Jason Kelley

An ultramicro method of amino acid analyses: Application to studies of protein metabolism in cultured cells

Abstract Analysis of the quantity and specific radioactivity of amino acids derived from intra-cellular pools, aminoacyl-transfer RNA, and protein hydrolysates of cultured cells has been achieved using a radiolabeled amino group ligand, dansyl chloride. Speeific activities of 14 C- or 3 H-labeled amino acids are calculated after reaction with appropriately labeled dansyl chloride of known specific activity. The quantity of amino acid is determined as a function of its diluting influence on a radioactive standard. The specific activity of as little as 2 pmol of amino acid can be measured using [ 14 C]dansyl chloride the less sensitive of the two isotopic species available. Thus, cells from a single 60-mm culture dish provide sufficient material for analysis of both intracellular and transfer RNA amino acid pools, and one can easily analyze the amino acids in hydrolysates made from individual bands in polyacrylamide gels. The method offers significant improvement in speed, sensitivity, and economy over conventional methods of amino acid analysis and, because of its double-label design, gives accurate results with a minimum of technical expertise and no major equipment other than a scintillation counter.

Bleomycin selectively elevates mRNA levels for procollagen and fibronectin following acute lung injury.

We employed the technique of dot blot hydridization of radiolabeled cDNA probes to examine the role of specific mRNA content in the control of extracellular matrix turnover in the remodeling rat lung. Following bleomycin instillation, total RNA content gradually doubled during the first 5 days following the initial lung injury, then rose much more rapidly during the ensuing 9 days. Individual mRNAs for procollagens I and III and for fibronectin were selectively enriched 2- to 4-fold above total RNA during the first week after bleomycin instillation. No comparable increases were observed in specific RNAs from liver, indicating that the response observed in the lung was not generalized to other organs. Moreover, the increases in mRNA species for procollagen types I or III in the lung could not be related to the influx of inflammatory cells which migrate into the lungs during acute injury, as cells obtained by alveolar lavage contained no mRNAs for procollagens.

Secretion of macrophage-derived growth factor during acute lung injury induced by bleomycin

Monocytes are capable of producing a growth factor which causes fibroblasts to replicate in vitro. This macrophage‐derived growth factor (MDGF) may be particularly important in controlling the proliferation of mesenchymal cells in the pulmonary interstitium following acute lung injury. To evaluate the role of macrophages in modulating the proliferative capacity of fibroblasts, we added serum‐free culture medium from lavage‐derived rat lung macrophages to confluent monolayers of quiescent adult rat lung fibroblasts and monitored their return to the cell cycle both by 3H‐thymidine incorporation and by increases in cell number. Growth factor activity secreted in 24 h by untreated macrophages was compared to that from normal macrophages stimulated in vitro by bacterial lipopolysaccharide (LPS) and from macrophages retrieved from lungs injured by intratracheal administration of bleomycin. Unstimulated macrophages from control animals secreted minimal amounts of MDGF; addition of LPS to normal macrophages increased MDGF secretion 3‐ to 4‐fold. Adherent cells obtained by lung lavage of bleomycin‐treated animals spontaneously secreted near maximal levels of nondialyzable MDGF with little further stimulation by LPS. The MDGF secreted by LPS‐stimulated control cells and by macrophages retrieved from injured lungs share identical elution profiles from DEAE‐Sepha‐ cel anionic exchange columns, and heat stability profiles. The substance acts as a competence factor when tested on rat lung fibroblasts. These shared physical and biologic properties suggest that the growth factor secreted by cells lavaged from injured lungs is identical to MDGF secreted in response to LPS. Because MDGF release could not be induced by direct exposure of freshly isolated macrophages to bleomycin, we suggest that augmented release of MDGF is an indirect feature of acute lung injury in this model.

Complex Cytokine Modulation of a Continuous Line of Mink Lung Epithelial Cells (Mv1Lu)

The continuous mink lung epithelial cell line Mv1Lu has proven to be a sensitive reporter line in the bioassay for purified TGF-beta, exhibiting a sigmoid-shaped concentration-response relationship with an EC50 of 12 pM (0.3 ng/mL). Maximal inhibition of Mv1Lu cells generates a 75-95% decrement in the number of adherent cells. However, this bioassay is not specific for TGF-beta as originally claimed. Mv1Lu cells are sensitive to other cytokines and substances found in complex biological fluids. In this study the effects of other biological response modifiers in this assay were tested and several were found to have important growth modulatory capacities that confound the quantitation of TGF-beta. EGF, TGF-alpha, fibronectin, and IGF-I all induce Mv1Lu cell proliferation. In contrast, neither PDGF (-AA, -AB, -BB) nor endotoxin (< or = 10 micrograms/mL) affect Mv1Lu cell number. TGF-beta and TNF-alpha at high concentrations (> or = 10 ng/mL) are the only cytokines examined that inhibit Mv1Lu proliferation. TGF-beta decreases final cell number both by preventing mitosis and by inhibition of adherence of cells to the uncoated dish. Several strategies are suggested to assure the specificity of this otherwise convenient bioassay for TGF-beta.

