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PubMed1972

Contact regions for dinitrophenyl and menadione haptens in an immunoglobulin binding more than one antigen.

Protein 460 is a mouse myeloma gamma A(2) protein that competitively binds two small haptens, 2,4-epsilon-dinitrophenyl-L-lysine (Dnp-Lys) and 2-methyl-1:4-naphthaquinone thioglycollate (MenTG), to the antibody-combining region. The intact protein has a relatively inaccessible sulfhydryl group on each heavy chain. When it is substituted with a bulky reagent the binding affinity for MenTG decreases, while the binding of Dnp-Lys remains the same. Guanidine.HCl selectively reduces binding of Dnp-Lys; dimethylsulfoxide selectively reduces binding of MenTG. Papain digestion of protein 460 followed by column chromatography gave two fractions: one contained both binding activities and the other contained the sulfhydryl group. The affinity for Dnp-Lys of the first fraction is the same as that of the whole molecule, while affinity for MenTG is decreased. Since selective alteration of one or the other binding activity can occur in different ways, it seems likely that even though the haptens compete with each other, there is some spatial separation between the groups of contact amino-acid residues involved in the binding of these two haptens. These findings do not support the hypothesis that an immunoglobulin molecule carries combining sites complementary only to a single hapten or to a structurally related series of haptens, but rather suggests that the antibody-combining site may be a polyfunctional region capable of binding several structurally dissimilar haptens. We discuss a mechanism whereby polyfunctional combining sites can give rise to an antibody population (immune serum) that has a high degree of specificity to a single hapten.

Alkylation↗

PubMed1995

Macrophage-released factor stimulates selectively myogenic cells in primary muscle culture.

Myofibers are reconstituted by the proliferation and fusion of muscle precursor cells when skeletal muscle is injured. One of the critical events is the peak accumulation of macrophages after 48 hours at the damage site before the satellite cell proliferation. In addition to their well-known role as a scavenger cell, there is now direct evidence of a mitogenic role of macrophages in regenerating muscle. We have utilized an in vitro model to directly investigate and prove that macrophages increase myoblast growth not only of satellite cells, but also of primary myoblasts. Rat muscle cells were cultured in the presence or absence of exudate macrophages obtained by peritoneal washing after thioglycollate broth injection. Macrophage coculture increases several times the myoblasts/myotubes yield. This effect is particularly evident in muscle culture conditions in which fibroblast growth is predominant over myoblast proliferation, suggesting a myoblast selective mitogenic effect of macrophages. The results are confirmed by quantitative analyses of both DNA and skeletal muscle-specific-contractile proteins by gel electrophoresis and immunocytochemistry. Experiments with macrophage-conditioned media show this effect is mediated by soluble factors. This growth factor-like activity, which has been shown to be acid-stable and heat-labile, labile, exerts its effects not only on specialized satellite cells during muscle regeneration, but also has a broader mitotic activity on all myogenic cells. In view of the role of muscle regeneration in muscle diseases and of the perspectives offered by gene therapy via myoblasts, we strongly believe that our results open new opportunities in removing many of the clinical constraints associated with repair and cell transplantation.

Animals↗

PubMed1979

Physiological characterization of nutritionally variant streptococci.

Twenty-five isolates of nutritionally variant streptococci submitted to the Streptococcus Laboratory of the Center for Disease Control over a 2-year period were tested for growth requirements and for biochemical reactions. After they were recovered from storage in blood at -170 degrees C, all isolates grew within 48 h in both thioglycollate broth and Todd-Hewitt broth supplemented with 0.001% pyridoxal.HCl. They grew better in the latter, even though they all grew on unsupplemented infusion agar, anaerobe blood agar, and chopped meat-glucose medium. Biochemical patterns of the isolates resemble those of five viridans streptococcal species. Two isolates had patterns which did not resemble those of any viridans species. Biochemical reactions obtained with heart infusion broth base biochemicals and carbohydrate fermentation media compared favorably for an overall agreement rate of 86.5% for key tests. Lactic acid and acetic acid were the major fermentation products detected with gas-liquid chromatography.

