Cytoplasm Of Cell Bodies Research Articles

Detection of Tissue Macrophages in Different Organs Using Antibodies to the Microglial Marker Iba-1.

Resident macrophages of different organs have structural and functional features, which can complicate their identification and analysis. A promising candidate for the role of a universal immunohistochemical marker of resident macrophages is the calcium-binding protein Iba-1, a well-known marker of brain microglia. The purpose of this work was to study the possibility of using one variant of antibodies to the Iba-1 protein for the immunohistochemical detection of resident macrophages in the liver, myocardium, lung, and choroid plexus of the rat brain. The study was performed on male Wistar rats (n = 15). It was shown that the use of rabbit monoclonal antibodies against Iba-1 allows highly effective detection of Kupffer cells in the liver, resident macrophages in the myocardium, alveolar and interstitial macrophages in the lung, and Kolmer cells in the choroid plexus of the rat brain. In all cases, the reaction is characterized by a high specificity and the absence of background staining. In contrast to the classical marker of macrophages, the CD68 molecule, the Iba-1 protein is evenly distributed in the cytoplasm of cell bodies and processes. This makes it possible to more fully identify cells using immunostaining for Iba-1, carry out their three-dimensional reconstructions, and study their structural and functional organization. Immunohistochemical reaction against Iba-1 can be successfully used as a universal alternative to other common methods for identifying resident macrophages.

Expression of p16 Within Myenteric Neurons of the Aged Colon: A Potential Marker of Declining Function.

Human colonic neuromuscular functions decline among the elderly. The aim was to explore the involvement of senescence. A preliminary PCR study looked for age-dependent differences in expression of CDKN1A (encoding the senescence-related p21 protein) and CDKN2A (encoding p16 and p14) in human ascending and descending colon (without mucosa) from 39 (approximately 50: 50 male: female) adult (aged 27–60 years) and elderly donors (70–89 years). Other genes from different aging pathways (e.g., inflammation, oxidative stress, autophagy) and cell-types (e.g., neurons, neuron axonal transport) were also examined. Unlike CDKN1A, CDKN2A (using primers for p16 and p14 but not when using p14-specific primers) was upregulated in both regions of colon. Compared with the number of genes appearing to upregulate in association with temporal age, more genes positively associated with increased CDKN2A expression (respectively, 16 and five of 44 genes studied for ascending and descending colon). Confirmation of increased expression of CDKN2A was sought by immunostaining for p16 in the myenteric plexus of colon from 52 patients, using a semi-automated software protocol. The results showed increased staining not within the glial cells (S100 stained), but in the cytoplasm of myenteric nerve cell bodies (MAP2 stained, with identified nucleus) of ascending, but not descending colon of the elderly, and not in the cell nucleus of either region or age group (5,710 neurons analyzed: n = 12–14 for each group). It was concluded that increased p16 staining within the cytoplasm of myenteric nerve cell bodies of elderly ascending (but not descending) colon, suggests a region-dependent, post-mitotic cellular senescence-like activity, perhaps involved with aging of enteric neurons within the colon.

The Y172 Monoclonal Antibody Against p-c-Jun (Ser63) Is a Marker of the Postsynaptic Compartment of C-Type Cholinergic Afferent Synapses on Motoneurons.

C-bouton-type cholinergic afferents exert an important function in controlling motoneuron (MN) excitability. During the immunocytochemical analysis of the role of c-Jun in MNs with a monoclonal (clone Y172) antibody against phospho (p)-c-Jun (serine [Ser]63), unexpected labeling was identified in the cell body cytoplasm. As predicted for c-Jun in adult spinal cord, very few, if any MNs exhibited nuclear immunoreactivity with the Y172 antibody; conversely, virtually all MNs displayed strong Y172 immunostaining in cytoplasmic structures scattered throughout the soma and proximal dendrites. The majority of these cytoplasmic Y172-positive profiles was closely associated with VAChT-positive C-boutons, but not with other types of nerve afferents contacting MNs. Ultrastructural analysis revealed that cytoplasmic Y172 immunostaining was selectively located at the subsurface cistern (SSC) of C-boutons and also in the inner areas of the endoplasmic reticulum (ER). We also described changes in cytoplasmic Y172 immunoreactivity in injured and degenerating MNs. Moreover, we noticed that MNs from NRG1 type III-overexpressing transgenic mice, which show abnormally expanded SSCs, exhibited an increase in the density and size of peripherally located Y172-positive profiles. A similar immunocytochemical pattern to that of the Y172 antibody in MNs was found with a polyclonal antibody against p-c-Jun (Ser63) but not with another polyclonal antibody that recognizes c-Jun phosphorylated at a different site. No differential band patterns were found by western blotting with any of the antibodies against c-Jun or p-c-Jun used in our study. In cultured MNs, Y172-positive oval profiles were distributed in the cell body and proximal dendrites. The in vitro lentiviral-based knockdown of c-Jun resulted in a dramatic decrease in nuclear Y172 immunostaining in MNs without any reduction in the density of cytoplasmic Y172-positive profiles, suggesting that the synaptic antigen recognized by the antibody corresponds to a C-bouton-specific protein other than p-c-Jun. Our results lay the foundation for further studies aimed at identifying this protein and determining its role in this particular type of synapse.

