Identification of a cysteine residue as the binding site for the dipyrromethane cofactor at the active site of Escherichia coli porphobilinogen deaminase

The dipyrromethane cofactor of Escherichia coli porphobilinogen deaminase was specifically labelled with 13C by growth of the bacteria in the presence of 5-amino[5-13C]levulinic acid. Using 13C-NMR spectroscopy, the structure of the cofactor was confirmed as a dipyrromethane made up of two linked pyrrole rings each derived from porphobilinogen. The chemical shift data indicate that one of the pyrrole rings of the cofactor is covalently linked to the deaminase enzyme through a cysteine residue. Evidence from protein chemistry studies suggest that cysteine-242 is the covalent binding site for the cofactor.     

Role of self carriers in the immune response and tolerance. XII. Effect of epitope density and antigen-presenting cell phenotype on the presentation of hapten-modified self for the induction of immunity or tolerance in vitro

The data presented in the accompanying paper (J. P. Cogswell, R. P. Phipps, and D. W. Scott, Cell. Immunol. 114, 55-70, 1988) indicate that certain macrophage-like and lymphoid dendritic-like (P388AD.2) tumor lines which express major histocompatibility encoded class II (Ia) antigens and produce interleukin 1 (IL-1) are uniquely able to present hapten-modified self (HMS) in an immunogenic fashion in vivo. In the current study, the relationship between phenotype and function has been confirmed utilizing a completely in vitro system. This investigation revealed that B-cell priming required T cells restricted to P388AD.2's I-A antigens. In addition, exogenous IL-1 reconstituted the response of an IL-1-deficient tumor (P388AD.2-ILd), although it had no effect on the other nonimmunogenic Ia+ tumor lines. Unlike the in vivo system, effective B-cell tolerance was induced when P388AD.2 was modified with high concentrations (10 mM) of hapten or when highly haptenated tumor was added to 0.1 mM TNBS-modified P388AD.2. These results suggest that positive regulation of in vitro immune responses to HMS is dependent upon the phenotype of the accessory cell carrier (with lymphoid dendritic-like cells being unusually potent), while negative regulation is associated with high epitope density. This system now allows the dissection of the properties of different accessory cells and the signals required for B-cell priming or tolerance induction.     

Role of self carriers in the immune response and tolerance. XI. Correlation of Ia expression and interleukin-1 production with delivery of immunogenic signals in vivo by hapten-modified accessory cell-like tumor lines

It was recently demonstrated that a lymphoid dendritic-like tumor, P388AD.2, presented hapten-modified self (HMS) in an immunogenic fashion even after injection via the normally "tolerogenic" intravenous (iv) route. To determine whether this property was unique to the P388AD.2 line, other hapten-modified tumors were administered iv and the result of their presentation was measured by changes in the number of splenic plaque-forming cells (PFC) following in vitro challenge with thymic-independent antigens. Of the six tumors tested, two (P388 and J774.5R) primed for augmented PFC responses, while four others (P388NA.10, P388D1, WEHI-231, and 70Z/3) did not. When these tumors were compared for Ia expression and production of interleukin-1 (IL-1), it was discovered that (1) all of the immunogenic tumors were Ia+ and IL-1 producing (IL-1+), although not all Ia+,IL-1+ tumors could elicit augmented PFC responses; (2) none of the tumors that were deficient in either Ia expression or IL-1 production could prime B-cell responses in vivo; and (3) the ability to augment PFC responses was proportional to the density of Ia on the immunogenic tumors. These results demonstrated that P388AD.2 was not the only tumor line capable of presenting HMS iv as an immunogen, and that the accessory cell phenotype is critical for the induction of an immunogenic response in vivo.     

