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NADPH-diaphorase-positive neurons in primate cerebral cortex colocalize with GABA and calcium-binding proteins.

Neurons in the monkey cerebral cortex containing nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) can be divided into two distinct types, both nonpyramidal. Type I neurons have a large soma (diameter 20-50 microm), a dense NADPH-d histochemical reaction, and are distributed throughout the cortex, but mainly in the subcortical white matter, and are mostly aspiny. Type II cells have a small soma ( Together with previous observations that almost all cortical NADPH-d cells in various subprimates are like type I cells, we suggest that type II cells may form a group of NADPH-d-rich neurons differentiated in higher mammalian cortex from a subpopulation of calbindin-containing GABAergic interneurons, and these nitric oxide-synthesizing cells may play a role in control of intracortical neuronal activity characteristic of higher cerebral functions in advanced mammals.

Animals↗

rDNA (18S-28S and 5S) colocalization and linkage between ribosomal genes and (TTAGGG)(n) telomeric sequence in the earthworm, Octodrilus complanatus (Annelida: Oligochaeta: Lumbricidae), revealed by single- and double-color FISH.

Spermatogonial and metaphase I chromosomes of the lumbricid earthworm Octodrilus complanatus (Annelida: Oligochaeta) were examined using fluorescent in situ hybridization (FISH) with three repetitive DNA probes-5S rDNA, 18S-26S rDNA, and (TTAGGG)(n). Single-color FISH consistently mapped one chromosome pair per spread using either 5S rDNA or 18S-26S rDNA as probes. Simultaneous (18S-26S)-5S and (18S-26S)-(TTAGGG)(n) FISH demonstrated that repeated units of the two ribosomal families were overlapped and closely associated with telomeric sequences.

Animals↗

Colocalization of tau and alpha-synuclein epitopes in Lewy bodies.

The major protein constituent of Lewy bodies (LBs), the pathological hallmark of Parkinson disease and dementia with Lewy bodies, is considered to be alpha-synuclein, but other proteins, in particular the microtubule-associated protein tau, have been implicated in the pathogenesis of LBs. Tau is the major structural component of neurofibrillary tangles (NFTs). Both direct immunochemical studies of partially purified LBs and indirect immunohistochemical studies have suggested that LBs may contain tau, but most of these studies were based upon a single tau antibody, and immunologic cross-reactivity was not completely excluded. To gain insight into the relation between tau and alpha-synuclein in LBs, double immunostaining was performed in Lewy body cases with a rabbit polyclonal antibody to alpha-synuclein and a panel of monoclonal antibodies to phospho- and nonphospho-tau epitopes (Alz50, CP9, CP13, PG5, TG3, PHFI) that spanned the length of the tau molecule. Tau-immunoreactive LBs were present in the medulla in 80% of the cases, irrespective of Braak stage. All tau antibodies recognized at least some LBs, arguing against nonspecific antibody cross-reactivity. In most lesions the tau immunostaining was present at the periphery of the LB. The phospho-tau antibody, TG3, detected more LBs than any of the other tau antibodies. The proportion of LBs with tau immunoreactivity was greatest in neurons vulnerable to NETs, such as those in the locus ceruleus and basal nucleus of Meynert, and least in neurons resistant to NFTs, such as the dorsal motor nucleus of the vagus in the medulla. The present results suggest that tau may coaggregate with alpha-synuclein in LBs, especially in neuronal populations vulnerable to both NFTs and LBs.

Aged↗

The ability to detoxify the mycotoxin deoxynivalenol colocalizes with a major quantitative trait locus for Fusarium head blight resistance in wheat.

