Lymphocyte Mitogenesis Research Articles

Application of a Lipopolysaccharide (LPS)-Stimulated Mitogenesis Assay in Smallmouth Bass (Micropterus dolomieu) to Augment Wild Fish Health Studies

The utility of a functional immune assay for smallmouth bass (Micropterus dolomieu) lymphocyte mitogenesis was evaluated. Wild populations in the Potomac River have faced disease and mortality with immunosuppression from exposure to chemical contaminants a suspected component. However, a validated set of immune parameters to screen for immunosuppression in wild fish populations is not available. Prior to use in ecotoxicology studies, ancillary factors influencing the mitogenic response need to be understood. The assay was field-tested with fish collected from three sites in West Virginia as part of health assessments occurring in spring (pre-spawn; April–May) and fall (recrudescence; October–November). Anterior kidney leukocytes were exposed to lipopolysaccharide (LPS) from E.coli O111:B4 or mitogen-free media and proliferation was measured using imaging flow cytometry with advanced machine learning to distinguish lymphocytes. An anti-smallmouth bass IgM monoclonal antibody was used to identify IgM+ lymphocytes. Lymphocyte mitogenesis, or proliferative responses, varied by site and season and positively and negatively correlated with factors such as sex, age, tissue parasites, and macrophage aggregates. Background proliferation of IgM− lymphocytes was negatively correlated to LPS-induced proliferation in both seasons at all sites, but only in spring for IgM+ lymphocytes. The results demonstrate that many factors, in addition to chemical contaminants, may influence lymphocyte proliferation.

Induction of both local and systemic immunity by in vivo injection of PHA into ginbuna carp fin

Phytohemagglutinin (PHA) is a well-known mitogen inducing activation and proliferation of lymphocytes, particularly T lymphocytes in vitro. PHA has also been used in vivo for assessing cell-mediated immunity in non-mammalian vertebrates, particularly in birds. However, it has been suggested that local inflammation as a direct result of tissue damage could be responsible for skin swelling after PHA injection, in addition to induction of T lymphocyte mitogenesis. In order to understand the complex nature of this response in fish we investigated the accumulation of cell types chronologically in dorsal fin of ginbuna crucian carp Carassius auratus langsdorfii after PHA injection. Neutrophils appeared first and showed a peak response on day 1, decreasing gradually and followed by macrophages and blast cells while lymphocytes increased later with a peak response on day 5. The number of accumulated cells was significantly higher in PHA-injected fish than controls in most cases. Lymphocytes identified as CD4-1+and CD8α+ were significantly more abundant in PHA-injected fish than in control fish throughout the 7-day experimental period except on day 1, while the number of IgM+ lymphocytes was higher in PHA-injected fish only on day 1. In the blast cell fraction, the number of CD4-1+ lymphocytes was significantly higher in PHA-injected fish than in control fish throughout experimental period, except on day 1. We also document the migration of neutrophils from the kidney to the fin through blood, followed by granulopoiesis in the kidney.These results suggest that adaptive immunity as well as innate immunity was induced by in vivo stimulation with PHA.

Minimal uranium immunotoxicity following a 60-day drinking water exposure to uranyl acetate in male and female C57BL/6J mice

Historical uranium (U) mining in the Southwestern United States resulted in significant environmental contamination throughout this region and presents a significant risk of chronic metal exposure and toxicity for communities living in close proximity to mine waste sites. Uranium exposure is associated with numerous deleterious health effects including immune dysfunction; however, its effects on the immune system have yet to be fully characterized. We recently published that drinking water exposure to U, in the form of uranyl acetate (UA), results in low overall tissue retention of U (<0.01%), with very little accumulation in immune organs (blood, bone marrow, spleen, and thymus) of male and female mice. In the present study we characterized the immunotoxicity of U, in the form of UA, following a 60-day drinking water exposure to 5 and 50 ppm in male and female C57BL/6J mice. The following immunotoxicity endpoints were evaluated: hematology, immune tissue weights and total cell recoveries, immunophenotying of the spleen and thymus, and immune cell function (lymphocyte mitogenesis and T-dependent antibody response). Uranium exposure had subtle impacts on the immune endpoints evaluated, likely due to low U accumulation at these sites. The only significant alterations were a slight decrease in the percentages of splenic natural killer T-cells and macrophages in exposed male mice. Despite minimal immunological effects, this study highlights the importance of investigating toxicological endpoints in both sexes and developing accurate animal models that model epidemiological exposures in the future.

