A facile tritium release assay for mammalian L-dihydroorotate dehydrogenase

Proteinase B activity in crude yeast extract is difficult to detect, unless the firmly bound peptide inhibitor is previously inactivated. Methods currently used to accomplish this inactivation suffer from serious limitations, particularly when applied to crude extracts obtained from yeast grown in complete media. These limitations are discussed on the basis of the kinetics of activation. A novel procedure to unmask proteinase B activity, based on pepsin treatment of crude extracts, is described. Specific activity of proteinase B, determined after pepsin A treatment, is always two to three times higher than that obtained by other methods. The method proposed is simple, reliable, and can be applied to crude extracts stored at ?20°C for up to 1 week, repeatedly giving the same specific activity for proteinase B.     

Uridine phosphorylase from Escherichia coli B.: kinetic studies on the mechanism of catalysis

Using a highly purified enzyme preparation of uridine phosphorylase from Escherichia coli B, we have performed detailed kinetic studies which include initial-velocity and product-inhibition experiments in the forward and reverse directions of the reaction. These studies indicate a rapid-equilibrium random mechanism for this enzyme with the formation of an enzyme . uracil phosphate abortive complex. Lack of formation of the enzyme . uridine . ribose-1-phosphate abortive complex suggests that the ribosyl moiety of the two ligands compete for the same binding site. The random mechanism is different from the ordered addition of substrates found for uridine phosphorylase from other sources. All the kinetic constants in the forward and reverse directions and the Keq of reaction for E. coli uridine phosphorylase are reported herein.     

Cytostatic and cytolytic activities of macrophages regulation by prostaglandins

Murine peritoneal macrophages (M phi), activated in vivo or in vitro, remarkably inhibited the uptake of thymidine by a lens epithelial cell line, while resident M phi, or M phi induced by thioglycollate, exhibited much lower or no cytostatic capacity. The target cells were partially protected from the cytostatic activity by the anti-inflammatory agents indomethacin, aspirin, and dexamethasone, but not by lipoxygenase inhibitors. The protective activity of indomethacin and aspirin, but not of dexamethasone, was completely counteracted by prostaglandin E2 (PGE2). Yet, PGE2 alone has no effect on the uptake of [3H]thymidine by lens epithelial cells. PGE1 resembled PGE2 in its effect on this system, whereas PGA2, PGB2, or PGF2 alpha had no detectable activity. The counteracting effect of PGE2 was mimicked by dibutyryl cAMP or by cholera toxin, an agent which increases cAMP levels. These findings suggest that PGEs are not direct cytostatic agents, but rather, are essential mediators for the development of the cytostasis. Activated M phi did not lyse cells of the original lens epithelial cell line, but caused substantial cytolysis of cells of a subline derived from it. In contrast to its aforementioned effect on the cytostasis, PGE2 inhibited the cytolytic activity of M phi. Thus, this study provides a first demonstration in a single system of the opposite effects of PGEs on M phi activity on target cells, i.e., mediating the cytostasis and inhibiting the cytolysis.     

Serratia marcescens chitinase: One-step purification and use for the determination of chitin

The extracellular chitinase produced by Serratia marcescens was obtained in highly purified form by adsorption-digestion on chitin. After gel electrophoresis in a nondenaturing system, the purified preparation exhibited two major protein bands that coincided with enzymatic activity. A study of the enzyme properties showed its suitability for the analysis of chitin. Thus, the chitinase exhibited excellent stability, a wide pH optimum, and linear kinetics over a much greater range than similar enzymes from other sources. The major product of chitin hydrolysis was chitobiose, which was slowly converted into free N-acetylglucosamine by traces of β-N-acetylglucosaminidase present in the purified preparation. The preparation was free from other polysaccharide hydrolases. Experiments with radiolabeled yeast cell walls showed that the chitinase was able to degrade wall chitin completely and specifically.     

