Pure gelatin microcarriers: Synthesis and use in cell attachment and growth of fibroblast and endothelial cells

Summary A new type of microcarrier was described using bead emulsion-polymerization techniques. An aqueous solution of gelatin and glutaraldehyde was dispersed in a hydrophobic phase of mineral oil, using Triton X-114 as an emulsifier, and polymerization was initiated. The resultant spherical beads, composed entirely of gelatin, showed excellent mechanical stability to ethanol drying, sterilization, and long-term use in microcarrier spinner cultures. The solid gelatin microcarriers supported the growth of L-929 fibroblast, swine aorta endothelial, human umbilical endothelial, and HeLa-S₃ cultures with no adverse effects on cell morphology or growth. The beads were transparent in growth medium and attached cells were clearly visualized without staining. The beads were also compatible with techniques for scanning electron microscopy. Collagenase could be used to entirely digest the gelatin beads, leaving the cells free from microcarriers and suspended in solution while retaining 98% cell viability. The results further showed that after collagenase treatment the cells would populate fresh gelatin microcarriers and grow to confluence. Cell attachment kinetics revealed that the endothelial cells attached to the gelatin beads at the same rate as to tissue culture plates, whereas the fibroblast cells attached to the beads more slowly. However, once the fibroblast cells were attached to the gelatin microcarriers they spread and grew normally. This research was supported in part by the National Institutes of Health (GN 29127) and Ventrex Laboratories, Portland, Maine.     

Induction of erythropoietin responsiveness in murine hematopoietic cells by the gag-myb-ets-containing ME26 virus.

ME26 virus, which was generated by inserting the coding region of the acute avian leukemia-inducing virus E26 into a murine retrovirus vector, encodes a 135-kDa gag-myb-ets fusion protein. Amphotropic murine leukemia virus pseudotypes of ME26 virus induce a high incidence of erythroleukemia 2 to 4 months after injection into newborn NFS/N mice. Spleen cells from the majority of these mice proliferate to high levels in the presence of the erythroid hormone erythropoietin (Epo) and can easily be established as permanent Epo-dependent cell lines. The cell lines contain multiple copies of ME26 viral DNA and express viral message and protein. An Epo receptor mRNA of normal size can be detected in these cells, and binding studies reveal a single class of lower-affinity Epo receptor with an affinity for Epo that is in the range of that previously reported for erythroid cells. The ME26 virus-induced Epo-dependent cell lines, however, appear more immature than previously described erythroid cell lines and more closely resemble early hematopoietic precursor cells, suggesting that the virus may be activating the Epo receptor in hematopoietic cells that do not normally express it. Consistent with this idea, we are able to infect an interleukin-3-dependent myeloid cell line, FDC-P2, with ME26 virus and convert it to Epo dependence. The ME26 virus-infected FDC-P2 cells, even before growth on Epo, showed a large increase in the amount of Epo receptor mRNA. However, no ME26 viral integrations can be detected adjacent to the Epo receptor gene, indicating that the virus is not activating the Epo receptor gene by promoter/enhancer insertion. Our results are more consistent with the hypothesis that the gag-myb-ets-encoded viral fusion protein, which is known to bind DNA, is directly or indirectly activating the expression of the Epo receptor gene in these cells.     

Analysis of candidate genes of spontaneous arthritis in mice deficient for interleukin-1 receptor antagonist

Previously, we identified a major quantitative trait locus (QTL) on mouse chromosome 1 that regulates the susceptibility to arthritis in an F2 population generated from arthritis-prone BALB/c and arthritis-resistant DBA/1 mice deficient for interleukin-1 receptor antagonist. To further select candidate genes for the QTL, we analyzed the expression patterns of arthritis in 38 F2 individuals and compared the expression levels of key candidate genes to the parental strains. Two distinct subpopulations of arthritic mice were identified in the 38 F2 mice. One subgroup of diseased mice was characterized by myeloid cell dominant inflammation, whereas the other was mainly associated with increased anti-apoptotic activities of inflammatory cells. Several differentially expressed important candidate genes in parental strains in the QTL region are relevant to myeloid cell, apoptotic activities, or to both. About one-quarter of those genes have been previously linked to arthritis in literature. The present study reveals two distinct subpopulations of arthritic mice with spontaneous arthritis due to deficiency for interleukin-1 receptor antagonist, suggesting that genes with function relevant to myeloid cell and/or apoptotic activities are most likely the key candidate genes for the QTL.     