Acetates↗

PubMed1992

Beta-VLDL-induced cholesterol ester deposition in macrophages may be regulated by neutral cholesterol esterase activity.

We examined the role of enzyme activities involved in cholesterol ester metabolism in the accumulation of cholesterol ester in macrophages. When [3H]cholesterol linoleate-beta-very low density lipoproteins (beta-VLDLs) were incubated with rat resident peritoneal macrophages (RPMs), thioglycolate-elicited macrophages (TEMs), or alveolar macrophages (AMs), cholesterol ester accumulation was the greatest in TEMs and the least in AMs. The uptake of [3H]cholesterol linoleate-beta-VLDL into AMs was four times that into RPMs, and the uptake into TEMs was twice that into RPMs. The [3H]cholesterol released from [3H]cholesterol linoleate-beta-VLDL-loaded AMs was five times higher than from TEMs and RPMs, whereas those from RPMs and TEMs were almost the same. In studies using cell homogenates, the acid cholesterol esterase activity in AMs was 1.7 times that in TEMs and six times that in RPMs. The acyl-coenzyme A:cholesterol acyltransferase (ACAT) activities in AMs and TEMs were similar but were higher than that in RPMs. The neutral cholesterol esterase activity was seven times higher in AMs than in RPMs and TEMs. In the study using intact cells, the hydrolysis of loaded [3H]cholesterol linoleate-beta-VLDL in the presence of the ACAT inhibitor CL277,082 and the cholesterol [14C]oleate synthesis from [14C]oleate were enhanced in AMs about twofold compared with RPMs and TEMs. The reduction of de novo synthesized cholesterol [14C]oleate in the presence of CL277,082 was enhanced in AMs four times compared with TEMs or RPMs.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1981

Chromatographic determination of thiodiglycolic acid - a metabolite of vinyl chloride.

The determination of thiodiglycolic acid (TDGA) In urine by gas chromatography (GC) is described. The analytical procedure includes addition of internal standard (o-phthalic acid), ethyl acetate extraction, evaporation of the solvent and silylation of TDGA with N-trimethylsilyl-diethylamine in pyridine (1:1). The results are calculated from the ratio of TDGA and internal standard peak heights. The detection limit of the method is 10 mg/l and the coefficient of various is +/- 5%.

Chromatography, Gas↗

PubMed2006

Nuclear status of immature and mature stallion spermatozoa.

'The highly packed chromatin of mature spermatozoa results from replacement of somatic-like histones by highly basic arginine- and cysteine-rich protamines during spermatogenesis, with additional conformational changes in chromatin structure during epididymal transit. The objective of the present study was to compare the nuclear characteristics of immature and mature epididymal stallion spermatozoa, using a variety of experimental approaches. Resistance to in vitro decondensation of chromatin, following exposure to SDS-DTT and alkaline thioglycolate, increased significantly in mature spermatozoa. Evaluation of the thiol-disulfide status (monobromobimane labeling) demonstrated that immature cells obtained from ductulli efferentes contained mostly thiol groups, whereas these groups were oxidized in mature cells collected from the cauda epididymidis. Based on atomic absorption spectrophotometry, maturation of stallion spermatozoa was accompanied by a 60% reduction in the Zn(2+) content of sperm cells, concomitant with increased concentrations of this ion in epididymal fluid. Furthermore, the degree of disulfide bonding was inversely correlated with susceptibility of chromatin to acid denaturation (SCSA). Collectively, these data were consistent with the hypothesis that maturation of stallion spermatozoa involves oxidation of sulphydryl groups to form intra- and intermolecular disulfide links between adjacent protamines, with loss of zinc as an integral feature. These changes endow mechanical and chemical resistance to the nucleus, ensuring efficient transmission of the paternal genome at fertilization.

Animals↗

PubMed1999

Determination of thiodiglycolic acid in urine by capillary electrophoresis.

