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Autopsy case of colocalized tumors of hepatocellular carcinoma and malignant lymphoma.

We present a rare case of colocalized tumors of hepatocellular carcinoma (HCC) and malignant lymphoma in the liver and bone marrow. A 64-year-old man with hepatitis B virus (HBV)-induced cirrhosis was admitted after detection of elevated serum alpha-fetoprotein level. He was diagnosed as having HCC and treated with chemotherapy and radiotherapy, but the size of the tumors did not diminish. After 11 months' hospitalization, he died due to rupture of esophageal varices. Autopsy revealed two heterogenic malignant tumors, HCC and malignant lymphoma (B cell type), in the liver. These tumors showed colocalization in the liver and bone marrow. The carcinogenetic process of colocalized tumors of HCC and lymphoma was not clarified, but the possibility that persistent HBV infection play a role in promoting hepatocarcinogenic and hematopoietic tumor growths in the reticuloendothelial organs could not be excluded.

Autopsy↗

Colocalization of [3H]muscimol and antisera to GABA and glutamic acid decarboxylase within the same neurons in monkey retina.

Two procedures have been used to test for the colocalization of different neuroanatomical gamma-aminobutyric acid (GABA)ergic markers within the same neurons in primate retina. First, sequential immunocytochemical processing of sections was done using antisera to glutamic acid decarboxylase and to GABA, and these antisera were visualized by peroxidase-antiperoxidase and fluorescein isothiocyanate techniques respectively. Colocalization of both antisera was found within the same neuron cell bodies. In the second experiment, immunocytochemical staining using GABA antiserum was performed on retinal tissue that had been previously incubated in vitro for neuronal uptake of [3H]muscimol. Both markers colocalized in 70% of the labeled cell body population.

Animals↗

The colocalization of substance P and prodynorphin immunoreactivity in neurons of the medial preoptic area, bed nucleus of the stria terminalis and medial nucleus of the amygdala of the Syrian hamster.

To determine the extent of colocalization of substance P (SP) and prodynorphin peptides within neurons of the medial nucleus of the amygdala (AMe), medial bed nucleus of the stria terminalis (BNSTm) and medial preoptic area (MPOA), we incubated colchicine-treated Syrian hamster brain tissue in an antiserum mixture containing rat anti-SP antibody combined with 1 of 3 rabbit antibodies against prodynorphin peptides: anti-dynorphin A(1-17), anti-dynorphin B(1-13) or anti-C-peptide. This was followed by incubation in a secondary antiserum mixture containing fluorescein-labelled anti-rabbit and rhodamine-labelled anti-rat antibodies. Sections were viewed with an epifluorescence microscope using blue light excitation for fluorescein and green light excitation for rhodamine. Colocalization of SP and prodynorphin labelling was observed in neurons of the caudal parts of AMe, BNSTm and MPOA, areas which are essential for male mating behavior. The colocalization was most extensive in the dorsolateral part of the caudal MPOA, the caudodorsal part of the BNSTm, and in the posterodorsal subdivision of AMe. Although all 3 dynorphin peptides coexisted with SP in these areas, dynorphin B did so less than C-peptide, and dynorphin A less than dynorphin B.

Amygdala↗

Neurotensin innervation of the human cerebral cortex: lack of colocalization with catecholamines.

We have localized neurotensin (NT) with immunocytochemical methods in the normal human cerebral cortex. Extensive areas of the frontal cortex, the hippocampal formation, and selected areas of the parietal, temporal and occipital lobes, were examined using post-mortem brain tissue. The peptidergic innervation was characteristically restricted to the limbic belt and to the dorsally contiguous regions. NT-labeled perikarya were found throughout the subiculum, including its dorsal supra-callosal continuation. NT terminal plexuses were particularly abundant in layers I-VI of the anterior cingulate cortex, in layer I of area 32 and of medical areas 9, 8, 6 and in layers II-III of area 29, of the presubiculum and entorhinal cortex. Elsewhere, NT fibers were scarce being more frequent in layer I. This regional and laminar pattern differed significantly from that of tyrosine hydroxylase (TH), which was used to label catecholaminergic axons, and preferentially the dopaminergic ones. Even in zones where TH and NT innervations were abundant, such as the anterior cingulate cortex or area 32, double-labeling procedures disclosed no colocalized fibers. The lack of NT-TH colocalization in human, contrasts with previous findings in the rodent cortex, where a contingent of the DA cortical afferents contains NT. The DA mesocortical neuronal population, labeled by TH antisera, thus seems to change its chemical phenotype, by losing the expression of an associated peptidergic neurotransmitter; this could be related to the predominant extension in the ascent of the phylogenetic scale of the non-colocalized, type of cortical DA innervation which is also found in rodents. The possible origins of the cortical, non-dopaminergic NT innervation in human are discussed: thalamo-cortical, subiculo-cortical or intrinsic. Such cortical NT innervation could be very important in limbic circuitry as a regulatory peptide in affective processes and could be involved in the physiology of pain and memory.

