Purification and properties of a plasminogen activator from cultured rat prostate adenocarcinoma cells

Zymographic analysis of the supernates from confluent cultures of a rat prostate adenocarcinoma cell line, PA-III, revealed the existence of two molecular forms of specific plasminogen activators, one of molecular weight of approximately 80 000 and another of approximate molecular weight of 45 000, in sodium dodecyl sulfate. The low molecular weight form has been purified 364-fold in 66% yield from the culture medium by a combination of gel filtration on Sephacryl S-200 and affinity chromatography on Sepharose 4B-benzamidine. The purified material possessed a specific activity of 192 000 urokinase CTA units mg-1. This enzyme displayed activity toward human Glu1-plasminogen, characterized by a Km of 1.7 +/- 0.2 microM and a Vmax of 0.53 +/- 0.1 pmol of plasmin min-1 unit-1. A synthetic chromogenic substrate, H-D-Ile-Pro-Arg-p-nitroanilide (S-2288), was found for the activator. The enzyme possessed a Km of 0.33 mM and a kcat of 55 s-1 for S-2288. The activator was found to be a serine protease, inhibited by diisopropyl fluorophosphate (iPr2PF). At a concentration of 1 mM iPr2PF, and 30 nM enzyme, the half-time of this inhibition was 3.8 min. The 45 000 molecular weight enzyme was found to be inhibited by rabbit antibodies to human urokinase, thus characterizing the activator as a member of the urokinase class. The 80 000 molecular weight enzyme was not neutralized by anti-human urokinase but was neutralized by rabbit anti-human melanoma activator, likely allowing it to be classified as the tissue activator type.     

Thymus cell differentiation and in vivo T-cell migration. I. Migration of lectin-selected thymocytes

The in vivo quantitative distribution and tissue positioning of mouse thymocytes selected in vitro by Lyt phenotype and lectin binding properties were examined. Lyt 1+2- thymocytes were selected for by cytotoxic elimination; peanut agglutinin (PNA) and soybean agglutinin (SBA) binding and nonbinding thymocyte fractions were separated by an agglutinin technique. Selected cell suspensions were labelled in vitro with 51chromium (51Cr) or [3H]adenosine. Labeled washed cells were injected intravenously into syngeneic recipients which were killed at 1, 24 or 48 hr. In recipients of 51Cr-labeled cells, tissues were collected for gamma counting, and the overall percentage recovery of injected radiolabel from the various tissues was assessed. Tissues collected from recipients of [3H]adenosine-labeled cells were fixed, sectioned, and processed for autoradiography; the positioning of labeled cells within the tissues was determined. Selected Lyt 1+2-, PNA-, and SBA- sets all showed significantly enhanced entry into lymph nodes and intestinal lymphoid tissues. Entry of SBA+ cells into these tissues was comparable to that of peripheral T cells. PNA- and SBA- selected sets, but not Lyt 1+2- selected cells, also showed increased localization to the spleen and lungs, and decreased localization to the liver. By autoradiography, PNA- cells entered lymphoid tissues much more than PNA+ cells, and at 1 hr fewer PNA+ cells in spleen were associated with lymphoid follicles. At 24 and 48 hr almost all labeled cells in lymphoid tissues were positioned in T-dependent areas. These results suggest that enrichment for thymocyte subpopulations described as "mature" also enriches for cells with the ability to enter lymphoid tissue. They also suggest that interactions at other tissue sites are important in the determination of in vivo migration, and that surface carbohydrate composition is an important factor in this determination.     

Induction of repairable DNA damage in Escherichia coli and interaction with DNA in vitro by the radical cation of chlorpromazine

Studies were performed to determine the DNA interactions of and the induction of cytotoxic effects by the radical cation (CPZ+.) formed enzymatically from chlorpromazine (CPZ): in the presence of native DNA the lifetime of CPZ+. is markedly increased. The decreased reactivity of CPZ+. in the presence of native DNA and the concomitant increased viscosity of CPZ+.-DNA complexes strongly support the assumption that CPZ+. does form intercalation complexes with DNA. The relative strong bacteriotoxicity of CPZ+. hindered the accurate determination of mutagenesis in various Salmonella indicator strains, but a test for repairable DNA damage in Escherichia coli using various repair-deficient strains indicated that the cytotoxic action of CPZ+. is in part due to DNA alterations which can be excised in wild-type DNA repair-proficient strains. After activation of CPZ with long wavelength UV light, genetic effects are observed in S. typhimurium strain TA98, as well as in the E. coli tester strains. The possible role of CPZ+. in the photosensitization of CPZ is discussed.     