A sensitive capillary electrophoretic method was developed for the determination of thiodiglycolic acid (TDA) in urine which avoids the pretreatment of the urine sample. Several carrier electrolytes were examined. The most suitable carrier electrolyte system consisted of potassium hydrogen phthalate (5 mM), 2-(N-morpholino)ethanesulfonic acid (50 mM) and tetradecyltrimethylammonium bromide (0.5 mM), pH 5.2. Ten times diluted fresh midstream void urine was used for the determination. In this way, the concentrations of TDA between 5 and 50 mg/l in undiluted urine samples can be determined.

Buffers↗

PubMed1983

A novel role for macrophages: the ability of macrophages to tolerize B cells.

We investigated the ability of macrophages (M phi) to present the tolerogen fluoresceinated sheep gamma-globulin (FL-SGG) to B cells. M phi pulsed with FL-SGG or murine FL-IgG2 were able to tolerize normal spleen B cells specifically as assessed by the plaque-forming cell (PFC) response to the antigens FL-Brucella abortus (FL-BrA) and FL-polymerized flagellin (FL-POL). Tolerance was not induced when M phi were pulsed with a variety of other FL antigens or the synthetic tolerogen FL-D-glutamic acid-D-lysine (FL-D[G,L]). The ability of M phi to tolerize B cells was not T cell-dependent, because populations of both T-depleted B cells and hapten-specific B cells were tolerizable. M phi-induced B cell tolerance did not exhibit genetic restriction with regard to the H-2 haplotype of the presenting M phi or the responding B cells. A variety of different types of peritoneal M phi, including normal resident M phi and those elicited by thioglycollate or concanavalin A (the latter are predominantly la+), could tolerize B cells after being pulsed with FL-SGG. We compared tolerogen-pulsed M phi to soluble tolerogen for the ability to tolerize B cells and found that tolerogen bound to M phi was more than 10 times as potent as an equivalent amount of soluble tolerogen. In contrast to the ability of M phi to present FL-SGG in a tolerogenic fashion to B cells, the P388AD lymphoid dendritic cell-like tumor line presented FL-SGG in an immunogenic mode to B cells. Tolerogen bound to P388AD cells could specifically augment a PFC response to both FL-BrA and FL-POL. We suggest that certain types of M phi may play a role in unresponsiveness by enhancing the tolerogenicity of soluble antigen, whereas other accessory cells may present the same moieties in an immunogenic fashion.

Animals↗

PubMed1962

Pharmacological actions of pepsitensin.

In confirmation of the results of Croxatto and his co-workers, plasma proteins incubated with pepsin yielded a substance (pepsitensin) with pressor activity. Euglobulin gave a much higher yield than the other plasma protein fractions. Incubation for 4 hr at pH 4 gave high yields of pressor activity (pepsitensin) but no antidiuretic activity; incubation of euglobulin for 4 hr at pH 2.5 yielded extracts with antidiuretic as well as pressor activity. Incubation for 8 to 11 hr at pH 2.5 produced the highest yield of both activities. Further incubation, at the same pH, up to 20 hr caused a rapid decline in the pressor activity of the extracts, but the antidiuretic activity was much more resistant to destruction by pepsin. Pepsitensin was found to be very soluble in water and poorly soluble in organic solvents. It is not inactivated by thioglycollate. In blood pressure assays some animals did not respond to pepsitensin, and nephrectomized (17 to 24 hr) rats were found to be more suitable preparations. Pepsitensin was shown to exert pressor effect by direct action on the blood vessels. Its pressor action could be differentiated from that of tyramine, dimethylphenylpiperazine, nicotine, noradrenaline and Pitressin but not from that of angiotensin. The isolated guinea-pig ileum and the rat uterus were equally sensitive to angiotensin and pepsitensin. In paper chromatograms, in the solvent system butanol-acetic acid-water, the R(F) of pepsitensin was very similar to that of angiotensin.

Angiotensin Amide↗

PubMed1992

Mouse MRP8 and MRP14, two intracellular calcium-binding proteins associated with the development of the myeloid lineage.

