The effect of tryptase from human mast cells on human prekallikrein

Tryptase, the dominant protease in human mast cells, was examined for its effect on human prekallikrein. Tryptase in the presence and absence of heparin failed to activate prekallikrein as shown in a spectrophotometric assay for kallikrein employing benzoy 1-pro-phe-arg-p-nitroanilide. Treated prekallikrein was converted to active kallikrein by bovine trypsin. Prekallikrein cleavage products were analyzed by electrophoresis in polyacrylamide gels under denaturing conditions (+/- reduction). Tryptase caused no apparent cleavage under conditions where trypsin caused complete cleavage. Thus, tryptase, which has previously been shown to lack kallikrein and kininase activities, neither activates nor destroys prekallikrein.     

Effect of hyaluronidase treatment of intact cells on hyaluronate synthetase activity

Hyaluronidase treatment of mouse oligodendroglioma cells in monolayer culture resulted in a 4-5-fold stimulation of hyaluronate synthetase, assayed in washed membrane preparations [Philipson, L., & Schwartz, N. B. (1984) J. Biol. Chem. 259, 5017-5023]. We now report studies on the mechanism of the hyaluronidase-induced increase in the specific activity of the membrane-bound synthetase complex. The stimulation was dependent on the concentration of hyaluronidase but not on the particular bond cleaved or the nature of the product generated. Analysis of chain growth during cell-free synthesis by the disaccharide ratio method suggested that substantial internal labeling of hyaluronate chains had occurred. With both treated and untreated membranes, greater than 90% of incorporated (and recovered) radioactivity appeared in unsaturated disaccharides. Further analysis showed that hyaluronidase treatment increased both the rate of elongation and the rate of release of elongated chains from the enzyme complex. Hyaluronidase treatment also caused a change in the apparent steady-state kinetic patterns of double-reciprocal plots from intersecting lines for membranes from control cells to a family of parallel lines. Both the overall stimulation of synthesis and the change in apparent kinetic pattern were reversed by brief incubation of washed cells in the absence of hyaluronidase. These results have led to the development of an explicit kinetic model for hyaluronate synthesis which suggests an explanation for the switch in apparent kinetic patterns based on changing concentrations of a postulated key intermediate.     

Reduction potential of iron in transferrin

The reduction potential of Fe3+ in transferrin was measured spectrophotometrically by equilibration with methyl viologen in the presence of sodium dithionite. For an ionic strength near 0.1 M at 25 degrees C and pH 7.3 under 0.048 atm. CO2, half of the iron is reduced at a potential near -0.40 V (vs. standard hydrogen electrode). At least one disulfide bond of the protein is partially reduced at a potential of -0.44 V, as evidenced by reaction with [14C]iodoacetate.     

Cervical intraepithelial neoplasia and associated condylomatous lesions. A preliminary report on 4,764 women from Northern Israel

During the period spanning the years 1973 to 1981, 4,764 women visited the Gynecology Out-Patient Clinics and Colposcopy Unit of the Nahariyya Hospital to be examined colposcopically and cytologically (and histologically whenever indicated) for precancerous and cancerous lesions of the cervix. Of these women, 2,614 (55%) were referred because of symptoms of cervical pathology and 2,150 (45%) for other (prophylactic) reasons. The subdivision of all women according to their demographic backgrounds afforded a comparison of the findings in Israeli-born Jewesses with those of foreign-born Jewesses and non-Jewish females living in the same geographic area of the Western Galilee district of Israel. Despite the low prevalence of cervical cancer in Jewesses throughout the world, the preliminary report of our pilot study demonstrated that the percentage rates of all degrees of dysplasia/cervical intraepithelial neoplasia (CIN I, II and III) of the uterine cervix of Israeli-born Jewesses was 5.4% in patients with cervical pathology and 3.24% in noncervical-pathology patients. These rates were the highest recorded for any of the demographic groups: 2.06% and 0.33%, respectively, in Moslem women; 1.23% in Christian women with cervical pathology; 2.38% and 1.78%, respectively, in European/American-born Jewesses; and 1.63% and 0.48%, respectively, in Asian/African-born Jewesses. The highest proportion of CIN lesions occurred in the 15- to 30-year-old age groups. Of 100 CIN lesions found in all patients, 45 were cytohistologically associated with the cells of condylomatous lesions. Of 36 patients in whom cervical squamous-cell carcinoma lesions were detected, 18 (50%) were staged (FIGO) as carcinoma in situ (stage 0); the remainder were in stages IA, IB, IIA and IIB, with none in stages III or IV.     