Adult↗

Localization of corticotropin-releasing hormone (CRH) neurons in the paraventricular nucleus of the human hypothalamus; age-dependent colocalization with vasopressin.

Immunocytochemical staining, using a monoclonal antibody against corticotropin-releasing hormone, was performed on hypothalami of 13 human subjects between 23 and 91 years of age who had not suffered from a primary neurological or psychiatric disease. Corticotropin-releasing hormone (CRH) immunoreactivity was present in neurons of the paraventricular nucleus (PVN) and in their fibers running to the median eminence. The CRH-positive neurons were scattered throughout the PVN, but in the rostral part relatively few cells were present. There were large individual differences in the number and staining intensity of CRH neurons in the PVN and in the staining intensity of the median eminence. These differences seemed not to be attributed to age, sex, postmortem delay, fixation time or hour of death. In the rat, too, no relationship was found between a postmortem delay of up to 24 h and CRH staining intensity of the median eminence. Since the distribution of CRH-immunoreactive neurons in the human PVN strongly overlap with vasopressin, colocalization of these peptides was investigated in a double label study and indeed found in subjects ranging between 43 and 91 years of age. However, cells staining for only one of the peptides were also observed. The vasopressin cells had a mean cellular profile area which was 2.3 times as large as the CRH cells and 2.2 times as large as the CRH and vasopressin containing neurons. In younger subjects (23-37 years of age) no colocalization of the two peptides was seen. The age-dependent colocalization of CRH with vasopressin is interpreted as a sign of increased activation of the CRH neurons with age.

Adult↗

NADPH-diaphorase activity and its colocalization with transmitters and neuropeptides in the postganglionic neurons of the rat superior cervical ganglion.

NADPH-diaphorase activity (NADPH-DA), a marker of neural nitric oxide synthase, was found in many postganglionic nerve cell bodies in the adult rat superior cervical ganglion (SCG) after colchicine treatment, postganglionic nerve trunk ligation or ganglion culture. NADPH-DA colocalized with immunoreactivity to tyrosine hydroxylase (TH), serotonin, vasoactive intestinal peptide (VIP), neuropeptide Y (NPY), methionine-enkephalin and somatostatin. Almost all cells showing NADPH-DA were TH-immunoreactive, although several TH-immunoreactive cells lacked NADPH-DA. While suggesting that nitric oxide has an important role in the neuronal modulation in the synaptic transmission in the rat SCG, our results point out that nitric oxide synthesis is confined to a subpopulation of ganglion neurons. Our findings confirm the idea that the superior cervical ganglion consists of several subpopulations in which noradrenaline is colocalized with other transmitter or neuropeptide. Only about one-fourth of serotonin-immunoreactive neurons contained NADPH-DA. Similarly, the neuropeptides studied showed only partial colocalization with NADPH-DA. Our results thus suggest that nitric oxide is not associated with any particular transmitter or peptide.

Animals↗

Colocalization of octopamine and FMRFamide related peptide in identified heart projecting (DUM) neurones in the locust revealed by immunocytochemistry.