Localization of collagen in the rat lung: biochemical quantitation of types I and III collagen in small airways, vessels, and parenchyma

The marked insolubility of pulmonary collagen has limited its accurate biochemical quantitation in small samples of lung and other tissues. We have recently developed a microassay based on radioisotope dilution techniques that we have used for the accurate determination of types I and III collagen in extremely small tissue samples. By applying this method to carefully dissected small airways and vessels and samples of parenchymal tissue of rat lungs, we have localized and quantitated biochemically the type I and III structural collagens of the lung. Large pulmonary arteries are the units richest in these interstitial collagen types on the basis of dried tissue weight (50 µg/100 µg dried tissue). Amounts of both types I and III collagen are considerably lower in the alveolar domain than in vessels and airways of the rat lung. The proportion of tissue composed of these collagen types decreases centripetally in rat pulmonary arteries, but increases in the bronchial tree. The relative proportions of type I and type III remain constant in all the structures tested. The higher total amount of collagen in the nonalveolar domain has implications for biochemical studies based on whole lung samples.

Microquantitation of insoluble tissue collagen (types I and III) by radiodilution assay.

The individual collagen types of the extracellular matrix of small tissue samples have been difficult to quantitate accurately both due to their marked insolubility and their relatively low immunogenicity. Thus no microassay with the sensitivity of a radioimmunoassay is currently available for quantitation of insoluble collagen types I and III in extremely small tissue samples. A radiochemical assay has been developed which allows direct processing of small tissue samples containing as little as 1-3 micrograms of a given collagen alpha chain. Unprocessed lyophilized tissues were digested with cyanogen bromide (CNBr) in the presence of a tritiated probe containing a soluble mixture of 3H-alpha 1(I) and 3H-alpha 1(III) collagen previously extracted and purified from tissue minces incubated with [3H]leucine. The resulting mix of radiolabeled peptides was separated on sodium dodecyl sulfate-polyacrylamide gradient gels. Reduction of the specific radioactivity of free leucine in acid hydrolysates of each individual CNBr peptide can be used to quantitate the amount of collagen types I or III in the original sample. Similar radiodilution analysis using a 3H-alpha 2(I) probe indicated a normal 2:1 ratio of alpha chains of type I collagen in the tissues tested. This method is also applicable to cell culture, easily measuring the collagen associated with fibroblast cell layers or medium in individual microtiter wells. When applied to various tissues of known collagen-type composition, it provides reproducible results which compare well with values published in the literature.

Protein Synthesis in the Growing Rat Lung

Developmental control of protein synthesis in the postnatal growth of the lung has not been systematically studied. In male Fischer 344 rats, lung growth continues linearly as a function of body weight (from 75 to 450 g body weight). To study total protein synthesis in lungs of growing rats, we used the technique of constant intravenous infusion of tritiated leucine, an essential amino acid. Lungs of sacrificed animals were used to determine the leucine incorporation rate into newly synthesized protein. The specific radioactivity of the leucine associated with tRNA extracted from the same lungs served as an absolute index of the precursor leucine pool used for lung protein synthesis. On the basis of these measurements, we were able to calculate the fractional synthesis rate (the proportion of total protein destroyed and replaced each day) of pulmonary proteins for each rat. Under the conditions of isotope infusion, leucyl-tRNA very rapidly equilibrates with free leucine of the plasma and of the extracellular space of the lung. Infusions lasting 30 minutes or less yielded linear rates of protein synthesis without evidence of contamination of lung proteins by newly labeled intravascular albumin. The fractional synthesis rate is considerably higher in juvenile animals (55% per day) than in adult rats (20% per day). After approximately 12 weeks of age, the fractional synthesis rate remains extremely constant in spite of continued slow growth of the lung. It is apparent from these data that in both young and adult rats the bulk of total protein synthesis is devoted to rapidly turning over proteins and that less than 4 percent of newly made protein is committed to tissue growth.

Platelet-Derived Growth Factor, Transforming Growth Factor-β and Connective Tissue Growth Factor

Cell-to-cell communication determines the compostition and hence function of the airways of the lung in health. Conversely, altered balances of cytokines, either primary or secondary are thought to play a determining role in acute and chronic pathological disorders. The protein cytokines are a unique group of extracellular signalling molecules with critical roles in these processes. Cytokines (also termed growth factors) are pleiotrophic molecules produced ubiquitously and which act on a wide array of target cells. They modulate a wide array of cellular functions including growth (stimulation or inhibition), differentiation, survival and a wide range of phenotypic expression. Although generally small multimeric proteins, the cytokines are considerably larger than other signalling molecules such as the kinins, prostaglandins, leukotrienes and other molecules involved in modulating cell phenotype.