Ultrastructural responses by Golgi apparatus of rat submandibular gland to beta-adrenergic, alpha-adrenergic, and cholinergic stimulation

Rat submandibular gland tissue pieces were stimulated in vitro for 30 min with a beta-adrenergic agent or a cyclic AMP analog to stimulate protein secretion, or with alpha-adrenergic or cholinergic agents or a Ca2+ ionophore to stimulate fluid secretion. Acinar cells were examined by transmission electron microscopy. In control tissue, acinar cells showed little evidence of secretory activity. The Golgi apparatus was sparse and was associated with a few small, immature secretory granules with fine fibrillar contents. Following secretory granule discharge stimulated by isoproterenol or dibutyryl cyclic AMP, acinar cells were constricted, and had extensive basolateral membrane folding and tightly packed rough endoplasmic reticulum. Golgi complexes were prominent and had multiple small granules with filamentous contents. After stimulation of fluid secretion by alpha-adrenergic agents (epinephrine, phenylephrine), or cholinergic agents (acetylcholine, carbachol, pilocarpine), or a Ca2+ ionophore (A23187), the Golgi apparatus had compact concave cisternae enclosing aggregates of tubulovesicles. Acinar cells were distended, basolateral membranes were expanded, and rough endoplasmic reticulum was dilated and vesiculated.     

Primary interaction between antibody and components of Alternaria. I. Immunology and chemical characteristics of labeled antigens.

Components were isolated from Alternaria tenuis and its culture filtrate, and were radiolabeled with 123-I. The labeled antigenic components had a high polysaccharide content as determined by staining patterns following electrophoresis in polyacrylamide gels and by inactivation with sodium metaperiodate. A primary binding test was employed to detect and measure serum antibodies to the components from A. tennuis. This procedure was more sensitive in detecting antibodies that bound to antigens than were comparable tests dependent upon precipitin types of reactions. The labeled components of A. tenuis cross-reacted or shared antigens with 3 other species of molds: Stemphylium sp., Curvularia sp., Aspergillus fumigatus, but not with a variety of other fungal and nonfungal materials.     

Variability of Alternaria alternata: Biochemical and immunological characteristics of culture filtrates from seven isolates

Biochemical variability of culture filtrates from the common allergenic mold Alternaria alternata was studied. Differences between culture filtrates from 7 different isolates and between 4 batches of culture filtrate from the same isolate were observed, suggesting the unreliability of presently employed biochemical methods in the routine standardization of mold allergens and the possibility of difficulties in developing standard techniques for their purification. Dextranases, cellulases, and agarases, found in one culture filtrate, may be a further source of problems in laboratory techniques employing A. alternata. The proportions of nitrogen: carbohydrate: dialyzed dry weight were different in culture filtrates from each isolate. Polysaccharides in culture filtrates from 3 of the 7 isolates contained glucose, mannose, xylose, galactose, and a fifth, unidentified sugar. Polysaccharides from one isolate lacked xylose, and the fifth sugar was not demonstrable in 4 of the isolates. Despite the biochemical differences, extensive antigenic cross-reactivity between different isolates was found in precipitin studies and inhibition of antibody binding.     

Primary interaction between antibody and components of Alternaria: II. Antibodies in sera from normal,allergic, and immunoglobulin-deficient children

Antibodies in sera from normal, allergic, and immunoglobulin-deficient children were studied for binding to radiolabeled components of Alternaria tenuis. Significant binding levels were found in 103 of 105 sera from normal children. The levels were age-dependent, rising from a low point in the 7- to 12-month age group to adult levels by the age of 8 years. Levels of binding to two antigens, a culture filtrate derivative (¹²⁵I-CLF) and a mycelial derivative (¹²⁵I-IIS), were similar. Sera from asthmatic children with strong immediate skin test reactions to Alternaria extracts bound significantly higher levels of ¹²⁵I-CLF than did sera from allergic children with negative skin tests or from control children. Binding levels in sera from children with hypogammaglobulinemia were significantly less than binding levels in sera from normal children in any age group. Sera from children with selective IgA deficiency bound ¹²⁵I-CLF at normal levels. The almost universal occurrence of anti-Alternaria antibodies in children was partly explained by the finding of partial cross-reactivity and/or shared antigens among several fungal species, including A. tenuis, Stemphylium sp., Curvularia sp., and Aspergillus fumigatus. The biological significance of these antibodies is not clear, but the procedures described lend themselves to further investigations.     

