Formation of 100 A filaments from purified glial fibrillary acidic protein in vitro.

Glial fibrillary acidic protein, which is specific to astroglia in the central nervous system, polymerizes in vitro into filaments similar to native ~ 100 Å filaments. Following purification from aqueous extracts of bovine brain by immunoaffinity chromatography, GFA ² protein is highly soluble in very low ionic strength solutions. Sedimentation equilibrium analysis of protein solutions in prefilament solvent conditions (2 mm-Tris · HCl, pH 7.8, 20 °C, containing 0.5 mm-dithiothreitol) indicates a paucidisperse mixture of species in solution with a typical range of apparent weight-average molecular weights from about 186,000 to 227,000. Between pH 6.0 and 8.0 the solubility is a function of pH and ionic strength as well as temperature, and precipitation is favored by lowering the pH or temperature and by raising the ionic strength. GFA protein associates in the form of filaments over a narrow range of pH and ionic strength; optimal conditions for polymerization of a 0.1 mg/ml protein solution are 100 mm-imidazole-HCl buffer (pH 6.8), at a temperature of 37 °C, and there is no requirement for co-factors. Filaments appear primarily as tangles of smooth curvilinear structures approximately 100 Å in diameter and of indefinite length, although some lateral association of filaments into thick bundles is also observed. While the formation of filaments is not affected by the presence or absence of reducing agent, under oxidizing conditions disulfide linkages form between protein subunits. Disassembly is achieved by dialysis against 2 mm-Tris · HCl buffer (pH 8.5), but this process is significantly enhanced by the addition of 0.5 mM-dithiothreitol during assembly and disassembly.These experiments clarify the role of GFA protein as the subunit of astroglialspecific intermediate filaments. In addition, they suggest that the 100 Å filament, as other components of the cytoskeleton, may assemble and disassemble in the glial cytoplasm.     

Cell-specific domains of glial- and muscle-type intermediate filament proteins : Immunoaffinity chromatography and immunoblotting study of GFA protein and desmin

In order to localize the cell specific domains of glial- and muscle-type intermediate filaments, the purified subunits (bovine GFA protein and chicken desmin) were fragmented and the digests passed through immunoaffinity columns or stained by the immunoblotting procedure to determine which fragments reacted with the monospecific polyvalent antisera. The following fragments were found immunoreactive according to these criteria: 30 K (GFA) and 33 K (desmin) N-bromosuccinimide fragments (tryptophan cleavage); 35 K (GFA) and 39 K (desmin) 2-nitro-5-thiocyanobenzoic acid fragments (cysteine cleavage); 18 K (GFA) and 9 K (desmin) cyanogen bromide fragments. Fragmentation of GFA protein was also accomplished using proteolytic digestion with chymotrypsin and trypsin. Two resistant core polypeptides, one about 37 K and stable in the chymotryptic digests and one about 21 K and stable in the tryptic digests bound specifically to the immunoaffinity columns. The 21 K tryptic fragment was found to contain the 18 K cyanogen bromide fragment. The fragmentation patterns support recently published structural domain models for intermediate filament proteins. The immunochemical findings indicate that the immunoreactive regions of GFA protein are located in the aminoterminal region of the middle domain of these models (coil I), while they appear to be situated in the aminoterminal headpiece of the protein in the case of desmin.     

Phosphorylation of cardiac sarcolemma by endogenous and exogenous protein kinases.

