Mapping of Histamine H1 Receptors in the Human Brain Using [11C]Pyrilamine and Positron Emission Tomography

We have studied the characteristics of carbon-11 labeled pyrilamine as a radioligand for investigating histamine H1 receptors in human brain with positron emission tomography (PET). [11C]Pyrilamine is distributed evenly in proportion to cerebral blood flow at initial PET images. Later (after 45-60 min), 11C radioactivity was observed at high concentrations in the frontal and temporal cortex, hippocampus, and thalamus, and at low concentrations in the cerebellum and pons. The regional distribution of the carbon-11 labeled compound in the brain corresponded well with that of the histamine H1 receptors determined in vitro in autopsied materials. In six controls, the frontal and temporal cortices/cerebellum ratio increased during the first 60 min to reach a value of 1.22 +/- 0.071. Intravenous administration of d-chlorpheniramine (5 mg) completely abolished the specific binding in vivo in the frontal cortex and temporal cortex (cortex/cerebellum ratio, 0.955 +/- 0.015). The availability of this method for measuring histamine H1 receptors in vivo in humans will facilitate studies on neurological and psychiatric disorders in which histamine H1 receptors are thought to be abnormal.     

Phenotypic progression of a rat lymphoid cell line immortalized by human T-lymphotropic virus type I to induce lymphoma/leukemia-like disease in rats.

Rat lymphoid cells, TARS-1, immortalized by coculture with adult T-cell leukemia cells, were intraperitoneally injected into 65 newborn, inbred WKAH/Hkm rats. In most of the rats, tumor nodules were discernible 7 to 15 days after transplantation but were completely rejected within 5 to 6 weeks. Two rats with no tumor nodules exhibited gait disturbances and paralysis of the hind legs 3 to 4 weeks after transplantation. Histological and hematological examinations revealed that a lymphoma/leukemia-like disease had developed in one of the two rats, and the T-lymphoid cell line WLeuk-1 was established from peripheral blood mononuclear cells from this rat. When the WLeuk-1 cells were transplanted into newborn WKAH/Hkm rats, the animals died of a lymphoma/leukemia-like disease within several weeks after transplantation, in contrast to their rejection of the TARS-1 cells. Southern blot and karyotype analyses revealed that WLeuk-1 cells had retained the marker chromosomes and human T-lymphotropic virus type I (HTLV-I) integration patterns of the parent cell line, TARS-1. The additional specific chromosome abnormalities 3p+,t (12;13), and Xq+ were found in the WLeuk-1 cells. Moreover, the expression of HTLV-I structural proteins was slightly depressed in WLeuk-1 cells, while that of the transacting factors p40tax and p21x, but not that of p27rex, was enhanced about fivefold compared with that in TARS-1. The transactivating function of p40tax was intact in WLeuk-1, as evidenced by enhanced interleukin-2 receptor alpha chain expression. These results suggest that aberrant expression of HTLV-I regulatory genes and alteration of cellular genes were associated with the phenotypic progression of the WLeuk-1 cell line.     

Recombinant transforming growth factor type beta 3: biological activities and receptor-binding properties in isolated bone cells.

We have recently cloned the cDNA for transforming growth factor type beta 3 (TGF-beta 3), a new member of the TGF-beta gene family. We examined the biological effects of recombinant TGF-beta 3 protein in osteoblast-enriched bone cell cultures. In this report we demonstrate that TGF-beta 3 is a potent regulator of functions associated with bone formation, i.e., mitogenesis, collagen synthesis, and alkaline phosphatase activity. In a direct comparison between TGF-beta 3 and TGF-beta 1, TGF-beta 3 appeared to be three- to fivefold more potent than TGF-beta 1. Our cross-linking experiments with iodinated TGF-beta showed that in osteoblast-enriched bone cell cultures, both TGF-beta 3 and TGF-beta 1 associated with the same three cell surface binding sites. Scatchard analysis of receptor competition studies indicated the presence of high-affinity binding sites for TGF-beta 3 in the picomolar range. TGF-beta 3 showed an approximately fourfold-higher apparent affinity than TGF-beta 1 in overall binding.     