Comparison of dipeptidyl carboxypeptidase and endopeptidase activities in the three enkephalin-hydrolysing metallopeptidases: "angiotensin-converting enzyme", thermolysin and "enkephalinase"

Angiotensin-converting enzyme (ACE), thermolysin and "enkephalinase", three metallopeptidases cleaving the Gly3-Phe4 amide bond of enkephalins, were compared regarding substrate specificity and effects of butanedione, an arginyl-directed reagent. The hydrolysis of enkephalins and analogues was more affected by the nature of P1 and P2 residues in the case of thermolysin than in those of ACE or "enkephalinase"; amidation of the C-terminal carboxylate decreased drastically the hydrolysis by ACE but only marginally by thermolysin and the effect was intermediate for "enkephalinase". With adequate model substrates, the ratio of dipeptidylcarboxypeptidase to tripeptidylcaroxypeptidase (endopeptidase) activities were of 25 for ACE, 3 for "enkephalinase" and only 0.3 for thermolysin. Finally a butanedione treatment increased thermolysin activity, but abolished ACE activity; it reduced "enkephalinase" activity by 80% when measured with a free C-terminal carboxylate enkephalin analogue but only slightly with the corresponding amidated derivative. A critical role of an Arg residue in ACE and, to a lesser extent, in "enkephalinase" (but not in thermolysin) is suggested to be responsible for the preferential dipeptidylcarboxypeptidase activity of these two enzymes.     

Effect of lysosomotropic amines on the secretory pathway and on the recycling of the asialoglycoprotein receptor in human hepatoma cells

We studied the intracellular transport of secretory and membrane proteins in the human hepatoma cell line HepG-2 infected with vesicular stomatitis virus. Cells were pulse-labeled in the presence of [35S]methionine and chased in the presence of the lysosomotropic agent primaquine. At a concentration of 0.3 mM primaquine effectively inhibited the secretion of albumin and, to a lesser extent, that of orosomucoid and transferrin. The drug also prevented the budding of virus particles at the cell surface. The intracellular transport to the Golgi complex of the membrane protein VSV-G was not affected by primaquine as it acquires resistance to endo-beta-N-acetylglucosaminidase H at the same rate as in control cells. Addition of primaquine at various times after the initiation of the chase period indicates that the effect of primaquine occurs just before secretion. In confirmation of the biochemical data, immunocytochemical localization of albumin in cells treated with NH4Cl demonstrated that albumin accumulated in vesicles at the trans side of the Golgi complex. The effect of primaquine on secretion was also compared with its effect on receptor recycling. The dose-response characteristics of the effect of primaquine on receptor recycling are identical to those of the effects on protein secretion and virus budding. These results indicate that both processes involve the same transport mechanism, and/or that they occur via at least one identical intracellular compartment.     

Lupus prone (SWR x NZB)F1 mice produce potentially nephritogenic autoantibodies inherited from the normal SWR parent

The incidence of nephritis in autoimmune NZB mice is low, but when they are crossed with normal SWR mice, almost 100% of the female F1 hybrids (SNF1) develop lethal glomerulonephritis. To define the contribution of the normal SWR strain to the development of nephritis, we analyzed 65 monoclonal anti-DNA autoantibodies derived from SNF1 mice and compared them with those obtained from the NZB parent. The majority of the SNF1-derived anti-DNA antibodies were IgG and cationic in charge. By contrast, 77% of the NZB-derived antibodies were IgM. Moreover, all three NZB-derived IgG anti-DNA antibodies were anionic. The cationic property of the SNF1-derived IgG autoantibodies was not restricted to any particular antigenic specificity pattern or IgG subclass, nor was there a preference for the allotype of either parent. However, we identified a set of highly cationic (pI at 8.2 to 8.8 pH) IgG2b anti-DNA antibodies from SNF1 hybrids that had the SWR allotype. Isoelectric focusing of intact antibodies and isolated heavy and light chains showed that the highly cationic charge of these antibodies was determined by the variable regions of their heavy chains. Because IgG anti-DNA antibodies with cationic charge are especially pathogenic, those antibodies bearing the allotype of the normal SWR parent may account for the high incidence of severe nephritis in the F1 hybrids. The results indicate that pathogenic autoantibodies, which are encoded by genes of the nonautoimmune SWR parent, are expressed in the SNF1 mice due to some cellular and genetic regulatory influence of the NZB parent.     