Sarcolemmal membranes isolated from guinea pig heart ventricles contained endogenous protein kinase activity and protein substrates for this enzyme. Phosphorylation of sarcolemma was modestly stimulated by cyclic AMP with the half-maximal stimulation at 0.5 μm cyclic AMP. The phosphorylation of sarcolemma due to endogenous kinase was dependent on Mg²⁺. The apparent affinity for Mg²⁺ was found to be 1.4 and 0.53 mm in the absence and presence of 1 μm cyclic AMP, respectively. The apparent affinity for ATP was 55 μm. Sarcolemmal membranes were also phosphorylated by exogenous (purified) cyclic AMP-dependent protein kinase(s). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of phosphorylated membranes, followed by slicing and determination of the radioactivity in the gel slices, showed that endogenous protein kinase activity promoted the phosphorylation of specific protein peaks, arbitrarily designated a–g in order of increasing relative mobility (relative molecular weights 125,000, 110,000, 86,000, 58,000, 48,000, 22,000, and 16,000, respectively); peak e (48,000) was the major phosphorylated band. Exogenous protein kinase stimulated the phosphorylation of all peaks. However, the degree of stimulation of the low molecular weight peaks f and g was more marked. Results obtained after treatment of phosphorylated membranes with hydroxylamine at acid pH indicated the absence of any significant amount of acyl phosphate-type incorporation of phosphate. Purified phosphoprotein phosphatase from rabbit liver effected dephosphorylation of previously phosphorylated sarcolemma; this treatment resulted in dephosphorylation of all peaks (a–g). Pretreatment of sarcolemma with trypsin (membrane to trypsin ratio of 100) was found to markedly reduce both the total membrane phosphorylation as well as relative phosphorylation of peaks c, f, and g. On the other hand, pretreatment of sarcolemma with phospholipase c slightly stimulated total membrane phosphorylation with nondiscriminatory enhancement of the phosphorylation of all peaks. Microsomal membrane vesicles (enriched in sarcoplasmic reticulum fragments) isolated from guinea pig heart ventricle also contained endogenous protein kinase activity. Cyclic AMP modestly increased the kinase. Polypeptides of molecular weights 56,000, 22,000, and 16,000 were found to be phosphorylated. Exogenous (purified) cyclic AMP-dependent protein kinase increased the phosphorylation of microsomes and of 22,000 and 16,000 molecular weight polypeptides.     

Enhanced purification of Mullerian inhibiting substance by lectin affinity chromatography

Mullerian inhibiting substance (MIS), a secreted testicular product responsible for regression of the Mullerian ducts in the male mammalian embryo, was purified 7000 fold, exploiting the glycoprotein nature of this important fetal regressor to achieve enhanced purification. The present procedure employs media incubation of newborn calf testis, passage through DEAE Bio-Gel A and CM Bio-Gel A and sequential lectin affinity chromatography on wheat germ lectin (WGL)-Sepharose 6MB and concanavalin A (Con A)-Sepharose 4B. Strongly bioactive MIS was released from both lectin columns in the bound glycoprotein fraction only after elution with lectin-specific sugar. Carbohydrate analysis of the highly purified glycoprotein fraction eluted from Con A indicated the presence of both N-acetyl glucosamine and mannose, as would be expected from its sequential lectin affinity, as well as of galactose, galactosamine and N-acetyl neuraminic acid. Electrophoresis of this fraction on polyacrylamide-SDS gels showed an identical band pattern after staining with either Coomassie blue or periodic acid-Schiff reagent, further indicating that MIS is a glycoprotein.     

Inhibition of lymphocyte transformation responses to phytohemagglutinin by normal human plasma.

Marked variability in lymphocyte transformation responses to a suboptimal phytohemagglutinin concentration (0.1 μg/ml) was observed when peripheral blood mononuclear cells of normal subjects were cultured in media containing 15% autologous plasma. Low responses were related to the plasma and were caused by direct inhibition rather than an inability to support the response. This inhibitory activity varied greatly among different plasma specimens and was also found in human and animal serum. It appeared to be specific for suboptimal concentrations of phytohemagglutinin and could be overcome by increasing the mitogen concentration. The inhibitory activity was heat stable, was not dialyzable, and appeared to affect a very early stage of the response since a delay in addition of the inhibitory plasma reversed its effect. Our interpretation of these results is that human plasmas vary greatly with respect to their ability to bind phytohemagglutinin so that the addition of different plasmas to lymphocyte cultures stimulated by a limiting concentration of phytohemagglutinin results in marked variability of the results obtained.     