Filaggrin breakdown to water binding compounds during development of the rat stratum corneum is controlled by the water activity of the environment

Filaggrin is a specific epidermal protein which is the precursor of the free amino acids, urocanic acid and pyrrolidone carboxylic acid which are largely responsible for the ability of the stratum corneum of the skin to remain hydrated at low environmental humidity. The distribution of filaggrin shown by immunofluorescence in the stratum corneum of the rat changed dramatically during the first hours of postnatal life. During late foetal development, filaggrin accumulated through the entire thickness of the stratum corneum, indicating that there was a block on the subsequent processing of the protein which normally would convert it to free amino acids. Immediately after birth this block was lifted and normal proteolysis of the filaggrin took place in the outer part of the stratum corneum, leaving the normal adult pattern of a thin zone of cells containing filaggrin at the bottom of the stratum corneum. This activation of filaggrin proteolysis was dependent on the drop in external water activity caused by the transition from an aqueous environment in utero to a dryer environment after birth and it could be blocked by maintaining a 100% humidity atmosphere around the newborn rat after birth. In isolated stratum corneum in vitro, filaggrin proteolysis took place only between 80 and 95% relative humidity, both higher and lower relative humidity blocked the proteolysis. Application of occlusive patches to adult rats prevented the normal proteolysis of filaggrin, indicating that this mechanism controls not only the massive filaggrin proteolysis occurring after birth but also the proteolysis occurring during normal stratum corneum maturation. The stratum corneum therefore has the ability to respond to changes in external humidity by altering the level of the stratum corneum where it converts its reserves of filaggrin into water binding amino acids, such that under humid conditions water binding components will be produced in only the most superficial stratum corneum, or even not produced at all.     

Distribution of Hydrogen-Metabolizing Bacteria in Alfalfa Field Soil

H₂ evolved by alfalfa root nodules during the process of N₂ fixation may be an important factor influencing the distribution of soil bacteria. To test this hypothesis under field conditions, over 700 bacterial isolates were obtained from fallow soil or from the 3-mm layer of soil surrounding alfalfa (Medicago sativa L.) root nodules, alfalfa roots, or bindweed (Convolvulus arvensis L.) roots. Bacteria were isolated under either aerobic or microaerophilic conditions and were tested for their capacity to metabolize H₂. Isolates showing net H₂ uptake and ³H₂ incorporation activity under laboratory conditions were assigned a Hup⁺ phenotype, whereas organisms with significant H₂ output capacity were designated as a Hout⁺ phenotype. Under aerobic isolation conditions two Hup⁺ isolates were obtained, whereas under microaerophilic conditions five Hup⁺ and two Hout⁺ isolates were found. The nine isolates differed on the basis of 24 standard bacteriological characteristics or fatty acid composition. Five of the nine organisms were isolated from soil around root nodules, whereas the other four were found distributed among the other three soil environments. On the basis of the microaerophilic isolations, 4.8% of the total procaryotic isolates from soil around root nodules were capable of oxidizing H₂, and 1.2% could produce H₂. Two of the Hup⁺ isolates were identified as Rhizobium meliloti by root nodulation tests, but the fact that none of the isolates reduced C₂H₂ under the assay conditions suggested that the H₂ metabolism traits were associated with various hydrogenase systems rather than with nitrogenase activity. Results from this study support the concept that H₂ evolution by alfalfa root nodules has a significant effect on the surrounding microenvironment and influences the number and diversity of bacteria occupying that region.     

Biosynthesis of porphyrins and corrins. 1. 1H and 13C NMR spectra of (hydroxymethyl)bilane and uroporphyrinogens I and III

High-field NMR spectroscopic methods have been applied to study the reactions catalyzed by porphobilinogen (PBG) deaminase and uroporphyrinogen III (uro'gen III) cosynthase, which are the enzymes responsible for the formation of the porphyrin macrocycle. The action of these enzymes in the conversion of PBG, [2,11-13C]PBG, and [3,5-13C]PBG to uro'gens I and III has been followed by 1H and 13C NMR, and assignments are presented. The principal intermediate that accumulated was the correspondingly labeled (hydroxymethyl)bilane (HMB), the assignments for which are also presented.     