The double bromodomain protein Brd2 promotes B cell expansion and mitogenesis

Bromodomain-containing transcriptional regulators represent new epigenetic targets in different hematologic malignancies. However, bromodomain-mediated mechanisms that couple histone acetylation to transcription in lymphopoiesis and govern mature lymphocyte mitogenesis are poorly understood. Brd2, a transcriptional coregulator that contains dual bromodomains and an extraterminal domain (the BET family), couples chromatin to cell-cycle progression. We reported previously the first functional characterization of a BET protein as an effector of mammalian mitogenic signal transduction: Eμ-Brd2 Tg mice develop "activated B cell" diffuse large B cell lymphoma. No other animal models exist for genetic or lentiviral expression of BET proteins, hampering testing of novel anti-BET anticancer drugs, such as JQ1. We transduced HSCs with Brd2 lentivirus and reconstituted recipient mice to test the hypothesis that Brd2 regulates hematopoiesis in BM and mitogenesis in the periphery. Forced expression of Brd2 provides an expansion advantage to the donor-derived B cell compartment in BM and increases mature B cell mitogenic responsiveness in vitro. Brd2 binds the cyclin A promoter in B cells, shown by ChIP, and increases cyclin A mRNA and protein levels, and S-phase progression in vitro in mitogen-stimulated primary B cells, but not T cells, reinforcing results from Eμ-Brd2 mice. The small molecule BET inhibitor JQ1 reduces B cell mitogenesis, consistent with the interpretation that BET inhibitors are antiproliferative. Brd2-specific knockdown experiments show that Brd2 is also required for hematopoiesis. We conclude that Brd2 plays a critical, independent role in regulation of mitogenic response genes, particularly cyclin A, in B cells.

Interrelationships among selenium status, antioxidant capacity and oxidative stress in the horse

Interrelationships among selenium status, antioxidant capacity and oxidative stress in the horse

Low-Selenium Diet Induces Cell Cycle Arrest of Thymocytes and Alters Serum IL-2 Content in Chickens

The purpose of this 42-day study was to investigate the effects of low selenium (Se) on cellular immune function by determining cell cycle of thymus, serum IL-2 content, and mitogenesis of peripheral blood T-lymphocytes. One hundred twenty 1-day-old Avian broilers were randomly assigned to two groups of 60 each and were fed on a low-Se diet (0.0342mg/kg Se) or a control diet (0.2mg/kg Se), respectively. Cell cycle analysis by flow cytometry showed that low-Se diet caused an increase in G(0)G(1) phase cells that corresponded to a decrease in S-phase cells in thymus. Ultrastructurally, mitochondria injury and increased apoptotic cells with condensed nuclei were observed. Low-Se diet decreased the serum IL-2 contents and mitogenesis of peripheral blood lymphocytes to concanavalin A in comparison with those of control group. These data indicate that low-Se diet inhibits the development of thymus by arresting the cell cycle and decreasing the IL-2 content.

Protective Role of Salidroside against Aging in A Mouse Model Induced by D-galactose

To investigate the protective effects of putative AGEs (advanced glycation endproducts) inhibitor salidroside against aging in an accelerated mouse aging model induced by D-galactose. A group of 5-month-old C57BL/6J mice were treated daily with D-galactose, D-galactose combined with salidroside, salidroside alone, and control buffer for 8 weeks. At the end of the treatment, serum AGEs levels, neurological activities, expression of glial fibrillary acidic protein (GFAP) and neurotrophin-3 (NT-3) in the cerebral cortex, as well as lymphocyte proliferation and IL-2 production were determined. D-galactose induced mouse aging model was developed as described before. As expected, salidroside blocked D-galactose induced increase of serum AGEs levels. It also reversed D-galactose induced aging effects in neural and immune system, as evidenced by improving motor activity, increasing memory latency time, and enhancing lymphocyte mitogenesis and interleukin-2 (IL-2) production. Furthermore, elevated expression of GFAP and NT-3 in the aged model mice was also reduced upon salidroside treatment. Salidroside inhibits AGEs formation in vivo, which at least partially contributes to its anti-aging effect in D-galactose induced aging model.