Interactions of prostaglandin E1 (PGE1) and LRH on anterior pituitary function

Numerous biochemical pathways influence the synthesis and release of anterior pituitary hormones. Releasing factors extracted from the hypothalamus and prostaglandins (PGs) appear to alter a common biochemical activity, adenyl cyclase, in pituitary cells. Luteinizing hormone releasing hormone (LRH), prostaglandin (PGE₁), 7 oxa-13-prostynoic acid and cycloheximide were tested for individual and interacting effects on the in vitro release of FSH, LH and prolactin from hemipituitaries of 15 day old female rats. LRH (10 ng/ml) consistently released both LH and FSH in all in vitro experiments and inhibited prolactin release in 1 of 2 experiments. Lower concentrations (5 and 1 ng/ml) also stimulated LH and FSH release but did not influence prolactin release. Concurrent depletion of stored LH and FSH in the gland was observed. PGE₁ in a 6.5 hour incubation increased the storage of LH within the gland in the absence of LRH. In a 1.5 hour incubation in the presence of LRH, storage of LH was also increased. PGE₁ had no effect on LH and FSH release; however, in 1 of 2 experiments it stimulated prolactin release in the absence of LRH. Prostynoic acid stimulated LH and FSH release but did not synergize with LRH action in the same tissue. Cycloheximide did not affect LH release during the first 30 minutes of incubation; however, the release during the subsequent 1 hour was significantly inhibited. Similar tissue also exposed to cycloheximide was still responsive to LRH during the latter 1 hour incubation period. Cycloheximide had no effect on prolactin storage and release from the same tissue.     

Cell-mediated lymphocytotoxicity following in vitro sensitization of frozen stored human mononuclear cells in heterologous serum

Human peripheral mononuclear cells were subjected to controlled-rate freezing and stored at−196 °C. Following rapid thawing and slow removal of DMSO, the cells exhibited normal immune responsiveness to phytohemagglutinin and in mixed lymphocyte culture. After in vitro sensitization to frozen allogeneic cells in heterologous serum they developed cell-mediated lymphocytotoxicity demonstrated by ⁵¹Crrelease from specific targets isogeneic with the sensitizing cells.     

Evidence for a shift from IgD to IgA synthesis in the maturation of human B lymphocytes.

A case of chronic lymphatic leukemia (CLL) is described in which IgD and IgA are the copredominant membrane immunoglobulins. Since CLL represents malignant proliferations of B lymphocytes arrested at discrete points during maturation, the findings in this case suggest that at least some of the developing cells destined to synthesize IgA for secretion pass through a stage in which immunoglobulins D and A are present together on the cell membrane.     

Hyaluronidase activity and hyaluronate content of the developing chick embryo heart.

Hyaluronidase activity and hyaluronate content were measured in the developing chick heart from embryonic day 3 through posthatching stages. High levels of both enzyme and substrate were found during the earliest stages examined. Hyaluronidase activity gradually declined to 63% of the initial (day 3) level by embryonic day 16. Enzyme activity decreased more sharply during the next 4 days to 30% of the initial level and remained constant through 2 weeks after hatching. Low levels of enzyme activity (about 10% initial levels) were still detectable in 10-week-old chicken hearts. The heart hyaluronidase is an endoglycosidase with an estimated molecular weight of 62,000, which degrades hyaluronate and, to a lesser extent, chondroitin sulfate at an acid pH optimum. Hyaluronate constituted approximately 50% of the total glycosaminoglycan content at embryonic day 5. Between embryonic days 5 and 12, the concentration of hyaluronate decreased to 25–30% of the initial level and remained constant thereafter. The level of other glycosaminoglycans decreased more gradually than hyaluronate and did not reach a constant level until hatching. This pattern of hyaluronidase activity and hyaluronate concentration presumably reflects the extensive tissue remodeling which transforms the developing heart from a thin-walled tube containing extensive regions of extracellular matrix to a compact, thick-walled myocardium having a limited extracellular compartment.     

Effector cells which mediate antibody-dependent cell-mediated cytotoxicity. II. Ontogeny of effector activity in murine spleen.

The specificity of T cells for syngeneic target cells is directed to both antigens and products of the major histocompatibility complex (MHC) on the target cell surface. This dual requirement is best accounted for by the altered-self hypothesis, which implies that the MHC products on a cell's surface are able to form complexes with many other proteins on the surface of the same cell. To account for the ability of MHC products to bind so many different cell surface antigens we propose that interactions in general among macromolecules on the surface of a membrane may be dramatically enhanced by a purely physical effect. This effect derives from the confinement of membrane macromolecules to an effective volume which is the product of membrane surface area times d, the distance over which the center of mass of the molecules can move in a vertical direction (perpendicular to the membrane surface). Because d is very small the effective concentrations of surface molecules are extremely high and their interactions are correspondingly enhanced.     

