The stimulation of collagen secretion by ascorbate as a result of increased proline hydroxylation in chick embryo fibroblasts.

Collagen secretion by chick embryo fibroblasts was measured by incorporating [¹⁴C]proline into proteins and then analyzing the amount of collagen in the cell and medium separately by using purified bacterial collagenase. In order to produce varying levels of hydroxylation, cells were incubated with varying concentrations of ascorbate or with varying concentrations of α,α′-dipyridyl in the presence of saturating ascorbate. Ascorbate stimulated both the hydroxylation of proline in collagen and the secretion of collagen; the concentration of ascorbate required for half-maximal stimulation of both proesses was approximately 4.5 × 10^?7, m. Since the cells could concentrate ascorbate 10-fold, this K_M for proline hydroxylation is 100-fold lower than values reported for purified prolyl hydroxylase (Abbot, M. T., and Udenfriend, S. (1974) in Molecular Mechanisms of Oxygen Activation (Hayaishi, O., ed.), p. 173, Academic Press New York; Kivirikko K. I., et al. (1968) Biochim. Biophys. Acta, 151, 558–567). Conversely, α,ga′-dipyridyl inhibited both proline hydroxylation and collagen secretion; half-maximal inhibition of both processes was observed at 7 × 10^?5, m. The results of the two types of experiments show that the secretion of collagen becomes directly proportional to proline hydroxylation when approximately 30% of the proline residues in collagen have been hydroxylated compared to maximal hydroxylation of 50%. Since the stability of triple-helical collagen at 37 °C has been shown to be dependent on the hydroxyproline content of the molecule (Rosenbloom, J., et al. (1973) Arch. Biochem. Biophys., 158, 478–484), we suggest that the observed proportionality between secretion and hydroxylation is a reflection of the increased amount of stable triple helical collagen at 37 °C. When the cells were incubated with a concentration of ascorbate that was saturating for secretion and hydroxylation, there was no significant activation of prolyl hydroxylase as measured in a cell-free extract. These experiments suggest that ascorbate effects collagen secretion by acting at the site of proline hydroxylation but not by increasing the activity of prolyl hydroxylase.     

Defective spontaneous but normal antibody-dependent cytotoxicity for an extracellular protozoan parasite, Giardia lamblia, by C3H/HeJ mouse macrophages

To understand murine host responses to extracellular protozoa, the capacity of peritoneal macrophages to exhibit cytotoxicity for [3H]thymidine-labeled Giardia lamblia trophozoites was investigated. Resident peritoneal macrophages from C3H/HeN mice expressed spontaneous cytotoxicity for G. lamblia in a manner that was dependent on both time and effector cell number; this cytotoxic activity was increased with cells elicited by an intraperitoneal injection of thio-glycollate. In contrast, spontaneous cytotoxicity for G. lamblia by resident and thioglycollate-elicited peritoneal macrophages from C3H/HeJ mice was markedly reduced. In the presence of anti-G. lamblia serum (ADCC), however, peritoneal macrophages from both C3H/HeN and C3H/HeJ mice exhibited striking augmentation of their cytotoxic activity for G. lamblia to equivalent levels. We conclude that macrophages from C3H/HeJ mice express defective spontaneous cytotoxicity but normal ADCC for the extracellular protozoan parasite, G. lamblia. The dissociation between the expression of these two effector cell functions suggests that macrophage spontaneous cytotoxicity and ADCC for extracellular protozoa are mediated by separate macrophage functions.     

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.     

Evidence that forskolin activates turkey erythrocyte adenylate cyclase through a noncatalytic site

