Lactogen enhances Nb2 cell GTPase activity after 4 hours incubation

The lactogen receptor has been suggested to associate with one or more G proteins despite the absence of a 7-transmembrane spanning sequence. These studies were designed to determine whether lactogens acutely increase GTP binding to or GTPase activity in Nb2 cell membrane. Incubation of Nb2 cell membrane with either ovine PRL (10 ng/ml) or diluent for 0–1 h resulted in a decrease in total³⁵S-GTP binding to both with no difference in GTP binding between PRL- and diluent-treated membranes. There was also no change in³⁵S-GTP binding to Nb2 cell membrane incubated with increasing oPRL concentrations (0.001–100 ng/ml) for 60 min. α-³²P-GTP photoaffinity labelling was used to evaluate changes in GTP binding to specific G proteins. Photoaffinity labelling of α-³²P-GTP to no G protein was changed after preincubation with oPRL (10 ng/ml) for 0–60 min or with oPRL (0.01–10 ng/ml) for 60 min. Finally, it was determined whether oPRL had any acute effect on GTPase activity, as determined by release of³²Pi from γ-³²P-GTP. When Nb2 cell membrane was preincubated for 0–60 min with oPRL (10 ng/ml) or a range of oPRL concentrations (0–10 ng/ml), no change in GTPase activity was observed. However, when Nb2 cells were incubated with lactogen for 0–7 h, GTPase activity in equal quantities of Nb2 cell membrane prepared from those cells increased over time. Increased GTPase activity (64.9–74.4%;P<0.03 compared to 0 h) was observed after 4–7 h incubation with lactogen. In summary, addition of lactogen to Nb2 cell membrane did not acutely increase either GTP binding or GTPase activity. Yet when Nb2 cells were incubated with lactogen for 4 h prior to preparation of membrane, GTPase activity was significantly increased. This evidence, in addition to our previous results showing that 4 h incubation with lactogen increased G protein β subunit concentration and pertussis toxin-stimulated ADP-ribosylation of Gi, support a role for delayed lactogen modulation of one or more G proteins in the Nb2 cell, requiring at least 4 h for maximal effect.     

Arthrogryposis,renal dysfunction and cholestasis syndrome: Report of five patients from three Italian families

We report on five patients from three families with neurogenic arthrogryposis, cholestasis and tubular renal dysfunction. Despite a similar clinical picture the liver histology showed a broad pathological spectrum, ranging from pigment storage to parenchymal giant cell transformation and ductopenia. The findings are compared with those of other cases from the literature in search of a correct nosology of the syndrome characterized by arthrogryposis, renal and liver disease.Conclusion We propose to consider the picture of arthrogryposis, renal tubular dysfunction and cholestasis as a single syndrome.     

Characterization of the epitope recognized by a mAb that reacts differentially with murine suppressor T cells

Although reliable antibodies are available that distinguishhuman suppressor T (T_s) cells from CTL and other T cells, feware available for murine T_s cells. We have developed a mAb (984D4.6.5)that, in the presence of complement, depletes alloantigen-specificT_s cells but not CTL. This antibody recognizes activated TT_scells but not their precursors. In these studies, flow cytometricanalysis demonstrates that 984D4.6.5 reacts with several T_scell hybridomas, cloned T_h cell lines and WEHI-3 (a myelomonocytictumor cell line). Reactivity was not detected with BW5147, T_hcell hybridomas, cloned T_h cells, CTL lines and hybridomas,B cell lines, thymocytes, splenocytes, bone marrow cells nora variety of tumor cells. Among 984D4.6.5 positive lines, expressionis heterogeneous and the number of cells expressing high levelsof the epitope is increased when the hybridomas are maintainedat a relatively high cell density. Neuriminidase and pronasedeplete the epitope recognized by mAb 984D4.6.5. Protein synthesisand glycosylation inhibitors also reduce expression of thisepitope. These observations suggest that the epitope recognizedby 984D4.6.5 is a carbohydrate linked to a polypeptide. Thisantibody was tested by ELISA for binding to a large panel ofcarbohydrates and glycollpids coupled to BSA. The only one thatbound 984D4.6.5 was LS tetrasaccharide c (NeuNAc2-6Galpß1-4GIcNAcß1-3GaIß1-4Glc),an O-linked carbohydrate. Comparative analysis shows that boththe sequence and the linkage of these sugars are essential tothe reactivity with the 984D4.6.5 antibody. This epitope isexpressed by a glycoprotein of-200 kDa, as shown by Westernblots. The identity of this glycoprotein remains to be determined,but indirect evidence suggests that it is not CD45.     

