Lymphocyte DNA synthesis inhibition.

A specific endogenous inhibitor for lymphocyte DNA synthesis that can be isolated from the lymphoid system and which is probably cell specific is described. The inhibitor is thermolabile, is destroyed by trypsin, and has a mass of about 30,000 to 50,000 daltons.

Induction of collagenolytic and proteolytic activities in rat and human fibroblasts by anti-inflammatory drugs.

Confluent monolayers of either mouse or diploid human fibroblasts contained no measurable amounts of either collagenolytic (pH 5.5) or proteolytic (pH 7.5) activities. Within a few hours after exposure of these cells to antiinflammatory drugs, significant amounts of these enzymatic activities were demonstrable extracellularly. These activities were profoundly decreased in cultures simultaneously treated with the anti-inflammatory drugs and cycloheximide or actinomycin D.

Placental beta-endorphin-like peptides.

Acid extract of human placental tissue contain, by both radioimmunoassay and radioreceptor assay, beta-endrophin-like material. Half of this material will not go through a 5000-dalton filter and on Sephadex G-200 has a molecular size between 25,00 and 50,000 daltons. Of the material going through a 5000-dalton ultrafilter, 80 percent is excluded on Sephadex G-25 and held back, very slightly, on Bio-Rad P6, indicating a molecular size of approximately 4500 to 4800 daltons. Thus, placenta appears to have macromolecular precursors from which a beta-endorphin-like material is released, with a size approximately 12 amino acids longer than half of the pituitary hormone.

Lymphocyte and fibroblast chalones: some chemical properties.

Experiments with lymphocyte and fibroblast chalones indicate that they are complexed strongly with RNA in tissue extracts; have a molecular weight of less than 10,000 and more than 1,000 daltons, and probably about 5,000 daltons; are strongly cationically charged; and contain mannose and perhaps other carbohydrates.

Evidence of morphological and physiological transformation of mammalian cells by strong magnetic fields.

Cultures of L-929 and WI-38 cells, frozen to 4.2 degrees K and exposed for 4 to 8 hours to 5000-oersted magnetic fields, were markedly inhibited in their growth as compared to controls. In cultures grown on cover slips, approximately 7 days after exposure, morphologically distinct cells emerged and were propagated from generation to generation; 3 weeks later, in flask cultures, contact inhibition was abolished. It is concluded that under certain experimental conditions, strong magnetic fields induce morphological and physiological transformations of target cells.

Evidence for a colon chalone.

The molecular weight fractions of 10,000-50,000 daltons prepared from "used" medium obtained during cultivation of human colon carcinoma cells (SW-48) in vitro inhibited the proliferation and DNA synthesis of these cells. Fractions exceeding 50,000 daltons were not inhibitory; those less than 10,000 daltons were cytotoxic. The inhibitory fraction did not affect either proliferation of human fibroblasts or transformation of human lymphocytes in vitro. Similar fractions from the colon mucosa of other species inhibited the proliferation of SW-48 cells, whereas extracts of dog jejunum or lung did not. This mitotic inhibition was completely reversible and could be destroyed by preincubation with trypsin. Therefore, colon cells appear to contain a cell- (but not species) specific, endogenous mitotic inhibitor or chalone.

Inflammation: a quarter century of progress.

This report summarizes the progress of studies on inflammation during the past quarter century; it is a personal overview of the most important and salient accomplishments in this field. Except for a few references to review articles and textbooks in the field, much of the literature surveyed is not cited.

The relevance of growth control (chalones) to the aging process.

A large amount of data suggests that a wide variety of cells and tissues contain cell-specific endogenous inhibitors of mitosis which are non species-specific. Increasingly, it appears that these chalone inhibitors are actually relatively small polypeptides capable of complexing with large molecular weight anionic molecules. The purification and chamical characterization of these inhibitors has been rendered extraordinarily difficult and the results have been very slow in forthcoming because of this ability to form complexes with anionic polyelectrolytes. Our recent understanding of this behavior should markedly accelerate our understanding and knowledge of chalone biochemistry in the future. If, in fact, chalones are ubiquitously important to the control of cell proliferation, then they will be important to our understanding of the development of post-mitotic cells and to our concepts of programmed senescence; both in turn might be important to our knowledge of the aging process.

