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1.
Osteoporos Int ; 24(4): 1455-70, 2013 Apr.
Article in English | MEDLINE | ID: mdl-22932734

ABSTRACT

UNLABELLED: Effects of cladrin treatment and withdrawal in osteopenic rats were studied. Cladrin improved trabecular microarchitecture, increased lumbar vertebral compressive strength, augmented coupled remodeling, and increased bone osteogenic genes. A significant skeletal gain was maintained 4 weeks after cladrin withdrawal. Findings suggest that cladrin has significant positive skeletal effects. INTRODUCTION: We showed that a standardized extract of Butea monosperma preserved trabecular bone mass in ovariectomized (OVx) rats. Cladrin, the most abundant bioactive compound of the extract, promoted peak bone mass achievement in growing rats by stimulating osteoblast function. Here, we studied the effects of cladrin treatment and withdrawal on the osteopenic bones. METHODS: Adult female Sprague-Dawley rats were OVx and left untreated for 12 weeks to allow for significant estrogen deficiency-induced bone loss, at which point cladrin (1 and 10 mg/kg/day) was administered orally for another 12 weeks. Half of the rats were killed at the end of the treatments and the other half at 4 weeks after treatment withdrawal. Sham-operated rats and OVx rats treated with PTH or 17ß-estradiol (E2) served as various controls. Efficacy was evaluated by bone microarchitecture using microcomputed tomographic analysis and fluorescent labeling of bone. qPCR and western blotting measured mRNA and protein levels in bone and uterus. Specific ELISA was used for measuring levels of serum PINP and urinary CTx. RESULTS: In osteopenic rats, cladrin treatment dose dependently improved trabecular microarchitecture, increased lumbar vertebral compression strength, bone formation rate (BFR), cortical thickness (Cs.Th), serum PINP levels, and expression of osteogenic genes in bones; and reduced expression of bone osteoclastogenic genes and urinary CTx levels. Cladrin had no uterine estrogenicity. Cladrin at 10 mg/kg maintained acquired skeletal gains 4 weeks after withdrawal. CONCLUSION: Cladrin had positive skeletal effects in osteopenic rats that were maintained after treatment withdrawal.


Subject(s)
Bone Density Conservation Agents/therapeutic use , Bone Diseases, Metabolic/drug therapy , Isoflavones/therapeutic use , Animals , Biomarkers/blood , Body Weight/drug effects , Bone Density Conservation Agents/administration & dosage , Bone Density Conservation Agents/pharmacology , Bone Diseases, Metabolic/physiopathology , Compressive Strength/drug effects , Dose-Response Relationship, Drug , Drug Administration Schedule , Drug Evaluation, Preclinical/methods , Female , Femur/drug effects , Femur/metabolism , Femur/physiopathology , Gene Expression Regulation/drug effects , Isoflavones/administration & dosage , Isoflavones/pharmacology , Lumbar Vertebrae/drug effects , Lumbar Vertebrae/physiopathology , Organ Size/drug effects , Osteoblasts/drug effects , Osteoblasts/metabolism , Osteoclasts/drug effects , Osteoclasts/metabolism , Osteogenesis/drug effects , Osteogenesis/genetics , Ovariectomy , RNA, Messenger/genetics , Rats , Rats, Sprague-Dawley , Tibia/drug effects , Tibia/physiopathology , Uterus/drug effects , Uterus/pathology , X-Ray Microtomography
2.
Osteoporos Int ; 22(12): 3013-27, 2011 Dec.
Article in English | MEDLINE | ID: mdl-21225417

