Localization of insulin-like immunoreactive neurons in the rat gracile nucleus

An insulin-like immunoreactivity (ILIR) was localized in the neuronal somata, dendrites and myelinated axons in the gracile nucleus of the male Wistar rat. In the neuronal somata, the reaction product was dispersed in the cell nucleus and cytoplasm. In the cell nucleus, the reaction product was scattered throughout the nucleoplasm, but not within the nucleolus. In the cytoplasm, the reaction product was evenly distributed, mainly in the vicinity of the cisternae of the rough endoplasmic reticulum. In labelled dendrites, the reaction product was closely associated with the parallel arrays of neurotubules and postsynaptic densities. Most of these labelled dendrites were postsynaptic to unlabelled axon terminals. A labelled dendrite often formed the central element of a synaptic glomerulus with several unlabelled axon terminals. Numerous labelled myelinated axons were also present in the neuropil. However, axon terminals appeared to be unlabelled. It is hypothesized that insulin-like substance(s) may be modulating nuclear activities as well as neurotransmission at the synapse in the gracile nucleus.     

Interleukin-2-activated killer cell activity in colorectal tumor patients: evaluation of in vitro effects by prothymosin alpha1

The effects of prothymosin alpha1 (Pro alpha1) in combination with interleukin-2 (IL-2) on peripheral blood lymphocytes from 50 colorectal tumor patients at different stages were studied with respect to immunocytotoxicity, adhesion to cultured SW620 colon carcinoma cells, secretion of cytokines and expression of adhesion and surface marker molecules. On average, the patients showed lower natural killer (NK) cell activity than healthy donors, which was associated with a lower adhesion capacity to the tumor target cells. The NK cell activity of the patients was inversely related to the tumor stage. The generation of lymphokine(IL-2)-activated killer (LAK) cell activity was found to be comparable on lymphocytes from healthy individuals and patients and was not correlated to tumor stage. Pro alpha1 stimulated patients' LAK cell activity only, primarily at the early stage (Dukes A/B). The Pro alpha1 effect was associated with an increased adhesion of lymphocytes to tumor target cells and an increased secretion of the deficient IL-2-induced IFN gamma secretion. No significant effects on the low level of TNF alpha secretion was noted. By flow cytometry, Pro alpha1 in combination with IL-2 augmented the expression of the NK cell markers CD56, CD16/56, the subset CD3/16/56 and CD25 on lymphocytes of the patients. In contrast, Pro alpha1 was equally effective by increasing the expression of CD18 and CD11a on lymphocytes from the patients and from normal controls. In conclusion, Pro alpha1, in combination with IL-2, can partially normalize lymphocyte deficiencies of colon cancer patients in vitro. This potential might provide an experimental basis for applying Pro alpha1 or related thymic peptides in selected immunotherapies against colorectal tumors.     

Localization and expression of the alpha1A-1, alpha1B and alpha1D-adrenoceptors in hyperplastic and non-hyperplastic human prostate

PURPOSE: To determine the expression and localization of the alpha1A-1, alpha1B and alpha1D-adrenoceptor (AR) subtypes in hyperplastic and non-hyperplastic human prostate tissue. MATERIALS AND METHODS: The expression of the alpha1-AR subtypes was examined at the mRNA level by quantitative solution hybridization, and at the protein level by immunohistochemistry using subtype selective antibodies. RESULTS: While the overall level of alpha1-AR mRNA was not significantly different between hyperplastic and non-hyperplastic tissue, there were significant differences in the ratio of the alpha1-AR subtypes expressed in the two tissue types. The most significant finding from these studies was the reduced expression of the alpha1b-AR mRNA in both glandular and stromal hyperplasia. By immunohistochemistry, the alpha1A-1-AR was detected in the stroma and not in the glandular epithelium. The alpha1B-AR was localized predominantly in the epithelium and was weakly present in the stroma. Lower levels of the alpha1B-AR were detected in the hyperplastic prostatic epithelium. The alpha1D-AR was detected in areas of stroma and was abundantly present in blood vessels. CONCLUSIONS: The alpha1A-1-, alpha1B- and alpha1D-AR subtypes are differentially localized in human prostate, and the expression levels of all three subtypes are altered in BPH. Alterations in a1-AR subtype expression (particularly the alpha1B-AR) in BPH cannot be solely attributed to changes in tissue morphometry resulting from hyperplasia and may be of significance in the pathogenesis of BPH.     