Methylglyoxal-mediated growth inhibition in an Escherichia coli cAMP receptor protein mutant

Under certain growth conditions, some strains of Escherichia coli accumulate toxic levels of methylglyoxal. This report characterizes a strain which synthesizes a mutant cAMP receptor protein in an adenylate cyclase deletion background. When cultured in glucose 6-phosphate minimal medium, this strain (222) was prematurely growth arrested due to methylglyoxal production; growth inhibition did not occur when the strain was grown in glucose minimal medium. A comparison of a variety of enzyme and cofactor levels in the related strains 222 (mutant) and 225 (wild-type) grown on either glucose or glucose 6-phosphate medium was carried out. The only difference found that might explain an increase in methylglyoxal accumulation was an elevated level of phosphofructokinase in strain 222 grown on glucose 6-phosphate. Since this enzyme activity probably limits hexose phosphate metabolism, it is suggested that growth inhibition in strain 222 may be due to increased production of triose phosphate, some of which is converted to methylglyoxal.     

Monoclonal antibodies directed against the sugar sequence of lacto-N-fucopentaose III are obtained from mice immunized with human tumors

Four hybridomas obtained from mice immunized with human adenocarcinomas of colon or stomach produce antibodies that bind specifically in solid-phase radioimmunoassay to the ceramide pentasaccharide that contains the lacto-N-fucopentaose III sequence of sugars. Binding of the antibodies to the glycolipid is inhibited by lacto-N-fucopentaose III,

but not by structurally related oligosaccharides. The antibodies bind to glycolipids of erythrocytes, granulocytes, and certain normal and malignant tissues.     

Mitogen-induced suppressor factor(s) from human lymphocytes: effects on lymphoid and nonlymphoid cells and biophysical properties

Concanavalin A (Con A) or phytohemagglutinin activate a population of human circulating lymphocytes to exert suppressive functions. We found that supernates from the activated human lymphocytes suppress lymphocyte responses to Con A, the mixed lymphocyte reaction and pokeweed mitogen-induced IgM production. Mitogen stimulated suppressor lymphocytes, or their supernates, inhibit also the spontaneous proliferation of human retinoblastoma cells (Y-79 line) and primary cultures of human keratocytes. A correlation was always noted between the levels of inhibitory activities of the lymphocytes and their supernates. Furthermore, a good correlation was found between the levels of inhibition by the supernates of lymphocyte functions (proliferation and IgM production) and of the nonlymphoid cells' proliferation. Some of the properties of this suppressor factor(s) are: (i) produced only by the T-cell population; (ii) appears after 8 hr of Con A stimulation, peaks at 24 to 48 hr and declines later on; (iii) stable at 56 °C and labile to 70 °C; (iv) nondialyzable and present in the 40K–100K dalton fraction of a G-200 Sephadex column; (v) labile to pH 2 treatment.     

Activation of human B lymphocytes. XVII. Synergy between nonspecific and specific signals in the antigen-specific responses of human B cells

The incubation of human peripheral blood lymphocytes (PBL) with the natural killer (NK)-sensitive MOLT-4 cell line results in PBL-target cell conjugate formation by certain lymphocyte subpopulations. Following velocity sedimentation, the PBL depleted of these conjugate-forming subpopulations are markedly diminished in the ability to mediate either antibody-dependent cellular cytotoxicity (ADCC) or NK activity. The immediate testing of highly pure PBL subpopulations isolated from the NK target conjugates does not reveal the expected recovery of augmented ADCC or NK levels. Following in vitro incubation, however, the PBL NK target-binding subpopulations do manifest augmented levels of both NK and ADCC, whereas the depleted PBL continue to display diminished NK and ADCC levels. In addition, the degree of augmented NK and ADCC levels recovered by the NK target-binding PBL subpopulations appears dependent on both the time and the temperature of in vitro incubation. Moreover, the ADCC recovery patterns are identical to those observed for NK activity regardless of the time and temperature of in vitro incubation. These results directly demonstrate that the PBL subpopulations isolated from certain NK target cells are functionally enriched in the ability to mediate from ADCC and NK activity.     

Uridine-cytidine kinase. Kinetic studies and reaction mechanism.