We investigated the hypothesis that resistance to deoxynivalenol (DON) is a major resistance factor in the Fusarium head blight (FHB) resistance complex of wheat. Ninety-six double haploid lines from a cross between 'CM-82036' and 'Remus' were examined. The lines were tested for DON resistance after application of the toxin in the ear, and for resistances to initial infection and spread of FHB after artificial inoculation with Fusarium spp. Toxin application to flowering ears induced typical FHB symptoms. Quantitative trait locus (QTL) analyses detected one locus with a major effect on DON resistance (logarithm of odds = 53.1, R2 = 92.6). The DON resistance phenotype was closely associated with an important FHB resistance QTL, Qfhs.ndsu-3BS, which previously was identified as governing resistance to spread of symptoms in the ear. Resistance to the toxin was correlated with resistance to spread of FHB (r = 0.74, P < 0.001). In resistant wheat lines, the applied toxin was converted to DON-3-O-glucoside as the detoxification product. There was a close relation between the DON-3-glucoside/DON ratio and DON resistance in the toxin-treated ears (R2 = 0.84). We conclude that resistance to DON is important in the FHB resistance complex and hypothesize that Qfhs.ndsu-3BS either encodes a DON-glucosyl-transferase or regulates the expression of such an enzyme.

Fusarium↗

Somatostatin-14 neurons in the ovine hypothalamus: colocalization with estrogen receptor alpha and somatostatin-28(1-12) immunoreactivity, and activation in response to estradiol.

Pituitary gland growth hormone (GH) secretion is influenced by two hypothalamic neuropeptides: growth hormone-releasing hormone (GHRH) and somatostatin. Recent data also suggest that estrogen modulates GH release, particularly at the time of the preovulatory luteinizing hormone surge, when a coincident surge of GH is observed in sheep. The GHRH neurons do not possess estrogen receptor alpha (ERalpha), suggesting that estrogen does not act directly on GHRH neurons. Similarly, few somatotropes express ERalpha, suggesting a weak pituitary effect of estradiol on GH. It was hypothesized, therefore, that estradiol may affect somatostatin neurons to modulate GH release from the pituitary. Using immunocytochemical approaches, the present study revealed that although somatostatin neurons were located in several hypothalamic sites, only those in the arcuate nucleus (13% +/- 2%) and ventromedial nucleus (VMN; 29% +/- 1%) expressed ERalpha. In addition, we found that all neurons immunoreactive for somatostatin-14 were also immunoreactive for somatostatin-28(1-12). To determine whether increased GH secretion in response to estradiol is through modulation of GHRH and/or somatostatin neuronal activity, a final study investigated whether c-fos expression increased in somatostatin- and GHRH-immunoreactive cells at the time of the estradiol-induced LH surge in intact anestrous ewes. Estradiol significantly (P < 0.05) increased the percentage of GHRH (estradiol, 75% +/- 3%; no estradiol, 19% +/- 2%) neurons expressing c-fos in the hypothalamus. The percentage of somatostatin-immunoreactive neurons coexpressing c-fos in the estradiol-treated animals was significantly (P < 0.05) higher (periventricular, 44% +/- 3%; arcuate, 72% +/- 5%; VMN, 81% +/- 5%) than in the control animals (periventricular, 22% +/- 1%; arcuate, 29% +/- 3%; VMN, 31% +/- 3%). The present study suggests that estradiol modulates the activity of GHRH and somatostatin neurons but that this effect is most likely mediated through an indirect interneuronal pathway.

Animals↗

Actin-binding properties and colocalization with actin during spermiogenesis of mammalian sperm calicin.

The nucleus of mammalian spermatozoa is surrounded by a rigid layer, the perinuclear theca, which is divided into a subacrosomal layer and a postacrosomal calyx. Among the proteins characterized in the perinuclear theca, calicin is one of the main components of the calyx. Its sequence contains three kelch repeats and a BTB/POZ domain. We have studied the association of boar calicin with F-actin and the distribution of boar and human calicin during spermiogenesis compared with the distribution of actin. Calicin was purified from boar sperm heads under nondenaturating conditions. The molecule bound actin with high affinity (K(d) = approximately 5 nM), and a stoichiometry of approximately one calicin per 12 actin monomers was observed. Gel filtration studies showed that calicin forms homomultimers (tetramers and higher polymers). According to immunocytochemical results, calicin is present (together with actin) in the acrosomal region of round spermatids and is mainly localized in the postacrosomal region of late spermatids and spermatozoa. Taken together, the results suggest that the affinity of calicin to F-actin allows targeting of calicin at the subacrosomal space of round spermatids, and that its ability to form homomultimers contributes to the formation of a rigid calyx.