Localization of granzyme B in the nucleus. A putative role in the mechanism of cytotoxic lymphocyte-mediated apoptosis

One mechanism used by cytotoxic T cells and natural killer cells to kill target cells involves synergy between the pore-forming protein, perforin, and a serine protease termed granzyme B, both constituents of the cytoplasmic granules of cytolytic lymphocytes. Exposing susceptible cells to perforin and granzyme B results in apoptosis, the morphological consequences of which are most clearly seen in the nucleus. It is conventionally accepted that perforin acts by perforating the target cell membrane; however, the site and mode of action of granzyme B are unknown. We have addressed this issue using Western blotting, proteolytic assays, and confocal laser scanning microscopy to demonstrate that purified human granzyme B can be taken up in large amounts and bound within nuclei. By contrast, perforin and nongranzyme serine proteases did not undergo nuclear uptake. Both unglycosylated human granzyme B (26 kDa) and that bearing high mannose glycosylation (32 kDa) were internalized and bound within nuclei, but forms greater than 32 kDa with complex carbohydrate addition were excluded. The uptake of granzymes was not dependent on net charge, as nuclei absorbed similar quantities of granzyme B at neutral pH and through a range of basic pHs but did not take up other very basic serine proteases such as the mouse mast cell protease 5. Confocal laser scanning microscopy indicated nuclear and nucleolar accumulation of fluoresceinated granzyme B by isolated nuclei. Measurement of the kinetics of nuclear import using an in vitro nuclear transport assay indicated maximal levels of nuclear accumulation of granzyme about 2.5-fold above those in the cytoplasm and nucleolar accumulation a further 3-4-fold higher. Nuclear and nucleolar accumulation were exceedingly rapid, reaching half-maximal levels within 3.3 and 7.5 min, respectively, implying that nuclear accumulation probably occurs prior to transport to the nucleolus. Our observations may provide a mechanism explaining how aspartate-specific cell death proteases access the nuclear substrate poly(ADP-ribose) polymerase, the cleavage of which is an early event in apoptosis.     

Localization of tyrosine hydroxylase in neuronal targets and efferents of the area postrema in the nucleus tractus solitarii of the rat

Catecholamines in the nucleus tractus solitarii (NTS) have been implicated in autonomic responses to circulating hormones that act on neurons in the area postrema, the most caudal circumventricular organ in brain. We combined immunoperoxidase labeling of the anterograde tracer, Phaseolus vulgaris leucoagglutinin (PHAL) with immunogold-silver labeling of tyrosine hydroxylase to determine whether this enzymatic marker for catecholamines was present in efferents from the area postrema or their targets in the rat NTS. At survival periods of 10-12 days after PHAL injections into the area postrema, light microscopy revealed numerous varicose processes containing peroxidase reaction product for PHAL in the dorsomedial, medial, and commissural NTS. Some of these labeled processes were located near neuronal perikarya and processes containing immunogold-silver intensified reaction product for tyrosine hydroxylase. Electron microscopy of the commissural and dorsomedial NTS established that the majority of the labeling for PHAL was in axon terminals, whereas immunogold labeling for tyrosine hydroxylase was mainly in soma and dendrites. Only 3 out of 579 PHAL-labeled terminals also contained detectable tyrosine hydroxylase immunoreactivity. Fifty-eight percent (335/579) of the PHAL-labeled terminals formed synapses with recognized symmetric junctions, whereas the remainder lacked synaptic specializations within the examined series of serial sections. Of those PHAL terminals forming recognized symmetric junctions, 22% were on tyrosine hydroxylase-immunoreactive dendrites, 74% on unlabeled dendrites and 4% on unlabeled axon terminals. From a total of 1,250 observed contacts on tyrosine hydroxylase labeled dendrites, 88 (7%) contained PHAL, 9 (< 1%) contained TH, and 1,180 (93%) lacked detectable immunoreactivity and formed primarily symmetric synapses. We conclude that a few catecholamine, but mainly noncatecholamine efferents from the area postrema provide a monosynaptic, and most likely inhibitory input to target neurons both with and without tyrosine hydroxylase immunoreactivity in the dorsomedial and commissural NTS. Synapses between the efferent terminals from the area postrema and tyrosine hydroxylase labeled and unlabeled dendrites as well as unlabeled axons in these specific subnuclei of the NTS suggest multiple sites for modulation of gastric and cardiovascular reflexes in response to circulating peptides.     