Ly-4.2: a cell membrane alloantigen of murine B lymphocytes. III. In vitro studies

The alloantigenic specificity Ly-4.2 is present on a restricted population of murine lymphocytes which have previously been shown to have some of the properties generally ascribed to B lymphocytes, both with regard to distribution and function. In the study reported herein, the effect of anti-Ly-4.2 and anti-Thy-1.2 (θ) antisera have been examined in various in vitro systems. (a) T cell-mediated lysis of ⁵¹Cr-labeled P815-X2 target cells by immune allogeneic peritoneal exudate cells is inhibited by anti-Thy-1.2, but not affected by the anti-B (Ly-4.2) reagent. (b) Antibody-dependent lymphocyte-mediated lysis of ⁵¹Cr-labeled sheep red cells was only slightly inhibited by anti-Ly-4.2 and anti-Ig antisera, and not at all by anti-Thy-1.2 antisera, indicating that this type of cell lysis is mediated by neither T (Thy-l⁺) nor B (Ly-4.2⁺,Ig⁺) cells. (c) The response of lymph node lymphocytes to various mitogens was affected thus: PHA, completely inhibited by anti-Thy-1.2 but not by anti-Ly-4.2; Con A, largely inhibited by anti-Thy-1.2, and slightly by anti-Ly-4.2; PWM (pokeweed mitogen), partially inhibited by both antisera; E. coli endotoxin lipopolysaccharide, greatly inhibited by anti-Ly-4.2 but only slightly by anti-Thy-1.2. The findings demonstrate that anti-Thy-1.2 reacts predominantly with T cells and anti-Ly-4.2 with B cells.     

The preservation of Mullerian inhibiting substance during long-term freezing of testicular fragments

Mullerian Inhibiting Substance, a fetal testicular hormone found in most mammalian species, causes regression in the male of the Mullerian duct, the anlagen of the fallopian tube, uterus, and upper vagina. Limitations to study of this substance in the past have been posed by its short period of production and by its localized and specific action. We have been able to store testicular fragments that continue to demonstrate detectable Mullerian Inhibiting Substance activity for up to 5 months by using techniques of slow freezing which approximate 1 °C/min, cryoprotective additives, storage in liquid nitrogen, and rapid thawing. These fragments then can be pooled for biochemical and endocrinological studies. In addition, unknown fragments can be transported long distances for assay of Mullerian Inhibiting Substance.     

BCG-induced chronic pulmonary inflammation and splenomegaly in mice: suppression of PHA-induced proliferation, delayed hypersensitivity to sheep erythrocytes, and chronic pulmonary inflammation by soluble factors from adherent spleen cells

C57BL/6 (B6), but not CBA, mice develop intense chronic granulomatous inflammation (CGI) in the lungs and spleen in response to an iv injection with killed BCG in an oil-in-saline emulsion (BCG-E). Concomitant with the development of CGI, these mice show diminished responsiveness to PHA and LPS, as well as suppression of antibody synthesis and production of delayed hypersensitivity (DH) to sheep erythrocytes (SRBC). Suppression results from the development of adherent, Thy-1^?, Ig^? spleen cells. The present study shows that cells from inflamed spleens of BCG-E-treated B6 mice elaborate factors in vitro which (a) inhibit PHA-induced proliferation of both normal syngeneic and allogencic cells, (b) suppress DH to SRBC in B6 mice, and (c) diminish the intensity of BCG-E-induced CGI in the lungs and spleens of B6 mice. These factors are produced by adherent Thy-1^? cells in BCG-injected mice but not in similarly treated CBA mice. These factors may be important in understanding the control of immunologically mediated chronic inflammation.     

Optimization of in vitro protein synthesis by isolated mouse adrenal mitochondria