MRP8 and MRP14 are two S100-like calcium-binding proteins of unknown function, associated with numbers of human inflammatory disorders. Both molecules have been described as L1 complex, cystic fibrosis antigen, or p8 and p14. We report here the cloning of mouse MRP8 and MRP14 and their pattern of expression during hematopoiesis. Mouse MRP8 and MRP14 proteins share 59% identity with their human counterparts, but they are more divergent than the other members of the S100 protein family. Mouse MRP proteins are coexpressed in fetal myeloid progenitors, where they are detected as early as day 11 of gestation. In fetal liver and yolk sac, MRP+ cell populations increased in number, in association with the development of the myeloid lineage. In adult mouse, we identified MRP8 and MRP14 proteins in immature myeloid cells of the bone marrow, myeloid cells in the splenic red pulp and marginal zone, in addition to monocytes and blood neutrophils. However, MRP expression is lost as cells terminally differentiate into tissue macrophages. In addition, using thioglycollate-induced peritoneal inflammatory exudates, we showed that MRP8 and MRP14 proteins are highly expressed in recruited neutrophils and monocytes.

Animals↗

PubMed1990

Control of lipoprotein lipase secretion by macrophages: effect of macrophage differentiation agents.

The effect of macrophage differentiation agents on lipoprotein lipase (LPL) secretion by macrophages at different stages of differentiation/maturation was investigated. Phorbol myristate acetate (TPA) had an augmenting effect on LPL secretion by in vitro-derived bone marrow macrophages (BMMs), thioglycollate-elicited peritoneal macrophages (TgM phi), and resistant macrophages. Augmentation was time dependent and reached approximately two-fold and approximately threefold increase over control cells within 16 and 96 hr, respectively. TPA did not affect LPL secretion from J774.1 cells treated with the agent for 16-72 hr. L-cell conditioned medium (L-CM), a source of macrophage colony-stimulating activity, augmented LPL secretion by BMMs and Tg-M phi, and when added together with TPA had an additive augmenting effect on LPL secretion in these cells. Retinoic acid (RA) exerted a time-dependent suppressive effect on LPL secretion by BMMs (46% within 16 hr and 83% within 6 d), had a relatively small effect on secretion from J774.1 cells (approximately 20% in 72 hr) and had no effect on LPL secretion by Tg-M phi. Dexamethasone suppressed LPL secretion by BMMs, Tg-M phi, and J774.1 cells. Optimal suppression of LPL secretion by BMMs required more than 24 hr. Thus, TPA and L-CM, agents that exert a mitogenic effect on BMMs and Tg-M phi, augmented the secretion of LPL in these cell types, and RA and dexamethasone, agents which induce differentiation patterns in myeloid cells, suppressed LPL secretion.

Animals↗

PubMed1994

Nitric oxide production in mouse peritoneal macrophages enhanced with glycyrrhizin.

The enhancement of nitric oxide (NO) production in glycyrrhizin (GL)-induced macrophages (M phi) in response to lipopolysaccharide (LPS) was investigated. No production in GL-induced macrophage culture supernatants was stimulated in response to LPS (10 micrograms/ml) for 24- or 48- h cultures, and these levels were compared three times with the levels in saline-induced peritoneal exudate cell cultures. Furthermore, M phi induced with proteose peptone (PP) containing GL could generate greater NO production than M phi induced with PP alone. However, no stimulation of NO production was observed by addition of GL in the cultures of M phi induced with thioglycollate or Bacillus Calmette Guerin. Moreover, GL-induced M phi showed cytostasis against such tumor target cells as L 1210 and P 388 lymphoma cell lines. These observations indicate that GL can activate the M phi in vivo system and stimulate NO production in response to LPS.

Animals↗

PubMed1984

African trypanosomiasis alters prostaglandin production by murine peritoneal macrophages.