Export of importin alpha from the nucleus is mediated by a specific nuclear transport factor

NLS proteins are transported into the nucleus by the importin alpha/beta heterodimer. Importin alpha binds the NLS, while importin beta mediates translocation through the nuclear pore complex. After translocation, RanGTP, whose predicted concentration is high in the nucleus and low in the cytoplasm, binds importin beta and displaces importin alpha. Importin alpha must then be returned to the cytoplasm, leaving the NLS protein behind. Here, we report that the previously identified CAS protein mediates importin alpha re-export. CAS binds strongly to importin alpha only in the presence of RanGTP, forming an importin alpha/CAS/RanGTP complex. Importin alpha is released from this complex in the cytoplasm by the combined action of RanBP1 and RanGAP1. CAS binds preferentially to NLS-free importin alpha, explaining why import substrates stay in the nucleus.     

The effect of alpha2-adrenergic drugs on the activity of neurons in the rat nucleus raphe magnus in vitro

The nucleus raphe magnus (NRM) is an important descending inhibitory system for pain transmission. We tested whether clonidine, an alpha2-adrenergic agonist, and yohimbine, an alpha2-adrenergic antagonist, modulate the activity of NRM neurons using extracellular recording in a rat brainstem slice preparation. Clonidine 1-20 microM increased firing frequencies (FF) in 22 (37%) and decreased FF in 6 (10%) spontaneously active neurons. Correlation between the concentrations of clonidine and FF changes was unremarkable. Eight spontaneously active neurons (13%) showed increases followed by decreases in FF with increasing doses of clonidine. The remaining 24 neurons (40%) showed no change in FF. Yohimbine 1 microM decreased FF in 38 spontaneously active neurons (58%), whereas the remaining 27 neurons (42%) showed no change in FF. In some neurons, yohimbine antagonized the increase or decrease in FF by application of clonidine. In three silent neurons (25%), clonidine (5 or 10 microM) induced firing activity, which stopped or decreased with the increasing doses of clonidine. In the remaining nine neurons (75%), clonidine did not induce firing activity. We conclude that activation and inhibition of alpha2-adrenergic receptors of NRM neurons augments and suppresses output of the descending inhibitory pain pathway. Implications: The nucleus raphe magnus is implicated in descending control of the nociceptive processes. We found that clonidine and yohimbine increased and decreased, respectively, the firing activity of a substantial number of nucleus raphe magnus neurons. Clonidine and may facilitate and yohimbine may reduce the outflow of the descending inhibitory pathway.     

Localization and characterization of nitric oxide synthase in the rat suprachiasmatic nucleus: evidence for a nitrergic plexus in the biological clock

Behavioral and electrophysiological evidence indicates that the biological clock in the hypothalamic suprachiasmatic nuclei (SCN) can be reset at night through release of glutamate from the retinohypothalamic tract and subsequent activation of nitric oxide synthase (NOS). However, previous studies using NADPH-diaphorase staining or immunocytochemistry to localize NOS found either no or only a few positive cells in the SCN. By monitoring conversion of L-[3H]arginine to L-[3H]-citrulline, this study demonstrates that extracts of SCN tissue exhibit NOS specific activity comparable to that of rat cerebellum. The enzymatic reaction requires the presence of NADPH and is Ca2+/calmodulin-dependent. To distinguish the neuronal isoform (nNOS; type I) from the endothelial isoform (type III), the enzyme activity was assayed over a range of pH values. The optimal pH for the reaction was 6.7, a characteristic value for nNOS. No difference in nNOS levels was seen between SCN collected in day versus night, either by western blot or by enzyme activity measurement. Confocal microscopy revealed for the first time a dense plexus of cell processes stained for nNOS. These data demonstrate that neuronal fibers within the rat SCN express abundant nNOS and that the level of the enzyme does not vary temporally. The distribution and quantity of nNOS support a prominent regulatory role for this nitrergic component in the SCN.     