Role of Dynein and Dynactin (DCTN-1) in Neurodegenerative Diseases

The pathophysiology and concept of degeneration in central nervous system is very complex and overwhelming at times. There is a complex mechanism which exists among different molecules in the cytoplasm of cell bodies of neurons, antegrade and retrograde axonal transport of cargoes and accumulation of certain substances and proteins which can influence the excitatory neurotransmitter like glutamate initiating the process of neurodegeneration. Neurons have extensive processes and communication between those processes and the cell body is crucial to neuronal function, viability and survival over time with progression of age. Researchers believe neurons are uniquely dependent on microtubule-based cargo transport. There is enough evidence to support that deficits in retrograde axonal transport contribute to pathogenesis in multiple neurodegenerative diseases. Cytoplasmic dynein and its regulation by Dynactin (DCTN1) is the major molecular motor cargo involved in autophagy, mitosis and neuronal cell survival. Mutation in dynactin gene located in 2p13.1,is indeed studied very extensively and is considered to be involved directly or indirectly to various conditions like Perry syndrome, familial and sporadic Amyotrophic lateral sclerosis, Hereditary spastic paraplegia, Spinocerebellar Ataxia (SCA-5), Huntingtons disease, Alzheimers disease, Charcot marie tooth disease, Hereditary motor neuropathy 7B, prion disease, parkinsons disease, malformation of cortical development, polymicrogyria to name a few with exception of Multiple Sclerosis (MS).

Royal jelly increased map-2 expression in hippocampal neurons of hypothyroid rats: an immunohistochemical study

ABSTRACTWe investigated microtubule-associated protein 2 (MAP-2) immunoreactivity of hippocampal neurons and the potential role of royal jelly (RJ) in regulating MAP-2 during experimental hypothyroidism (HT). Thirty adult female Wistar albino rats were randomized into five groups: the control group was untreated, the sham control group was treated with 10 mg/kg 0.9% sterile saline injected intraperitoneally (i.p.), The RJ group was treated with 100 mg/kg RJ by oral gavage, the HT group was treated with 10 mg/kg propylthiouracil (PTU) i.p. to induce experimental hypothyroidism, and the HT + RJ group was treated with 10 mg/kg PTU i.p. + 100 mg/kg RJ by oral gavage. Oral and i.p. administrations were performed once daily for 20 days. Thyroid stimulating hormone (TSH) and free thyroxine (fT4) levels in the serum were measured biochemically, MAP-2 was measured immunohistochemically in the hippocampus and an immunohistochemical H score was calculated. MAP-2 immunoreactivity appeared in the cytoplasm of neuron cell bodies and dendrites in the hippocampal CA3 and CA1 regions in the control, sham control and RJ groups. MAP-2 immunoreactivity decreased significiantly in the HT group compared to control, sham control and RJ groups. Also, vascular dilation and swollen cells were observed following PTU administration. The degeneration that was observed in the HT group decreased by RJ administration. By contrast, MAP-2 immunoreactivity increased following administration of RJ. Experimental hypothyroidism reduced significiantly MAP-2 immunoreactivity in both the CA3 and CA1 regions and caused degeneration, including edema and vascular dilation, in the hippocampus. RJ increased MAP-2 expression and exhibited a therapeutic effect on the degenerative changes.

Effects of rhBMP-2 gene transfection to periodontal ligament cells on osteogenesis.