Many factors contribute to the severe immunosuppression associated with African trypanosomiasis. Macrophages have been shown to be important target cells which after uptake of parasites, mediate immune dysfunction in vivo. We observed that infection of mice with Trypanosome brucei brucei (clone NIM 6) induces profound changes in arachidonic acid metabolism and prostaglandin (PG) secretion by macrophages. Normal macrophages release more PGI2 than PGE2 and production of both these prostaglandins is stimulated equally by endotoxin (LPS). Macrophages taken from NIM 6 infected mice at the peak of the first parasitaemia, release increased amounts of PGE2 and are hyperresponsive to LPS stimulation, while PGI2 secretion remains normal. As the infection progresses, there is a striking decrease in both basal PGE2 and PGI2 secretion and the ability of macrophages to respond to LPS. By the third week of infection, shortly before death, peritoneal macrophages resemble thioglycollate elicited macrophages in their inability to be stimulated by LPS to synthesize prostaglandins. Infection with a more virulent clone of T. brucei (NIM9) results in suppression of both PGE2 and PGI2 release by day 9 of infection. The increased production of PGE2 by macrophages during the height of infection is likely to contribute to the general immunosuppression associated with African trypanosomiasis.

Animals↗

PubMed2004

Bis(2-chloroethoxy)methane-induced mitochondrial and myofibrillar damage: short-term time-course study.

Cardiotoxicity induced by 2-, 3-, 5-, and 12-day dermal administration of 400 and 600 mg/kg/day of bis(2-chloroethoxy)methane to F344/N male and female rats was characterized. The severity and incidence of lesions were similar among males and females and in all three regions of the heart examined (atrium, ventricle, interventricular septum). Damage induced by bis(2-chloroethoxy)methane consisted of time-related development of myofiber vacuolation, necrosis, mononuclear-cell infiltration, fibrosis, and atrial thrombosis. Changes were pronounced at day 2, increased in severity at day 3, appeared to decrease at day 5, and resolved by study-day 16 that corresponded to 12 dosings. Ultrastructural analysis of 2- and 5-day 600 mg/kg/day-treated females elucidated the primary site of damage, the mitochondrion, and two types of vacuolation, one that formed as damaged mitochondria became devoid of cristae and their bounding double membranes became reduced to singleness, and the other manifested as distention of the sarcoplasmic reticulum. After the initial damage induced by bis(2-chloroethoxy)methane, or its metabolite, thiodiglycolic acid, protective mechanisms within the heart were apparently initiated, enabling it to cope with the continued exposure to the toxicant while eliminating some damaged myofibers.

Animals↗

PubMed1991

Chemoselective one-step purification method for peptides synthesized by the solid-phase technique.

The specific reaction between SH and iodoacetamide groups has been explored as the basis of an affinity-type purification procedure for peptides synthesized by the solid-phase technique. For this affinity-type purification procedure, we synthesized an SH precursor reagent bearing an acid-labile S-protecting group, pMB-SCH2CONHCH2CH2-SO2CH2CH2OCO2pNP (compound I), in which pMB is p-methoxybenzyl and pNP is p-nitrophenyl. Using this reagent, the procedure involves the following sequence of four reactions: (i) attachment of the SH function of compound I to the alpha-amino group of a peptide-resin through a base-labile sulfonylethoxycarbonyl linkage in the final step of solid-phase peptide synthesis, (ii) acid treatment to remove the S-pMB and side-chain-protecting groups employed and cleave the modified peptide from the resin, (iii) immobilization of the derived SH-peptide on an iodoacetamide-resin column, and (iv) base (5% NH4OH) treatment to release the desired peptide from the resin in nearly pure form. To facilitate this purification procedure, unreacted amino groups were acetylated in each step during solid-phase synthesis. The usefulness of this method was demonstrated by the purification of several peptides (18 to approximately 44 amino acids in length) synthesized by the 9-fluorenylmethoxycarbonyl (Fmoc)-based solid-phase technique. The principle of this affinity-type purification procedure may also be applied to the tert-butoxycarbonyl (Boc)-based solid-phase technique.