Suppression of lymphocyte proliferation by ovarian cavity fluid from the viviparous fish Neoditrema ransonnetii (Perciformes; Embiotocidae)

As the fetus expresses paternal major histocompatibility complex molecules, viviparous vertebrates require sophisticated mechanisms to modulate maternal immunology to ensure successful pregnancy. We anticipated that ovarian cavity fluid (OCF) is likely to feature significantly in the modulation of ovarian cavity immunology. Consequently, we examined the effects of OCF upon leukocyte function in Neoditrema ransonnetii. OCF did not affect phagocytosis or superoxide production by phagocytes. However, OCF suppressed lymphocyte proliferation induced by ConA almost completely. As OCF contained PGE 2 at high levels during late pregnancy, we also investigated the effect of PGE 2 upon lymphocyte expansion. PGE 2 exhibited negative effects upon lymphocyte mitogenesis in a dose-dependent manner (10–1000 ng/ml). PGE 2 significantly suppressed lymphocyte proliferation when present at levels equivalent to that seen in OCF (30.2 ± 16.1 ∼ 185.4 ± 107.4 ng/ml). Data indicate that PGE 2 is one of the key modulatory molecules of the maternal immune system ensuring successful pregnancy in this viviparous species.

Effects of In Ovo Exposure to PCBs 126 and 77 on Mortality, Deformities and Post-hatch Immune Function in Chickens

In laboratory experiments, planar PCBs produce immune organ atrophy in chicken embryos. To study the immunotoxic effects of PCBs in birds, the coplanar congeners 3,3′,4,4′,5-pentachlorobiphenyl (PCB 126) and 3,3′,4,4′-tetrachlorobiphenyl (PCB 77) were injected into the air cell of fertile white leghorn chicken eggs before incubation at doses of 0.25 and 0.5 ng/g egg PCB 126 and 0.64 ng/g egg PCB 77. Mortality and deformities were assessed during incubation of the eggs, and immune function was analyzed post-hatch using phytohemagglutinin (PHA) skin test for T-cell mediated immunity, antibody titers to sheep red blood cells (SRBC), mitogenesis of peripheral blood lymphocytes, and immune organ mass and cellularity. Exposure to 0.25 ng/g PCB 126 elevated mortality (61% and 69%) and deformities (31% and 32%), three or more times higher than controls. Two-fold suppression of antibody titers was observed in 28 day old chicks exposed to PCB 126 or PCB 77. No consistent alterations in PHA skin response or lymphocyte proliferation were observed. In 14 day old chicks in experiment two, PCB 126 decreased thymus and bursa cellularity by 33% and 35%, respectively. Immune organ atrophy was transient, recovering to control levels by 42 days of age. These experiments demonstrate that PCB 126 and 77 suppress antibody responses in juvenile chickens following an in ovo PCB exposure. Results reinforce the need for measuring multiple immune endpoints to detect immunotoxicity.

Mitogenesis of PHA-stimulated pig lymphocytes in phenol-added medium. A note.