ABSTRACT

UNLABELLED: The effect of quercetin C-glucoside (QCG) on osteoblast function in vitro and bone formation in vivo was investigated. QCG supplementation promoted peak bone mass achievement in growing rats and new bone formation in osteopenic rats. QCG has substantial oral bioavailability. Findings suggest a significant bone anabolic effect of QCG. INTRODUCTION: Recently, we showed that extracts of Ulmus wallichiana promoted peak bone mass achievement in growing rats and preserved trabecular bone mass and cortical bone strength in ovariectomized (OVx) rats. 3,3',4',5,7-Pentahydroxyflavone-6-C-ß-D-glucopyranoside, a QCG, is the most abundant bioactive compound of U. wallichiana extract. We hypothesize that QCG exerts bone anabolic effects by stimulating osteoblast function. METHODS: Osteoblast cultures were harvested from rat calvaria and bone marrow (BM) to study differentiation and mineralization. In vivo, growing female Sprague Dawley rats and OVx rats with osteopenia were administered QCG (5.0 or 10.0 mg kg(-1) day(-1)) orally for 12 weeks. Efficacy was evaluated by examining changes in bone microarchitecture using histomorphometric and microcomputed tomographic analyses and by determination of new bone formation by fluorescent labeling of bone. Plasma and BM levels of QCG were determined by high-performance liquid chromatography. RESULTS: QCG was much more potent than quercetin (Q) in stimulating osteoblast differentiation, and the effect of QCG was not mediated by estrogen receptors. In growing rats, QCG increased BM osteoprogenitors, bone mineral density, bone formation rate, and cortical deposition. In osteopenic rats, QCG treatment increased bone formation rate and improved trabecular microarchitecture. Comparison with the sham group (ovary intact) revealed significant restoration of trabecular bone in osteopenic rats treated with QCG. QCG levels in the BM were ~50% of that of the plasma levels. CONCLUSION: QCG stimulated modeling-directed bone accrual and exerted anabolic effects on osteopenic rats by direct stimulatory effect on osteoprogenitors likely due to substantial QCG delivery at tissue level following oral administration.


Subject(s)
Bone Density/drug effects , Bone Diseases, Metabolic/drug therapy , Glucosides/isolation & purification , Osteoblasts/drug effects , Osteogenesis/drug effects , Quercetin/analogs & derivatives , Animals , Bone Marrow/chemistry , Chromatography, Liquid , Female , Femur/diagnostic imaging , Femur/drug effects , Glucosides/chemistry , Glucosides/pharmacology , Hindlimb , Ovariectomy , Quercetin/blood , Quercetin/chemistry , Quercetin/pharmacology , Rats , Rats, Sprague-Dawley , Tibia/diagnostic imaging , Tibia/drug effects , X-Ray Microtomography
3.
Br J Pharmacol ; 158(7): 1820-34, 2009 Dec.
Article in English | MEDLINE | ID: mdl-19912231

ABSTRACT

BACKGROUND AND PURPOSE: The current clinical strategy to protect the auditory organ against inflammatory damage by migrating leukocytes is the local delivery of glucocorticoids. However, the mechanism by which glucocorticoids confer this protection remains unknown. Therefore, we investigated the cellular and molecular targets of glucocorticoids in the cochlea that could be involved in preventing leukocyte migration. EXPERIMENTAL APPROACH: We used microscopy as well as immunocytochemical and microfluidic techniques to elucidate the effect of dexamethasone, hydrocortisone and prednisolone on the cellular and intracellular distribution of annexin A1 (ANXA1) - a glucocorticoid target known to inhibit leukocyte migration by receptor-mediated signalling - in the cochlea and isolated cochlear cells of guinea pigs. KEY RESULTS: All the cells lining the scala media - the cochlear compartment containing the auditory organ - express ANXA1 and the ANXA1 receptor FPR2/ALX is present in the scala media, as well as in other cochlear ducts. The majority of ANXA1 in the scala media is stored inside lipid droplets within cochlear Hensen cells. Glucocorticoids activate a myosin IIC-mediated mechanism that drives ANXA1 from the lipid droplets to the apical region of the Hensen cells, where ANXA1 is released to the external milieu by a process involving ABC transporters. CONCLUSIONS AND IMPLICATIONS: These findings suggest that ANXA1 could be a major mediator of the anti-inflammatory effects of glucocorticoids in the cochlea and identify new molecular targets for prevention of sudden sensorineural hearing loss.