Immunocytochemical techniques are employed to reveal colocalization of octopamine with FMRFamide related peptide in the locust ventral nervous system. In each unfused pregenital abdominal ganglia (A4-A6) there are 3 octopamine-like immunoreactive neurones. By combining intracellular Lucifer yellow staining with subsequent immunocytochemistry these are individually identified as the efferent dorsal unpaired median (DUM) neurones DUM-1 and DUM-2, which innervate abdominal tergal and respectively sternal skeletal muscles, and DUM heart-1, an FMRFamide-like immunoreactive neurone which projects to the heart and associated alary muscles. Colocalization of octopamine- and FMRFamide-like immunoreactivity in DUM heart-1 is verified by alternate staining of consecutive sections. With respect to locust ventral ganglia, this investigation shows that colocalization of octopamine with an FMRFamide related peptide is restricted to a single DUM cell occurring in each abdominal ganglion 2-7, which most likely corresponds to segmental homologues of DUM heart-1.

Abdomen↗

Colocalization of somatostatin, neuropeptide Y, neuronal nitric oxide synthase and NADPH-diaphorase in striatal interneurons in rats.

The neuropeptides somatostatin (SS), neuropeptide Y (NPY), the enzyme neuronal nitric oxide synthase (nNOS) and enzymatic activity for NADPH diaphorase (NADPHd) are extensively colocalized in striatal interneurons, which has led to the widespread tendency to operationally treat all four substances as being completely colocalized within a single class of striatal interneurons. We have explored the validity of this assumption in rat striatum using multiple-labeling methods. Conventional epi-illumination fluorescence microscopy was used to examine tissue triple labeled for SS, NPY and nNOS, or double-labeled for SS and nNOS or for SS and NPY. In tissue double-labeled for SS and nNOs, confocal laser scanning microscopy (CLSM) images of SS and nNOS labeling were compared to subsequent NADPHd labeling. We found that SS, NPY and nNOS co-occurred extensively, but a moderately abundant population of neurons containing SS and nNOS but not NPY was also observed, as were small populations of SS only and nNOS only neurons. About 80% of SS+ neurons contained NPY, and no NPY neurons were devoid of SS or nNOS. All neurons containing nNOS in rat striatum were found to contain NADPHd. Combining our various quantitative observations, we found that of those striatal neurons containing any combination of SS, NPY, nNOS and NADPHd in rats, about 73% contained all four, 16% contained SS, nNOS and NADPHd, 5% contained SS only, and 6% contained only nNOS and NADPHd. These results indicate that while there is a large population of striatal neurons in which SS, NPY, nNOS and NADPHd are colocalized in rats, there may be smaller populations of striatal neurons devoid of NPY in which SS or nNOS/NADPHd are found individually or together.

Animals↗

Ultrastructural studies of the ontogeny of fetal human and porcine endocrine pancreas, with special reference to colocalization of the four major islet hormones.

Most, if not all, endocrine cells seem capable of synthesizing and storing more than one hormone. Such cellular colocalization of hormones can be due either to the presence of two or more specific granules within the cells or to colocalization of the hormones within a single granule. The present study was performed to clarify the subcellular localization of insulin, glucagon, somatostatin, and pancreatic polypeptide within the endocrine cells of the human and porcine pancreas during fetal development, with special reference to possible colocalization of the hormones. The tissue specimens were processed for ultrastructural cytochemistry using Lowicryl as embedding medium. An immunogold labeling technique was used with two parallel, but not interacting, antibody chains. Sections from each specimen were double labeled in different combinations giving a complete covering of the four major islet hormones. During fetal life (50-90 days prenatally in porcine pancreas, 14 weeks gestation in the human pancreas) several hormones were demonstrated, not only in the same endocrine cells, but also in the same secretory granules (polyhormonal granules). Costorage of insulin, glucagon, somatostatin, and pancreatic polypeptide was demonstrated in granules in pancreatic endocrine fetal cells. At an early fetal stage, the endocrine cells contained either dense, round granules or pale, heteromorphous granules. With increasing age and maturation of the endocrine cells, structural differentiation of the secretory granules was found to be associated with a gradual disappearance of the polyhormonal granules. The first genuine monohormonal cell to appear in the porcine fetus was the pancreatic polypeptide cell (at 70 days gestation); it was followed by the somatostatin-producing endocrine cell. Mature insulin- and glucagon-producing cells were only demonstrated after birth. Thus, in the adult pancreatic endocrine cells, each specific endocrine cell type produced only one of the four classical hormones. The present investigation demonstrated that the endocrine cells of the fetal, but not the adult, pancreas are able to synthesize all the major islet hormones, and that these peptides are costored in the same granule. The data obtained support the concept of a common precursor stem cell for pancreatic hormone-producing cells.