Lymphocytes infiltrating the CNS during inflammation display a distinctive phenotype and bind to VCAM-1 but not to MAdCAM-1

The nature of inflammatory lymphocytes recruited to the CNShas been studied in a model of chronic inflammation. Injectionof killed Corynebacterlum parvum into the cortex of the mousebrain produces a circumscribed inflammatory cellular infiltratearound the injection site, and recruited mononuclear inflammatorycells (IC) can be isolated for flow cytometric analysis. Themajority of IC were T cells. In comparison with the predominantnaive population of mesenteric lymph node T cells, IC T cellsexpress much higher levels of CD44, LFA-1 and ICAM-1, and lowerlevels of CD45RB, features commonly associated with memory (previouslyactivated) cells. In addition, in contrast to the L-selectin⁺6-integrin^low phenotype of naive lymph node T cells, IC T cellslacked L-selectin and were 6-integrin^–. Mac-1, recentlyproposed as another marker of memory T cell differentation,was not displayed by IC T cells, suggesting that Mac-1 expressionmay be heterogeneous among memory T cell subsets. A subset ofmesenteric lymph node (MLN) T cells, probably representing activatedT cells undergoing the naive to memory transition, but not ofIC T cells, expressed high levels of 6-, ß7- and E-integrin.IC and MLN naive T cells expressed comparable levels of 4-integrin,but IC T cells stain poorly with anti-ß7 mAbs andwith mAb DATK 32, specific for the 4ß7 heterodimericlymphocyte homing receptor for the mucosal addressin MAdCAM-1,suggesting that these inflammatory cells express more 4ß1than 4ß7. Consistent with this, in in vitro adhesionassays, brain IC bound better than MLN cells to the 4ß1integrin ligand VCAM-1 and the LFA-1 ligand ICAM-1 but adheredvery poorly to the 4ß7 ligand MAdCAM-1. These findingsare consistent with and extend previous immunohistological studiesof T cells in murine experimental autoimmune encephalomyelitis,and demonstrate a distinctive phenotype for lymphocytes beingpresent in the chronically inflamed brain.     

Tumor necrosis factor-{alpha} up-regulates Bcl-2 expression and decreases calcium-dependent apoptosis in human B cell lines

Group I and Epstein–Barr virus-negative Burkitt's lymphomacell lines and the B104 lymphoma cell line which expresses aphenotype of immature B cells undergo apoptosis after cross-linkingof their surface Ig receptors or after exposure to a calciumionophore. We show here that tumor necrosis factor (TNF)- protectsthese B cell lines against Ca²⁺-dependent apoptosis. Protectionwas associated with up-regulatlon of bcl-2 mRNA and proteinexpression. The increase of Bcl-2 expression induced by TNF-was inhibited by chelerythrine, a specific inhibitor of proteinkinase C (PKC), suggesting that Bcl-2 expression was dependenton PKC activation. Furthermore, we show that phorbol estersand cyclosporin A (CsA), which prevent Ca²⁺-dependent apoptosis,up-regulated Bcl-2 expression. The effect of CsA on Bcl-2 expressionis controlled by calcineurin since we have shown that FK506but not rapamycin had the same effect on Bcl-2 expression, whereasokadaic acid, an inhibitor of phosphatases 1, 2A and 2C, wasineffective. These data provide direct evidence that TNF- preventsCa²⁺-dependent apoptosis by a Bcl-2-dependent mechanism mediatedby PKC.     

In situ dormancy of B lymphocytes programmed for an IgE antibody response and their sudden release from unresponsiveness under in vitro conditions

Priming of CBA/J mice with different doses of antigen has aprofound effect on the ratio of IgE versus IgG antibodies appearingupon Immunization. Repeated injections of minute doses induceIgG and high titers of IgE antibodies. Large doses elicit ahigh IgG but a very low IgE antibody titer. In order to studythe modalities for activation and inactivation of IgE-producingB cells, an in vitro culture system was established in whichspleen cells from animals primed with keyhole limpet hemocyaninwere re-stliulated with antigen. In contrast to the expectationfrom the in vivo situation, spleen cells from animals Immunizedwith large doses of antigen and virtually lacking IgE antibodiesproduce high amounts of IgE antibodies upon re-stimulation invitro. The titers in spleen cell cultures from mice primed withminute doses remain proportional to the response measured asserum antibodies. In accordance with the induction of high amountsof IgE antibodies in spleen cell cultures from mice primed withlarge doses, the frequency of IgE antibody-secreting cells wasraised drastically, 1000-fold. The in vitro response is a trueanamnestic response. The sudden appearance in high frequencyof IgE antibody-forming cells among spleen cells isolated fromprimed mice which have high IgG but virtually no IgE antibodytiters is as yet unexplained and the origin of the B memorycells has not yet been traced. The answer might be crucial forour understanding of the down-regulation of the IgE Immune responses.     