Expression, purification, and properties of the plasma membrane Ca2+ pump and of its N-terminally truncated 105-kDa fragment.

Isoform 4b of the human plasma membrane Ca2+ pump was expressed in COS cells and in the baculovirus system (Sf9 cells). A 105-kDa pump fragment lacking the first two transmembrane domains and the so-called transduction domain was also expressed. The expression level was 2-4 times the background in COS cells and at least 7 times in the baculovirus system. Tests on membranes from both systems showed that the expressed pump was active. The expressed pump and the 105-kDa fragment were isolated from Sf9 cell membranes by calmodulin affinity chromatography. The pump had Ca(2+)-dependent ATPase activity with a calmodulin stimulation factor of 3, formed a La(3+)-stabilized phosphoenzyme, and had a KM (Ca2+) in the presence of calmodulin of about 1 microM. The 105-kDa fragment, assayed by the phosphoenzyme test on COS or Sf9 cell membranes or by ATPase measurements after isolation from Sf9 cells, proved inactive. Laser confocal microscopy on Sf9 cells showed that both the pump and the 105-kDa fragment were apparently associated with the plasma membrane. The expressed pump in COS and Sf9 cells and the endogenous pump in a number of other cell lines had a slower gel mobility (i.e. a higher apparent molecular mass) than the erythrocyte pump.     

Inhibitory effect of silibinin on hepatitis B virus entry

Hepatitis B virus (HBV) infection is a worldwide health problem because of its potential to cause liver cirrhosis and hepatocellular carcinoma. Silibinin is a constituent of an extract of milk thistle, which is empirically used as a herbal medicine for the protection of liver, but its detailed effects on HBV are unknown. Because a previous study reported that silibinin hinders clathlin-mediated endocytosis (CME), we aimed to test whether silibinin inhibits the entry of HBV into hepatocytes. Using HepG2-NTCP-C4 cells, which overexpress sodium taurocholate cotransporting polypeptide (NTCP), it was shown that silibinin inhibited HBV infection dose-dependently. Similar effects were observed using human primary hepatocytes (PXB-cells). Additionally, a combination of silibinin and entecavir reduced HBV DNA in the culture supernatant more than either mono-treatment alone in HepG2-NTCP-C4 cells that had already been infected with HBV. Silibinin decreased transferrin uptake but did not affect the interaction between the HBV envelope and NTCP, suggesting that silibinin might inhibit HBV infection by hindering CME. In conclusion, this study showed that silibinin inhibits HBV entry in vitro.     

Modulation of the biologic activities of IgE binding factor. VI. The activation of phospholipase by glycosylation enhancing factor

Glycosylation enhancing factor (GEF) from rat T cells is a kallikrein-like enzyme and enhances the assembly of N-linked oligosaccharides to IgE binding factors during their biosynthesis, whereas another T cell factor, i.e., glycosylation inhibiting factor (GIF), is a fragment of phosphorylated lipomodulin (i.e., phospholipase inhibitor), which when dephosphorylated inhibits phospholipase and the glycosylation process. The two T cell factors compete with each other when they are added to normal mesenteric lymph node cells during the formation of IgE binding factors. The addition of GEF to T cell hybridoma 23A4 cell switches the cells from the formation of unglycosylated IgE binding factor to the formation of N-glycosylated IgE binding factor. However, GEF neither inactivated GIF nor inhibited the formation of GIF by the T cell hybridoma. Stimulation of the T cell hybridoma with either affinity-purified GEF or bradykinin resulted in the release of GIF from the cells. GIF released by GEF stimulation had a m.w. of approximately 15,000 and bound to monoclonal antibody against lipomodulin. GEF and bradykinin also induced normal mesenteric lymph node cells to release GIF. Incorporation of 14C-arachidonic acid into 23A4 cells, followed by stimulation of the cells with GEF, resulted in the release of 14C-arachidonate. The results suggest that lipomodulin, a phospholipase inhibitory protein, is present in lymphocytes, and indicate that GEF and bradykinin induce the activation of phospholipase by stimulating cells to release lipomodulin.     