The diterpene forskolin has been reported to activate adenylate cyclase in a manner consistent with an interaction at the catalytic unit. However, some of its actions are more consistent with an interaction at the coupling unit that links the hormone receptor to the adenylate cyclase activity. This report adds support to the latter possibility. Under conditions that lead to stimulation of adenylate cyclase in turkey erythrocyte membranes by GTP, forskolin also becomes more active. Additional evidence to support an influence of forskolin upon adenylate cyclase via the GTP-coupling protein N includes the following: (i) forskolin, at submaximal concentrations, leads to enhanced sensitivity and responsiveness of isoproterenol-dependent adenylate cyclase activity in turkey erythrocyte membranes; (ii) under specified conditions, the nucleotide GDP, an inhibitor of the stimulating nucleotide GTP and its analog, guanyl imidodiphosphate (Gpp(NH)p), also markedly inhibits the action of forskolin; (iii) both Gpp(NH)p and forskolin are associated with a decrease in agonist affinity for the beta-adrenergic receptor. However, actions of forskolin in the turkey erythrocyte are not identical to those of GTP: (i) forskolin is never as potent as Gpp(NH)p in activating adenylate cyclase; (ii) the magnitude of synergism between isoproterenol and forskolin is not equal to that observed with isoproterenol and Gpp(NH)p; (iii) at high concentrations, forskolin inhibits antagonist binding to the beta-receptor. Forskolin appears to have several sites of action in the turkey erythrocyte membrane, including an influence upon the adenylate cyclase regulatory protein N.     

Inhibition of mycoplasma cell division by cytochalasin B

Mycoplasma gallisepticum has subcellular organelles which may function as a primitive "mitotic-like" apparatus. To investigate these further, we have studied the effects of cytochalasin B (CB) on M. gallisepticum. We found that CB inhibits cell division; this is the only procaryote thus far reported to be inhibited by CB. CB does not inhibit glucose or macromolecule precursor uptake. It stops cellular DNA synthesis, however, although RNA and protein synthesis continue (at a reduced rate). CB removal results in a resumption of DNA synthesis, followed by cell division. There appears to be some degree of cell synchrony in this first division after CB removal. These results, together with morphological data, indicate that CB blocks at two points in the cell cycle: at the time "mitotic-like" structures are formed and at the time of cell division. It is suggested that the CB blocks may result from a disruption of actin-like protein structures required at these points in the cell cycle.     

Relaxation studies on complex formation of macrocyclic and open chain antibiotics with monovalent cations

The stability constants for the 1 : 1 complexes of macrocyclic antibiotics (nonactin, monactin, dinactin and trinactin) with Li⁺, Na⁺, K⁺, Rb⁺, Cs⁺, NH⁺₄ and for the Na⁺-complexes with the open chain compounds nigericin and monensin in methanol solution have been determined. The relaxation amplitude method was employed to obtain both the equilibrium constants and the enthalpies of reaction. The kinetics were studied with the help of temperature-jump, electric-field pulse and ultrasonic absorption techniques. Although complex formation of the metal ions with the antibiotics involves multidentate ligand chelation, the formation rates are in general very high, i.e. close to the limits imposed for diffusion controlled processes. The data for the macrotetrolides indicate the existence of conformational transition prior to complexation. A sequential substitution or “redressing” mechanism is proposed which is in accord with the high rates of complex formation. The selectivity patterns, as expressed by the equilibrium constants, are similar to those observed for the transport of metal ions across membranes in presence of the antibiotics. Selectivity results from an optimal balance between the strength of metal ion solvation and the stability of the individual metal complex, which in turn is governed by the conformational flexibility of the antibiotics.     

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.     

Antigen-specific human B-cell responses: modulation by immunoregulatory T-cell subsets

In vitro T-cell requirements for and modulation of human B-cell responses were studied in individuals immunized in vivo to the protein antigen keyhole limpet hemocyanin or tetanus toxoid. T cells were required for antibody synthesis in both antigen-driven and pokeweed mitogen (PWM)-driven cultures. T cells were separated into T4+ and T8+ subpopulations using monoclonal antibodies, and their modulation of antibody synthesis was studied. T4+ cells functioned as helper cells in both antigen-driven and PWM-driven cultures in a dose-dependent manner. Whereas T8+ cells suppress both total and specific immunoglobulin secretion in PWM-stimulated cultures, in antigen-stimulated cultures T8+ cells do not suppress unless activated by another cell population present in peripheral blood mononuclear cells (PBMNC). This cellular requirement was further investigated by prestimulation of cells prior to addition to optimally stimulated antigen-driven cultures of PBMNC or B cells, monocytes, and helper T cells. No suppression of these optimally stimulated cultures was seen when T8+ cells were precultured with antigen or PWM. However, after 3-5 days preculture of total T cells with PWM or antigen and then selection of T4+ cells, these cells were able to induce fresh autologous T8+ cells to suppress optimally stimulated antigen-driven cultures. Addition of a precultured mixture of T8+ cells with 20% T4+ cells also resulted in antigen-induced suppression. These data indicate that T8+ cells can suppress antigen-driven cultures but require the presence of preactivated T4+ cells for induction of this suppression of antigen-specific T-cell-dependent human B-cell responses.     