Subject(s)
Annexin A1/drug effects , Cochlea/drug effects , Glucocorticoids/pharmacology , Animals , Annexin A1/metabolism , Cochlea/cytology , Cochlea/metabolism , Dexamethasone/pharmacology , Drug Delivery Systems , Guinea Pigs , Hydrocortisone/pharmacology , Microscopy , Myosin Type II/metabolism , Prednisolone/pharmacology , Receptors, Formyl Peptide/metabolism , Signal Transduction/drug effects
5.
Mech Ageing Dev ; 108(3): 183-206, 1999 May 17.
Article in English | MEDLINE | ID: mdl-10405980

ABSTRACT

Changes in the T-lymphocyte compartment represent the most critical component of immunological aging. Recent studies have demonstrated that the age-related decline in T-cell-mediated immunity is a multifactorial phenomenon affecting T-cell subset composition as well as several proximal events such as protein tyrosine phosphorylation, generation of second messengers, calcium mobilization and translocation of protein kinase C, and distal events such as lymphocyte proliferation and cytokine production of the T-cell activation pathway. Age-related T-cell immune deficiency is preceded by thymic involution and is influenced by several intrinsic as well as extrinsic factors. Further, the role of monocytes and macrophages in T-cell activation changes with advancing age. This brief review will summarize the current knowledge of the cellular as well as molecular aspects of immunodeficiency of T cells due to aging, some of the paradoxes of aging as related to T-cell-mediated immunity, and possible factors which contribute to this paradox. Finally, experimental approaches will be suggested that might resolve these controversies and that might provide insights into the diverse and complex mechanisms that contribute to immunodeficiency of T cells. Ultimately these studies may suggest possible therapeutic interventions to enhance immune function in the elderly.


Subject(s)
Aging/immunology , Immunity, Cellular , T-Lymphocytes/immunology , Animals , Humans
6.
Mech Ageing Dev ; 104(1): 41-58, 1998 Aug 01.
Article in English | MEDLINE | ID: mdl-9751431

ABSTRACT

Several events of T cell activation have been reported to decline in humans with age. Since protein tyrosine phosphorylation is an early critical event of T cell activation, we performed a systematic analysis of the age-associated changes in the mitogen induced protein tyrosine phosphorylation of human T lymphocytes using SDS-PAGE and Western blotting techniques. Following stimulation with Con A and PHA, an identical pattern of protein tyrosine phosphorylation was observed in the lysates of T cells prepared from seven healthy young adults and eight healthy elderly human subjects. Five different high molecular mass proteins (75, 115, 120, 140 and 170 kDa) were consistently tyrosine phosphorylated in all of the donors from both age groups and peaked between 3 and 10 min. Tyrosine phosphorylation of the above substrates was observed in both CD4 and CD8 subsets. When compared for individual donors from both age groups, variations in the T cell response with regard to net tyrosine phosphorylation for all the substrates was observed. However, the mitogen induced level of tyrosine phosphorylation of only p75 was found to be significantly lower in unfractionated T cells as well as CD4 and CD8 subsets of older subjects than that of young subjects. Using immunoblotting, p75 was identified as ZAP-70, a member of the syk family of protein tyrosine kinases. Understanding of the biochemical basis of the reduced level of tyrosine phosphorylation of ZAP-70 will be helpful in delineating the molecular basis of age-associated impairment of T cell activation.


Subject(s)
Aging/metabolism , Protein-Tyrosine Kinases/metabolism , T-Lymphocytes/metabolism , Tyrosine/metabolism , Adult , Aged , CD4-Positive T-Lymphocytes/drug effects , CD4-Positive T-Lymphocytes/enzymology , CD4-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/drug effects , CD8-Positive T-Lymphocytes/enzymology , CD8-Positive T-Lymphocytes/metabolism , Concanavalin A/pharmacology , Down-Regulation , Humans , Mitogens/pharmacology , Phosphorylation , Phytohemagglutinins/pharmacology , T-Lymphocytes/drug effects , T-Lymphocytes/enzymology , ZAP-70 Protein-Tyrosine Kinase
7.
Biochem Biophys Res Commun ; 243(2): 591-7, 1998 Feb 13.
Article in English | MEDLINE | ID: mdl-9480853