Inhibition of lymphocyte DNA-synthetic responses by spermine-derived polycations.

The polyamines putrescine, spermidine, and spermine are oxidized by the enzyme diamine oxidase to form the corresponding aldehyde derivatives. These aldehydes have been shown to undergo a variety of spontaneous reactions, some of which result in polycationic addition compounds. We have chemically synthesized some spermine-derived polycations by reaction with the dialdehyde glutaraldehyde followed by reduction of te resulting Schiff base with sodium borohydride. Their migration on ion exchange and gel filtration columns was consistent with the formation of polycations with properties similar to those reported for the spontaneous reaction products. When added to cultures of alloantigen or mitogen stimulated lymphocytes, these polycations were potent inhibitors of the incorporation of tritiated thymidine and blast cell formation. This inhibition was reversible, non-cytotoxic, and only apparent if the polycation was added early in the culture period. The concentration of polycation necessary to achieve 50% inhibition of the lymphocyte response decreased as the cationic nature relative to spermine increased.

Purification of thymic macrophage migration inhibitory factor (MIF).

Aqueous extracts of the thymus of animals which had been challenged immunologically have been shown to contain MIF activity. This MIF could be purified by precipitation with 70% ethanol, concentrated by ultrafiltration between 30,000 and 50,000 daltons, isoelectrically focused at pH 6.8-7.1, and electrophoresed on preparative acrylamide gels. The resulting product is electrophoretically homogeneous at pH 4.3 in polyacrylamide-gel electrophoresis and SDS-gel electrophoresis. It has a molecular weight of 36,000 daltons. It is trypsin- and neuraminidase-labile and is thermostable. It degrades and reassembles in electrophoresis at pH 7.0. It is not chemotactic for macrophages but apparently activates them phagocytically. It has no proteolytic activity.

Properties of lymphocyte chemotactic factor (lymphotactin) in thymus.

Aqueous extracts of calf thymus from immunologically stimulated animals contain trypsin- and neuraminidase-labile macromolecules with molecular weights of less than 30,000 daltons which will specifically attract chemotactically lymphocytes. This activity we ascribe to "lymphotactin."

The purification and characterization of a lymphokine chemotactic for lymphocytes--lymphotactin.

Aqueous extracts of the thymus from vaccinated calves contain a protein specifically chemotactic for lymphocytes (lymphotactin). Lymphotactin has been isolated and purified by ethanol fractionation, ultrafiltration, isoelectric focusing, and preparative acrylamide gel electrophoresis. Its isoelectric point is 5.9, and the molecular weight by both SDS electrophoresis and exclusion chromatography is about 10,500. The purified sialoprotein is homogeneous at three different pH's in acrylamide gel electrophoresis. As little as 1 microgram of this material intraperitoneally wlll produce massive lymphocyte infiltration within 4 h in vivo.

Permeability factor contaminating hyaluronidase preparations.

Both crude and highly purified testicular hyaluronidase preparations have been shown to contain a component which increases the permeability of the microcirculation in rat skin. This permeability activity had an isoelectric point of 7.4 while hyaluronidase was 9.4. It also could be separated from hyaluronidase by acrylamide gel electrophoresis. The permeability factor was not inhibited by serum and may explain previous observations that hyaluronidase preparations decrease the ischemia and necrosis appropriate to experimental myocardial infarction in vivo.

Pulmonary macrophage growth factor.

Pulmonary macrophage growth factor (MCF) can be purified from the "used" serum-free medium of cultures of mouse lung cells by concentration and dialysis over a 30,000-dalton Amicon ultrafilter and subjecting the retentate to isoelectric focusing. This yields a protein fraction with an isolelectric point of pH 4.2, which contains all the MGF activity. Upon electrophoresis of this fraction in analytical gels, one large and four small bands could be visualized. All bands were biologically active. This same "heterogenous" fraction ran as one band (mol wt 68,000) in SDS analytical gel electrophoresis, suggesting the ordered aggregation of this monomer. This was confirmed by reelectrophoresis of the first (and largest) band demonstrating the same 5-banded pattern as the initial material.

Inhibition of mast-cell degranulation by chemotactic peptides.