Chimeric tick-borne encephalitis and dengue type 4 viruses: effects of mutations on neurovirulence in mice.

Two new chimeric flaviviruses were constructed from full-length cDNAs that contained tick-borne encephalitis virus (TBEV) CME or ME structural protein genes and the remaining genes derived from dengue type 4 virus (DEN4). Studies involving mice inoculated intracerebrally with the ME chimeric virus indicated that it retained the neurovirulence of its TBEV parent from which its pre-M and E genes were derived. However, unlike parental TBEV, the chimeric virus did not produce encephalitis when mice were inoculated peripherally, indicating a loss of neuroinvasiveness. In the present study, the ME chimeric virus (vME) was subjected to mutational analysis in an attempt to reduce or ablate neurovirulence measured by direct inoculation of virus into the brain. We identified three distinct mutations that were each associated independently with a significant reduction of mouse neurovirulence of vME. These mutations ablated (i) the TBEV pre-M cleavage site, (ii) the TBEV E glycosylation site, or (iii) the first DEN4 NS1 glycosylation site. In contrast, ablation of the second DEN4 NS1 glycosylation site or the TBE pre-M glycosylation site or amino acid substitution at two positions in the TBEV E protein increased neurovirulence. The only conserved feature of the three attenuated mutants was restriction of virus yield in both simian and mosquito cells. Following parenteral inoculation, these attenuated mutants induced complete resistance in mice to fatal encephalitis caused by the highly neurovirulent vME.     

Proton NMR and fast-atom bombardment mass spectrometry analysis of the melanoma-associated ganglioside 9-O-acetyl-GD3

A glycolipid antigen, detected by a monoclonal antibody (ME 311) obtained by immunizing mice with a human metastatic melanoma cell line (WM 46), was isolated and structurally characterized. Using immunostaining on thin-layer chromatograms for monitoring, 1.0 mg of a pure alkali-labile disialoganglioside was obtained from 23 g of packed melanoma cells (WM 164). Fractionation of the lipid extract was done on DEAE-Sepharose columns into total disialogangliosides which were repeatedly separated by high-pressure liquid chromatography. On mild alkaline treatment, the ganglioside was converted to a slower migrating species identical with a ganglioside GD3 isolated from the same source (Neu5Ac alpha 2----8Neu5Ac alpha 2----3Gal beta 1----4Glc beta 1----1-cer-amide) and specifically detected by monoclonal antibody R24. Comparison of the two gangliosides by fast-atom bombardment mass spectrometry (revealing an acetyl group on the terminal sialic acid on the alkali-labile species) and by 1H NMR (indicating the position of the acetyl group) suggested the following structure: Neu5,9Ac2 alpha 2----8Neu5Ac alpha 2----3Gal beta 1----4Glc beta 1----1-ceramide. This is identical with a ganglioside proposed earlier to exist in melanoma cells (Cheresh, D. A., Varki, A. P., Varki, N. M., Stallcup, W. B., Levine, J., and Reisfeld, R. A. (1984) J. Biol. Chem. 259, 7453-7459). Immunostaining with ME 311 antibody of cell extracts on thin-layer chromatography chromatograms revealed only this ganglioside in the melanoma cells, while normal human brain was negative. However, in one of the total ganglioside extracts tested for presence of binding with antibody ME 311, three gangliosides were found to bind. No evidence was obtained for the presence of the antigenic epitope in mucins or glycoproteins of the melanoma cells.     

Endotoxin-induced susceptibility to staphylococcal infection and its reversal by adrenergic blocking agents

Sultzer, Barnet M. (Princeton Laboratories, Inc., Princeton, N.J.), and Henry H. Freedman. Endotoxin-induced susceptibility to staphylococcal infection and its reversal by adrenergic blocking agents. J. Bacteriol. 90:1001-1006. 1965.-The transient phase of increased susceptibility to bacterial infection in mice provoked by prior administration of small doses of endotoxin was investigated for possible mediation by vasoactive substances. Animals were given endotoxin intravenously shortly before intraperitoneal injection of Staphylococcus aureus Smith, thereby lowering the lethal inoculum 10-fold. To determine whether this susceptibility state could be obviated, mice were pretreated with phenoxybenzamine or dibenzylchlorethylamine. Mortality decreased from an average of 81% in the endotoxin control groups to about 23% in the treated mice, closely approximating the mortality in control mice injected with saline and staphylococci. Neither antiadrenergic agent independently altered the resistance of mice to a higher lethal staphylococcal challenge, nor did these materials induce extravascular leukocyte mobilization into the peritoneal cavity. The results suggest a possible role of vasoactive staphylococcal alpha-toxin, as well as epinephrine or epinephrine-like factors, in this altered state of resistance to staphylococcal infection.     