ABSTRACT

The presence of two different signaling molecules is essential to induce opsonin-independent phagocytosis of particulate activators of the human alternative complement pathway by human monocytes. In addition to the involvement of a low M(r) peptide cytokine or phagocytosis inducing factor (PIF), we have now established that the participation of bacterial lipopolysaccharide is also required. PIF has been demonstrated to be present in human cell lines of different origins, e.g., WISH cells, Raji cells, U937 cells, HL-60 cells and M21 cells. PIF has been purified to apparent homogeneity from the U937 cell line by anion-exchange chromatography on DEAE-Sephacel followed by gel filtration chromatography on Sephadex G-50. On the basis of Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the apparent molecular mass has been estimated to be approximately 1,600 Da. PIF also plays an important role in the regulation of cell-substratum adherence.


Subject(s)
Cell Adhesion/drug effects , Peptides/isolation & purification , Phagocytosis/physiology , Cell Division , Cell Line , Erythrocytes/metabolism , Humans , Lipopolysaccharides/pharmacology , Molecular Weight , Monocytes/drug effects , Peptides/metabolism , Peptides/pharmacology , Phagocytosis/drug effects
8.
Immunol Lett ; 54(1): 59-64, 1996 Dec 01.
Article in English | MEDLINE | ID: mdl-9030983

ABSTRACT

Sclerosing keratitis is the major cause of blindness due to onchocerciasis which results from chronic infection with the filarial parasite Onchocerca volvulus. Using a murine model of onchocercal sclerosing keratitis, we have demonstrated previously that predominantly (> 85%) CD3 + /CD4+ T-cells as well as the IL-2 receptor bearing cells infiltrate into the cornea in vivo during development and progress of the disease. The identification of CD4+ subsets TH1 and TH2 based on the cytokine secretion patterns of murine T-lymphocytes has been useful for understanding the immune basis of resistance and pathogenesis in murine models of several parasitic diseases. The present investigation was carried out to demonstrate whether the local immune response at the corneal lesion due to onchocercal interstitial keratitis correlated with such distinct patterns of cytokine production. For that purpose, mRNA was extracted separately from corneas obtained from the diseased eyes and the normal eyes of A/J mice with onchocercal interstitial keratitis, reverse transcribed and amplified by the polymerase chain reaction with four different cytokine specific primers. In corneas obtained from the eyes affected with onchocercal interstitial keratitis, mRNAs coding for IL-4 and IL-5 were up-regulated compared to the normal eyes having no lesions from the same animals. However, the levels of mRNAs for IL-2 and IFN gamma were found to be the same in the diseased and normal eyes. Taken together, these data suggest that IL-4 and IL-5 producing TH2-lymphocytes are active at the corneal lesion due to onchocercal interstitial keratitis.


Subject(s)
Cornea/immunology , Cytokines/biosynthesis , Onchocerca volvulus/immunology , Onchocerciasis, Ocular/immunology , Animals , Antigens, Helminth/immunology , Cornea/parasitology , Cytokines/genetics , Disease Models, Animal , Interferon-gamma/genetics , Interferon-gamma/immunology , Interleukin-2/biosynthesis , Interleukin-2/genetics , Interleukin-4/biosynthesis , Interleukin-4/genetics , Interleukin-5/biosynthesis , Interleukin-5/genetics , Keratitis/immunology , Mice , Mice, Inbred A , Neovascularization, Pathologic , RNA, Messenger , Th1 Cells/immunology , Th2 Cells/immunology , Up-Regulation
9.
Exp Parasitol ; 81(4): 445-52, 1995 Dec.
Article in English | MEDLINE | ID: mdl-8542985