Pepstatin and f-methionyl peptides which are potent chemotactic agents for neutrophils were found to inhibit the increase in vascular permeability of rat skin which follows the injection of 48/80, anti-rat IgE serum, or pulmonary permeability factor. These latter compounds are known to act by releasing histamine from mast cells. f-Met-Leu-Phe, which is the most active chemotactically, was also found to be the most active inhibitor.

Macromolecular, anionic pulmonary permeability factor.

Aqueous extracts of fresh or acetone-powdered calf lung have been found to contain a factor which increases the permeability of the microcirculation when injected into the skin of rats. This permeability factor, which is not found in similar extracts of muscle or kidney, was concentrated by Amicon ultrafiltration at a molecular weight range of between 50,000 and 100,000 daltons and via isoelectric focusing at an IEP of pH 4.2. After preparative acrylamide gel electrophoresis, this permeability factor was homogeneous by electrophoretic criteria and in SDS acrylamide gel electrophoresis had a molecular weight of approximately 82,000 daltons. This apparently homogeneous permeability factor from lung was inhibited by pepstatin, and yet possessed no acid proteolytic activity against any substrate. Its activity was completely inhibited by pretreatment of the animals with antihistamines. This pepstatin-inhibitable permeability factor was found largely in the lysosomal fraction of fresh lung and could also be obtained by extraction of large amounts of alveolar "washout" macrophages. Since macrophages, during phagocytosis, are known to leak significant quantities of their lysosomal contents, the fact that one of these components is a material which could release histamine from mast cells and thereby increase locally the permeability of the microcirculation may be of importance in the defense system of the lung.

Pulmonary polyamine permeability factor.

Acid extracts of calf lung have been found to contain low-molecular-weight factors which increase the permeability of the microcirculation when injected into the skin of rats. These factors, which were present in very low levels in aqueous extracts, were purified by gel filtration and ion-exchange chromatography. High-voltage paper electrophoresis revealed two active compounds with mobilities identical to the polyamines spermine and spermidine. Authentic samples of these compounds were as active in the "blueing" reaction as the isolated compounds. The permeability activity of both the isolated factors and the synthetic ones was inhibited by pepstatin and by pretreatment of the animals with pyrilamine maleate. If the normally low extracellular levels of these polyamines is increased by tissue damage, they could increase vascular permeability within the lung by releasing histamine from adjacent mast cells.

An Intercomparison Between NPL (India) and NIST (USA) Pressure Standards in the Hydraulic Pressure Region up to 26 MPa.

Results are presented of an intercomparison of pressure measurements between the National Physical Laboratory (NPL), India, and the National Institute of Standards and Technology (NIST), USA, using piston gauge pressure standards over the range 6 MPa to 26 MPa. The intercomparison, using the NPL piston gauge pressure standard, with a nominal effective area of 8.4×10-5 m2 and the NIST piston gauge pressure standard, with a nominal effective area of 2.0× 10-5 m2 was carried out at the NPL. The intercomparison data obtained show a relative difference of 1 × 10-6 in the zero-pressure effective area (A0) of the NPL standard as obtained by the NIST standard. At 6 MPa the relative difference in effective areas is 3.5×10-6; at the full scale pressure of 26 MPa, the relative difference is 12×10-6. These differences are in excellent agreement with the statements of uncertainty of the respective standards as obtained from the primary standards of these two laboratories.

Isolation of an effective debriding agent from the stems of pineapple plants.

The stem of the pineapple plant contains, in addition to a number of proteases, particularly bromelain, a non-proteolytic component which is responsible for the complete bridement of experimental burns by an "enzymatic dissection" between the viable native and the non-viable denatured burn tissue. With very little scraping, using a tongue depressor, all of the eschar can be removed and a bed suitable for grafting results. This "Escharase" has a molecular weight of 45,000 daltons and is a trimer made up of three identical subunits weighing 15,000 daltons each; it has an isoelectric point of pH 6.04 and has no hydrolytic enzyme activity against normal protein substrates or various glycosaminoglycan substrates. It can be concentrated by membrane ultrafiltration in a molecular weight range between 30,000 and 50,000 daltons and be purified by isoelectric focusing. The biological activity of the Escharase is not dependent on sulfhydryl groups nor upon the contaminating bromelain activity. The Escharase activity varies enormously from preparation to preparation, but the ultrafiltration procedure tends to concentrate Escharase activity to a fairly constant amount.