The initial velocity pattern has been determined for uridine-cytidine kinase purified from the murine mast cell neoplasm P815. With either uridine or cytidine as phosphate acceptor, and ATP as phosphate donor, the pattern observed was one of intersecting lines, ruling out a ping-pong reaction mechanism, and suggesting that the reaction probably proceeds by the sequential addition of both substrates to the enzyme to form a ternary complex, followed by the sequential release of the two products. This pattern was obtained whether the reaction was run in 0.01 m potassium phosphate buffer, pH 7.5, or in 0.1 m Tris-HCl, pH 7.2. When analyzed by the Sequen computer program, the data indicated an apparent K_m of the enzyme for uridine of 1.5 × 10^?4m, an apparent K_m for cytidine of 4.5 × 10^?5m, and a K_m for ATP, with uridine or cytidine as phosphate acceptor, of 3.6 × 10^?3m or 2.1 × 10^?3m, respectively. The V was 1.83 μmol phosphorylated/min/mg enzyme protein for the uridine kinase reaction and 0.91 μmol for the cytidine kinase reaction.     

Atrial tissue contains a metallo dipeptidyl carboxyhydrolase not present in ventricular tissue: partial purification and characterization

A new membrane-bound dipeptidyl carboxyhydrolase has been identified in bovine atrial tissue, and has been partially purified after extraction with Triton X-100. This enzyme, found in quantities of 0.01-0.03 units/g tissue assayed with Bz-Gly-His-Leu, is potentially capable of hydrolyzing atriopeptin II to atriopeptin I. The enzyme is located in the microsomal fraction and in sucrose density fractions enriched for atrial granules. The enzyme is completely inhibited by reagents for heavy metals such as EDTA, o-phenanthroline, dithiothreitol, and mercaptoethanol. The latter two compounds are also disulfide reagents. The atrial enzyme is also inhibited by D-2-methyl-3-mercaptopropanoyl-L-Pro(Captopril), 3-mercaptopropanoyl-L-Pro, 2-D-methylsuccinyl-L-Pro, and bradykinin potentiating factor, all inhibitors of the angiotensin I-converting enzyme. However, the atrial enzyme differs from the converting enzyme in a number of kinetic and molecular properties. Its activity increases with ionic strength, but the atrial enzyme does not have a chloride dependence for Bz-Gly-His-Leu hydrolysis; the pH optimum, 7.3, is slightly lower, and it is 5500 times less sensitive to the very potent converting enzyme inhibitor, D-Cys-L-Pro. The strokes radius of the atrial enzyme is 5.00 nm as compared to 4.10 nm, and its molecular weight is 240,000 compared to 145,000. Ventricular tissue, which does not contain the atrial peptides, does not contain the dipeptidyl carboxyhydrolase enzyme.     

Suppression of lymphokine production: I. Macrophage-mediated inhibition of MIF production

Macrophages have been found to suppress the in vitro production by stimulated T lymphocytes of a lymphokine, migration inhibitory factor. When macrophages isolated from primary MSV-induced tumors were added to antigen-stimulated MSV-immune spleen cells, a complete suppression of MIF production was observed. This suppression was nonspecific, since MIF production by antigen-stimulated alloimmune spleen cells and by PHA-stimulated normal spleen cells was also inhibited. Suppressor macrophages could also be induced by inoculation with Corynebacterium parvum, whereas light mineral oil-induced peritoneal macrophages had no detectable effect on MIF production. The failure to detect MIF in the supernatants of stimulated cultures containing activated macrophages appeared to be due to inhibition of lymphokine production rather than to absorption or inactivation of MIF or to interference with the assay for detection of MIF. Macrophages were able to suppress MIF production only when added during the first 4–5 hr of culture and they had no effect when added later. These data show that activated macrophages can nonspecifically suppress lymphokine production and that this appears to be due to inhibition of an early step in lymphocyte stimulation.     