ABSTRACT

A major antigen recognized by human sera in Onchocerca volvulus infections is a parasite eggshell protein. The cDNA clone for this antigen was isolated from a lambda gt11 O. volvulus cDNA library using antisera from patients with high microfilarial counts. Sequence analysis of the cDNA clone predicts a polyglutamine repeat near the 5' end of the cDNA, and a motif of four arginines near the 3' end, reminiscent of that found in many regulatory proteins. The cDNA was subcloned into a yeast expression vector and reagent quantities of recombinant antigen produced in Saccharomyces cerevisiae. Antisera produced to the recombinant purified protein localized the antigen to the eggshell of developing microfilariae within the adult female uterus. No other sites of Oveg1 expression were noted in adult worms, but labeling was seen in internal membrane structures of L3 larvae. Sera from infected chimps recognized Oveg1 only after infections became patent. Sera from infected humans showed reactivity to Oveg1 that varied from 39 to 95%, depending upon the geographic location.


Subject(s)
Antigens, Helminth/genetics , Egg Proteins/genetics , Helminth Proteins/genetics , Onchocerca volvulus/immunology , Ovum/immunology , Amino Acid Sequence , Animals , Antibodies, Helminth/blood , Antigens, Helminth/immunology , Base Sequence , Egg Proteins/immunology , Egg Proteins/isolation & purification , Female , Gene Library , Helminth Proteins/immunology , Helminth Proteins/isolation & purification , Humans , Immunohistochemistry , Microscopy, Immunoelectron , Molecular Sequence Data , Onchocerca volvulus/genetics , Onchocerciasis/blood , Onchocerciasis/immunology , Ovum/ultrastructure , Pan troglodytes , Recombinant Proteins/immunology , Saccharomyces cerevisiae/genetics
10.
Exp Eye Res ; 60(4): 347-58, 1995 Apr.
Article in English | MEDLINE | ID: mdl-7789415

ABSTRACT

Sclerosing keratitis is the predominant cause of blindness due to onchocerciasis which is a major human parasitic disease caused by the filarial parasite Onchocerca volvulus. In the present investigation, native pathogenic antigens of O. volvulus which are particularly potent in causing interstitial keratitis were characterized utilizing a guinea pig model. Following demonstration of the protein nature of these antigens using pronase digestion, the crude O. volvulus antigen extract was subjected to stepwise procedures of protein purification. At each stage of purification, pooled antigen fractions were injected into one cornea of presensitized guinea pigs followed by clinical evaluation of stromal inflammation and vascularization at different intervals of time after intrastromal challenge. Initial purification of the pathogenic antigens was carried out in the following order: molecular sieve chromatography on Bio-gel A-5m. anion exchange chromatography on Mono Q followed by DEAE-Sepharose CL-6B and cation exchange chromatography on Mono S. Two out of six different pools from the Mono S column (pool a eluted unbound at 10 mM-NaCl and pool e eluted between 130 mM and 475 mM-NaCl) were found to be most pathogenic. Further purification of Mono S pool a and pool e separately by gel filtration chromatography using Superose 12 demonstrated that the fractions which were most potent in inducing interstitial keratitis contained proteins with approximate molecular masses between 100 and 200 kDa. These results show that minor subfractions of total crude antigens of O. volvulus are largely responsible for induction of experimental interstitial keratitis. We have demonstrated the presence of these antigens in O. volvulus microfilariae by their cross-reactivities with anti-microfilarial antibodies, and hence the relevance of the purified antigens to ocular onchocerciasis in man since sclerosing keratitis is associated with invasion of the cornea by O. volvulus microfilariae. Isolation of these two pathogenic antigen pools represents the practical limits of purification and subsequent animal experiments possible with the available amounts of native parasite material obtained from infected human individuals in the absence of a suitable non-human host or of an in vitro culture system for O. volvulus.