Animals↗

Manganese superoxide dismutase and reduced nicotinamide adenine dinucleotide diaphorase colocalize in the rat gut.

BACKGROUND & AIMS: Superoxide and other free radicals participate in inflammatory bowel disease and ischemia-reperfusion injury. Manganese superoxide dismutase (SOD) scavenges superoxide. Mn SOD is colocalized with reduced nicotinamide adenine dinucleotide (NADH) diaphorase in some tissues. NADH diaphorase histochemistry selectively stains enteric nerves. The aim of this study was to seek colocalization of Mn SOD with NADH diaphorase in the gut, especially in enteric nerves. METHODS: Indirect immunofluorescent staining and histochemistry were used to localize Mn SOD and NADH diaphorase in rat gut. RESULTS: Strong Mn SOD immunoreactivity was found in parietal cells, most intramural nerve cell bodies, the colonic interstitial cells of Cajal (at the submucosa-circular muscle layer interface), and intestinal epithelium cells. Weak to moderate Mn SOD immunoreactivity characterized smooth muscle cells, small submucosal arteries, esophageal striated muscle, esophageal epithelium, gastric epithelium, and intestinal glands. NADH diaphorase histochemistry (with Triton X-100) resulted in identical staining. CONCLUSIONS: Mn SOD and NADH diaphorase are colocalized throughout rat gut with strong activity in enteric nerves and colonic interstitial cells of Cajal.

Animals↗

Cooperation of EBV DNA polymerase and EA-D(BMRF1) in vitro and colocalization in nuclei of infected cells.

Expression of the Epstein-Barr virus (EBV) DNA polymerase (EBVpol) open reading frame (BALF5) by in vitro transcription-translation yielded a 116-kDa primary translation product. Enzymatic DNA polymerase activity of the in vitro translated polypeptide required the presence of the 47-kDa BMRF1 (EA-D) gene product. Antiserum raised to the BALF5 gene product expressed in Escherichia coli specifically precipitated a 116-kDa polypeptide in extracts of latently infected lymphoblastoid cells induced for EBV replication. Immunofluorescence microscopy revealed colocalization of the EBVpol and EA-D(BMRF1) to discrete foci within the nuclei of induced cells; however, the blockade of viral DNA synthesis resulted in diffuse nuclear staining patterns for both antigens. Bromodeoxyuridine staining of these discrete foci colocalizing with EBVpol suggests that they are sites of early viral DNA synthesis. These observations suggest that EA-D(BMRF1) may be an accessory protein of the EBV DNA polymerase which colocalizes in vivo with EBVpol to sites of viral DNA replication and cooperates in vitro to form an active EBVpol holoenzyme.

Antigens, Viral↗

Ontogeny of the distribution and colocalization of calbindin D28K within neural and endocrine cells of the gastrointestinal tract of fetal and neonatal sheep.

Using immunocytochemical techniques we have demonstrated that Calbindin D28K (CaBP) is present in the gastrointestinal tract of ovine fetuses early in development (by day 45). At day 45, CaBP was limited to neuronal elements in the developing intestine. By day 100, CaBP immunoreactivity was abundant in both epithelial endocrine cells and nerves of the submucous and myenteric ganglia. The location of CaBP containing cells and fibers was similar in duodenal sections taken from day 100 and term (145 days), as well as those taken from 24-48 h postnatal lambs. CaBP is colocalized in endocrine cells containing gastrin, glucagon, somatostatin and neurotensin, but not glucose dependent insulinotrophic peptide (GIP). Furthermore, it is extensively colocalized in nerve fibers and cells containing neurotensin but not somatostatin or vasoactive intestinal peptide. The colocalization of CaBP within various endocrine and nerve cells does not change in fetal sheep over the last one-third of gestation and there is no difference between fetal and neonatal sheep.