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.     

Structural studies of human chorionic gonadotropin and its subunits using tyrosine fluorescence.

The fluorescence properties of the tyrosyl residues of human chorionic gonadotropin (hCG) and its α and β subunits have been examined. The effects of pH, guanidine, and disulfide cleavage on the intensity and polarization of the fluorescence suggest that the isolated subunits possess little, if any, tertiary structure beyond that which is stabilized by the disulfide bonds. Essentially all of the fluorescence of hCG and its subunits was accessible to quenching by iodide ions. Similar results were observed for several other proteins whose fluorescence originates from tyrosyl residues. Thus, we have confirmed and extended the conclusion of R. W. Cowgill ((1966) Biochim. Biophys. Acta120, 196) that the buried tyrosyl residues in ribonuclease fluoresce with a much lower quantum yield than those which are exposed. The dissociation of hCG into its subunits was accompanied by an increase in fluorescence, suggesting the exposure of tyrosyl residues. This was confirmed by difference absorption measurements which indicate a net exposure of two to three tyrosyl residues upon dissociation of the subunits. An additional 0.6 tyrosine was exposed when the disulfide bonds of the β-subunit were cleaved. The polarization of the fluorescence of hCG-β was high (P = 0.19) and, unlike several other proteins with high polarization, could not be lowered by denaturing conditions. Only by cleavage of the disulfide bonds could the fluorescence polarization of either subunit be lowered to a value (P = 0.08) characteristic of a random polypeptide. It appears that the disulfide bonds play an important role in maintaining the rigidity of the fluorescent tyrosyl residues, located at or near the surface of the protein.     

Stimulation of T-cell activation by UV-treated, antigen-pulsed macrophages: evidence for a requirement for antigen processing and interleukin 1 secretion

The nature of the defect(s) in the ability of UV-treated guinea pig macrophages to stimulate the proliferative response of guinea pig T cells to soluble protein antigens was investigated. T cells proliferated vigorously when cultured with peritoneal exudate cells (PEC) which had been pulsed with soluble protein antigens, but failed to proliferate when cultured with soluble antigen or with antigen-pulsed, UV-treated PEC. UV-treated macrophages were unable to secrete interleukin 1 (IL-1). Addition of IL-1 partially restored the T-cell proliferative response stimulated by antigen-pulsed, UV-treated PEC. However, IL-1 was able to restore such a response only when the PEC were pulsed with antigen before being exposed to UV. Similar results were obtained when antigen-pulsed PEC were used to stimulate T cells to secrete interleukin 2 (IL-2). These results demonstrate that UV-treated macrophages are defective both in their ability to properly process and present antigen for T-cell recognition and in their ability to secrete IL-1.     

Ability of dialysates containing transfer factor to induce lymphocyte activating factor by human mononuclear cells.

Dialysates of human leukocyte lysates containing transfer factor (TF_d) stimulated human mononuclear cells (MNL) to produce lymphocyte activating factor (LAF). Both unfractionated and adherent MNL cultures were stimulated by TF_d to produce a factor which was mitogenic for murine thymocytes and had the biochemical characteristics of LAF as determined by Bio-Gel P-100, DEAE cellulose, and hydroxylapatite chromatography. Fractionation of TF_d on Sephadex G-25 showed that the specific transfer factor activity of converting in vivo skin tests was present in the major uv-absorbing peak, whereas the substance(s) that induced LAF activity was present in a number of the other fractions. Therefore, the capacity of TF_d to induce monocytes to produce LAF is not a measure of classical transfer factor activity. However, this effect of TF_d may instead participate in the nonspecific immunoenhancing effects of TF_d.     

Dimeric intermediates of recombination in phage lambda

Biparental lambda phage DNA dimers formed by the Rec recombination system of E. coli were isolated in the absence of DNA replication and phage maturation. The RecA but not the RecB gene is required for dimer formation. Dimers are primarily circular but can also be branched circular or linear. In circular dimers the crossover points are distributed uniformly along the chromosome, even in the presence of the RecB-dependent Chi recombinational hotspots. Thus in the absence of DNA synthesis and maturation, the Rec system can act reciprocally both in the presence and absence of the RecB gene; this lack of RecB participation accounts for the observed lack of Chi activity.     

