platelets and monocytes (anti-monocyte platelet antigen and FA6 152) has been investigated by an ultrastructural immunogold method. Only with FA6 152, which identifies a structure shared by erythroblasts, platelets, and monocytes, was labeling obtained on membranes of both platelets and monocytes from normal blood. With all the other monoclonal antibodies, platelets were highly labeled but monocytes lacked quantitatively significant label; however, focal microparticles which exhibited gold particles were adherent to the membrane of monocytes. This localized labeling, interpreted as resulting from the fragmentation of platelet membranes during monocyte isolation with adhesion of the fragments to monocyte surfaces, was verified by two approaches. First, double staining with C17 visualized by an anti-IgG coupled to 40 nm gold particles and MO2 recognizing exclusively a surface monocyte antigen, as visualized by an anti-IgG coupled to 15 nm gold particles, was performed. The absence of colocalization of large and small gold particles either on monocytes or on microparticles confirmed the exclusive cell origin. Second, when analyzed by quantitative x-ray analysis, monocyte associated gold following C17 treatment was restricted to platelet pseudopods and fragments on whole mount spread cells. Finally, when monocytes were spread immediately after blood collection in the absence of sedimentation and centrifugation to prevent platelet activation, platelet rosetting was avoided and the number of microparticles markedly decreased. Thus, the attachment to monocyte membranes of microparticles originating from platelets may be confused with true labeling of monocytes by antibodies to platelet glycoproteins if analysis is limited to immunofluorescence.

Adsorption

rSec6 and rSec8, mammalian homologs of yeast proteins essential for secretion.

Many of the molecules necessary for neurotransmission are homologous to proteins involved in the Golgi-to-plasma membrane stage of the yeast secretory pathway. Of 15 genes known to be essential for the later stages of vesicle trafficking in yeast, 7 have no identified mammalian homologs. These include the yeast SEC6, SEC8, and SEC15 genes, whose products are constituents of a 19.5S particle that interacts with the GTP-binding protein Sec4p. Here we report the sequences of rSec6 and rSec8, rat homologs of Sec6p and Sec8p. The rSec6 cDNA is predicted to encode an 87-kDa protein with 22% amino acid identity to Sec6p, and the rSec8 cDNA is predicted to encode a 110-kDa protein which is 20% identical to Sec8p. Northern blot analysis indicates that rSec6 and rSec8 are expressed in similar tissues. Immunodetection reveals that rSec8 is part of a soluble 17S particle in brain. COS cell cotransfection studies demonstrate that rSec8 colocalizes with the GTP-binding protein Rab3a and syntaxin 1a, two proteins involved in synaptic vesicle docking and fusion at the presynaptic terminal. These data suggest that rSec8 is a component of a high molecular weight complex which may participate in the regulation of vesicle docking and fusion in brain.

Amino Acid Sequence

Sphingolipid metabolism in cultured fibroblasts: microscopic and biochemical studies employing a fluorescent ceramide analogue.

A fluorescent analogue of ceramide, N-[7-(4-nitrobenzo-2-oxa-1,3-diazole)]-epsilon-aminocaproyl sphingosine (C6-NBD-ceramide), was used to investigate sphingolipid metabolism in Chinese hamster fibroblasts. C6-NBD-ceramide was incorporated into small unilamellar dioleoyl phosphatidylcholine vesicles and incubated with cells in monolayer culture at 2 degrees C, resulting in rapid and preferential transfer of the labeled ceramide from vesicles to cells. The cells were then washed and subsequently incubated at 37 degrees C for various intervals. The metabolism of C6-NBD-ceramide was monitored by lipid extraction and analysis, and the intracellular distribution of the labeled molecule was followed by fluorescence microscopy. Initially, fluorescence was detected almost exclusively in mitochondria, with over 90% of the extractable lipid fluorescence due to C6-NBD-ceramide. After 30 min at 37 degrees C, intense fluorescence appeared in the Golgi apparatus. This organelle was identified by colocalization of NBD fluorescence with a Golgi-apparatus-specific stain. At later times the plasma membrane became visibly labeled as well, at which point 90% of the cell-associated fluorescence was recovered as NBD-labeled sphingomyelin and NBD-labeled cerebroside. These metabolites were identified by enzymatic and biochemical analysis and by thin-layer chromatography of the fluorescent lipid extracts. The finding that C6-NBD-ceramide is used by these cells in standard pathways of sphingolipid biosynthesis suggests that this fluorescent precursor will be a valuable tool for correlating the metabolism of sphingolipids with their intracellular distribution and translocation. In addition, during its metabolism by Chinese hamster fibroblasts, this compound acts as a vital stain for the Golgi apparatus.