The present study aims to investigate the effect of recombinant human bone morphogenetic protein-2 (rhBMP-2) on the osteogenesis of periodontal ligament (PDL) cells. The expression vector of rhBMP-2 (pcDNA3.1-rhBMP-2) was established. PDL cells were obtained through the enzymatic digestion and tissue explant methods and verified by immunohistochemistry. Cells were classified into experimental (cells transfected with pcDNA3.1/rhBMP-2-EGFP), blank (cells with no transfection) and control group (cells transfected with empty plasmid). rhBMP-2 expression was assessed via Western blotting analysis. The mineralization ability, alkaline phosphatase (ALP) activity and level of related osteogenic biomarkers were detected to evaluate the osteogenic characteristics of PDL cells. The rhBMP-2 expression vector (pcDNA3.1-rhBMP-2) was successfully established. Primary PDL cells displayed a star or long, spindle shape. The cultured cells were long, spindle-shaped, had a plump cell body and homogeneous cytoplasm and the ellipse nucleus contained two or three nucleoli. Cells displayed a radial, sheaf-like or eddy-like arrangement after adherence growth. Immunohistochemical staining confirmed that cells originated from mesenchymal opposed to epithelium. The experimental group exhibited an enhanced mineralization ability, higher ALP activity and increased expression of rhBMP-2 and osteogenic biomarkers (Runx2, collagen type I and osteocalcin) than the blank and control group. The present study demonstrated that rhBMP-2 transfection enhances the osteogenesis of PDL cells and provides a possibility for the application of rhBMP-2 expression products in dental disease treatment.

INTRAHEPATIC CHOLANGIOCARCINOMA AND ENCEPHALOMALACIA IN A BUDGERIGAR (MELOPSITTACUS UNDULATUS)

Encephalomalacia is softening or loss of brain tissue following cerebral infarction; cerebral ischemia infection, craniocerebral trauma or other injuries. Cholangiocarcinomas are malignant neoplasms of biliary epithelium, which usually arise from the intrahepatic ducts. A budgerigar was presented with a history of ataxia, decreased activity, neck weakness and weight loss. Supportive care was attempted, but the bird did not improve and euthanasia was elected. At necropsy, the liver was pale and large. There were two symmetrical pale areas on the cortical surface of cerebrum. Representative specimens from all tissues were fixed in 10% buffered formalin, embedded in paraffin and stained with hematoxylin and eosin (HE). In gray matter of cerebral tissue, there were small clear vacuoles of varied size that formed in the cytoplasm of neuron cell bodies. Proliferation of capillaries was seen. The fluid-filled clear spaces were in the white matter especially in peduncles. There were a few thromboses in the vessels and neuronal necrosis of cerebrum. Neoplastic tissue was limited to the liver. There were numerous epithelial lined ducts with an associated dense fibrous stroma. Cholangiocarcinoma and encephalomalacia were confirmed based on the macroscopic and microscopic evidences. It seems that chronic exposure to a mycotoxin and vitamin E deficiency are probable causes of encephalpmalacia and cholangiocarcinoma in this bird.

Huntingtin Aggregation Kinetics and Their Pathological Role in aDrosophilaHuntington’s Disease Model

Huntington's disease is a neurodegenerative disorder resulting from expansion of a polyglutamine tract in the Huntingtin protein. Mutant Huntingtin forms intracellular aggregates within neurons, although it is unclear whether aggregates or more soluble forms of the protein represent the pathogenic species. To examine the link between aggregation and neurodegeneration, we generated Drosophila melanogaster transgenic strains expressing fluorescently tagged human huntingtin encoding pathogenic (Q138) or nonpathogenic (Q15) proteins, allowing in vivo imaging of Huntingtin expression and aggregation in live animals. Neuronal expression of pathogenic Huntingtin leads to pharate adult lethality, accompanied by formation of large aggregates within the cytoplasm of neuronal cell bodies and neurites. Live imaging and Fluorescence Recovery After Photobleaching (FRAP) analysis of pathogenic Huntingtin demonstrated that new aggregates can form in neurons within 12 hr, while preexisting aggregates rapidly accumulate new Huntingtin protein within minutes. To examine the role of aggregates in pathology, we conducted haplo-insufficiency suppressor screens for Huntingtin-Q138 aggregation or Huntingtin-Q138-induced lethality, using deficiencies covering ~80% of the Drosophila genome. We identified two classes of interacting suppressors in our screen: those that rescue viability while decreasing Huntingtin expression and aggregation and those that rescue viability without disrupting Huntingtin aggregation. The most robust suppressors reduced both soluble and aggregated Huntingtin levels, suggesting toxicity is likely to be associated with both forms of the mutant protein in Huntington's disease.