Comparison of commercial kits for apoprotein A-I and apoprotein B with standardized apoprotein A-I and B radioimmunoassays performed at the Northwest Lipid Research Center

There has recently been a proliferation of commercial kits available for apoproteins A-I and B. Since reference procedures for apoproteins have not yet been established we have elected to compare apoprotein kit methods with highly standardized apoprotein B and A-I radioimmunoassays developed at the Northwest Lipid Research Center. Commercial radial immunodiffusion kits for apoproteins A-I and B were obtained from three separate companies, Calbiochem, Daiichi Pure Chemicals, and Tago, and a commercial radioimmunoassay kit for apoprotein A-I was obtained from Ventrex Laboratories. Considerable differences were observed between the commercial kit methods and the Northwest Lipid Research radioimmunoassay methods. Some of the differences between methods were related to the assigned value of the reference materials. Other differences between methods were clearly method-dependent.     

A comparison of isozymes of five axenic Giardia isolates

The relative mobilities of six enzymes from the trophozoites of five axenically-cultured isolates of Giardia from human, cat, and guinea pig hosts were compared by starch and polyacrylamide gel electrophoresis. The six enzymes compared were malate dehydrogenase (NAD+) (MDH) (EC 1.1.1.37), malate dehydrogenase (decarboxylating) (ME) (EC 1.1.1.40), hexokinase (EC 2.7.1.1), 6-phosphogluconate dehydrogenase (EC 1.1.1.44), glucose-6-phosphate dehydrogenase (G6P) (EC 1.1.1.49), and alpha-glycerophosphate dehydrogenase (EC 1.1.1.8). The latter three enzymes have not been previously reported in Giardia. On the basis of zymogram patterns, the five Giardia isolates were divided into three zymodemes. Zymodeme I comprised human-1/England, human-1/Bethesda, and cat-1/Portland, Zymodeme II the guinea pig-1/Portland isolate, and Zymodeme III the human-1/Portland isolate. These zymodemes were further substantiated when several physical and kinetic properties of three of the enzymes, MDH, ME, and G6P, were examined. Our results, in which Giardia isolated from different mammalian hosts share multiple isoenzymes, question the validity of the practice of assigning Giardia species names on the basis of the animal host from which the protozoan was obtained.     

Accessibility of low‐molecular‐mass molecules to the median eminence and arcuate hypothalamic nucleus of adult mouse

Blood‐derived molecules are able to access to the median eminence (ME) and arcuate hypothalamic nucleus (Arc) due to the lack of the blood–brain barrier. In the present study, we examined the accessibility of low‐molecular‐mass (LMM) molecules into parenchyma in the ME and Arc of adult mice by administration of Dextran 3000 (Dex3k), Dex10k, Evans blue (EB) and fluorescein isothiocyanate (FITC). In the external zone of the ME, the fluorescence of Dex3k, EB and FITC tracers generated an intensity gradient from fenestrated capillary, but that of Dex10k was detected only between the inner and outer basement membrane of pericapillary space. The fluorescence of FITC in the external zone of the ME was closely associated with axonal terminals and surrounded by cellular processes of tanycytes‐like cells and astrocytes. In the ependymal/internal zone of the ME and Arc, the fluorescence of all LMM tracers was seen at tanycytes‐like cells and neurons. The fluorescence of EB and FITC in these regions was not detected when brains were fixed during or before the administration of tracers. The inhomogeneity of accessibility for fluorescent tracers depended on routes for tracer administration. Thus, the present study indicates that the accessibility of LMM blood‐derived molecules to parenchyma depends on fenestration of the capillary in the external zone of the ME and active transport of ependymal cells in the ependymal/internal zone of the ME and Arc. Copyright © 2013 John Wiley & Sons, Ltd.     