The requirements for in vitro mitochondrial protein synthesis have been studied using isolated mitochondria from cultured adrenal Y-1 tumor cells from mice. By reducing the reaction volume to 50 microliter we were able to assay in replicate the requirements for various reaction components using trichloroacetic acid (TCA)-precipitable counts for a quantitative evaluation with time of incubation. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis followed by autoradiography was also used for a qualitative and quantitative evaluation of the translation products. With the optimized system, 1 to 3% of added [35S]methionine was incorporated. The products of mitochondrial protein synthesis range from 70,000 to 5000 molecular weight. Major autoradiographic bands were observed at 38,000, 31,000, 23,000, 20,000, and 5600 molecular weight as separated on 10 to 20% gradient SDS-polyacrylamide gels; however, 20 to 30 protein products of various molecular weights were discernible. Mitochondrial concentrations of 0.8 to 1.4 mg/ml of incubation gave the better incorporation of [35S]methionine per milligram of protein. Total [35S]methionine incorporated into mitochondrial protein was greatest at 25 degrees C after 90 min. Chloramphenicol at 10 micrograms/ml inhibited mitochondrial protein synthesis by more than 50% and at 100 micrograms/ml inhibited incorporation by more than 95%. Cycloheximide had no effect on incorporation at less than 1.0 mg/ml. Magnesium and ATP in a molar ratio of one to one at 5 mM gave optimal incorporation. Other energy generating systems using oxidative phosphorylation to supply ATP for protein synthesis were not as effective as ATP and 5 mM phosphoenol pyruvate, 20 micrograms/ml pyruvate kinase and 5 mM a-ketoglutarate. In contrast to in vitro yeast mitochondrial protein synthesis, no enhancement of in vitro adrenal cell mitochondrial protein synthesis was found with GTP or its analogs. The buffers N,N-bis(2-hydroxyethyl)glycine, N-(tris(hydroxymethyl)methyl)glycine, and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid were superior to Tris-HCl for mitochondrial protein synthesis. Optimal pH for [35S]methionine incorporation into mitochondrial proteins was pH 7.0 to 7.6. Potassium at 50 to 90 mM gave the best incorporation of [35S]methionine, and the higher molecular weight products of translation were enhanced at these concentrations. Sodium at 10 to 40 mM had no effect; however, 100 mM sodium inhibited label incorporation by 30%. Calcium at 100 microM inhibited mitochondrial protein synthesis by approximately 50%, and at 1.0 mM little if any incorporation occurred.(ABSTRACT TRUNCATED AT 400 WORDS)     

Myogenesis in vitro. Enhancement by dibutyryl cAMP

Cholera enterotoxin (CT) increased the concentration of adenosine 3′-5′-cyclic monophosphate (cAMP) in monolayer cultures of adrenal tumor cells after a 60 min lag phase in contrast to the rapid effect of adrenocorticotropin (ACTH). The change in intracellular cAMP was accompanied by the release of steroids into the culture medium and a reversible alteration of monolayer morphology.     

Sensitivity of pigmented mucosa and skin to freezing injury.

Pigmented areas of canine skin and oral mucosa were subjected to freezing in situ at various temperatures for the purpose of investigating possible differences in the sensitivity of epidermal cells to cold injury. Destruction of melanocytes occurred in the range of ?4 to ?7 °C, while squamous cells resisted injury even at ?20 °C. Replacement of the lost pigmented cells occurred from the normal tissue at the periphery of the injured area. The experiments suggest that selective destruction of pigmented lesions in clinical conditions may be achieved by freezing tissue to ?4 to ?7 °C or colder, but not to exceed ?20 °C in order to avoid destruction of squamous cells. The experiments also support wider use of cryosurgery for pigmented lesions of the skin and oral cavity.     

Adenylate cyclase activity in normal and leukemic human leukocytes as determined by a radioimmunoassay for cyclic AMP

Adenylate cyclase in human leukocytes is a labile enzyme easily damaged during rupture. Homogenization in a glass homogenizer in the presence of 0.25 M sucrose preceded by swelling in the slightly hypotonic media retained the cyclase activity. Other conditions such as sonication or homogenization in the absence of sucrose resulted in a variable loss of activity. The cyclase activity was determined by a radioimmunoassay which, with some modifications, proved more sensitive than the labelled ATP assay method. Adenylate cyclase from lymphocytes of patients with chronic lymphatic leukemia exhibited an altered response to prostaglandins E₁, E₂ and F_2α when compared with cyclase from normal human lymphocytes.     

Purification of a progesterone receptor from rabbit uterus

A progesterone receptor has been purified to homogeneity from rabbit uterus by steroid affinity chromatography. The receptor was obtained in 5% yield, with a specific activity for [³H]progesterone binding of 14,580 pmol/mg protein. The pure receptor migrated as a single band on SDS-polyacrylamide electrophoresis, with a MW of 70,000. Progesterone binding to the receptor was heat labile and was displaced by an excess of R5020. Photoaffinity labeling of the pure receptor with [³H]R5020 corresponded to the major photoaffinity labeled species in crude cytosol.