4-Chloro-7-nitrobenzofurazan

Colocalization of glycine and GABA in synapses on spinomedullary neurons.

Spinomedullary neurons of the postsynaptic dorsal column pathway in adult cats were retrogradely labelled with horseradish peroxidase. Postembedding immunogold reactions were performed with antisera which recognise GABA or glycine to determine if synaptic boutons in contact with these neurons contain both transmitters. Analysis of series of ultrathin sections revealed that synaptic profiles with strong immunogold reactions for GABA usually also displayed strong immunogold reactions for glycine. Pre-embedding immunocytochemistry was performed on sections containing labelled cells with a monoclonal antibody which recognises the glycine receptor-associated protein, gephyrin. Many synapses onto postsynaptic dorsal column neurons were associated with gephyrin-like immunoreactivity and these typically contained irregularly shaped vesicles. Immunogold reactions showed that synaptic profiles apposed to gephyrin-immunoreactive junctions contained GABA and glycine. The evidence suggests that glycine is a neurotransmitter at synapses on spinomedullary neurons and that it is colocalized with GABA.

Animals

Multi-omic characterization of the Hispanic/Latino blood lipidome reveals an additional locus and attenuated genetic prediction.

While lipids have been extensively investigated, genetic regulation of the circulating lipidome in diverse populations remains poorly understood. We conducted a lipidome-wide genome-wide association study (GWAS) of 830 lipid species in 2,287 Hispanic/Latino participants and performed predictive modeling across omics layers. We identified 7,593 genome-wide significant SNPs mapping to 208 genes. Conditional analysis disentangled the long-range linkage disequilibrium artifacts from the pleiotropic FADS1/2/3 cluster. Separately, we discovered an association at the GPLD1 locus for a circulating ceramide. Colocalization revealed shared genetic architecture with conventional lipids alongside distinct, species-specific pathways. Incorporating Native/Indigenous American expression quantitative trait loci (eQTLs) within a multi-omic framework uncovered 62 likely regulatory genes missed by European-centric gene expression models. Finally, genetically regulated predictive models demonstrated performance declining from transcriptomics to proteomics to lipidomics, reflecting increased distance from gene action along the molecular cascade. Our study provides a genetic landscape of lipid metabolism in a highly burdened population and highlights the challenges in predicting lipid abundance.

Hispanic/Latino population

Expression, secretion, and age-related downregulation of pigment epithelium-derived factor, a serpin with neurotrophic activity.

Retinal pigment epithelial (RPE) cells form a functional complex with photoreceptor neurons of the retina, interacting through the interphotoreceptor matrix (IPM). We now provide evidence that the gene for pigment epithelium-derived factor (PEDF), a protein possessing neurotrophic and neuronal-survival activities, is highly expressed by both fetal and young adult RPE cells. PEDF mRNA is present in RPE cells of the human eye at 17 weeks of gestation, demonstrating its potential for action in vivo during early retinal development. The PEDF protein is secreted in vivo where it constitutes a part of the fetal and adult IPM surrounding photoreceptor outer segments. A polyclonal PEDF antibody recognizes at least four isoforms of secreted human and bovine PEDF by two dimensional gel analysis, and detects a similar 50 kDa protein in the IPM of several other vertebrate species. Within soluble extracts of RPE cells, however, where little, if any, of the 50 kDa species can be detected, an immunoreactive 36 kDa protein is observed by Western blot analysis. By immunofluorescence, PEDF is localized intracellularly in association with the nucleus, presumptive secretory granules, and cytoskeletal elements of cultured RPE cells with PEDF and actin antibodies colocalizing to the same cytoskeletal structures. During initial stages of attachment, PEDF and actin also concentrate at the tips of pseudopods extended by the cultured RPE cells. However, with successive passages, synthesis, and secretion of the PEDF protein as well as transcription of its mRNA decrease and are lost by about 10 passages. In parallel, cultured RPE cells lose their proliferative potential and change from an epithelial-like morphology in early passages to a more fibroblast-like appearance by about the 10th passage. PEDF is thus apparently present intracellularly and extracellularly in both fetal and early adult periods where it could be involved in cellular differentiation and survival and with its loss, in the onset of senescence.