Animals↗

Visualization of cholinoceptive neurons in the rat neocortex: colocalization of muscarinic and nicotinic acetylcholine receptors.

The present investigation analyzes the cellular distribution of muscarinic and nicotinic acetylcholine receptors in rat neocortex, by use of monoclonal antibodies raised against purified receptor proteins. The degree of colocalization of both types of receptors was determined by way of immunofluorescent double-labeling techniques. For both classes of receptors, pyramidal and nonpyramidal cells were found immunostained and an identical laminar distribution pattern of immunopositive neurons in the rat neocortex became apparent. A striking similarity in distribution of the two cholinergic receptor types was found in the frontal/motor and parietal cortex. Accordingly, we observed a high degree of colocalization of muscarinic and nicotinic acetylcholine receptors within immunopositive cortical neurons. Approximately 90% of the cholinoceptive neurons expressed both types of receptors. The current data demonstrate that (i) the distribution of muscarinic and nicotinic cholinoceptive neurons in the neocortex is present in identical laminar patterns and represent the same type of cells, (ii) both classes of cholinergic receptors are highly colocalized within cholinoceptive neurons, which points at individual neurons as a likely site of interaction between muscarinic and nicotinic acetylcholine receptor-mediated processes.

Animals↗

Colocalization of gamma-aminobutyric acid and acetylcholinesterase in rodent cortical neurons.

We have previously demonstrated that neurons of the rat cerebral cortex which stain positively for acetylcholinesterase are not likely to be cholinergic since they do not colocalize with choline acetyltransferase immunoreactivity [Levey, Rye, Wainer, Mufson and Mesulam (1984) Neuroscience 9, 9-22]. These noncholinergic acetylcholinesterase-positive cells were similar in morphology to cortical neurons which localize gamma-aminobutyric acid or glutamate decarboxylase immunoreactivity. In order to investigate the possibility that the two substances may be colocalized to the same cortical neurons, gamma-aminobutyric acid immunohistochemistry and acetylcholinesterase histochemistry were combined in single sections of rat cerebral cortex. We found that 18% of gamma-aminobutyric acid-immunoreactive cortical neurons are also acetylcholinesterase-positive, and about 36% of acetylcholinesterase-positive cells are gamma-aminobutyric acid-immunoreactive. Neurons which colocalized both substances were multipolar and bipolar neurons in cortical laminae II-VI and were observed in every cortical area examined. The possibility that gamma-aminobutyric acid-immunoreactive/acetylcholinesterase-positive cortical neurons may be postsynaptic targets of cholinergic afferents to the cerebral cortex is discussed.

Acetylcholinesterase↗

The effect on osteoblast function of colocalized RGD and PHSRN epitopes on PEG surfaces.

Poly(ethylene glycol) hydrogels were synthesized with pendant peptide functionalities to examine the influence of synergistic peptide sequences on osteoblast adhesion, spreading, and function. Specifically, acrylated monomers were prepared that contained the peptide sequence, Arg-Gly Asp (RGD), as well as monomers with RGD plus its synergy site, Pro-His-Ser-Arg-Asn (PHSRN), linked via a polyglycine sequence to recapitulate the native spacing of fibronectin. The colocalized RGD-PHSRN sequence improved osteoblast adhesion, spreading, and focal contact formation when compared to RGD alone. In addition, proliferation, metabolic activity, and levels of alkaline phosphatase production, a common marker for osteoblast function, were statistically higher for the colocalized peptide sequences at 1 day, 1 week, and 2 weeks, when compared to control surfaces. Interestingly, increases were not observed in all areas of cell function, as extracellular matrix (ECM) production was the lowest on gels functionalized with the colocalized peptide sequence. This result was attributed to strong receptor-ligand interactions initiating signal transduction cascades that down-regulate ECM production.

Alkaline Phosphatase↗

Colocalization of phosphorylated CREB with calcium/calmodulin-dependent protein kinase IV in hippocampal neurons induced by ohmfentanyl stereoisomers.