Deletions of the Synenkephalin Domain Which Do Not Alter Cell-Specific Proteolytic Processing or Secretory Targeting of Human Proenkephalin

Abstract: To identify signals that direct the proteolytic processing and regulated secretion of human proenkephalin (hPE), we have transfected the hPE gene or minigene constructs into pituitary tumor cells, either rat GH4C1 cells or mouse AtT-20 cells. Cells transfected with either the hPE gene or minigene contained similar levels of methionine-enkephalin (ME)-containing peptides and hPE mRNA. In the GH4C1 clones, ME was present predominantly in high-molecular-mass forms (5–25 kDa). In contrast, the AtT-20 clones contained almost exclusively free ME and low-molecular-mass forms (<5 kDa), with very little high-molecular-mass species present. Thus, among pituitary cells, corticotroph-derived cells appear better equipped to process hPE than lactotroph-derived cells. Despite limited proteolytic processing, GH4C1 clones secreted large amounts of unprocessed (>20 kDa) hPE into the medium, making up to 10% of endogenous rat prolactin secretion. Both precursor and processed forms of ME were cosecreted acutely (<1 h) with rat prolactin, and release of both polypeptides was stimulated up to 12-fold by secretagogues. Thus, complete proteolytic processing was not required for accurate targeting of hPE to the regulated secretory pathway. When transfected with constructs bearing deletions of amino-terminal amino acids 2–43 or 2–67, i.e., part or nearly all of the synenkephalin moiety, GH4C1 cells handled the modified protein much like cells expressing the complete protein. They did not process the modified hPE extensively, but the protein was correctly targeted to the regulated secretory pathway. AtT-20 cells transfected with truncated hPE cDNA constructs expressed and processed the protein as efficiently as cells expressing unmodified hPE and expressed predominantly low-molecular-mass forms of ME. Therefore, the structural features required for correct targeting and processing are not present in the cysteine-rich amino-terminal third of the prohormone. It is interesting that the deletions did not include the SHLL peptide motif in synenkephalin, a motif that has been proposed as a sorting signal.     

Mitochondrial malic enzyme (ME2) in pancreatic islets of the human, rat and mouse and clonal insulinoma cells: Simple enzyme assay for mitochondrial malic enzyme 2

Despite interest in malic enzyme(ME)s in insulin cells, mitochondrial malic enzyme (ME2) has only been studied with estimates of mRNA or with mRNA knockdown. Because an mRNA’s level does not necessarily reflect the level of its cognate enzyme, we designed a simple spectrophotometric enzyme assay to measure ME2 activity of insulin cells by utilizing the distinct kinetic properties of ME2. Mitochondrial ME2 uses either NAD or NADP as a cofactor, has a high K_m for malate and is allosterically activated by fumarate and inhibited by ATP. Cytosolic ME (ME1) and the other mitochondrial ME (ME3) use only NADP as a cofactor and have lower K_ms for malate. The assay easily showed for the first time that substantial ME2 activity is present in pancreatic islets of humans, rats and mice and INS-1 832/13 cells. ME2’s presence was confirmed with immunoblotting. There was no evidence that ME3 is present in these tissues.     

Growth of Chinese hamster ovary (CHO) cells in the presence of MMC in low-serum medium.

Chinese hamster ovary (CHO-WBLT) cells growing in McCoy's 5a with 10% fetal bovine serum (FBS) were adapted to 0.5% FBS in CHO-1 Complete Media System, a serum-free medium from Ventrex. Cells in these two media were exposed to 10(-7) M and 10(-8) M mitomycin C (MMC) for 24 h. Comparison of cell growth over 10 days showed that cells in 0.5% serum proliferate, though at a slower rate than cells in 10% serum. Treatment with MMC revealed that at 10(-7) M, MMC is cytotoxic to cells to both the media; at 10(-8) M, MMC is non-cytotoxic to cells in both media.     