B04 A genetic screen targeting suppressors of aggregation and neurodegeneration in a Drosophila Huntington's disease model

BackgroundHuntington's disease is a neurodegenerative disorder resulting from expansion of a polyglutamine tract in the huntingtin protein (Htt). Polyglutamine expansion in Htt leads to intracellular aggregates within neurons although it...

Expression of GFAT1 and OGT in podocytes: Transport of glucosamine and the implications for glucose uptake into these cells

Glutamine:fructose-6-phosphate amidotransferase (GFAT) and N-acetylglucosaminyltransferase (OGT) participate in glucosamine (GlcN) production and its utilization in O-glycosylation, one of key post-translational modifications of nuclear and cytoplasmic proteins. For this purpose, cells require a high rate of intracellular production of GlcN and/or significant GlcN delivery. We studied the expression of GFAT1 and OGT and measured uptake of glucose and GlcN in cultured rat podocytes, the main cellular component of glomerular filtration barrier. RT-PCR revealed the presence of both GFAT1 and OGT mRNA. Immunofluorescence of GFAT1 has shown staining signal diffused within the cytoplasm of the cell body and processes. However, OGT was distinctly visible around the nucleus and, in diffuse form, within the cytoplasm of cell bodies and processes. Glucose was transported (1.3 +/- 0.2 nmol/min/mg protein) mainly by facilitative transporter systems whilst GlcN uptake (1.1 +/- 0.2 nmol/min/mg protein) in a significant part, involved a sodium-dependent transporter. There was interplay between glucose and GlcN uptake. In the presence of GlcN (50 microM), the rate of glucose uptake decreased by about 50%. The rate of GlcN uptake decreased by 28% in the presence of 5.6 mM glucose. Our results suggest that cultured podocytes possess limited ability to synthesize GlcN internally and therefore may need to receive GlcN from the extracellular environment.

Accumulation of tumor-suppressor PTEN in Alzheimer neurofibrillary tangles

The phosphatase and tensin homologue deleted on chromosome 10 (PTEN) negatively regulates intracellular levels of PIP3 and antagonizes the PI3K signaling pathway important for cell survival. The present study determined whether altered distribution of PTEN occurs in Alzheimer's disease (AD) brains. We investigated a possible role for PTEN in postmortem brain tissues from elderly controls and patients with AD using immunoblotting and microscopic analyses. Intense immunolabeling was found in the large neurons such as pyramidal cells. In normal neurons, PTEN was located in the nucleus, the cytoplasm of cell bodies and the proximal portion of apical dendrites. Reduced expression and redistribution of PTEN was seen in the remaining neurons in AD. In addition, PTEN was redistributed in damaged neurons from the nucleus and cytoplasm to neuritic pathology such as intracellular neurofibrillary tangles (NFTs), neuropil threads and dystrophic neurites within senile plaques in AD hippocampus, subiculum, entorhinal cortex and angular gyrus. Furthermore, double immunofluorescence staining showed dual labeling of intracellular NFTs for PTEN and tau, labeling of some axons for PTEN and phosphorylated neurofilament, and weak labeling of a few reactive astrocytes around senile plaques for PTEN and GFAP. Double labeling of NFTs was observed in a subset of tangle-bearing neurons either for PTEN and GSK3β or for PTEN and MEK. Thus our results suggest that PTEN delocalized from the nucleus to the cytoplasm and to intracellular NFTs may cause a deregulation of PI3K pathway in the cytoplasm and may induce the nuclear dysfunction of PTEN in AD degenerating neurons.

Differential expression of ATP7A, ATP7B and CTR1 in adult rat dorsal root ganglion tissue.