The transcription factor cAMP response element-binding protein (CREB) plays an important role in opioids dependence. To better understand the role of CREB in opioids dependence and underlying signal pathways, we compared the effects of three ohmfentanyl stereoisomers ((-)-cis-(3R,4S,2'R) OMF (F9202), (+)-cis-(3R,4S,2'S) OMF (F9204), (-)-cis-(3S,4S,2'R) OMF (F9203)) and morphine on CREB phosphorylation and the expression of Ca2+/calmodulin-dependent protein kinase IV (CaMKIV) in hippocampus derived from mice which displayed conditioned place preference (CPP) behavior by Western blot, and immunohistochemistry analyses. Moreover, we studied the effects of OMF and morphine on CREB phosphorylation and colocalization of phosphorylated CREB (P-CREB) with CaMKIV in cultured rat hippocampal neurons by Western blot, and confocal fluorescence microscopy analyses. The results showed that F9202, F9204 or morphine, which could induce CPP, enhanced CREB phosphorylation and the expression of CaMKIV in hippocampus from CPP mice without affecting total CREB protein level. The CREB phosphorylation of cultured hippocampal neurons was also enhanced and reached its peak level at 30 min upon exposure to F9202 (100 nM), F9204 (100 nM) or morphine (1 microM), while the total CREB protein level was not altered. KN-62 (10 microM), an inhibitor of CaM kinases, prevented CREB phosphorylation induced by morphine, F9202, and F9204 without change of total CREB level. The results of confocal fluorescence microscopy further demonstrated that the activated CREB (P-CREB) was colocalized with CaMKIV in nucleus. F9203, which could not induce CPP, failed to increase the CREB phosphorylation and the colocalization of P-CREB with CaMKIV both in hippocampus from CPP mice and in cultured hippocampal neurons. This is the first evidence to suggest that the increased CREB phosphorylation via CaMKIV signal pathway in hippocampus is relevant to opioids psychological dependence.

Animals↗

Colocalization of nitric oxide synthase and monoamines in neurons of the amphibian brain.

By means of double immunohistofluorescence techniques, we have investigated the colocalization of nitric oxide synthase and tyrosine hydroxylase (TH) or serotonin (5-HT) in the central nervous system of the anurans Rana perezi and Xenopus laevis and the urodele Pleurodeles waltl. A wide codistribution of neuronal populations, expressing these markers, was found throughout the brain and spinal cord. In contrast, colocalization of these markers was rather restricted. Only in the caudal portion of the brainstem raphe column in anurans, approximately 80% of the 5-HT-positive cells were also NOS-immunoreactive, whereas in the urodele brain, about 40% of the serotonergic cells at the level of the glossopharyngeal motor nucleus were simultaneously NOS-positive. In various brain regions, a wide codistribution of NOS- and TH-containing neurons was observed, but real colocalization of nitrergic and catecholaminergic cells was only found in a small neuron population in the posterior tubercle of anuran amphibians. Therefore, in amphibians, only a distinct and small cell population within the serotonergic raphe column (anurans and urodele) and in the catecholaminergic posterior tubercle (anurans) seem to produce simultaneously nitric oxide.

Amphibians↗

Colocalization studies of Arp1 and p150Glued to spindle microtubules during mitosis: the effect of cytochalasin on the organization of microtubules and motor proteins in PtK1 cells.

Motor proteins play a fundamental role in the congression and segregation of chromosomes in mitosis as well as the formation of the mitotic spindle. In particular, the dynein/dynactin complex is involved in the maintenance of the spindle, formation of astral microtubules, chromosome motion, and chromosome segregation. Dynactin is a multisubunit, high molecular weight protein that is responsible for the attachment of cargo to dynein. There are a number of major subunits in dynactin that are presumed to be important during mitosis. Arp1 is thought to be the attachment site for cargo to the complex while p150(Glued), a side arm of this complex regulates binding to MTs and the binding of dynactin to dynein. We performed colocalization studies of Arp1 and p150(Glued) to spindle microtubules. Both Arp1 and p150(Glued) colocalize with spindle MTs as well as cytoplasmic components. When treated with cytochalasin J, Arp1 concentrates at the centrosomes and is less co-localized with spindle MTs. Cytochalasin J has less of an effect on the colocalization of p150(Glued) with spindle MTs, suggesting that Arp1 may have a cytochalasin J sensitive site.

Animals↗