Actins↗

Mitochondrial DNA deletion mutations colocalize with segmental electron transport system abnormalities, muscle fiber atrophy, fiber splitting, and oxidative damage in sarcopenia.

The in vivo cellular impact of age-associated mitochondrial DNA mutations is unknown. We hypothesized that mitochondrial DNA deletion mutations contribute to the fiber atrophy and loss that cause sarcopenia, the age-related decline of muscle mass and function. We examined 82,713 rectus femoris muscle fibers from Fischer 344 x Brown Norway F1 hybrid rats of ages 5, 18, and 38 months through 1000 microns by serial cryosectioning and histochemical staining for cytochrome c oxidase and succinate dehydrogenase. Between 5 and 38 months of age, the rectus femoris muscle in the hybrid rat demonstrated a 33% decrease in mass concomitant with a 30% decrease in total fibers at the muscle mid-belly. We observed significant increases in the number of mitochondrial abnormalities with age from 289 +/- 8 ETS abnormal fibers in the entire 5-month-old rectus femoris to 1094 +/- 126 in the 38-month-old as calculated from the volume density of these abnormalities. Segmental mitochondrial abnormalities contained mitochondrial DNA deletion mutations as revealed by laser capture microdissection and whole mitochondrial genome amplification. Muscle fibers harboring mitochondrial deletions often displayed atrophy, splitting and increased steady-state levels of oxidative nucleic damage. These data suggest a causal role for age-associated mitochondrial DNA deletion mutations in sarcopenia.

Aging↗

Membrane lipids, EGF receptors, and intracellular signals colocalize and are polarized in epithelial cells moving directionally in a physiological electric field.

Directed cell migration is essential for tissue formation, inflammation, and wound healing. Chemotaxis plays a major role in these situations and is underpinned by asymmetric intracellular signaling. Endogenous electric fields (EFs) are common where cell movement occurs, such as in wound healing, and cells respond to electric field gradients by reorienting and migrating directionally (galvanotaxis/electrotaxis). We show that a physiological EF redistributed both EGF (epidermal growth factor) receptors and detergent-insoluble membrane lipids asymmetrically, leading to cathodal polarization and enhanced activation of the MAP kinase, ERK1/2. This induced leading-edge actin polymerization in directionally migrating mammalian epithelial cells. Inhibiting the EGF receptor-MAP kinase signaling pathway significantly decreased leading edge actin asymmetry and directional migration. We propose a model in which EF-polarized membrane lipid domains and EGF receptors cause asymmetric signaling through MAP kinase, which drives directional cell migration. A comparison is made with the mechanisms underpinning chemotaxis.

Animals↗

Colocalization of nitric oxide synthase and NADPH-diaphorase in cultured myenteric neurones.

Nitric oxide synthase immunoreactivity and NADPH-diaphorase activity were examined in explant culture preparations of the myenteric plexus from beneath the taenia coli of the guinea-pig caecum. Nitric oxide synthase immunoreactive neurones formed approximately one third of the total neuronal population. NADPH-diaphorase positive neurones, demonstrated histochemically, constituted a similar proportion of the total number of neurones. Immunocytochemistry and NADPH-diaphorase histochemistry performed on the same preparations revealed that all nitric oxide synthase immunoreactive neurones expressed NADPH-diaphorase activity. This histochemical evidence is consistent with the view that nitric oxide may act as a regulatory agent in the guinea-pig caecum.

Amino Acid Oxidoreductases↗

Colocalization of NGF receptor with VIP in rat suprachiasmatic neurones.

Low-affinity nerve growth factor receptor (p75NGFR) and vasoactive intestinal polypeptide (VIP) immunoreactive neuronal structures and their interrelationship were investigated at light and electron microscopic levels in rat suprachiasmatic nucleus (SCN). p75NGFR immunoreactive neuronal perikarya were detected in the ventrolateral part, while the dorsolateral region contained mainly receptor positive fibres. Double-label immunocytochemistry showed that nearly all p75NGFR positive neurones in the ventrolateral region of the SCN also contained VIP. The axons of the receptor positive neurones terminated on unidentified neurones. In single-label experiments, the axons of the VIP containing neurones formed axodendritic synapses on cells containing the same peptide. The findings provide further insight on the chemical structural organization of the SCN.