BackgroundATP7A, ATP7B and CTR1 are metal transporting proteins that control the cellular disposition of copper and platinum drugs, but their expression in dorsal root ganglion (DRG) tissue and their role in platinum-induced neurotoxicity are unknown. To investigate the DRG expression of ATP7A, ATP7B and CTR1, lumbar DRG and reference tissues were collected for real time quantitative PCR, RT-PCR, immunohistochemistry and Western blot analysis from healthy control adult rats or from animals treated with intraperitoneal oxaliplatin (1.85 mg/kg) or drug vehicle twice weekly for 8 weeks.ResultsIn DRG tissue from healthy control animals, ATP7A mRNA was clearly detectable at levels similar to those found in the brain and spinal cord, and intense ATP7A immunoreactivity was localised to the cytoplasm of cell bodies of smaller DRG neurons without staining of satellite cells, nerve fibres or co-localisation with phosphorylated heavy neurofilament subunit (pNF-H). High levels of CTR1 mRNA were detected in all tissues from healthy control animals, and strong CTR1 immunoreactivity was associated with plasma membranes and vesicular cytoplasmic structures of the cell bodies of larger-sized DRG neurons without co-localization with ATP7A. DRG neurons with strong expression of ATP7A or CTR1 had distinct cell body size profiles with minimal overlap between them. Oxaliplatin treatment did not alter the size profile of strongly ATP7A-immunoreactive neurons but significantly reduced the size profile of strongly CTR1-immunoreactive neurons. ATP7B mRNA was barely detectable, and no specific immunoreactivity for ATP7B was found, in DRG tissue from healthy control animals.ConclusionsIn conclusion, adult rat DRG tissue exhibits a specific pattern of expression of copper transporters with distinct subsets of peripheral sensory neurons intensely expressing either ATP7A or CTR1, but not both or ATP7B. The neuron subtype-specific and largely non-overlapping distribution of ATP7A and CTR1 within rat DRG tissue may be required to support the potentially differing cuproenzyme requirements of distinct subsets of sensory neurons, and could influence the transport and neurotoxicity of oxaliplatin.

Fixation conditions affect the immunoreactivity of gustducin in rat vallate taste buds

This study aimed to examine gustducin immunoreactivities when adopting various immunostaining conditions in rat vallate taste buds. The occurrence and intensity of the immunoreactivities exhibited specific patterns in accordance with the fixation time. The immunoreactions were localized to only taste hairs, the upper part of the taste bud, after short fixation periods but then to the cell-body cytoplasm excluding the taste hairs after long fixation periods. These immunohistochemical data suggest that the staining protocols, especially the fixation time, cause discrepancies in gustducin immunoreactivities.

Drebrin (developmentally regulated brain protein) is associated with axo-somatic synapses and neuronal gap junctions in rat mesencephalic trigeminal nucleus

Drebrin (developmentally regulated brain protein)-like immunoreactivity was investigated in the adult rat mesencephalic trigeminal nucleus (MTN) using light and electron microscope. Intense immunoreactive puncta were observed on the cytoplasmic membrane and within the cytoplasm. The cytoplasm was also faintly immunopositive for drebrin, and thus MTN somata other than multipolar cells were distinguishable from non-MTN somata. These immunoreactive cell bodies were localized from the level of the superior colliculus to the pons. Electron microscopic observation showed that the post-synaptic cytoplasmic membrane at axo-somatic synapses was immunoreactive for drebrin. Drebrin-like immunoreactivity was also observed on spine-like processes emanating from MTN somata. In addition, the post-synaptic cytoplasmic membrane at axo-somatic synapses was also immunopositive for drebrin. Within the cytoplasm of MTN cell bodies, a part of the rough endoplasmic reticulum and neighboring structures were also immunopositive. Further, both ends of the somato-somatic close appositions that contained neuronal gap junctions harbored immunoreactive structures. We can infer from the results that drebrin is an ideal marker protein for MTN cell bodies. The abundance of drebrin-like immunoreactivity in the MTN neurons suggests that the MTN has highly flexible synaptogenesis.

The Caenorhabditis elegans AMP-activated Protein Kinase AAK-2 Is Phosphorylated by LKB1 and Is Required for Resistance to Oxidative Stress and for Normal Motility and Foraging Behavior

AAK-2 is one of two alpha isoforms of the AMP-activated protein kinase in Caenorhabditis elegans and is involved in life span maintenance, stress responses, and germ cell cycle arrest upon dauer entry. We found that AAK-2 was phosphorylated at threonine 243 in response to paraquat treatment and that this phosphorylation depends on PAR-4, the C. elegans LKB1 homologue. Both aak-2 mutation and par-4 knockdown increased the sensitivity of C. elegans worms to paraquat, and the double deficiency did not further increase sensitivity, indicating that aak-2 and par-4 act in a linear pathway. Both mutations also slowed body bending during locomotion and failed to reduce head oscillation in response to anterior touch. Consistent with this abnormal motility and behavioral response, expression of the AAK-2::green fluorescent protein fusion protein was observed in the ventral cord, some neurons, body wall muscle, pharynx, vulva, somatic gonad, and excretory cell. Our study suggests that AMPK can influence the behavior of C. elegans worms in addition to its well known function in metabolic control.