Phenol (Ph) was added ab initio in 72 h-blood cultures, from 3 piglets, in 1640 RPMI medium supplemented with 10% fetal bovine serum and gentamicin (50 ml/ml). Lymphocyte mitogenesis was induced with phytohaemagglutinin (PHA-M, 30 mg/ml). The lymphocyte response was evaluated by microscopy (mitosis on slide). As compared with PHA- stimulated variant, lymphocyte proliferation in PHA- stimulated and Phenol- treated variants represented 0; 17.4; 20.8; 32.0 and 122.4% at concentrations of 1/100, 1/1000, 1/5000, 1/10000 and 1/15000 respectively (g/ml). At the concentration of 1/20000, the lymphocyte proliferation was normal. According to experimental results, Phenol elicits a progressive toxic/ suppressor effect on pig lymphocyte mitogenesis at concentrations higher than 1g/ 15000 ml culture medium

17β-Estradiol enhances contact hypersensitivity and IFN-γ expression in inflamed skin of BALB/c mice

The effects of 17β-estradiol (E 2) on mouse contact hypersensitivity (CHS) elicited at the ears by 4-ethoxymethylene-2-phenyl-2-oxazolin-5-one (OXA) were examined. Male and female BALB/c mice were sham-treated or gonadectomized, and then subcutaneously injected with E 2 twice a week for 4 weeks. The mice were sensitized by OXA application to their back and CHS was elicited at the ears. E 2 enhanced the ear swelling of all groups at 6 h after the elicitation. E 2 had no effect on the mitogenesis of splenic lymphocytes or nitric oxide synthesis by peritoneal macrophages. E 2 increased the number of thymic cells in female mice, but not male mice, and had no effect on the splenic cells of either female or male mice. Evaluation of the cytokine expressions in the inflamed skin revealed that E 2 enhanced the expression of interferon-γ, but had no effect on the expression of interleukin-4. These results suggest that E 2 affects the thymus and enhances the production of interferon-γ in skin to augment the skin swelling in CHS elicited by OXA.

Isolation, Cryopreservation, and Mitogenesis of Peripheral Blood Lymphocytes from Chickens (Gallus domesticus) and Wild Herring Gulls (Larus argentatus)

Monitoring the toxicity of environmental contaminants on the physiologic function of wild birds often includes measures of immune function. The purpose of this study was to apply methods of isolation, cryopreservation, and cell culture of chicken lymphocytes to blood samples from herring gulls, which are a sentinel species for biomonitoring studies in the Great Lakes and northern North America. Slow-spin centrifugation and density gradient isolation of lymphocytes were compared using chicken blood. Significant thrombocyte contamination of density gradient isolated samples (40% to 77% thrombocytes) led to the use of slow-spin centrifugation (2% thrombocytes) for blood from herring gulls. Cryopreserved blood samples were collected from adult and prefledgling herring gulls at sites of low environmental contamination around the Great Lakes and the Bay of Fundy between 1999 and 2002. Cryopreservation decreased the viability of lymphocytes from wild birds, but a high proportion of samples yielded enough live lymphocytes to assess function in culture. Cryopreserved lymphocytes from herring gulls proliferated in response to phytohemagglutinin (PHA), concanavalin-A, phorbol myristate acetate (PMA), PHA plus PMA, and lipopolysaccharide stimulation. Weber and Roswell Park Memorial Institute medium (RPMI) 1640 media were compared for culture of lymphocytes. Weber medium better supported chicken B-lymphocyte proliferation than RPMI 1640 and supported chicken T-lymphocyte proliferation of a similar magnitude as RPMI. Proliferation responses for lymphocytes from prefledgling herring gulls were stronger in Weber medium than RPMI medium, especially for PHA, for which there was no stimulation in RPMI. Proliferation responses of lymphocytes from adult herring gulls were up to twofold greater than that for prefledgling herring gulls. The magnitudes of proliferation responses were similar to that for chicken lymphocytes. These methods have subsequently proven useful in ecotoxicology studies that involve species in remote locations.