Aging

Overexpression of acidic and basic fibroblast growth factors in human pancreatic cancer correlates with advanced tumor stage.

Acidic fibroblast growth factor (aFGF) and basic FGF (bFGF) are mitogenic polypeptides that may contribute to cancer cell proliferation. In the present study we examined aFGF and bFGF expression in human pancreatic cancer. Northern blot analysis of total RNA isolated from 12 pancreatic cancers revealed elevated aFGF and bFGF mRNA levels in 12 and 10 samples, respectively, by comparison with the normal human pancreas. Immunostaining demonstrated the presence of aFGF and bFGF in many cancer cells and in the atrophic acini and ducts adjacent to the cancer cells, but to a much lesser extent in the surrounding fibroblasts. By in situ hybridization, both mRNA moieties colocalized with their respective proteins and were abundant in many cancer cells. Immunoblotting confirmed that cancer tissues with increased aFGF and bFGF immunoreactivity contained elevated levels of both proteins. To determine the significance of aFGF and bFGF expression in the pancreatic cancer cells, immunohistochemical analysis of 78 human pancreatic carcinomas was performed. aFGF and bFGF immunoreactivity was present in the cancer cells in 47 (60%) and 44 (56%) of the tumors, respectively. There was a significant correlation between the presence of either aFGF or bFGF in the cancer cells and advanced tumor stage, and the presence of bFGF and shorter patient survival. These data suggest that aFGF and bFGF are overexpressed in a significant proportion of human pancreatic carcinoma cells and that this overexpression may contribute to disease progression.

Adult

Comparative distribution of 3H-GABA uptake and GAD immunoreactivity in goldfish retinal amacrine cells: a double-label analysis.

The comparative distribution of 3H-GABA uptake and glutamic acid decarboxylase immunoreactivity (GAD-IR) in amacrine cells of goldfish retina was studied simultaneously by using a combined autoradiographic/immunocytochemical technique in order to determine the degree of colocalization of these two markers of GABAergic neurons; 3H-GABA was taken up most intensely by large pyriform Ab amacrine cells (3% of inner nuclear layer (INL) somata), and less intensely by smaller, polyform amacrine cells (12% of INL somata), cell bodies in the ganglion cell layer (one-half as common as Ab cells), and cell bodies in the inner plexiform layer (very rare). GAD-IR was observed in 25% of amacrine cells in the INL, the vast majority of which were polyform in shape, and cell bodies in the ganglion cell layer. Twice as many cells were labeled for GAD-IR as for 3H-GABA uptake. Of the cells that took up 3H-GABA, colocalization of 3H-GABA uptake and GAD-IR was observed in 90% of the polyform amacrine cells, 80% of the cells in the ganglion cell layer, and none of the pyriform Ab amacrine cells or cells in the inner plexiform layer. We suggest that the polyform cells compose the major population of GABAergic amacrine cells in the goldfish retina, rather than the pyriform Ab cells. GABAergic displaced amacrine cells or ganglion cells are also indicated by our data. The implications of these data with regard to the physiology of the goldfish retina are discussed as well.

Animals

Characterization and colocalization of steroid binding and dimerization activities in the mouse estrogen receptor.