Fibronectin-induced migration of melanophores in vitro in scales of medaka,Oryzias latipes

Summary The effects of fibronectin on melanophores were examined in two mutant strains of medaka, Oryzias latipes: mm (BmmR), which has condensed melanophores and normal dendritic melanophores; and cm (BcmR), which has condensed melanophores. When medaka scales were cultured in the presence of fibronectin, melanophores of the wild type and dendritic melanophores of the mm mutant changed their shape and migrated, whereas melanophore migration was rarely seen in the absence of fibronectin. Melanophores of the cm mutant and condensed melanophores of the mm mutant did not migrate even in the presence of fibronectin. When melanophores of the wild type and mm mutant were condensed by adrenalin, they did not migrate. On the other hand, when melanophores of the cm mutant were dispersed by theophylline, they were able to migrate. These results indicate that fibronectin induces the migration of melanophores and that dispersion of melanin granules may be requisite for such migration.     

Effect of taurine on a benzodiazepine-GABA-chloride ionophore receptor complex in rat brain membranes

Taurine at 10 mM had no effect on basal binding of [³H]diazepam to the membranes, while it significantly inhibited a GABA-stimulated binding of [³H]diazepam in cerebral cortex, hippocampus, but not in cerebellum. The inhibition by taurine in the presence of GABA (1M to 1 mM) was not competitive. At low concentrations (0.04 to 0.2 nM) the binding of [³H]propyl--carboline-3-carboxylate, a ligand exhibiting higher affinity for type I than type II benzodiazepine receptors, was not enhanced by GABA, while the binding of higher concentrations (0.5 nM) was. This GABA enhancement of [³H]propyl--carboline-3-carboxylate binding was also selectively blocked by taurine. Pentobarbital increased the binding of [³H]diazepam in a medium containing chloride and this effect was potentiated by taurine at 1–10 mM. These findings may be relevant to the modulatory role of taurine in the central nervous system.     

KINETICS OF ENTRY OF PROTEINS INTO THE MYELIN MEMBRANE1

Brain slices were prepared from 17-day old rats, and incubated with [³H]glycine or [³H]-leucine to label proteins. Myelin was isolated from the slices, and the proteins were separated by discontinuous gel electrophoresis in buffers containing sodium dodecyl sulfate. Radioactive basic and Wolfgram proteins appeared in myelin at similar initial rates, and their entry was nearly linear between 15 and 120 min with no detectable lag. Radioactive proteolipid protein appeared in myelin at one-fourth the rate of the basic and Wolfgram proteins between 0 and 30 min, then entered at a rate comparable to the other proteins between 45 and 120 min. When cycloheximide (0.2 mM) or puromycin (1.0 mM) was added, appearance of newly labeled basic and Wolfgram proteins in myelin stopped while proteolipid protein continued to appear in myelin at a normal rate for at least 30 min. Chase experiments with unlabeled glycine had similar effects. These results indicate the existence of a previously synthesized precursor pool of proteolipid protein with a 30-min interval between synthesis of proteolipid protein and its appearance in myelin. Incorporation of [³H]fucose into glycoprotein of the myelin sheath was studied, as was inhibition of incorporation of radioactivity by the use of either cycloheximide, or dilution with unlabeled fucose. The results indicated fucosylation of a sizable pool of presynthesized protein and a delay of 30 min between fucosylation of these polypeptides and their subsequent appearance in myelin as glycoproteins.     

Interaction between thymopoietin and facteur thymique serique in the rosette inhibition assay.

In the rosette inhibition assay, thymopoietin was active and its activity was enhanced in the presence of high concentrations of ubiquitin. Facteur thymique serique (FTS) was active in the same assay, but its activity was completely inhibited by high concentrations of ubiquitin. Mixtures of thymopoietin and FTS showed activity both in the presence or absence of ubiquitin depending on the concentration of thymopoietin or FTS in the mixtures. There seemed to be an important biologic interaction of thymopoietin and FTS when presented as a mixture, suggesting that thymopoietin and FTS might interact $\text{in}\text{vitro}$ and possibly $\text{in}\text{vivo}$.