Duck hepatitis B virus is tropic for exocrine cells of the pancreas

Earlier observations had established that duck hepatitis B virus (DHBV) is tropic for pancreatic endocrine cells, including cells localized to islets and to acini. Because cells identifiable as endocrine represented only a minor fraction of the total acinar-associated, infected subpopulation, the possibility was addressed in the present study that this subpopulation also comprises exocrine cells. Fixed preparations of cells from pancreas of congenitally DHBV-infected young ducks were reacted in double immunofluorescence assay with anti-virus serum and either anti-avian pancreatic polypeptide (APP) serum, a probe for a major subclass of acinar-associated endocrine cells, or anti-chymotrypsin serum, a probe for exocrine cells. Approximately 2-5% of the cells in these preparations were viral antigen-positive, comprising a minor fraction positive for APP and a much larger fraction positive for chymotrypsinogen. The detection of the latter establishes that DHBV is tropic for exocrine cells.     

Upper motor neurone modulation of charge movement and mechanical activation in rat skeletal muscle fibres

Intraperitoneal injection of rat prolactin to cycling female rats results in neuroendocrinological alterations which depend on the strain of animals. Specifically, preovulatory surges of luteinizing hormone and follicle-stimulating hormone were suppressed in Sprague-Dawley rats while elevated levels of serum luteinizing hormone were observed in Wistar animals.     

Separation of rat T and B lymphocytes by density gradient electrophoresis.

Density gradient electrophoresis has been employed for the preparative separation of T and B lymphocytes from rat spleen and peripheral blood. The high mobility cells were found to be predominantly T lymphocytes, as determined by their response to phytohemagglutinin and the relative absence of immunoglobulin-positive cells. The low mobility cells were predominantly B cells, as determined by the high percentage of immunoglobulin-positive cells and the total lack of response to PHA, an exclusive T cell mitogen. A better separation of peripheral blood T and B lymphocytes was achieved than with spleen T and B cells.     

Common surface receptors on both mouse and rat cells distinguish different classes of mouse mammary tumor viruses

Cell surface receptors which bind mouse mammary tumor virus (MMTV) were detected on mouse and rat cells. Virus binding was quantitated by measuring ¹²⁵T-protein A binding to immune complexes composed of a C3H MMTV gp52 type-specific monoclonal antibody and receptor-bound MMTV. C3H MMTV binding to normal mouse mammary epithelial cells (NMuMG) was dose dependent and was ≥50% inhibited by GR MMTV, but not by endogenous C3Hf MMTV or Gross murine leukemia virus. These results were confirmed in [³H]leucine MMTV binding inhibition assays in which GR MMTV and C3H MMTV blocked C3H [³H]MMTV binding while C3Hf MMTV did not block. The affinities of C3H [³H]MMTV binding to receptors on NMuMG, NIH Swiss (SLP), and Fischer rat embryo (FRE) cells were identical by Scatchard analysis. C3Hf [³H]MMTV also bound these cells, but with an affinity approximately 10 times weaker than the C3H [³H]MMTV binding. Interference assays using a Kirsten sarcoma virus (C3H MMTV) pseudotype confirmed the importance of C3H MMTV specific binding to viral penetration and expression. Pretreatment of SLP or FRE cells with 100 μg of C3H MMTV or GR MMTV inhibited focus formation by ≥50% while C3Hf MMTV and RIII MMTV did not inhibit. Therefore, the functional C3H MMTV cell receptors on mouse and rat cells were related and were able to distinguish C3H MMTV and GR MMTV from C3Hf MMTV and RIII MMTV. These comparable receptors may represent evolutionarily conserved surface components of murine cells.     

X-ray microanalysis of spontaneously formed urinary calculi in a jaundiced strain of rat

A high incidence of spontaneously formed urinary stone was found in the females of a jaundiced strain of rat developed from a cross between Gunn's rat and Wistar-Imamichi rat. In this colony, 42.3% of the females had urinary calculi. Elemental analyses of these urinary calculi were carried out with an analytical electron microscope, a high-resolution transmission electron microscope fitted with an energy dispersive type X-ray microanalyzer and a scanning device. In the surface and middle areas of the stone, the main components were recognized as magnesium (Mg) and phosphorus (P). In the central region of the stone, calcium (Ca) and phosphorus (P) were found as the main elements with trace amounts of sodium (Na), chlorine (Cl), and potassium (K). The analyses indicated that the spontaneous urinary stone consisted of phosphate salts with calcium or magnesium. In addition, the mass ratio of the $\text{Mg}\text{P}$ or $\text{Ca}\text{P}$ in the stones was calculated from X-ray pulse intensity ratios and compared with the mass ratio of a standard sample. The results suggested that the magnesium and phosphorus in the urinary stones existed as ammonium magnesium phosphate, MgNH₄PO₄, and the calcium and phosphorus as tribasic calcium phosphate, Ca₃(PO₄)₂.     