The molecular basis of the requirement for antigen processing of pigeon cytochrome c prior to T cell activation

Antigen-induced activation of T lymphocytes that co-recognize Ia molecules has been shown to require an antigen-processing step by the presenting cell before T cell stimulation can occur. In this report, we demonstrate that antigen presentation of pigeon cytochrome c to an E kappa beta:E kappa alpha-restricted T cell hybridoma, 2C2, is inhibited by pretreatment of the antigen-presenting cells (APC) either with chloroquine or with fixation by paraformaldehyde. The chloroquine effect was partially reversible after 22 hr; the paraformaldehyde effect was not. In contrast, these treatments had little or no effect on the presentation of the carboxy-terminal cyanogen bromide cleavage fragment of pigeon cytochrome c, residues 81 to 104. There was at least a 50-fold greater potency of the fragment, as compared to that of the intact molecule, when paraformaldehyde-fixed APC were used. In addition, the fixed cells did not present synthetic fragments of the cytochrome c that were nonstimulatory when presented by unfixed cells. This observation showed that the loss of potency, demonstrated previously for analogs of pigeon cytochrome c with single amino acid substitutions at positions such as 99, was not a consequence of an alteration in the rate of antigen-processing. This result is consistent with our earlier hypothesis that these residues are contact amino acids with the antigen-specific T cell receptor or the Ia molecule. The major goal of these experiments was to define the molecular transition that occurred as a result of antigen processing. To achieve this end, we tested a variety of pigeon cytochrome c molecules and fragments for their ability to be presented by paraformaldehyde-fixed APC. Apocytochrome c, the denatured form of the molecule with the heme removed, could not be presented by the fixed cells, nor could the fragment 60-104, derived by acid cleavage of the tryptophan at position 59. Both molecules stimulated an IL 2 response from the T cell hybridoma when unfixed APC were utilized, demonstrating that the conditions used to prepare these two molecules did not destroy their antigenic determinant. In contrast, carboxy-terminal fragments, both native and synthetic, ranging in size from 16 to 39 amino acids, were capable of stimulating in the presence of paraformaldehyde-fixed APC. In particular, the partial-digest cyanogen bromide fragment, residues 66 to 104, was only twofold less potent than the pigeon fragment 81-104.(ABSTRACT TRUNCATED AT 400 WORDS)     

A minimum mechanism for Na+−Ca++ exchange: Net and unidirectional Ca++ fluxes as functions of ion composition and membrane potential

Summary Both simultaneous and consecutive mechanisms for Na⁺–Ca⁺⁺ exchange are formulated and the associated systems of steady-state equations are solved numerically, and the net and unidirectional Ca⁺⁺ fluxes computed for a variety of ionic and electrical boundary conditions. A simultaneous mechanism is shown to be consistent with a broad range of experimental data from the squid giant axon, cardiac muscle and isolated sarcolemmal vesicles. In this mechanism, random binding of three Na⁺ ions and one Ca⁺⁺ on apposing sides of a membrane are required before a conformational change can occur, translocating the binding sites to the opposite sides of the membranes. A similar (return) translocation step is also permitted if all the sites are empty. None of the other states of binding can undergo such translocating conformational changes. The resulting reaction scheme has 22 reaction steps involving 16 ion-binding intermediates. The voltage dependence of the equilibrium constant for the overall reaction, required by the 31 Na⁺Ca⁺⁺ stoichiometry was obtained by multiplying and dividing, respectively, the forward and reverse rate constants of one of the translocational steps by exp(–FV/2RT). With reasonable values for the membrane density of the enzyme (120 sites m²) and an upper limit for the rate constants of both translocational steps of 10⁵·sec^–1, satisfactory behavior was obtainable with identical binding constants for Ca⁺⁺ on the two sides of the membrane (10⁶ m ^–1), similar symmetry also being assumed for the Na⁺ binding constant (12 to 60m ^–1). Introduction of order into the ion-binding process eliminates behavior that is consistent with experimental findings.     

Computer-Assisted Image Analysis of Plant Growth, Thigmomorphogenesis, and Gravitropism

A nonintrusive auxonometric system, based on the DARWIN image processor (Telewski et al. 1983 Plant Physiol 72: 177-181), is described and demonstrated in the analysis of gravitropism and thigmomorphogenesis in corn seedlings (Zea mays). Using this system, growth and bending of regularly shaped plants or organs can be quickly and accurately measured without, in any way, interfering with the plant. Furthermore, the growth and bending curves are automatically plotted. Thigmomorphogenesis in the aerial part of corn seedlings involves growth promotion at a low force load and growth retardation at higher force loads. The time courses of the two kinds of response are somewhat different, with retardation occurring immeditely after mechanical perturbation and growth promotion taking somewhat longer to begin. Gravitropic experiments show that when dark-grown corn seedlings are placed on their side in the light, the resulting curvature is due to two consecutive morphological mechanisms. In the first instance, lasting for about 15 minutes, the elongation of the bottom edge of the plant accelerates, while the elongation of the top edge remains constant. After that, for the next 1.75 hours, the elongation of the top edge decelerates and stops while that of the bottom edge remains constant at the increased rate for most of the period. The measurements taken from both experiments at relatively high resolution (0.08-0.1 millimeter) show that the growth curves are not smooth but show many small irregularities which may or may not involve micronutations.