Subject(s)
Antigens, Helminth/isolation & purification , Keratitis/immunology , Onchocerca volvulus/immunology , Onchocerciasis, Ocular/immunology , Animals , Chromatography, Gel , Chromatography, Ion Exchange , Cornea/immunology , Electrophoresis, Polyacrylamide Gel , Female , Guinea Pigs , Molecular Weight
11.
Cell Immunol ; 159(2): 306-14, 1994 Dec.
Article in English | MEDLINE | ID: mdl-7994762

ABSTRACT

Sclerosing keratitis is the major cause of blindness due to onchocerciasis caused by the parasite Onchocerca volvulus. Although the importance of T cells in the pathogenesis of onchocerciasis has been suggested, their precise role in onchocercal sclerosing keratitis has not yet been defined. Using immunohistological techniques and a murine model of onchocercal sclerosing keratitis, we have performed a temporal analysis of the inflammatory T cells infiltrating into the cornea at Days 4, 7, and 21 following intrastromal challenge with soluble O. volvulus antigens into presensitized mice. The maximum number of CD3+ T cells were observed in the corneal stroma at Day 21 when sclerosing keratitis was most severe. The majority (> 85%) of the CD3+ T cells were CD4+ at all time points. A few infiltrating cells bore IL-2 receptors indicating possible activation of a small fraction of the T cells. These results suggest that CD4+ T cells play an important role in onchocercal sclerosing keratitis.


Subject(s)
CD4-Positive T-Lymphocytes/immunology , Cornea/immunology , Keratitis/immunology , Keratitis/parasitology , Onchocerca volvulus/immunology , Animals , Cell Movement/immunology , Immunoenzyme Techniques , Mice , Mice, Inbred A , Onchocerciasis/immunology , Sclerosis/immunology , Sclerosis/parasitology
12.
Cell Immunol ; 154(1): 134-42, 1994 Mar.
Article in English | MEDLINE | ID: mdl-8118882

ABSTRACT

Human peripheral blood lymphocytes or the soluble (105,000g) supernatant of the lymphocyte lysate can increase the percentage of human monocytes ingesting particulate activators of the human alternative complement pathway, e.g., rabbit erythrocytes (Chakravarti et al., J. Immunol. 137, 880, 1986). We now show that preincubation of the lymphocytes led to an increase in their augmenting ability. However, no such increase in the augmenting ability of the lymphocytes or soluble supernatant made from these lymphocytes was observed when they were preincubated and added to monocytes in the presence of cycloheximide; rather there was a significant reduction in the ingesting ability of the monocytes. Our results demonstrate that in the case of intact lymphocytes, the observed inhibition was because of (i) inhibition of de novo synthesis of protein(s) in the monocytes during their adherence and (ii) inhibition of de novo synthesis of protein(s) in the lymphocytes, possibly of a signaling molecule necessary for release of the peptide cytokine, phagocytosis-inducing factor (PIF), from the lymphocytes. However, the inhibition observed with soluble supernatant made from lymphocytes preincubated in the presence of cycloheximide was because of the above phenomenon (i) only and indicated that there was no de novo synthesis of PIF during preincubation of the lymphocytes.


Subject(s)
Leukocytes, Mononuclear/immunology , Leukocytes, Mononuclear/metabolism , Phagocytosis/physiology , Protein Biosynthesis , Blood Proteins/biosynthesis , Complement Pathway, Alternative , Cycloheximide/pharmacology , Humans , In Vitro Techniques , Leukocytes, Mononuclear/drug effects , Lymphocytes/drug effects , Lymphocytes/immunology , Lymphocytes/metabolism , Monocytes/drug effects , Monocytes/immunology , Monocytes/metabolism , Opsonin Proteins , Phagocytosis/drug effects , Phagocytosis/immunology
13.
Exp Eye Res ; 57(1): 21-7, 1993 Jul.
Article in English | MEDLINE | ID: mdl-7691631