Differential expression of neuronal ACE2 in transgenic mice with overexpression of the brain renin-angiotensin system

Angiotensin-converting enzyme 2 (ACE2) is a newly discovered carboxy-peptidase responsible for the formation of vasodilatory peptides such as angiotensin-(1-7). We hypothesized that ACE2 is part of the brain renin-angiotensin system, and its expression is regulated by the other elements of this system. ACE2 immunostaining was performed in transgenic mouse brain sections from neuron-specific enolase-AT(1A) (overexpressing AT(1A) receptors), R(+)A(+) (overexpressing angiotensinogen and renin), and control (nontransgenic littermates) mice. Results show that ACE2 staining is widely distributed throughout the brain. Using cell-type-specific antibodies, we observed that ACE2 staining is present in the cytoplasm of neuronal cell bodies but not in glial cells. In the subfornical organ, an area lacking the blood-brain barrier and sensitive to blood-borne angiotensin II, ACE2 was significantly increased in transgenic mice. Interestingly, ACE2 mRNA and protein expression were inversely correlated in the nucleus of tractus solitarius/dorsal motor nucleus of the vagus and the ventrolateral medulla, when comparing transgenic to nontransgenic mice. These results suggest that ACE2 is localized to the cytoplasm of neuronal cells in the brain and that ACE2 levels appear highly regulated by other components of the renin-angiotensin system, confirming its involvement in this system. Moreover, ACE2 expression in brain structures involved in the control of cardiovascular function suggests that the carboxypeptidase may have a role in the central regulation of blood pressure and diseases involving the autonomic nervous system, such as hypertension.

A novel Krüppel related factor consisting of only a KRAB domain is expressed in the murine trigeminal ganglion

The largest family of zinc-finger (ZnF) transcription factors is that containing the Krüppel-associated box, or KRAB domain. The amino-terminal KRAB domain of these proteins functions as a transcriptional repressor with the downstream ZnF motifs providing DNA-binding specificity. Here we report the identification and characterization of a novel murine Krüppel-related factor (KLF), MIF1, which contains a KRAB domain but lacks a ZnF motif. Western blot analysis identified MIF1-like proteins in the murine trigeminal ganglion (TG) and immunostaining localized these proteins primarily to the cytoplasm of TG neuronal cell bodies. In situ hybridization for Mif1 transcripts confirms the selective expression of Mif1 in TG neurons. Consistent with the non-nuclear localization of MIF1 we could detect no transcriptional repressor activity of the MIF1 protein. However MIF1 appears to be capable of interacting with the co-repressor TIF1β and exhibits transcription repressor activity when fused to yeast GAL4 binding domain protein. Genomic analysis of Mif1 sequence suggests that the Mif1 transcript may result from splicing of a longer KRAB-ZnF containing transcript.

Organization and translation of mRNA in sympathetic axons.

Many axons carry out the synthesis of macromolecules independent of their cell bodies but the nature, organization and magnitude of axonal protein synthesis remain unclear. We have examined these features in axons of chick sympathetic neurons in cell culture. In situ hybridization showed that poly(A) mRNA is abundant and non-uniformly distributed in nearly all axons. The specific transcripts for beta-actin and actin-depolymerizing factor (ADF) were also present and non-uniformly distributed in axons, with an approximately hundredfold higher concentration in growth cones, branch points and axonal varicosities than in the axon shaft. Immunoprecipitation using specific antibodies indicates that beta-actin, ADF and neurofilament protein (NF) are translated in axons independently of cell bodies. Quantification of the distribution of beta-actin and ADF mRNAs showed that their ability to enter the axon was likely to be a property of the neuron as a whole rather than of individual axons. To compare the distribution of axonally translated protein to that of mRNA, we performed 35S metabolic labeling with axons separated from their cell bodies. Axonally synthesized proteins were distributed throughout the axons and their synthesis was inhibited by cycloheximide but not by chloramphenicol. Proteins translated mainly or exclusively in axons or cell bodies were both detected by metabolic labeling. Axons separated from their cell bodies synthesized up to 5% as much protein in a 3-hour period as did intact neurons. Because axons in our culture conditions contain approximately 50% of the non-nuclear volume of the neurons, we estimate that axoplasm of sympathetic neurons has a protein synthetic capacity per unit volume equal to 10% that of cell body cytoplasm.