Accessory elements,flanking DNA sequence,and promoter context play key roles in determining the efficacy of insulin and phorbol ester signaling through the malic enzyme and collagenase-1 AP-1 motifs

Insulin stimulates malic enzyme (ME)-chloramphenicol acetyltransferase (CAT) and collagenase-1-CAT fusion gene expression in H4IIE cells through identical activator protein-1 (AP-1) motifs. In contrast, insulin and phorbol esters only stimulate collagenase-1-CAT and not ME-CAT fusion gene expression in HeLa cells. The experiments in this article were designed to explore the molecular basis for this differential cell type- and gene-specific regulation. The results highlight the influence of three variables, namely promoter context, AP-1 flanking sequence, and accessory elements that modulate insulin and phorbol ester signaling through the AP-1 motif. Thus, fusion gene transfection and proteolytic clipping gel retardation assays suggest that the AP-1 flanking sequence affects the conformation of AP-1 binding to the collagenase-1 and ME AP-1 motifs such that it selectively binds the latter in a fully activated state. However, this influence of ME AP-1 flanking sequence is dependent on promoter context. Thus, the ME AP-1 motif will mediate both an insulin and phorbol ester response in HeLa cells when introduced into either the collagenase-1 promoter or a specific heterologous promoter. But even in the context of the collagenase-1 promoter, the effects of both insulin and phorbol esters, mediated through the ME AP-1 motif are dependent on accessory factors.     

In vitro selection of a cell line for resistance to lysis by tumor necrosis factor-alpha selects for reduced tumorigenicity

Experimentally, TNF-alpha can mediate the hemorrhagic necrosis of certain tumors. Furthermore, evidence indicates that natural cytotoxic (NC) activity, a cell-mediated cytolytic activity that utilizes TNF-alpha in the lysis of target cells, is involved in preventing the outgrowth of certain NC/TNF-alpha-sensitive tumor cells. These observations raise the issue of whether soluble TNF-alpha normally serves as a tumor surveillance mechanism preventing the outgrowth of some tumors. To address this issue, we have used TNF-alpha to select TNF-alpha-resistant variants from the NC/TNF-alpha-sensitive mouse fibroblast cell line 10ME. Previously, we have demonstrated that 10ME is tumorigenic in immune-deficient mice but fails to form tumors in normal mice. Moreover, selection of NC-resistant variants from 10ME selects for both TNF-alpha resistance and tumorigenicity in normal mice. As cells that have been selected for NC resistance form tumors in normal mice, whereas the NC-sensitive parental cell line does not, it seems that escape from NC activity is sufficient to significantly increase the tumorigenic potential of the cell line. We show that the selection with TNF-alpha, although associated with NC resistance, does not increase the tumorigenic potential of 10ME cells but reduces it. Thus, NC activity appears to function as a mechanism to prevent tumor formation, and escape from NC activity allows for tumor formation; TNF-alpha does not have similar activity. Moreover, this suggests that NC activity is not equivalent to soluble TNF-alpha activity, but utilizes TNF-alpha more efficiently than soluble TNF-alpha, or NC activity involves both TNF-alpha and other effector mechanisms.     

Mutants of neuroserpin that cause dementia accumulate as polymers within the endoplasmic reticulum

The dementia familial encephalopathy with neuroserpin inclusion bodies (FENIB) is caused by the accumulation of mutant neuroserpin within neurons (Davis, R. L., Shrimpton, A. E., Holohan, P. D., Bradshaw, C., Feiglin, D., Sonderegger, P., Kinter, J., Becker, L. M., Lacbawan, F., Krasnewich, D., Muenke, M., Lawrence, D. A., Yerby, M. S., Shaw, C.-M., Gooptu, B., Elliott, P. R., Finch, J. T., Carrell, R. W., and Lomas, D. A. (1999) Nature 401, 376-379), but little is known about the trafficking of wild type and mutant neuroserpins. We have established a cell model to study the processing of wild type neuroserpin and the Syracuse (S49P) and Portland (S52R) mutants that cause FENIB. Here we show that Syracuse and Portland neuroserpin are retained soon after their synthesis in the endoplasmic reticulum and that the limiting step in their processing is the transport to the Golgi complex. This is in contrast to the wild type protein, which is secreted into the culture medium. Mutant neuroserpin is retained within the endoplasmic reticulum as polymers, similar to those isolated from the intraneuronal inclusions in the brains of individuals with FENIB. Remarkably, the Portland mutant showed faster accumulation and slower secretion compared with the Syracuse mutant, in keeping with the more severe clinical phenotype found in patients with the Portland variant of neuroserpin. Both mutant and wild type neuroserpin were partially degraded by proteasomes. Taken together, our results provide further understanding of how cells handle defective but ordered mutant proteins and provide strong support for a common mechanism of disease caused by mutants of the serine protease inhibitor superfamily.     