The effects of different concentrations of glycerol and dimethylsulfoxide on the metabolic activities of kidney slices

Kidney slices either were exposed to the cryoprotectants for 1 hr at room temperature and subsequently washed and incubated in fresh KR buffer containing only the radioactive metabolic tracers, or were immediately incubated for 2 hr at 37 °C in KR buffer containing the cryoprotectant and the tracers. Exposure to glycerol by incubation of kidney slices in Krebs-Ringer bicarbonate buffer containing varying concentrations of glycerol from 0 to 70% ($\text{v}\text{v}$) resulted in a pronounced inhibitory effect on the protein synthesizing activity, while thymidine incorporation into DNA and the α-aminoisobutyric acid uptake through the cell membranes were less affected. Exposure of the tissue to buffer containing dimethylsulfoxide (Me₂SO) in concentrations of 10 to 20% ($\text{v}\text{v}$) resulted in a stimulatory effect on metabolism. At higher concentrations, Me₂SO was toxic resulting in damaging effects on protein and DNA synthesis as well as on membrane integrity. The stimulatory effects of exposure to low concentrations of Me₂SO on protein and DNA synthesis in kidney slices were concluded to be the result of an increased transport of precursors through the cell membranes.     

Mammary ductal elongation: differentiation of myoepithelium and basal lamina during branching morphogenesis

Elongation of mammary ducts in the immature mouse takes place as a result of rapid growth in end buds. These structures proliferate at the apex of elongating ducts and are responsible for penetration of the surrounding adipose stroma; by turning and branching, end buds give rise to the characteristic open pattern of the mammary ductal tree. We have used a variety of techniques to determine the cellular and structural basis for certain of these end bud activities, and now report the following. (1) The end bud tip is covered with a monolayer of epithelium, the "cap cells," which are characterized by a relative lack of intercellular junctions and other specialized features. (2) The cap cell layer extends along the end bud flank and neck regions where it is continuous with the myoepithelium which surrounds the subtending mature duct. A linear sequence of differentiative changes occur in the cap cells in this region as they progressively alter in shape and accumulate the cytological features of mature myoepithelium. Cap cells may therefore be defined as a stem cell population providing new myoepithelial cells for ductal morphogenesis and elongation. (3) Differentiation of cap cells into myoepithelium is associated with conspicuous changes in the basal lamina. At the tip, cap cells form a 104-nm lamina similar to that described in expanding mammary alveoli and in embryonic tissues. Along the end bud flanks the basal lamina is raised from the cell surface and extensively folded, resulting in a greatly thickened lamina, measuring as much as 1.4 microns. At the surface of the subtending ducts the lamina becomes structurally simplified and resembles that at the tip, but has a significantly greater thickness, averaging 130 nm. (4) The codifferentiation of myoepithelium and its basement membrane is associated with changes in the surrounding stroma. Undifferentiated mesenchymal-like cells attach to the surface of the basal lamina in the midportion of the end buds and become increasingly numerous in the neck region, forming a monolayer over the myoepithelial basal lamina. These stromal cells progressively differentiated into fibrocytes which participate in collagen fibrillogenesis and give rise to the fibrous components of the stroma surrounding the mature duct.     

Correlation between endogenous nucleosomal hyper(ADP-ribosyl)ation of histone H1 and the induction of chromatin relaxation.

The effect of poly(ADP-ribose) synthesis on chromatin structure was investigated by velocity sedimentation and electron microscopy. We demonstrate that locally relaxed regions can be generated within polynucleosome chains by the activity of their intrinsic poly(ADP-ribose)polymerase. This relaxation phenomenon is also shown to be NAD dependent and to be correlated with the formation of hyper(ADP-ribosyl)ated forms of histone H1. Evidence is also presented which suggests that hyper(ADP-ribosyl)ated histone H1 is neither released from the relaxed chromatin, nor does it seem to participate in polynucleosomal aggregation.     