Localization of glutathione S-transferases alpha and pi in human embryonic tissues at 8 weeks gestational age

Glutathione S-transferases (GST) are a family of enzymes involved in the detoxification of xenobiotics. In humans, GST are divided into four different classes, alpha, mu, pi and theta, with partly overlapping substrate specificity and a tissue-specific expression pattern. We studied the cellular distribution of GST alpha and pi in a variety of human embryonic organs obtained from an extra-uterine monozygotic twin pregnancy at 8 weeks' gestational age. Tissues were fixed in 4% paraformaldehyde and embedded in paraffin. Three 4 microm thick sections were used, one for routine haematein and eosin staining, the others for immunohistochemical determination using monoclonal and polyclonal antibodies against GST alpha and pi, respectively. Both GST alpha and pi were present in hepatocytes, gastrointestinal epithelium, adrenal gland medulla, and tela chorioidea in the telencephalon. GST pi, but not alpha, was found in the epithelium of pancreatic and pulmonary glands, trachea, nephrons and urinary collecting ducts, as well as in the pia mater of the telencephalon and in developing nerve tissue in the gastrointestinal muscularis mucosae. In summary, we have demonstrated that immunoreactive protein for both GST alpha and pi is expressed in the human embryo at 8 weeks' gestational age. The early expression of GST alpha and pi in the epithelia of the urinary and digestive tracts and the respiratory system supports the importance of GST in the detoxification of potentially toxic or carcinogenic compounds. Our results suggest that the embryo itself is capable of detoxifying noxious compounds that are generated intracellularly or that cross the trophoblastic tissue.     

Noradrenaline modifies the spontaneous spiking activity of red nucleus neurons in the rat by activation of alpha 2- and beta-adrenoceptors

We have investigated the effects of noradrenaline (NA) on the spontaneous firing activity of red nucleus (RN) neurons recorded extracellularly in anesthetized rats by using an in vivo electrophysiological technique. Microiontophoretic applications of NA (5-100 nA for 30 s) modified the background firing rate in 99 out of 124 neurons and three different patterns of response were observed in distinct cells. In 61% of the responding neurons NA decreased the mean firing rate, whereas 22% of the neurons responded to NA application with an increase of their spiking activity; in a smaller group of cells (17%) NA exerted a biphasic inhibitory/excitatory effect on the spontaneous firing rate. The effects of NA were reversible and dose-dependent. From histological examination, the neurons responding to NA with a purely inhibitory effect were scattered throughout the RN. On the other hand, the neurons responding to NA with an excitation were found to be more numerous in the dorso-medial part of the RN, whereas the neurons in which NA induced biphasic effects appeared to be segregated in the outer lateral portion of the RN. The alpha 2-adrenoceptor antagonist yohimbine completely blocked the inhibitory effect of NA but was unable to antagonize the excitatory response. In addition, the inhibitory effect of NA was mimicked by clonidine, a selective agonist of alpha 2-adrenoceptors; clonidine had no effect on those cells which responded to NA with an increase of the mean firing rate. The excitatory effect of NA was mimicked by the beta-receptor agonist isoprenaline and was antagonized by timolol, a selective antagonist of beta-adrenoceptors. Isoprenaline was ineffective in those cells in which NA exerted inhibitory responses. Taken together, our results indicate that the inhibitory effect of NA on the firing activity of rat RN neurons were mediated by alpha 2-adrenoceptors, whereas beta-adrenoceptors were responsible for the excitatory effects.     