We have identified a region within the steroid binding domain of the mouse estrogen receptor that is required for both receptor dimerization and high affinity DNA binding. Analysis of sequences in this region revealed that a heptad repeat of hydrophobic residues was conserved in all members of the nuclear receptor superfamily. Single amino acid substitutions of residues in the N-terminal half, but not the C-terminal half, of the repeat prevented receptor dimerization. Steroid binding was abolished by point mutations in the center of the conserved region, implying that the steroid binding and dimerization domains overlap. The role of this region in steroid receptor function is discussed in relation to other models of protein dimerization and DNA binding.

Amino Acid Sequence

B-50 (GAP-43) immunoreactivity is rarely detected within intact catecholaminergic and serotonergic axons innervating the brain and spinal cord of the adult rat, but is associated with these axons following lesion.

The persistence of high levels of B-50 (GAP-43) in fibers innervating various regions of the adult central nervous system is generally thought to characterize neuronal systems capable of undergoing morphological plasticity. In a recent series of in situ hybridization studies, it has been shown that most catecholaminergic and serotonergic neurons of the adult rat brain express high levels of B-50 mRNA. The present study addresses the question whether high expression of B-50 mRNA in the catecholaminergic and serotonergic perikarya corresponds with detectable high levels of the B-50 protein in the efferent axonal fibers that innervate various regions of the adult rat brain and spinal cord. For this purpose, vibratome sections were doubly immunostained for B-50 and for tyrosine hydroxylase or serotonin and were analyzed by laser scanning confocal microscope. Colocalizations were investigated either (1) in regions of intact rat brain and spinal cord in which particular concentrations of B-50 immunoreactive fibers appeared codistributed with catecholaminergic or serotonergic fibers or (2) in intrahypothalamic portions of the medial forebrain bundle in which a surgical lesion was made. In the intact brain, frequent colocalizations of B-50 and tyrosine hydroxylase were detected in fibers innervating both the mediobasal hypothalamus and the neurointermediate hypophysial lobe. In all the other regions examined, the analysis of thin optical sections demonstrated that immunoreactivity to B-50 was only rarely associated with axonal profiles immunoreactive to tyrosine hydroxylase or to serotonin. By contrast, in the lesioned medial forebrain bundle B-50 immunoreactivity was found to be associated with numerous catecholaminergic and serotonergic axonal sprouts that regenerate around the surgical lesion. These data indicate that the majority of intact catecholaminergic and serotonergic axons innervating the adult rat brain and spinal cord contains low levels of B-50. However, following axotomy, B-50 is immunocytochemically detectable in the regenerating sprouts produced by both types of axonal fibers. This suggests that under basal conditions the relatively high content of B-50 mRNA in monoaminergic perikarya does not lead to appreciable accumulation of B-50 within corresponding axonal fibers and terminals, whereas conditions of morphological reorganization induce increased production of B-50 that accumulates within monoaminergic axonal sprouts.

Animals

[Conformation analysis of modified tetrasaccharide sequences of the N-glycoprotein type--problem of the alpha-(1 to 6)-glycosidic bond].

The conformational analysis of the recently synthesized tetrasaccharides alpha-D-Manp (1----3)-[alpha-D-Manp-(1----6)]-4-deoxy-beta-D-lyx-hexp+ ++-(1----4)-D-GlcNAc (2) and alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-beta-D-Talp -(1----4)-D-GlcNAc (3) will be described. The preferred solution conformation of 2 and 3 is a gt-conformation, which is nearly identical with the preferred conformation of the naturally occurring tetrasaccharide alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-beta-D-Manp -(1----4)-D-GlcNAc (1). The main structural feature is the backfolding of the alpha-(1----6)-linked D-Man to the reducing D-GlcNAc unit. Conformational analysis of the tetrasaccharides alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-beta-D-Manp -(1----4)-1,6- anhydro-beta-D-GlcNAc (4), alpha-D-Manp-(1----3)-alpha-D-Manp-(1----6)]-4-deoxy-beta-D- lyx-hexp-(1----4)- 1,6-anhydro-beta-D-GlcNAc (5), and alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-beta-D-Talp -(1----4)- 1,6-anhydro-beta-D-GlcNAc (6) gave additional proof for this backfolding. The substitution of the reducing unit leads to a smaller amount of gt- and a greater amount of gg-conformers. The method used for conformational analysis of 2-6 is a combination of n.m.r.-experiments and HSEA-calculations with the program GESA. Concerning the application of new 2D-techniques, the COLOC-experiment turned out to be extremely useful in sequencing oligosaccharides.