Apoptosis Induction of Mouse Splenic Cells by Exposure to High-Level 17.BETA.-Estradiol and Endocrine Disrupting Chemicals

It is commonly known that the sex hormones play important roles not only in the sexual organs, but also in the nervous system and immune systems. 17β-Estradiol affects the differentiation and the maturation of T cells in the thymus and B cells in the bone marrow, which are concerned with the homeostasis of variable immune actions such as the weight of the spleen and thymus, the secretion of thymic hormones, the secretion of cytokines and the production of antibodies. A number of endocrine disrupting chemicals (EDCs) exist in the environment and have hormonal, mainly estrogenic, effects. Because endogenous 17βestradiol modulates the immune functions as mentioned above, EDCs may be also assumed to affect the immune system as well. However, little is known about their modification of immune responses because EDCs have usually been studied in respect to developmental toxicity, teratogenicity or influence on sexual organs. The lymphocyte mitogenesis test is one of the in vitro immunotoxicity tests used to evaluate the safety of drugs. In this test, lymphocytes are stimulated by a polyclonal mitogen specific for either B cells or T cells as an indication of humoral immunity or cell-mediated immunity, respectively. We preliminarily evaluated the effect of 17β-estradiol on the immune function using this lymphocyte mitogenesis test and found that this chemical suppressed the lymphocyte proliferation. We further investigated the immunosuppressive activity of estrogenic compounds focused on cell death. Apoptosis, which refers to programmed death, is supposed to be triggered by cellular functional mechanisms, and necrosis is supposed to result from extrinsic damage. Apoptosis is achieved through several steps. A Fas ligand binds to the Fas protein on the surface of the cell, then several kinases and proteases including caspase are activated, and finally endonucleases cut the DNA between nucleosomes to make fragments in lengths of some integer times 180 base pairs. Then, the electrophoresed genomic DNA will show a ladder-like pattern. At apoptosis, cell membrane structure also changes. Phosphatidyl serine, which usually exists on the inner side of the *To whom correspondence should be addressed: Division of Environmental Health, Faculty of Pharmaceutical Sciences, Setsunan University, 45–1 Nagaotoge-cho, Hirakata, Osaka 573– 0101, Japan. Tel.: +81-72-866-3122; Fax: +81-72-866-3123; Email: nakamuro@pharm.setsunan.ac.jp Apoptosis Induction of Mouse Splenic Cells by Exposure to High-Level 17 -Estradiol and Endocrine Disrupting Chemicals

A 1.7 Å Structure of Fve, a Member of the New Fungal Immunomodulatory Protein Family

Fve, a major fruiting body protein from Flammulina velutipes, a mushroom possessing immunomodulatory activity, stimulates lymphocyte mitogenesis, suppresses systemic anaphylaxis reactions and edema, enhances transcription of IL-2, IFN-γ and TNF-α, and hemagglutinates red blood cells. It appears to be a lectin with specificity for complex cell-surface carbohydrates. Fve is a non-covalently linked homodimer containing no Cys, His or Met residues. It shares sequence similarity only to the other fungal immunomodulatory proteins (FIPs) LZ-8, Gts, Vvo and Vvl, all of unknown structure. The 1.7 Å structure of Fve solved by single anomalous diffraction of NaBr-soaked crystals is novel: each monomer consists of an N-terminal α-helix followed by a fibronectin III (FNIII) fold. The FNIII fold is the first instance of “pseudo-h-type” topology, a transition between the seven β-stranded s-type and the eight β-stranded h-type topologies. The structure suggests that dimerization, critical for the activity of FIPs, occurs by 3-D domain swapping of the N-terminal helices and is stabilized predominantly by hydrophobic interactions. The structure of Fve is the first in this lectin family to be reported, and the first of an FNIII domain-containing protein of fungal origin.

Estrogen receptor α in mouse splenic lymphocytes: possible involvement in immunity

To elucidate relevance of estrogens to immune responses, we investigated whether estrogen receptor α (ER α) exists in mouse splenic B cells and T cells and the effect of 17β-estradiol and endocrine disrupting chemicals (EDCs) on lymphocyte mitogenesis. ER α was identified in both male and female mouse splenic cells using RT-PCR. Crude splenic cells were stained with anti-ER antibody, and the distribution of ER α in the splenic B cells and part of the splenic T cells was confirmed by flow cytometry. 17β-Estradiol inhibited B cell mitogenesis at the concentration of 10 −8 M and T cell mitogenesis at the concentration of 10 −6 M. Some EDCs, diethylstilbestrol, bisphenol A, p-nonylphenol and di-2-ethylhexylphthalate, suppressed lymphocyte mitogenesis at the concentration of 10 −6–10 −5 M. We therefore suggest that estrogen may suppress lymphocyte mitogenesis through ER α in B and T cells.