Localization of Myf-5, MRF4 and alpha cardiac actin mRNAs in regenerating Xenopus skeletal muscle

Dendrotoxin I (DTX-I) is a 60-residue peptide from the venom of the black mamba snake Dendroaspis polylepis, which binds to neuronal K+ channels. The structure reported previously for DTX-I was synthesized for the first time by a solution procedure. The synthetic product was confirmed to have the correct primary and disulfide structure determined by peptide mapping, sequence analysis and mass measurements. Comparison of synthetic DTX-I with the natural one by high-performance liquid chromatography and capillary zone electrophoresis, as well as by sequence analysis, revealed that the Asn residue at position 12 in the synthetic peptide was Asp in the natural product. Synthesis of DTX-I with Asp at position 12 gave a peptide identical with the natural product in all aspects. NMR analysis of synthetic [Asn12]- and [Asp12]-DTX-I also supported our findings that the Asn residue at position 12 in the DTX-I molecule should be revised as Asp. [Asn12]- and [Asp12]-DTX-I had very similar binding affinities when tested against radiolabeled dendrotoxin binding to rat brain synaptosomal membranes.     

Localization of myosin-V in the centrosome

The perinuclear localization of myosin-V was investigated in a variety of cultured mammalian cells and in primary cultures of rat hippocampus. In all cells investigated, myosin-V immunoreactivity was associated with the centrosome. In interphase cells, myosin-V was found in pericentriolar material, and in both mother and daughter centrioles. These results were obtained by using two different fixation protocols with three different affinity-purified antibodies that recognized a single band in Western blots. During cell division, myosin-V staining was intense throughout the cytoplasm and was concentrated in a trail between migrating centrioles and in the mitotic spindle poles and spindle fibers. The centrosome targeting site was determined to reside within the globular tail domain, because centrosome association also was observed in living cells transfected with DNA encoding the tail domain fused with a green fluorescent protein tag, but not in cells transfected with the vector encoding green fluorescent protein by itself.     

Differential expression of the metabotropic glutamate receptor mGluR1alpha by neurons and axons in the cochlear nucleus: in situ hybridization and immunohistochemistry

mGluR1alpha is a metabotropic glutamate receptor involved in synaptic modifiability. A differential expression in specific neuronal types could reflect their different connections and response properties in central auditory processing. Using in situ hybridization and immunohistochemistry, we studied mGluR1alpha receptor expression throughout the cochlear nucleus. Robust labeling occurred in the dorsal cochlear nucleus and small cell shell, with less in the ventral cochlear nucleus. Among the most intensely labeled were the granule cells of the small cell shell. In the dorsal cochlear nucleus, most cell types expressed message and receptor protein, except granule cells. High levels of receptor were expressed by corn cells and cartwheel cells. The terminal dendrites and synaptic spines of cartwheel and fusiform cells contained receptor protein in the molecular layer, where they could synapse with parallel fibers. Fusiform dendrites also expressed mRNA for mGluR1alpha. The basal dendrites of fusiform cells contained receptor protein in the region where they receive cochlear nerve synapses. Immunostaining of terminal axons was prominent in the molecular layer and the small cell shell, where they were associated with synaptic nests, structures thought to provide long-term changes in excitability. Differential expression levels may reflect different functional requirements of specific cell types, including inhibitory interneurons, like corn cells and cartwheel cells, and excitatory interneurons, like granule cells in the small cell shell, which may participate in local circuits involved in modulatory or gating functions, such as stimulus enhancement or suppression. In presynaptic axons, mGluR1alpha may relate to the long-term signaling requirements of their modulatory functions.     

Localization of matrix metalloproteinase MMP-2 to the surface of invasive cells by interaction with integrin alpha v beta 3

SUMMARY: Cellular invasion depends on cooperation between adhesive and proteolytic mechanisms. Evidence is provided that the matrix metalloproteinase MMP-2 can be localized in a proteolytically active form on the surface of invasive cells, based on its ability to bind directly integrin alpha v beta 3. MMP-2 and alpha v beta 3 were specifically colocalized on angiogenic blood vessels and melanoma cells in vivo. Expression of alpha v beta 3 on cultured melanoma cells enabled their binding to MMP-2 in a proteolytically active form, facilitating cell-mediated collagen degradation. In vitro, these proteins formed an SDS-stable complex that depended on the noncatalytic C-terminus of MMP-2, since a truncation mutant lost the ability to bind alpha v beta 3. These findings define a single cell-surface receptor that regulates both matrix degradation and motility, thereby facilitating directed cellular invasion.