Carbohydrate Conformation

Distribution and co-localization of calbindin D28k with VIP and neuropeptide Y but not somatostatin, galanin and substance P in the enteric nervous system of the rat.

Calbindin D28k, previously demonstrated in the mammalian central nervous system, has been localized to discrete neurons in the enteric nervous system of the rat. Calbindin D28k is present in cell bodies in both the myenteric and submucous plexi and in interganglionic nerve fibers in all regions of the gastrointestinal tract. Immunoreactive nerve fibers were also detected in the mucosal region, although none were observed in the pyloric sphincter, circular or longitudinal muscle layers. The highest concentration of immunoreactivity was present in the submucosal plexus and mucosa of the colon. Western blot analysis of the protein detected by the antiserum confirmed that it comigrated with purified calbindin D28k and the single immunoreactive band seen in extracts from rat brain. The colocalization of calbindin D28k with components of the peptidergic innervation was also investigated. Of the peptides studied the neurons containing both vasoactive intestinal polypeptide and neuropeptide Y in the submucous plexus were seen to exhibit calbindin D28k immunoreactivity. The neurons containing somatostatin, galanin and substance P did not demonstrate co-localization. In the stomach, calbindin D28k was detected within a small number of epithelial cells which were found to correspond to a sub-population of the somatostatin-immunoreactive endocrine cells.

Animals

Release of Ca2+ from individual plant vacuoles by both InsP3 and cyclic ADP-ribose.

Calcium mobilization from intracellular pools couples many stimuli to responses in plant cells. Cyclic adenosine 5'-diphosphoribose (cADPR), which interacts with a ryanodine receptor in certain animal cells, was shown to elicit calcium release at the vacuolar membrane of beet storage root. The vacuolar calcium release pathway showed similarities to cADPR-gated calcium release in animal cells, including inhibition by ruthenium red, ryanodine activation, and high affinity for cADPR [Michaelis constant (Km) = 24 +/- 7 nanomolar]. Analysis by patch-clamping demonstrated that the cADPR-gated pathway in beet is voltage-dependent over the physiological range, does not spontaneously desensitize, and is colocalized with an inositol 1,4,5-trisphosphate (InsP3)-gated calcium release pathway in individual vacuoles.

Adenosine Diphosphate Ribose

FANCI promotes esophageal squamous cell carcinoma progression and cell cycle regulation and interacts with FANCD2.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is an aggressive malignancy with poor clinical outcomes, and reliable molecular biomarkers and therapeutic targets remain limited. Fanconi anemia group I protein (FANCI) is a core component of the Fanconi anemia (FA) pathway, but its expression pattern, clinical significance, and functional role in ESCC have not been comprehensively defined. This study aimed to investigate FANCI expression and prognostic value in ESCC, assess its effects on malignant cellular phenotypes and tumor growth, and explore its potential mechanistic relationship with Fanconi anemia group D2 protein (FANCD2) and cell-cycle regulation. METHODS: Multi-cohort analyses were performed using The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) datasets, together with ESCC single-cell RNA sequencing (RNA-seq) data. FANCI functions were assessed by bidirectional gain- and loss-of-function experiments in vitro (proliferation, colony formation, migration, invasion, apoptosis, and cell-cycle assays) and by xenograft models in vivo. Mechanistic studies included protein-protein interaction (PPI) analyses, co-immunoprecipitation (Co-IP), and immunofluorescence (IF) colocalization. RESULTS: FANCI was consistently upregulated in ESCC across bulk transcriptomic datasets and was further supported by quantitative polymerase chain reaction (qPCR), Western blotting, and immunohistochemistry (IHC). FANCI discriminated ESCC from normal tissues in TCGA-ESCC and was independently validated in GSE53624 [area under the curve (AUC) =0.940 and 0.975, respectively]. FANCI was associated with poorer overall survival (OS) and shorter disease-free interval (DFI), and these findings were validated in an independent GEO cohort. Functionally, FANCI promoted ESCC cell proliferation, migration, and invasion, while inhibiting apoptosis; FANCI knockdown suppressed tumor growth in vivo and induced G2/M cell-cycle arrest. Mechanistically, FANCI physically interacted with FANCD2, colocalized with FANCD2 in the nucleus, and was associated with altered FANCD2 protein abundance, consistent with cell-cycle and DNA repair-related programs. Single-cell analysis indicated that FANCI was enriched in epithelial cells and associated with higher activity of malignant functional programs. In TCGA-ESCC, FANCI-high tumors showed distinct mutation profiles, a trend toward increased tumor mutation burden (TMB), and altered immune-associated signatures. CONCLUSIONS: FANCI is upregulated in ESCC and is associated with diagnostic and prognostic value. It promotes malignant phenotypes and tumor growth, potentially through a FANCI-FANCD2-linked cell-cycle/DNA repair program, supporting FANCI as a candidate biomarker and therapeutic target in ESCC.