Occurrence of molybdenum in the nicotinic acid hydroxylase from Clostridium barkeri

Molybdenum, assayed by atomic absorption spectrometry, copurifies with the selenium-containing nicotinic acid hydroxylase from Clostridium barkeri. Fluorescence spectral studies on the enzyme indicate the presence, along with flavin, of another component. The fluorescence spectra of this component obtained after the aerobic denaturation of the nicotinic acid hydroxylase are similar to the fluorescence properties reported for the “pterin-like” cofactor from xanthine oxidase and several other molybdoproteins. Nicotinic acid hydroxylase from C. barkeri contains molybdenum, selenium, iron, acid-labile sulfur, and flavin with the occurrence of a “pterin-like” cofactor also a likely component.     

Many monoclonal antibodies with an apparent specificity for certain lung cancers are directed against a sugar sequence found in lacto-N-fucopentaose III

Monoclonal antibodies with an apparent specificity for human small-cell carcinoma, adenocarcinoma, and squamous carcinoma of the lung are produced by some hybridomas obtained from mice and rats immunized with an established line of human small cell lung cancer. Out of 85 of these antibodies produced by independently isolated hybridomas from 15 different fusions, 21 are directed against the sugar sequence which occurs in lacto-N-fucopentaose III ceramide, in several higher glycolipids and in glycoproteins. Specificity was determined by autoradiography of thin-layer chromatograms of glycolipids, by solid-phase radioimmunoassays, and by hapten inhibition studies. All 21 antibodies are of the immunoglobulin M type.     

Biological electron microscopy: By Barbara L. Gabriel. Van Nostrand-Reinhold,Princeton, N. J., 1982. 240 pp. $29.95

Detergents containing either a cholic acid, a deoxycholic acid, or an octanoic acid-like hydrophobic moiety and a bisgluconamidopropyl polar group were synthesized. Extinction coefficients, partial specific volumes, critical micelle concentrations, and aggregation numbers were determined for each of the detergents. The two bile acid derivatives are capable of solubilizing functional opiate receptor, while the octanoic acid derivative is not.     

Development and characterization of interleukin-2-independent antigen-specific human T cell clones that produce multiple lymphokines

The development of antigen-specific T lymphocyte lines and clones has greatly facilitated the investigation of T-cell recognition of and response to foreign antigens. In the present study, human antigen-specific helper T cell lines and clones which are completely independent of exogenous interleukin-2 (IL-2) have been developed by cyclic restimulation with the soluble antigen keyhole limpet hemocyanin (KLH) to which the T cell donor had previously been immunized. These T cells uniformly bear the OKT4 phenotype and were shown to require both histocompatible antigen-presenting cells (APC) and antigen for optimal proliferation. The T cell line was composed of a highly antigen-specific and clonable T cell population. Following four cycles of antigen stimulation, limiting dilution cloning analysis showed a Poisson distribution of clonable T cells with a precursor frequency of 0.62, and from 88 to 92% of viable clones were specific for the stimulating antigen. Individual clones were obtained which recognized KLH with either DR 1 (one parental Ia haplotype of the donor) or DR 2 (the other parental Ia haplotype) allogeneic APC, but not both. Following stimulation with KLH, the T cell clones produced IL-2. Peak amounts of IL-2 were assayable in the first 6 to 24 hr after stimulation. In contrast, virtually no IL-2 was detectable in supernatants at 72 to 96 hr, suggesting autoutilization by the proliferating T cells. In addition, some clones were also capable of producing both B cell growth factor and IL-2 following KLH stimulation. These IL-2-independent T cells appeared to be derived from a discrete Leu 8-negative subclass of T4⁺ cells and expressed the full complement of Ia antigen of the donor. Thus, soluble antigen-specific human helper T cell clones have been produced which can be maintained in the absence of exogenous IL-2, elaborate their own growth factors and other immunoregulatory lymphokines, and show fine DR-related restriction to either one or the other parental DR haplotypes in antigen-stimulated proliferative responses.     