Selective localization of gelatinase A,an enzyme degrading β-amyloid protein,in white matter microglia and in Schwann cells

Gelatinase A is an enzyme capable of cleaving soluble -amyloid protein (AP), and may function as an -secretase to produce secretory forms of amyloid precursor protein. We examined gelatinase A immunoreactivity in the brains and posterior roots of neurologically normal, lacunar stroke, Alzheimer disease (AD), amyotrophic lateral sclerosis, progressive supranuclear palsy and myasthenia gravis cases. The gelatinase A antibody stained only microglial cells in the white matter in all the brain tissues. In AD brain, the reactive microglia located in the center of classical senile plaques, as well as in other microglial cells in the gray matter, showed no immunoreactivity. Gelatinase A in white matter microglial cells may play a role in preventing local deposition of AP. In the posterior root, Schwann cells had positive immunoreactivity. As with other metalloproteases, gelatinase A in Schwann cells may play an antiproliferative role.     

The dynamics of β1 integrin expression during peripheral nerve regeneration

The aim of the present study was to examine the expression of 1 integrin subunit after peripheral nerve transection. After sciatic nerve transection two experimental procedures were used; changes in the freely regenerating rat sciatic nerve were compared to a situation in which spontaneous regeneration was prevented by suturing both ends of the nerve to the muscle next to the point of transection. Specimens for morphological analysis were collected 6 h, 1, 3, 5, 7 days and 2, 4, 6 and 8 weeks after the axotomy. Sections from the proximal (two zones) and distal (three zones) stumps next to the point of transection were stained with antibodies against 1 integrin subunit, macrophages, collagen types I and III, and S-100 protein. The control nerves showed 1 integrin-stained cells in the perineurium and vasa nervorum but the endoncurium was negative. Positively stained endoneurial fibroblast-like cells could be seen in the proximal part of the nerve already at 24 h after transection. The number of these positively stained cells increased steadily; they were most numerous 4 weeks after transection in the distal zone 2. Subsequently, the number of positively stained endoneural cells declined sharply and 8 weeks after transection no positively stained cells could be found. The morphological appearance and the immunohistochemical properties of the cells suggest that the majority of 1 integrin-positive cells are endoneurial fibroblast-like cells. Thus, the process appeared to be dynamic, starting from the proximal part and continuing to the distal parts, and was similar in both experimental groups. The positive staining in perineurial cells indicate that 1 integrin, which is an important mediator of the cell-matrix interaction, may have an essential role in the formation and strengthening of the normal peripheral nerve structures. Furthermore, 1 integrin seems to have an active role in reactions which occur during the early phases of peripheral nerve regeneration.     

Effects of coexisting neurochemicals on the release of serotonin from the intermediate area of rat thoracic spinal cord

Serotonin (5-HT), substance P (SP), neurokinin A (NKA), and thyrotropin-releasing hormone (TRH) coexist in the nerve terminals of the intermediolateral cell column (IML) of the thoracic spinal cord. The Ca2⁺-dependent release of 5-HT from the microdissected intermediate area (including the IML) of the rat thoracic spinal cord, and the 5-HT_1B autoreceptor regulator of 5-HT release, were previously demonstrated. In this paper, the effects of SP, NKA, TRH, and/or their analogs on the release of [³H]5-HT from the intermediate area were investigated using an in vitro superfusion system. Both SP (the endogenous ligand for neurokinin-1 (NK₁) receptor) and an NK₁, agonist (GR 73632) significantly increased the basal release of [³3H]5-HT. SP and GR 73632 did not change the K⁺-stimulated release of [³H]5-HT. The effect of the NK₁ agonist on the basal release of [³H]5-HT was dose-dependent, was reduced by an NK₁ antagonist (GR 82334), and was not dependent on extracellular Ca²⁺. Neither NKA, an NK₂, agonist (GR 64349), nor a TRH analog (MK-771) altered the basal or stimulated release of [³H]5-HT. These data suggest that basal release of 5-HT from the intermediate area of the rat thoracic spinal cord is regulated by SP (acting through an NK₁ receptor), but not by NKA or TRH. These results provide evidence for the role of SP as a modulator of serotoninergic neurons in the intermediate area of the thoracic spinal cord, and may help to clarify the role of coexisting neurochemicals in the spinal regulation of the sympathetic nervous system. © 1995 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Intracisternal injection of TRH antibody blocks gastric emptying stimulated by 2-deoxy-d-glucose in rats

We evaluated the effect of 2-deoxy-d-glucose (2-DG) on gastric emptying of a non nutrient solution in conscious rats using a Phenol red method. Intravenous injection of 2-deoxy-d-glucose dose-dependently increased the rate of gastric emptying. This stimulatory action of 2-DG was abolished by subdiaphragmatic vagotomy. Intracisternal injection of thyrotropin-releasing hormone (TRH) antibody blocked intracisternal TRH and intravenous 2-DG-induced enhancement of gastric empyting but not the stimulation of gastric emptying induced by intracisternal pancreatic polypeptide. The TRH antibody injected intraperitoneally had no effect. These results suggest that endogenous TRH in the brain is involved in vagal-dependent stimulation of gastric emptying by 2-DG.