Effect of pH on binding of agonists and antagonists to rat heart muscarinic receptors.

Insulin, epidermal growth factor (EGF), platelet-derived growth factor, multiplication-stimulating activity and 10% foetal-calf serum each stimulated the phosphorylation of a cytosolic Mr-22000 acidic heat-stable protein in Swiss mouse 3T3-L1 adipocytes. Phosphorylation of this protein was not stimulated by isoprenaline or dibutyryl cyclic AMP. The effect of insulin was maximal (3-fold increase) by 10 min; half-maximal stimulation was observed at 70 pM-insulin. Both [32P]phosphoserine and [32P]phosphothreonine residues were present in the Mr-22000 protein after insulin- and growth-factor-stimulated phosphorylation, but no [32P]phosphotyrosine. The major site of insulin- and EGF-stimulated phosphorylation appeared to be a threonine residue, in contrast with previously studied insulin-stimulated phosphorylation of serine residues. Insulin treatment appeared to result in a shift of the protein toward the anode on isoelectric focusing. Insulin and EGF present simultaneously did not lead to phosphorylation beyond that seen with each hormone singly. We surmise that insulin, EGF and perhaps other growth factors may activate a common protein kinase or inhibit a common protein phosphatase in 3T3-L1 adipocytes which acts on the Mr-22000 protein.     

High-frequency conversion to a "fluffy" developmental phenotype in Aspergillus spp. by 5-azacytidine treatment: evidence for involvement of a single nuclear gene.

Transient exposure of mycelia from Aspergillus niger and Aspergillus nidulans to the cytidine analog 5-azacytidine, leading to no more than 0.3 to 0.5% substitution for cytosine by 5-azacytosine in A. nidulans DNA, resulted in the conversion of a high fraction of the cell population (more than 20%) to a mitotically and meiotically stable "fluffy" developmental phenotype. The phenotypic variants are characterized by the developmentally timed production of a profuse fluffy network of undifferentiated aerial hyphae that seem to escape signals governing vegetative growth. Genetic analysis with six different fluffy clones reveals that this trait is not cytoplasmically coded, is recessive in heterozygous diploids but codominant in heterokaryons, and exhibits a 1:1 Mendelian segregation pattern upon sexual sporulation of heterozygous diploids. Complementation and mitotic haploidization studies indicated that all variants are affected in the same gene, which can be tentatively located on chromosome VIII of A. nidulans. Molecular analysis to search for modified bases showed that DNA methylation is negligible in in both A. niger and A. nidulans and that no differences could be detected among DNAs from wild-type cells, fluffy clones, or mycelia exposed to 5-azacytidine. It thus appears that high-frequency conversion of fungal mycelia to a stable, variant developmental phenotype by 5-azacytidine is the result of some kind of target action on a single nuclear gene and that this conversion can occur in organisms virtually devoid of DNA methylation.     

Sayl andghayl: The ecology of water allocation in Yemen

In this article, comparison is made between the two major types of water allocation systems in Yemen: seasonal flood (sayl)and highland spring flow (ghayl).Constraints in the nature of water as a flowing resource are defined for each system. The major distinctions between the two types of systems are variability in water flow (which influences the determination of access rights), techniques of water control, measurement of water turns, the need for supervision of irrigation activities, and the potential for economic expansion of the production system. It is argued that tribal political organization is an adaptive response to highland spring flow allocation in Yemen, but undergoes stress in coastal flood systems where competition for the same water source extends across tribal boundaries in upstream-downstream conflict.     

Transport properties of rigid, symmetrical oligomeric structures composed of prolate, ellipsoidal subunits

We have calculated translational and rotational diffusion coefficients and intrinsic viscosities of oligomeric structures composed of n identical subunits having a prolate ellipsoidal shape with axial ratio p. Results are presented for p = 1-6 for a variety of structures with n = 1-6. We compare our results with those obtained by a different modeling procedure, proposed by other workers, in which the monomeric subunit is represented as a string of touching, colinear spheres. If n and an estimate of p are known, the structure of the oligomer can be. in most cases, unambiguously determined by comparison of the experimental oligomer-to-monomer ratios of a given property with the numerical results of this work. As examples of the applicability of our results, we examine the relationship between structure and properties for neurophysin. bovine serum albumin, hemoglobin and phycocyanin.