A convenient procedure for purification of thymidylate synthase from L1210 cells

Several laboratories have described procedures for purification of thymidylate synthase (TMP synthase) that utilize folates or folate analogs covalently attached to a matrix. The principle of separation is the formation of a ternary complex between dUMP, TMP synthase, and the bound ligand and the subsequent elution of the enzyme with buffers that do not contain dUMP. We have successfully used 10-formylfolic acid as the bound ligand for the purification of TMP synthase. As compared to other ligands that have been used, 10-formylfolic acid has the advantages that it can be easily synthesized, it is stable, and the enzyme is eluted as a sharp peak. Application of this procedure to L1210 leukemia cells gave 1765-fold purification of TMP synthase with a recovery of 39%. The native enzyme had a molecular weight of 78,000, which is about the same as that reported.     

Induction of monocytic suppression after stimulation of peripheral human mononuclear cells with staphylococcal protein A and Staphylococcus aureus

Staphylococcal protein-A (SPA) and Staphylococcus aureus are known to be polyclonal human B-cell activators. It was noted that they induced plaque-forming-cell (PFC) responses lower than those induced by pokeweed mitogen (PWM) and the possibility of early triggering of a suppressor cell was investigated in the present series of experiments. Peripheral mononuclear cells (MNC) were passed through Sephadex G-10 columns to eliminate monocytes. The PFC responses to SPA and S. aureus were thereby increased. PWM-driven PFC responses are suppressed by the simultaneous presence of SPA in a dose-related way, if present in the early phases of the cultures. MNC precultured with SPA or S. aureus have the ability to suppress the PFC response of autologous MNC to PWM. Interestingly this suppressor cell activity was radiation resistant and could not be abrogated by treatment with anti-T-cell monoclonal antibody plus complement. The above experiments clearly demonstrate that the observed low PFC responses of MNC after stimulation with SPA and S. aureus are due to the induction of suppressor cells by these stimulants. The suppressor cells are apparently of monocytic origin.     

Multiple expressions of the activity of guanine nucleotide regulatory protein in human thyroid adenylate cyclase

Adenylate cyclase (ATP pyrophosphate-lyase, EC 4.6.1.1) in plasma membranes from human thyroid was highly responsive to thyrotropin. Pretreatment of thyroid plasma membranes with 5′-guanylylimidodiphosphate (Gpp(NH)p) in the presence of Mg²⁺ led to a temperature-dependent activation, which was seen neither in the absence of Mg²⁺ nor at 4 °C. By contrast, thyrotropin bound to its receptors regardless of the temperature and produced its maximal effect after 2 min of preincubation in the absence or presence of Mg²⁺. Furthermore, activation was seen after treatment with thyrotropin and Gpp(NH)p even carried out in the absence of Mg²⁺ or at 4 °C. However, the full activation by Gpp(NH)p required Mg²⁺, hormone, and elevated temperature. These observations suggest that there appears to be two types of nucleotide interaction responsible for the Gpp(NH)p activation in human thyroid membrane; one type seen in the absence of hormone may represent the system uncoupled from hormone receptor, while the fully coupled hormone-sensitive adenylate cyclase accounts for the second type of interaction which requires the presence of hormone.     

Effects of lipid peroxidation products on the rat lens in organ culture: a possible mechanism of cataract initiation in retinal degenerative disease

Rat lenses in organ culture which are exposed to bovine rod outer segments (ROS) or to the major fatty acid of ROS, docosahexaenoic acid, are impaired in their ability to accumulate radiolabeled compounds which lenses normally accumulate by active processes. The extent of lens damage correlates well with the extent of lipid peroxidation in the culture medium as assessed by the thiobarbituric acid assay. Addition of vitamin E to the medium inhibits the effect on the lens while addition of Fe-ADP complexes potentiates the effect. Thus, the lens damage appears to be attributable to toxic species generated by peroxidation of the polyunsaturated lipid added to the culture medium. Toxic aldehyde products appear to be major mediators of the lens damage, since semi-carbazide, which avidly reacts with aldehydes, can protect lenses in this system. These findings may have relevance to the cataracts clinically associated with retinal degenerative diseases such as retinitis pigmentosa. The highly membranous photoreceptor cells are extremely rich in polyunsaturated lipid. Degeneration of these cells, which is the primary pathology in such diseases, would likely lead to peroxidation with generation of toxic products within the eye. Such products could potentially produce secondary damage to other ocular structures including the lens.