Hydrocortisone-mediated inhibition of monocyte antigen presentation: Dissociation of inhibitory effect and expression of DR antigens

The suppressive effects of hydrocortisone (HC) on the human immune system are well known. The mediation of the immunosuppressive effects of HC on lymphocyte responses via inhibition of monocyte function has been examined by monocyte-dependent, antigen-induced lymphocyte proliferation. Monocytes that were first treated with HC and then washed were unaffected in their subsequent ability to present antigen. However, there was a dramatic inhibition of lymphocyte proliferative responses if HC was present while monocytes were pulsed with antigen. This was directly related to the dose of HC present. HC-mediated inhibition of monocyte antigen presentation could not be overcome by the addition of interleukin-1 (IL-1) to the cultures, and thus inhibition of monocyte IL-1 secretion cannot totally account for the inhibition of monocyte antigen presentation. Although HC inhibits monocyte antigen presentation, HC increases the expression of HLA-DR antigens on monocytes. Other monocyte stimulants, including lipopolysaccharide (LPS), lymphokine, and gamma interferon, were examined for their effect on monocyte DR expression and their effect on monocyte antigen presentation. No correlation was found between the ability to increase monocyte DR antigen expression and the effect on antigen presentation. While HC, lymphokine, and gamma interferon all increased the expression of DR antigens on monocytes, HC, LPS, and lymphokine, but not gamma interferon, inhibited monocyte antigen presentation. Although HC can exert profound immunosuppressive effects via monocytes, it is not the only mechanism of inhibition. HC added to cultures after monocytes had been pulsed with antigen was also inhibitory.     

Differential effect of monoclonal anti-DR antibody on monocytes in antigen- and mitogen-stimulated responses: Mechanism of inhibition and relationship to interleukin 1 secretion

A differential role for DR antigens on monocytes in antigen-stimulated as opposed to mitogen-stimulated human lymphocyte responses has been observed. A monoclonal anti-DR antibody used to treat monocytes caused inhibition of antigen-induced T-cell responses and of T-cell-dependent B-cell responses. However, anti-DR antibody treatment of monocytes did not inhibit mitogen-induced responses. Anti-DR treatment of monocytes did not induce suppression, as antigen-induced responses could be reconstituted with untreated monocytes. Anti-DR treatment of monocytes did not merely block interleukin 1 (IL-1) secretion since addition of IL-1 could not restore antigen-induced responses. Monoclonal anti-DR antibody did not directly inhibit monocyte secretion of IL-1. DR-negative monocytes, selected by antibody and complement, could not present antigen, even though they were capable of secreting IL-1. Thus, this monoclonal anti-DR antibody sterically blocks antigen presentation by monocytes without induction of suppression or inhibition of IL-1 secretion. Monocyte DR antigens appear essential for stimulation of antigen-induced responses, but DR antigens on monocytes may not be essential for mitogen-stimulated responses and do not appear to be related to the ability of monocytes to secrete IL-1.     

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.     

The relationship between immunization and circulating antigen-specific plaque-forming cells

The relationship between the appearance of cells producing antibody to tetanus toxoid (TT) in the circulation and the serum titers of anti-TT IgG following booster immunization has been studied. It was found that cells producing anti-TT antibody can be detected in the circulation in a hemolytic plaque assay using sheep red blood cells (SRBC) coated with TT by the chromic chloride method. In symmetric inhibition studies using cells from TT or keyhole limpet hemocyanin (KLH)-immune donors, the homologous antigen inhibited 100% of the PFC with no cross-inhibition. Thus, the plaque-forming cells (PFC) detected in this assay are specific for the immunizing antigen. No evidence of polyclonal B-cell activation in response to TT was found, as shown by a failure to detect any PFC against unmodified or KLH or human serum albumin-treated SRBC. In addition, the increase in total Ig-secreting cells observed in a staphylococcal protein A reverse hemolytic plaque assay was always accounted for by the number of anti-TT antibody-producing cells observed. The peak number of anti-TT antibody-producing cells varied between donors, but the kinetics of their appearance was highly reproducible--none before Day 5, peak numbers between Days 6 and 8, and a sharp decline with only rare anti-TT Ig-secreting cells in the circulation by Day 15 postimmunization. Anti-TT antibody-producing cells appeared in the circulation prior to any detectable increase in serum anti-TT antibody titers, and following the disappearance of PFC from the circulation, there was no further increase in serum IgG anti-TT levels. These observations demonstrate a marked specificity of B-cell activation on boosting with a recall antigen, and a parallelism between the appearance of activated B cells in the circulation and of IgG anti-TT synthesis by the subject as a whole.     