Effects of cholecystokinin octapeptide and hydrocortisone on the exocrine pancreas of fed and fasted 24-hour-old rats: an electron microscopic study

We evaluated by electron microscopy the effects of two hormones, cholecystokinin octapeptide (CCK-8) and hydrocortisone, on the development of the pancreatic acinar cell in newborn rats. The newborn rat pancreas contains abundant zymogen granules of variable size but is unresponsive to secretagogues. In contrast the 24-hr-old pancreas is depleted of granules, indicating emptying of the acinocytes in response to feeding. Zymogen granule content is moderate in the 24-hr-old rat that is fasted from birth. The pancreas from the 24-hr-old rat that is fasted and treated with either hydrocortisone or CCK-8 has an intermediate number of granules. In the animals fasted and treated with hormones, the rough endoplasmic reticulum is arranged into parallel lamellae suggesting a higher level of protein synthesis which may contribute to the formation of abundant zymogen stores. We conclude that zymogen stores are lost earlier postnatally than previously understood, and that zymogen seen in the 24-hr-old animals may represent newly synthesized materials.     

Envelope proteins of the BALB/c myeloma mink cell focus-inducing (MCF) viruses

A recent report from this laboratory demonstrated that the BALB/c myeloma retroviruses, MO-21 and FL-1, have a polytropic host range and exhibit “mink cell focus-inducing” (MCF) activity. Because previous MCF isolates have been shown to possess a recombinant envelope (env) gene, we undertook neutralization and peptide mapping studies to analyze the envelope proteins of MO-21 and FL-1 viruses and compare them to various prototype murine retroviruses. Like other MCF viruses, the infectivity of the myeloma viruses was inhibited by normal mouse serum. Two-dimensional peptide mapping data show that the viral envelope protein (gp70) of MO-21 and FL-1 has several distinct peptides which distinguish them from the gp70 of any of the prototype viruses we analyzed. Unlike other MCF viruses, the gp70s of MO-21 and FL-1 are indistinguishable from one another. These results suggest that these MCF retroviruses may not be (env) gene recombinants. MO-21 FL-1 viruses also contain another viral-coded envelope protein which has an apparent higher molecular weight than the gp70. The possible significance of this gp70-like protein is discussed.     

Lack of correlation between thymidine kinase activity and the antiviral or antiproliferative response to interferon

Murine L cells lacking thymidine kinase activity (Ltk-) do not respond to the antiviral or antiproliferative activity of interferon, whereas human tk- cells show a normal response. Furthermore, a clone of tk+ cells derived from Ltk- cells by DNA-mediated transfer of the tk herpes gene does not respond to interferon. Cells of this clone and Ltk- cells do not produce interferon when infected with Newcastle disease virus or treated with poly(I) X poly(C) and DEAE-dextran. Possible reasons for this inability to produce interferon and the lack of response to interferon are discussed.     

Characterization of a regulator of host cell permissiveness to a specific enterovirus

The efficiency of echovirus 6 infection in cultured simian cells is enhanced significantly by a protein isolated from uninfected cultured cells. The enhancer protein was purified and characterized utilizing chromatographic and electrophoretic techniques. The enhancer was eluted from DEAE-cellulose columns with 0.3 M NaCl. Fractionation of these eluates by electrophoresis and isoelectric focusing in polyacrylamide gels indicated that enhancer activity was confined to one or two distinct protein bands. The mobility of the major enhancer band (protein a) in gels of different acrylamide concentrations and under nondenaturing conditions indicated a molecular weight of approximately 68,000. As shown by two-dimensional electrophoresis utilizing sodium dodecyl sulfate in the second dimension, this protein was further dissociated into two subunits, each with a molecular weight of approximately 32,000. The isoelectric point of the enhancer protein was estimated at pH 5.3. This enhancer protein a was detected in extracts from both permissive WISH and semipermissive Vero cells. The presence of the enhancer protein a in Vero cell extracts was demonstrated only in highly concentrated (70x or greater) preparations. There was a direct correlation between concentration of this protein and extent of enhancer activity in cell extracts. These results along with the previous observation that semipermissive cells can be rendered fully permissive by addition of exogenous enhancer suggest that the quantity of enhancer protein regulates host cell permissiveness.     

Interspecies reactivity of type C and D retrovirus p 15E and p 15C proteins

Two low-molecular-weight structural components of mammalian type C viruses p15E and p15C were studied in terms of their content of interspecies antigenic reactivity. We find that both components possess broadly cross-reacting determinants, which in the case of p15E extend to primate type D viruses.     

An immunochemical investigation of SV40 T antigens. 1. Production properties and specificity of rabbit antibody to purified simian virus 40 large-T antigen.