ABSTRACT

Sclerosing keratitis is the major cause of blindness due to onchocerciasis; its pathogenesis is poorly understood. We have previously reported an immune-mediated model of experimental interstitial keratitis in guinea pigs following intrastromal challenge with soluble antigens from Onchocerca volvulus. This model system is ideal for evaluation of pathogenicity of multiple purified antigen preparations; however, reagents necessary for detailed immunologic analysis of the inflammatory cellular infiltrate are not yet available for guinea pigs. Because of the ready availability of these reagents for mice, a mouse model has been developed. The inflammatory response observed in this model was analogous to that seen in human onchocercal sclerosing keratitis as well as in the guinea pig model of onchocercal sclerosing keratitis. Granulocytes were present in the acute inflammatory response, whereas the chronic response showed lymphocytes, plasma cells, and histiocytes. Neovascularization and scarring of the corneal stroma was also observed. This model will be helpful in examining the mechanisms of immunopathogenesis and the contribution of the host genetic background to the disease.


Subject(s)
Keratitis/immunology , Onchocerca volvulus/pathogenicity , Onchocerciasis, Ocular/immunology , Animals , Cornea/pathology , Female , Granulocytes/pathology , Histiocytes/pathology , Keratitis/pathology , Mice , Mice, Inbred A , Neovascularization, Pathologic/pathology , Onchocerciasis, Ocular/pathology , Plasma Cells/pathology , Time Factors
14.
Proc Natl Acad Sci U S A ; 86(8): 2799-803, 1989 Apr.
Article in English | MEDLINE | ID: mdl-2468158

ABSTRACT

The amino acid sequence of the amino-terminal half of the complement protein C6 has been found to show overall structural homology with the homologous regions of the channel-forming proteins C7, C8 alpha, C8 beta, and C9. In addition, two specific cysteine-rich segments common to the amino-terminal regions of C7, C8 alpha, C8 beta, and C9 also occur in their expected positions in C6, suggesting functional significance. Two cDNA clones encoding C6 were isolated from a human liver library in the bacteriophage vector lambda gt11. The predicted protein sequence contains an apparent initiation methionine and a putative signal peptide of 21 residues, as well as a site for N-glycosylation at residue 303. The sequence of the C6 protein reported here has 47-52% similarity with C7, C8 alpha, C8 beta, and C9, as well as 31-38% similarity with thrombospondin, thrombomodulin, and low density lipoprotein receptor. The sequence data have been interpreted by using computer algorithms for estimation of average hydrophobicity and secondary structure.


Subject(s)
Complement C6/ultrastructure , Complement System Proteins/genetics , Ion Channels/ultrastructure , Amino Acid Sequence , Base Sequence , Cloning, Molecular , Complement Membrane Attack Complex , Cysteine , DNA/genetics , Humans , Macromolecular Substances , Membrane Glycoproteins/ultrastructure , Molecular Sequence Data , Nucleic Acid Conformation , Solubility
15.
Cell Immunol ; 113(2): 251-60, 1988 May.
Article in English | MEDLINE | ID: mdl-2834070

ABSTRACT

Monolayers of freshly isolated human monocytes are known to ingest particulate activators of the human alternative complement pathway. The ingestion of rabbit erythrocytes, ER, by human monocytes in serum-free medium was studied. The process is Mg2+-dependent and optimum phagocytic activity was obtained at approximately 20 mM MgCl2. Preincubation of mononuclear leukocytes increased the number of monocytes ingesting ER by at least twofold and this involved de novo protein synthesis, as evidenced by inhibition with cycloheximide. However, preincubation of the mononuclear leukocytes for longer periods (greater than 4 hr) caused a decrease in the percentage of ingesting monocytes. No inhibition of ingestion of ER was observed by cobra venom factor (CVF) or F(ab')2 rabbit anti-human C3 of F(ab')2 murine monoclonal anti-human Bb, known to inhibit C3 convertase activity. The ingestion was also not inhibited by (a) rabbit anti-human CR1, (b) OKM1 or anti-MO1, two monoclonal anti-CR3 antibodies, (c) goat anti-human IgG Fc receptor, or (d) mannan, a competitive inhibitor of ligand uptake by the mannosyl-fucosyl receptor (MFR). In contrast, ingestion was inhibited by glucan particles of yeast.