HPLC/RIA analysis of bioactive methionine enkephalin content in the seizure-susceptible El mouse brain

We previously reported a deficit of methionine enkephalin-like immunoreactivity (ME-LI), in the cerebral cortex, septal area, hippocampus, and striatum and the abnormal metabolism of opioid peptides in the hippocampus and striatum of seizure-susceptible El mice, which are involved in the pathogenesis of seizures. However, these findings suggest that the ME-LI does not necessarily reflect the bioactive methionine enkephalin (ME). Herein, we measured the biologically active peptide, ME excluding cross-reactive substances by using HPLC coupled with radioimmunoassay to clarify the abnormal function of enkephalinergic neurons in the El mouse brain. The ME content in 25-day-old El mice that had no seizures was significantly decreased in the hippocampus and septal area, as compared with corresponding regions in ddY mice (seizure-nonsusceptible; the mother strain of El). At the age of 50 days when El mice displayed abortive seizures, this content in both stimulated El[s] and nonstimulated El[ns] was significantly reduced in the septal area and cerebral cortex. At the age of 150 days when El mice exhibit tonic-clonic seizures, this content in both El[s] and El[ns] was significantly reduced in the septal area, cerebral cortex and striatum. These findings were generally compatible with our previous findings. This study further supports our hypothesis that a deficit of anticonvulsant endogenous ME, in the cerebral cortex, septal area, and hippocampus of seizuresusceptible El mice play an important role in the pathogenesis of seizures.     

Circadian Dependent Effect of Epidermal Growth Factor,Insulin and Glucagon on Hepatic Pyruvate Kinase and Malic Enzyme of Mice

The influence of epidermal growth factor (EGF), 0.75 μg g^?1; insulin, 1.5 μg g^?1; glucagon, 1.25 ygg^?1 and their combinations on the activities of hepatic pyruvate kinase (PK) and malic enzymes (ME) was monitored. Male CD2F₁ mice were treated toward the end of the light or dark periods, 9 or 23 /tours after /ights on (9 or 23 HALO), and subgroups of six mice were killed at 4,8 or 12 hr post-treatment. PK and ME activities from control mice were well characterized by cosine curves. The PK activity was maximal when ME activity was minimal at the transition from light to dark (9 HALO plus 4 hr) and PK was at a minimum when ME was highest (23 HALO plus 4 hr). Both enzymes were influenced by at least one peptide hormone, and the effects were strongly circadian -stage dependent. The only effect attributed to EGF was an increase of PK activity (23%) 12 hr after injection at 23 HALO. PK activity was increased by insulin (23%) at 23 HALO (4 hr after injection), but not at 9 HALO, and decreased (17%) by glucagon 12 hr after injection at 9 HALO. Several reductions in PK activity in response to various combinations of peptides were observed, and appeared to be caused by glucagon but influenced by insulin. The activity of ME was decreased (33%) in response to insulin 4 hr after injection at 23 HALO but not at 9 HALO and increased (60-70%) by glucagon alone or in combinations with insulin or EGF, or both, at 4 hr after injection at 9 HALO but not at 23 HALO. In general, when ME activity was altered by either insulin or glucagon, PK activity was also altered in the opposite direction, and the effects of glucagon were opposed by insulin.     

Regulatory mechanisms of mast cell differentiation

Mast cells are a progeny of the multipotential hematopoietic stem cell. Most of progenies of the stem cell complete their differentiation within the bone marrow, but precursors of mast cells leave the bone marrow, migrate in blood, and invade into tissues. After the invasion, precursors proliferate and differentiate into mast cells. An appreciable proportion of mast cells retain proliferative potential after differentiation, and even after degranulation, some mast cells can proliferate and recover the original morphology. Proliferation of mast cells are regulated by both T cell-derived factors (i.e., IL-3 and IL-4) and fibroblast-derived factor(s). Mice of either W/Wv or Sl/Sld genotype lack mast cells, but mast cells do develop when bone marrow cells of W/Wv or Sl/Sld mice were cultured in the presence of T cell-derived factors. Mast cells derived from W/Wv mice cannot respond fibroblast-derived factor(s) and fibroblasts derived from Sl/Sld mice cannot support mast cells of normal mouse origin. Phenotypes of mast cells are determined by the environment in which the mast cells differentiated. However, when mast cells are transplanted into a new environment which is different from the original one, the mast cells acquire the phenotype which are dependent on the second environment.     