Large quantities of a species of T antigen with an apparent molecular weight of 84,000 have been isolated from monkey kidney cells infected with SV40 by using the protein A Antibody Adsorbent (P.A.A.) technique and preparative SDS-polyacrylamide gel electrophoresis. The purified polypeptide was found to be immunogenic, inducing a specific antibody response in a rabbit. The resulting antiserum was 10 times as potent as a hamster anti-tumor serum and reacted with native as well as SDS- and DTT-treated T antigens from SV40-transformed or lytically infected cells. It failed to show any reaction with T antigen from polyoma-infected cells and showed similar specificity to antitumor serum obtained from hamsters which had been inoculated with cells of an SV40-transformed, virus-free cell line. In both cases two distinct polypeptides, large T (84,000 and 94,000) and small t (19,000) were precipitated from extracts of SV40-transformed or lytically infected cells. The rabbit antiserum was shown to be capable of specifically precipitating small-t antigen in the absence of large-T antigen and therefore these two polypeptides must share common antigenic determinants. A radioimmunoassay showed large-T antigen to be very heat stable in direct contrast to earlier results obtained using the complement fixation test. The reasons for this discrepancy and its functional significance are discussed.     

Monoclonal antibodies to baculovirus structural proteins: determination of specificities by Western blot analysis

Conventional mouse hybridoma technology was utilized to produce a panel of monoclonal antibodies which reacted with baculovirus proteins. Using an enzyme-linked immunosorbent assay (ELISA), the hybridomas which were raised against polyhedrin from Autographa californica nuclear polyhedrosis virus (AcNPV) and Choristoeura fumiferana nuclear polyhedrosis virus (CfNPV) were found to cross-react differentially with polyhedrins and granulins from several species of baculoviruses. Hybridoma antibodies which reacted against the nonoccluded form (NOV) of AcNPV in an ELISA test expressed different specificities for the occluded form of the virus (OV), a mutant strain of AcNPV, and CfNPV. Four hybridoma clones produced antibody which neutralized the infectivity of AcNPV NOV. One hybridoma antibody reacted strongly with the uninfected Spodoptera frugiperda host cell line. Using Western blot analysis, it was shown that hybridoma antibodies against polyhedrin reacted differentially with the complete polypeptide and protease-generated fragments of polyhedrin. The polypeptide specificity of 19 of 28 hybridoma antibodies which reacted with OV and NOV of AcNPV was assigned using Western blot analysis.     

Amino acid sequence of southern bean mosaic virus coat protein and its relation to the three-dimensional structure of the virus

The amino acid sequence of the coat protein of southern bean mosaic virus has been determined. Chemical techniques were supplemented by a 2.8-A-resolution electron-density map which helped in assigning absolute positions for each peptide. Four amino acids and the placement of one heptapeptide near the amino terminus of the protein were interpolated by comparison with the partially known southern bean mosaic virus RNA sequence. The subunit is heavily lined with basic residues on the side facing the RNA. Subunit-subunit interactions are hydrophobic and ionic. The calcium site on the quasi-threefold axis has three glutamic residues as ligands. There is a disulfide bond, between Cys 168 and 176, within a sequence which is inserted in southern bean mosaic virus relative to the tomato bushy stunt virus structure. This portion is a helix nestling between the "beta-barrel" subunit structure and the RNA interior.     

2-5A synthetase activity induced by interferon alpha, beta, and gamma in human cell lines differing in their sensitivity to the anticellular and antiviral activities of these interferons

Two human cell lines (HEC-1, IF^r) which are resistant to the anticellular and/or antiviral action of HuIFN-α or -β have been tested with respect to the anticellular and antiviral activities of HuIFN-γ. HEC-1 cells were totally resistant to the antiviral activity of the IFN-γ, with either EMC or VSV as challenge virus. Per antiviral unit HuIFN-γ exerted a much stronger inhibitory effect on the growth of the interferon-sensitive cell line RSa than did IFN-α. IF^r cells were more resistant to the anticellular effect of IFN-γ than the wild-type cells (RSa), and HEC-1 cells were totally resistant to the anticellular effect of HuIFN-γ. 2-5A synthetase was present at a high basic level in untreated HEC-1 cells, as previously reported. This activity was not influenced by either HuIFN-β or -γ. Similar levels of 2-5A synthetase were induced by HuIFN-γ in IF^r and RSa cells; however, this induction was 10 times lower than that obtained for HuIFN-β. A 2′-phosphodiesterase activity was detected in control extracts from RSa and was not induced by either HuIFN-β or -γ. There was no correlation between the amplitude of 2-5A synthetase induction and extent of anticellular effect of either HuIFN-γ in different cell lines or of different interferons in a given cell line. Finally, HEC-1 represents the first example of a human cell line which is resistant to all three types of interferon (HuIFN-α, -β, and -γ).