Animals↗

NOS is present in the brain of Triatoma infestans and is colocalized with CCK.

Immunohistochemistry was used to establish the presence of nitric oxide synthase (NOS) in the central nervous system of Triatoma infestans which is the main vector for Chagas' disease in Argentina and neighbouring countries. In addition, we have investigated the presence of cholecystokinin (CCK) and studied the possible coexistence of these molecules. The results show NOS-like immunoreactivity (LI) in neurones of the soma rind of the protocerebrum, the optic lobe and in the lateral part of the sensory deutocerebrum with a few cells in the suboesophageal and the prothoracic ganglia. The distribution of CCK-LI was similar to that of NOS-LI and in several areas both molecules coexisted in neurones and fibres. The results suggest that nitric oxide may act as a neurotransmitter in the brain of insects.

Amino Acid Oxidoreductases↗

Ubiquitin and Alzheimer's amyloid beta precursor protein colocalize to endosomes-lysosomes in cultured human cells.

Chloroquine (CHQ)-sensitive cellular compartments, identified as endosomes-lysosomes (ELs), have been implicated in the proteolysis of amyloid beta precursor protein (A beta PP) in Alzheimer's disease. Here we show using immunocytochemistry and immunogold electron microscopy that not only A beta PP but also ubiquitin (Ub) co-localize to ELs in CHQ-treated human neuroblastoma (SK-N-SH) and glioblastoma (U-373). Immunoblotting analysis of cell lysates indicated a significant degree of CHQ-mediated interference in A beta PP metabolism in a time- and concentration-dependent manner. The implication is that abnormal intracellular accumulation of A beta PP and its C-terminal fragments beyond a certain threshold may trigger the Ub response. We hypothesize that Ub may play a role in A beta PP processing and/or trafficking to ELs, particularly in stress-related conditions.

Alzheimer Disease↗

Colocalization of calcitonin gene-related peptide and somatostatin in pancreatic islet cells and inhibition of insulin secretion by calcitonin gene-related peptide in the rat.

Calcitonin gene-related peptide (CGRP)- and somatostatin (SRIF)-containing cells were identified by immunocytochemical techniques in pancreatic islet cells of the rat. CGRP-containing cells were found primarily in the peripheral portion of the pancreatic islets. In addition, CGRP-containing cells also contained somatostatin, which identifies the islet CGRP-containing cells as D cells. In the present study, we also tested the effect of CGRP on gastrin-releasing peptide (GRP; 10(-9) M)- or cholecystokinin (CCK-8, 10(-9) M)-stimulated release of insulin from isolated rat islets in vitro. At concentrations of 10(-8)-10(-11) M, CGRP inhibited GRP- and CCK-8-stimulated release of insulin significantly when compared with GRP or CCK-8 alone. At the lowest concentration of CGRP (10(-11) M), the inhibitory effect of CGRP on CCK-8-stimulated release of insulin was statistically significant (p less than 0.05) and exceptionally potent (65-90% inhibition). We have also found that CGRP does not stimulate the release of SRIF from isolated islet cells. These findings suggest that CGRP may play a regulatory role in the release of insulin.

Animals↗

Adenovirus core protein VII contains distinct sequences that mediate targeting to the nucleus and nucleolus, and colocalization with human chromosomes.

During adenovirus infection, following capsid dissociation, core protein VII enters the host cell nucleus complexed with adenovirus DNA. In order to determine whether protein VII may have an active role in this nuclear import, regions of the preVII gene were amplified by PCR, and further oligonucleotide mutants were designed with site-directed mutation of codons for the basic amino acids arginine and lysine. Fragments were cloned into a mammalian expression plasmid to express the peptides as N-terminal fusions to enhanced green fluorescent protein. Results demonstrate that preVII protein contains both nuclear and nucleolar targeting sequences. Such signals may be important in the delivery of adenovirus DNA to the host cell nucleus during adenovirus infection. Furthermore, the data suggest that protein VII may bind to human chromosomes by means of two distinct domains, one sharing homology with the N-terminal regulatory tail of histone H3.

Adenoviruses, Human↗