Esophageal squamous cell carcinoma (ESCC)

Use of enhanced silver staining combined with electron microscopical immunolabelling to demonstrate the colocalization of neuropeptide Y and vasoactive intestinal polypeptide in cerebrovascular nerves.

The combination of immunolabelling at the electron microscope level and enhanced silver staining has been used to demonstrate the colocalization of neuropeptide Y and vasoactive intestinal polypeptide in perivascular nerves supplying cerebral arteries of the rat. This has been shown in control tissue, but it is easier to demonstrate after long-term sympathectomy since that leads to an enhancement of neuropeptide Y in vasoactive intestinal polypeptide-containing parasympathetic nerves supplying these vessels. Immunolabelling of the antigens for these peptides was performed sequentially with the biotin streptavidin diaminobenzidine method, and the end product to the first antiserum was gold-silver intensified before the visualization of the second antigen. Using this technique, it was shown that all the neuropeptide Y immunoreactivity present in the rat cerebral vessels after long-term sympathectomy with guanethidine was localized in vasoactive intestinal polypeptide-containing nerves. Furthermore, an immunohistochemical analysis of the parasympathetic pterygopalatine ganglia in guanethidine-treated rats showed an increase in the percentage of neurons displaying neuropeptide Y immunoreactivity. In order to clarify if the pterygopalatine ganglion was the origin of those neuropeptide Y/vasoactive intestinal polypeptide-immunoreactive cerebrovascular nerves, which had increased in number after sympathectomy, a fluorescent neuronal tracer (Fast Blue) was applied to the right middle cerebral artery of rats which had undergone guanethidine treatment for six weeks. Immunohistochemical analysis of the ipsilateral ganglion 72 h after application of the tracer revealed the presence of immunoreactivity to both these peptides in retrogradely labelled neurons. It is concluded that neuropeptide Y and vasoactive intestinal polypeptide are colocalized in perivascular parasympathetic nerves supplying the middle cerebral artery of the rat, which have their origin in the pterygopalatine ganglion. Furthermore, long-term sympathectomy with guanethidine leads to an increase in the expression of neuropeptide Y in these vasoactive intestinal polypeptide-immunoreactive neurons.

Animals

Receptor-mediated endocytosis of alpha 2 macroglobulin by a glioma cell line.

Previous experiments have shown that human neoplastic and embryonic glial cell lines synthesize and secrete in culture, alpha 2 macroglobulin (alpha 2M), a broad spectrum proteinase inhibitor present in serum and extracellular fluids. The present study was aimed to investigate the presence of alpha 2M receptors on glial cell membrane, since several non-neural cell types producing alpha 2M also express alpha 2M receptors. By flow cytometric analysis, immunofluorescence and immunoelectronmicroscopy techniques we demonstrate an alpha 2M receptor-related immunoreactivity on the plasma membrane of a human glioma cell line. Ultrastructural experiments reveal a close colocalization of immunoreactivities for alpha 2M and its receptor in clathrin-coated pits and vesicles, structures typically involved in receptor-mediated endocytic pathways.

Endocytosis