琵琶湖‐淀川水系河川水のバイオアッセイによる包括的毒性評価

The present study was carried out to evaluate comprehensively the estrogenicity, immunotoxicity and mutagenicity of environmental water concentrates prepared by the XAD-2 resin column concentration method. This investigation focused on water from the river basin of Lake Biwa-Yodo River. Estrogenicity was detected by the yeast two-hybrid system and the measurement of alkaline phosphatase activity in human endometrial adenocarcinoma cell line (Ishikawa cells). Although the two methods differed in sensitivity and magnitude of estrogenic activity for lake water and river water containing endocrine-disrupting chemicals (EDCs), estrogenicity was detected in all sampling points except the Katsura River (Miyamae bridge). These results suggested the co-existence of materials with EDCs in environmental water, which inhibited the detection of estrogenic activity. Immunotoxicity was evaluated by the mouse splenic lymphocyte mitogenesis test using lipopolysaccharide and concanavalin A as specific mitogens for B cells and T cells, respectively. Some of the water samples showed specific depression effect on B cell and T cell mitogenesis. Furthermore, the Salmonella/microsome assay (Ames test) with TA98 and TA100 strain series was used to determine the mutagenicity. In all the sampling points, the direct/indirect-frameshift mutagenicity was detected. These results indicated that the yeast two-hybrid system and the measurement of alkaline phosphatase activity in Ishikawa cells, the lymphocyte mitogenesis test and the Ames test are important for evaluating comprehensively each endpoint because there is no relevance among these different toxicities.

Immunotoxicological Evaluation of Environmental Chemicals Utilizing Mouse Lymphocyte Mitogenesis Test.

To establish a useful and convenient bioassay for evaluating immunotoxicological effects of environmental chemicals, lymphocyte mitogenesis tests of about 255 chemicals and environmental water were performed. We determined the growth-inhibitory effect of the environmental chemicals on the mouse splenic lymphocyte mitogenesis using lipopolysaccharide and concanavalin A as the specific mitogen for B cells and T cells, and evaluated toxicity on humoral immunity and cell-mediated immunity, respectively. The DNA content of grown cells was determined by the novel ethidium bromide-fluorophotometry method, with 96-well microplates. Of the 255 chemicals tested, 173 chemicals showed inhibitory effects on the mitogenesis. The data were classified into four typical inhibition patterns from the dose-response curves. The chemicals were categorized into six groups in the respect of inhibition selectivity between B cell mitogenesis and T cell mitogenesis. Some chemicals, such as metallic compounds, showed nonspecific effects for B cell and T cell-mitogenesis, which was attributed to the cytotoxicity. The previous reports relating to immunotoxicity found about 123 chemicals; 78 chemicals (63.4%) were positive and one chemical (0.8%) was negative for both experimental results and reported results. In total, the concordance between experimental results and reported results is 64.2%. In the application of river water concentrates for the lymphocyte mitogenesis test, some of the samples inhibited both B cell and T cell mitogenesis at the tested volume of 7.2 ml. These results indicated that the lymphocyte mitogenesis test is applicable in estimating immunotoxicity of pollutants in environmental water as well as authentic environmental chemicals.

Up-regulation of the IKCa1 Potassium Channel during T-cell Activation: MOLECULAR MECHANISM AND FUNCTIONAL CONSEQUENCES