A Calcium-Activated Phytase from Pollen of Lilium longiflorum

A phytase was isolated and partially purified from the pollen of Lilium longiflorum Thumb. Optimum activity was at pH 8.0. The phytase was activated by Ca²⁺ and Sr²⁺ but not by the other divalent cations tested. Activity was inhibited by ethylenediaminetetraacetate. The phytase had a temperature optimum of 55 to 60°C and an activation energy of about 12,700 calories/mole. Extraction of L. longiflorum pollen with 0.1% Triton X-100 increased recovery of the phytase by nearly 4-fold. The phytase had a molecular weight of about 88,000 as determined by gel filtration chromatography and a K_m value of 7.2 micromolar for phytic acid in the presence of Ca²⁺.     

Regulation of the terminal event in cellular differentiation: biological mechanisms of the loss of proliferative potential

Human plasma has been demonstrated to contain factors that induce the sequential expression of nonterminal and terminal adipocyte differentiation in 3T3 T mesenchymal stem cells. We now report the development of methods for the isolation of purified populations of nonterminally differentiated cells and terminally differentiated cells, and we show that it is possible to experimentally induce transition from the nonterminal to the terminal state of differentiation. With this model system it is therefore now possible to examine the biological and molecular processes associated with the terminal event in differentiation, i.e., the irreversible loss of proliferative potential. In this regard, we demonstrate that transition from the nonterminal to terminal state of differentiation is a complex metabolic process that consists of at least two steps and that this process can be triggered by pulse exposure to an inducer for approximately 12 h but that approximately 24-48 h is required for the process to be completed. The data also establish that induction of the terminal event in differentiation requires protein synthesis but not RNA and DNA synthesis. These and additional results suggest that loss of proliferative potential associated with the terminal event in cellular differentiation is a distinct regulatory process, and we suggest that defects in this regulatory process may be of etiological significance in the pathogenesis of specific human diseases, especially cancer.     

Changes in Insulin Binding to Developing Embryonic Chick Neural Retina Cells

Specific cell surface insulin binding to embryonic chick neural retina cells has been demonstrated in vivo. Kinetics of insulin binding as well as hormonal specificity were similar to those reported for other vertebrate cells and tissues, both neural and nonneural. When surface insulin binding to retinal cells was studied as a function of embryonic age, a developmental relationship was observed. Scatchard analysis revealed that the number of cell surface insulin receptors decreased approximately 75% between days 10 and 16 of embryonic development. Receptor affinities remained fairly constant for this period.     

Lymphoma models for B-cell activation and tolerance. II. Growth inhibition by anti-mu of WEHI-231 and the selection and properties of resistant mutants

Regulation of the growth of murine B-cell lymphomas has been used as a model for tolerance induction. The inhibition by anti-immunoglobulin reagents of the growth of WEHI-231 and several variant clones has now been studied. The parental line is exquisitely sensitive to growth inhibition by heterologous or monoclonal anti-mu or anti-k reagents and ceases to incorporate thymidine within 24-48 hr of exposure to anti-immunoglobulin reagents. Growth inhibition is initially reversible, but prolonged exposure to anti-mu results in cell death. This inhibition is specific for immunoglobulin light and heavy chains since growth is not inhibited by antibodies directed at either class I or class II histocompatibility antigens. In order to study the mechanism of growth inhibition, we have mutagenized WEHI-231 with ethylmethane sulfonate and cloned the surviving colonies in the presence of anti-mu. Such variants, which have been repeatedly recloned, are able to grow normally in the presence of anti-mu up to 100 micrograms/ml. These "resistant" clones, while expressing amounts of surface IgM similar to that observed on WEHI-231, do not differ markedly in their ability to cap their immunoglobulin receptors compared to the parental line but appear to have lost class II antigens. Cell cycle analysis revealed that anti-mu causes a block in the transition of WEHI-231 from G1 to S phase. The relevance of these processes to models of B-cell tolerance induction are discussed.