Chromatography, High Pressure Liquid↗

PubMed1993

Antibacterial effects of various endodontic irrigants on selected anaerobic bacteria.

This study was undertaken to determine the antibacterial effects of various endodontic irrigants against six selected anaerobic bacteria. The organisms were mixed separately with dilutions of each of the irrigants in tubes containing fluid thioglycollate broth and allowed to remain in contact for specific time periods before being evaluated for growth. Of the six irrigants tested, chlorhexidine appeared to be the most effective antibacterial substance. Hydrogen peroxide, sodium hypochlorite and REDTA were less effective while the calcium hydroxide and saline solutions proved to be totally ineffective.

Bacteria, Anaerobic↗

PubMed1986

Tumor associated macrophage mediated lysis of autologous tumor cells.

Lysis of autologous tumor cells (ATC) mediated by the tumor associated macrophages (TAM) was studied by 4 h 51Cr-release assay at different days of growth of a transplantable ascites tumor, S-180, inoculated in adult male Swiss mice. The ATC were coated with serum of tumor bearing mice. On day 3 after tumor inoculation the TAM mediated a remarkable lysis of the tumor serum coated ATC. With advancement of tumor growth the cytolysis by TAM decreased gradually to the basal level at day 10 and totally abrogated at day 12. Also the binding of the tumor serum coated ATC by TAM varied at different days of tumor growth. At early phases of tumor growth high lytic activity of the TAM was correlated with augmented target binding. The observed lysis may be considered as antibody dependent cell mediated cytotoxicity (ADCC) extended by the TAM, since presence of specific tumor antibodies were demonstrated in the tumor serum used for coating ATC. No significant lysis was recorded when the ATC was coated with normal serum or used as such for direct lysis by the TAM. In vitro treatment of the TAM with suitable doses of Levamisole and Polyionosinic-polycyticyclic acid further augmented the TAM mediated high level of ADCC at day 3 with no change in its binding of ATC. The immunopotentiators enhanced both ADCC and target binding ability of the functionally depressed TAM at day 10.

Animals↗

PubMed1984

Phospholipase A2 activity of Fc gamma 2b receptors of thioglycollate-elicited murine peritoneal macrophages.

The detergent lysate of plastic adherent cell population of thioglycollate-elicited peritoneal exudate cells from 100 individual Swiss mice was subjected to affinity chromatography on two different media, Sepharose coupled to heat-aggregated human IgG (IgG-Sepharose) and Sepharose coupled to the phosphatidylcholine analog, rac-1-(9-carboxyl)nonyl-2-hexadecylglycero-3 -phosphorylcholine (PC-Sepharose). Both IgG- and PC-binding proteins were further purified by Sephadex G-100 gel filtration and isoelectric focusing in the presence of 6M urea. Both IgG- and PC-binding proteins thus purified appear to be homogeneous in size as well as charge properties. The IgG-binding proteins of a molecular weight of 25,000 had an isoelectric point of 4.8, whereas the PC-binding proteins of a molecular weight of 42,000 were more basic and had an isoelectric point of 6.0. Both materials retained their IgG-binding capabilities as judged by their inhibitory capacity of murine EA gamma rosetting systems. The subclass specificities of the IgG- and the PC-binding proteins were for IgG2a and IgG2b, respectively. The PC-binding proteins possessed a typical phospholipase A2 activity, which was maximal at pH 9.5, depended on Ca++, and was specific for cleavage of fatty acid from the sn-2 position of phosphatidylcholine. The binding of aggregated IgG2b to the PC-binding proteins caused the ninefold increase in noted enzymatic activity in the presence of, but not in the absence of, Ca++ (5mM). The IgG-binding proteins on the other hand, lacked any detectable phospholipase A2 activity. Thus, the biochemical and biological properties of the PC- and IgG-binding proteins isolated from murine peritoneal macrophages are essentially identical to those homologous proteins previously isolated from P388D1 cells [20].

Androgen-Binding Protein↗