An immunofluorescent study of the distribution of fibronectin and laminin during limb regeneration in the adult newt

Fibronectin and laminin are two extracellular glycoproteins which are involved in various processes of cellular development and differentiation. The present investigation describes changes in their distribution during regeneration of the newt forelimb, as determined by indirect immunofluorescence. The distribution of fibronectin and laminin was similar in normal limb tissue components. These glycoproteins were localized in the pericellular region of the myofibers corresponding to its basement membrane; the perineurium and endoneurium of the nerves; and the basement membranes of blood vessels, skin epithelium, and dermal glands. The cytoplasm of myofibers, axons, skin epithelium, and bone matrix lacked fluorescence for both glycoproteins. After limb amputation in the regenerating blastema, extensive presence of fibronectin, but not laminin, was seen in and around the undifferentiated blastemal cells. Increased fluorescence for fibronectin was also seen during blastema growth, blastemal cell aggregation, and early stages of redifferentiation. As redifferentiation continued, staining for fibronectin slowly disappeared from the cartilage matrix and the myoblast fusion zone. Laminin was first observed around the regenerated myotubes; this was followed by the appearance of fibronectin suggesting a sequential formation of these two components of the new myotube basement membrane. In the regenerated limb, the distribution of laminin and fibronectin was similar to that seen in normal limb. Based on the distribution pattern of these glycoproteins, it is concluded that fibronectin may play an important role in blastemal cell aggregation, cell alignment, and initiation of redifferentiation. After redifferentiation, both laminin and fibronectin may be important in the determination of the architecture of the regenerated limb.     

Cell-mediated immunity in experimental filariasis: lymphocyte reactivity to filarial stage-specific antigens and to B- and T-cell mitogens during acute and chronic infection.

To study immunological responses in chronic filarial infections, a model utilizing inbred Lewis rats infected with Brugia pahangi was developed. Microfilaria were found in the bloodstream of over 90% of the rats by 16 weeks of infection. Using in vitro lymphocyte blastogenesis, cell-mediated immune responses of blood, splenic, and mesenteric node lymphocytes were followed during 1.5 years of infection. Lymphocyte responses to antigen prepared from infective stage filarial larvae were detectable in the early weeks of infection, whereas responses to microfilarial antigen only developed late as microfilaremia waned. Lymphocyte responses to antigen from adult filaria vacillated during the infection. With the mitogens, phytohemagglutinin, pokeweed mitogen, and bacterial lipopolysaccharide, periods of B and T-cell hyporesponsiveness were demonstrable. Between 16 and 36 weeks of infection node lymphocytes from many rats were unresponsive to all mitogens and antigens. The model of B. pahangi in inbred rats offers advantages for immunological studies of filarial infections.     

Conformation studies of 13 trinucleoside diphosphates by 360 MHz PMR spectroscopy. A bulged base conformation. I. Base protons and H1' protons

The 360 MHz NMR spectra of the base protons and the H1 protons of thirteen trinucleoside diphosphates have been analyzed. The sequences chosen represent all purine-pyrimidine sequences. The chemical shifts of the base protons give evidence for strong next nearest-neighbor effects in some oligonucleotides. Although increasing chain length usually increases nearest-neighbor base-base stacking, it is not always so. Comparing ApCpG, ApUpG and GpUpG to their component dimers, one finds a decrease in stacking of the center pyrimidine with the purine on either side. The coupling constants J 1'2' also show that these three trimers show less stacking for their terminal residues than expected from their component dimers. We conclude that the sequence Pu-Py-Pu favors a conformation in which the pyrimidine is bulged out and the two purines stack on each other.