Subject(s)
Monocytes/physiology , Animals , Complement System Proteins/physiology , Cycloheximide/pharmacology , Erythrocytes , Glucans/pharmacology , Humans , In Vitro Techniques , Kinetics , Magnesium/pharmacology , Magnesium Chloride , Mannans/pharmacology , Opsonin Proteins , Phagocytosis , Rabbits , Receptors, Cell Surface/physiology , Receptors, Complement/physiology , Receptors, Fc/physiology , Receptors, IgG , Time Factors
17.
J Immunol ; 137(3): 880-6, 1986 Aug 01.
Article in English | MEDLINE | ID: mdl-3088115

ABSTRACT

Monocytes isolated from human blood by centrifugal elutriation exhibited little ability to ingest rabbit erythrocytes (ER), zymosan particles, or desialated sheep erythrocytes. In contrast, 85 to 95% of these cells rosetted with C3b- or C3bi-bearing sheep erythrocytes (ES) or ingested IgG-coated ES. Preincubation of the monocytes with human lymphocytes increased their ability to ingest ER. the ER phagocytosis-inducing activity was contained in the 105,000 X G supernatant of lymphocyte lysates. These supernatants increased the percentage of ingesting monocytes from 5 to 15% to 80% within 60 min. The soluble factor was found to be relatively heat stable, inactivated by trypsin, and distinct from IFN-gamma. Its m.w. is less than 13,000. It was present in B and T lymphocytes and also in U937 cells. These results suggest that the ability of human monocytes to ingest nonopsonized particulate activators of the alternative complement pathway is a cytokine-inducible property and that the effect of the cytokine on complement receptor- or Fc receptor-dependent adherence or ingestion of opsonized particles is minor.


Subject(s)
Complement Activation , Complement Pathway, Alternative , Lymphokines/physiology , Monocytes/immunology , Phagocytosis , Animals , Cell Adhesion , Cell-Free System , Chemical Phenomena , Chemistry, Physical , Erythrocytes/immunology , Humans , Interferon-gamma/pharmacology , Kinetics , Lymphocytes/immunology , Macrophage-Activating Factors , Opsonin Proteins/physiology , Rabbits , Rosette Formation , Subcellular Fractions/analysis
18.
Jpn J Physiol ; 36(1): 151-62, 1986.
Article in English | MEDLINE | ID: mdl-3723874

ABSTRACT

The maximum aerobic capacity (VO2 max) of young untrained Bengali girls aged between 22-24 years after maximum workload as determined by bicycle ergometer, standard step test and a treadmill is studied in 22 subjects. It was found that the VO2 peak in the treadmill exercise was significantly higher than that obtained by the step test and bicycle ergometer, in that order. On each type of ergometer, VO2 max was found to be positively correlated with body weight, body surface area, maximal pulmonary ventilation and maximal oxygen pulse.


Subject(s)
Exercise Test , Oxygen/blood , Physical Fitness , Adult , Body Surface Area , Body Weight , Cross-Cultural Comparison , Female , Heart Rate , Humans , India , Pulmonary Gas Exchange
19.
Biochem J ; 222(3): 789-95, 1984 Sep 15.
Article in English | MEDLINE | ID: mdl-6435606

ABSTRACT

Isopenicillin N synthetase was extracted from Cephalosporium acremonium and purified about 200-fold. The product showed one major protein band, coinciding with synthetase activity, when subjected to electrophoresis in polyacrylamide gel. An isopenicillin N synthetase from Penicillium chrysogenum was purified about 70-fold by similar procedures. The two enzymes resemble each other closely in their Mr, in their mobility on electrophoresis in polyacrylamide gel and in their requirement for Fe2+ and ascorbate for maximum activity. Preliminary experiments have shown that a similar isopenicillin N synthetase can be extracted from Streptomyces clavuligerus.


Subject(s)
Enzymes/isolation & purification , Oxidoreductases , Acremonium/enzymology , Chromatography, Affinity , Chromatography, Gel , Electrophoresis, Polyacrylamide Gel , Penicillium chrysogenum/enzymology , Streptomyces/enzymology
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