Cellular compartmentalization of phosphorylated eIF2α and neuronal NOS in human temporal lobe epilepsy with hippocampal sclerosis

Hippocampal sclerosis (HS) is the most common neuropathologic finding in patients with medically refractory temporal lobe epilepsy (TLE). The mechanisms resulting in neuronal injury and cell loss in HS are incompletely understood, but inhibition of protein synthesis may play a pivotal role in these processes. This study examined the relationships between two molecules known to be involved in reduced protein synthesis in animals subjected to traumatic brain injury. Translational initiation of protein synthesis is inhibited when 2α (eIF2α) is phosphorylated. Recently, nitric oxide (NO) has been shown to reduce protein synthesis by inducing phosphorylation of eIF2α. We performed immunocytochemistry for eIF2α(P) and histochemistry (NADPH-D reaction) for nitric oxide synthase (NOS) to determine the distribution of these molecules in hippocampi removed from patients undergoing anterior temporal lobectomy (ATL) for medically intractable TLE due to HS. The greatest number of eIF2α(P) positive cells was in the CA1 sector of the hippocampus, followed by the hilus of the dentate gyrus. NADPH-D positive neurons were observed most often in the hilus. Labeling in both instances involved neuronal cell body cytoplasm and varicose processes. Combination of both staining procedures revealed close relationships between differentially labeled neurons within the hilus. The results suggest that NO participates in the phosphorylation of eIF2α since we demonstrated that nNOS processes are closely related to eIF2α(P) positive cells. This may occur through activation of kinases such as PERK, which was recently revealed. In human, TLE protein synthesis inhibition may occur at the translational level since the eIF2α (P) labeling is cytoplasmic. Protein synthesis inhibition may contribute to neuronal cell injury and death in HS.

Cellular localization and distribution of neurokinin-1 receptors in the rat stomach

In the stomach, the majority of substance P's effects are mediated by the activation of neurokinin-1 (NK1) receptors. The gastric cellular distribution of these receptors in Wistar and Sprague–Dawley rats was determined using immunocytochemistry. The localization of the NK1 receptors with respect to von Willebrand's factor, protein gene product 9.5, substance P, vasoactive intestinal peptide, and calcitonin gene-related peptide was also determined. Results show that NK1 receptor immunoreactivity was dependent on the duration of fixation. In corpus and antrum tissues that were fixed in 4% paraformaldehyde for 30 min, the presence of NK1 receptor immunoreactivity was demonstrated on nerve fibers throughout the stomach, on the surface and in the cytoplasm of myenteric cell bodies, on circular smooth muscle cells, and on vascular endothelial cells. This was observed in tissues from both rodent strains. Overnight fixation in the same fixative, however, demonstrated the presence of NK1 receptor immunoreactivity only on nerve fibers and cell bodies of the myenteric plexus, and on circular smooth muscle cells. In 30-min fixed tissues, the localization of NK1R immunoreactivity on vascular endothelial cells and nerve fibers was confirmed by co-localization with von Willebrand's factor and protein gene product 9.5 immunoreactivity, respectively. In both rodent strains, NK1 receptor immunoreactivity was co-localized with substance P immunoreactivity on nerve fibers of the longitudinal and circular muscle. In the Wistar rat, NK1 receptor immunoreactivity was co-localized with vasoactive intestinal peptide immunoreactivity or calcitonin gene-related peptide immunoreactivity throughout the stomach. However, in the Sprague–Dawley rat, NK1 receptor immunoreactivity was only co-localized with calcitonin gene-related peptide immunoreactivity in a minority of fibers of the circular muscle. The overall results of this study show that the antigenic epitopes of the NK1 receptor are sensitive to overfixation. When tissues were not overfixed, NK1 receptor immunoreactivity was distributed more extensively throughout the rat stomach than has been described previously. The results of this study provide the anatomical basis for many of the actions of substance P in the rat stomach.