We used whole cell recording to evaluate functional expression of the intermediate conductance Ca(2+)-activated K(+) channel, IKCa1, in response to various mitogenic stimuli. One to two days following engagement of T-cell receptors to trigger both PKC- and Ca(2+)-dependent events, IKCa1 expression increased from an average of 8 to 300-800 channels/cell. Selective stimulation of the PKC pathway resulted in equivalent up-regulation, whereas a calcium ionophore was relatively ineffective. Enhancement in IKCa1 mRNA levels paralleled the increased channel number. The genomic organization of IKCa1, SKCa2, and SKCa3 were defined, and IK(Ca) and SK(Ca) genes were found to have a remarkably similar intron-exon structure. Mitogens enhanced IKCa1 promoter activity proportional to the increase in IKCa1 mRNA, suggesting that transcriptional mechanisms underlie channel up-regulation. Mutation of motifs for AP1 and Ikaros-2 in the promoter abolished this induction. Selective Kv1.3 inhibitors ShK-Dap(22), margatoxin, and correolide suppressed mitogenesis of resting T-cells but not preactivated T-cells with up-regulated IKCa1 channel expression. Selectively blocking IKCa1 channels with clotrimazole or TRAM-34 suppressed mitogenesis of preactivated lymphocytes, whereas resting T-cells were less sensitive. Thus, Kv1.3 channels are essential for activation of quiescent cells, but signaling through the PKC pathway enhances expression of IKCa1 channels that are required for continued proliferation.

Defining the Carbohydrate Specificities of AplysiaGonad Lectin Exhibiting a Peculiar d-Galacturonic Acid Affinity

Aplysia gonad lectin (AGL), which has been shown to stimulate mitogenesis in human peripheral lymphocytes, to suppress tumor cells, and to induce neurite outgrowth and improve cell viability in cultured Aplysia neurons, exhibits a peculiar galacturonic acid/galactose specificity. The carbohydrate binding site of this lectin was characterized by enzyme-linked lectino-sorbent assay and by inhibition of AGL-glycan interactions. Examination of the lectin binding with 34 glycans revealed that it reacted strongly with the following glycoforms: most human blood group precursor (equivalent) glycoproteins (gps), two Galalpha1-->4Gal-containing gps, and two d-galacturonic acid (GalUA)-containing polysaccharides (pectins from apple and citrus fruits), but poorly with most human blood group A and H active and sialylated gps. Among the GalUA and mammalian saccharides tested for inhibition of AGL-glycan binding, GalUA mono- to trisaccharides were the most potent ones. They were 8.5 x 10(4) times more active than Gal and about 1.5 x 10(3) more active than the human blood group P(k) active disaccharide (E, Galalpha1-->4Gal). This disaccharide was 6, 28, and 120 times more efficient than Galbeta1-->3GlcNAc(I), Galbeta1-->3GalNAc(T), and Galbeta1--> 4GlcNAc (II), respectively, and 35 and 80 times more active than melibiose (Galalpha1-->6Glc) and human blood group B active disaccharide (Galalpha1-->3Gal), respectively, showing that the decreasing order of the lectin affinity toward alpha-anomers of Gal is alpha1-->4 > alpha1-->6 > alpha1-->3. From the data provided, the carbohydrate specificity of AGL can be defined as GalUAalpha1-->4 trisaccharides to mono GalUA > branched or cluster forms of E, I, and II monomeric E, I, and II, whereas GalNAc is inactive.

Interaction of histamine and other bioamines with cytochromes P450: implications for cell growth modulation and chemopotentiation by drugs

We have characterized microsomal and nuclear histamine sites, designated H IC , through which this amine acts as an intracellular mediator of platelet aggregation and lymphocyte mitogenesis. A major proportion, at least, of the microsomal H IC sites are on cytochromes P450, an important family of microsomal enzymes that are present in all cells, but most abundant in the liver. These enzymes are involved in the metabolism of xenobiotics and natural substrates, including lipid hormones that modulate gene function and cell growth. We have shown that polyamines, hormones (including estrogen, testosterone and progesterone), antihormones (including tamoxifen and flutamide) and various antidepressants and antihistamines, all inhibit histamine binding to P450; we have postulated that, through binding to the heme moiety, intracellular histamine regulates cell function by modulating the catalytic activity of P450 enzymes, an action that may be perturbed by endogenous and exogenous substances. We now demonstrate that, in addition to histamine, melatonin and the biogenic amines dopamine, serotonin and noradrenaline bind to P450 isozymes and to cytochrome C. Thus, heme enzymes in general may represent common targets where multiple bioamines, hormones and drugs interact to influence cell function and growth.