Circadian Dependent Effect of Epidermal Growth Factor, Insulin and Glucagon on Hepatic Pyruvate Kinase and Malic Enzyme of Mice

The influence of epidermal growth factor (EGF), 0.75 μg g^-1; insulin, 1.5 μg g^-1; glucagon, 1.25 ygg^-1 and their combinations on the activities of hepatic pyruvate kinase (PK) and malic enzymes (ME) was monitored. Male CD2F₁ mice were treated toward the end of the light or dark periods, 9 or 23 /tours after /ights on (9 or 23 HALO), and subgroups of six mice were killed at 4,8 or 12 hr post-treatment. PK and ME activities from control mice were well characterized by cosine curves. The PK activity was maximal when ME activity was minimal at the transition from light to dark (9 HALO plus 4 hr) and PK was at a minimum when ME was highest (23 HALO plus 4 hr). Both enzymes were influenced by at least one peptide hormone, and the effects were strongly circadian -stage dependent. The only effect attributed to EGF was an increase of PK activity (23%) 12 hr after injection at 23 HALO. PK activity was increased by insulin (23%) at 23 HALO (4 hr after injection), but not at 9 HALO, and decreased (17%) by glucagon 12 hr after injection at 9 HALO. Several reductions in PK activity in response to various combinations of peptides were observed, and appeared to be caused by glucagon but influenced by insulin. The activity of ME was decreased (33%) in response to insulin 4 hr after injection at 23 HALO but not at 9 HALO and increased (60-70%) by glucagon alone or in combinations with insulin or EGF, or both, at 4 hr after injection at 9 HALO but not at 23 HALO. In general, when ME activity was altered by either insulin or glucagon, PK activity was also altered in the opposite direction, and the effects of glucagon were opposed by insulin.     

Antinociceptive properties of the methanolic extract and two triterpenes isolated from Epidendrum Mosenii stems (Orchidaceae)

The antinociceptive effect of the methanolic extract (ME) and two triterpenes isolated from E. mosenii (Orchidaceae) has been investigated in chemical and thermal models of nociception in mice. The ME of E. mosenii (0.3-30 mg kg(-1), i.p. or 50-400 mg kg(-1), p.o.) produced dose-related, significant and long-lasting (4 to 6 h) inhibition of acetic acid-induced abdominal constriction, with ID50 values of 3.9 and 137.0 mg kg(-1), respectively. Pholidotin and 24-methylenecycloartenol isolated from E. mosenii (0.1-3.0 mg kg(-1), i.p.) also produced marked and dose-related inhibition of acetic acid-induced pain, with ID50 values of 0.9 and 1.1 mg kg(-1). However, these compounds and the ME were about 3- to 13-fold more potent at the level of ID50 than diclofenac when assessed in acetic acid-induced abdominal constriction. The ME of E. mosenii in the same range of doses produced dose-related inhibition of both phases of formalin-induced licking, with mean ID50 values for the first and the second phases of 0.9, 122.0 mg kg(-1) and 0.7, 258.0 mg kg(-1), respectively by i.p. or p.o. routes. In addition, the ME (0.3-30 mg kg(-1), i.p., or 50-400 mg kg(-1), p.o.) also caused dose-related inhibition of capsaicin-induced neurogenic pain with mean ID50 values of 5.2 and 130.0 mg kg(-1), respectively. Treatment of animals with naloxone (5 mg kg(-1), i.p.) completely reversed the antinociceptive effect caused by morphine (5 mg kg(-1), s.c.) and that caused by ME of E. mosenii (1 mg kg(-1), i.p.) when assessed against either phase of the formalin-induced pain. Furthermore, when assessed in the hot-plate test, ME (100 mg kg(-1), i.p.) and morphine (10 mg kg(-1), s.c.) caused significant increase in response latency. However, ME given daily for to 7 consecutive days did not develop tolerance to itself nor did it induce cross-tolerance to morphine. Taken together these data demonstrate that the ME of E. mosenii elicited pronounced antinociception, when assessed by i.p. or p.o. routes, against several models of pain. Its actions involve, at least in part, an interaction with opioid system, seeming no to be related with a non-specific peripheral or central depressant actions. Finally, the active principle(s) responsible for the antinociceptive action of E. mosenii is likely related to the presence of the triterpenes.