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PubMed1984

Attachment and phagocytosis by salmon macrophages of agarose beads coated with human C3b and C3bi.

Agarose beads (diameter 5-10 micron) preincubated in human serum became associated (attached and ingested) to 50-60% of the salmon macrophages within 60 minutes. However, Beads preincubated in serum treated with heating (50 degrees C, 20 min) or with EDTA (10 mM) to inhibit the activation of alternative complement pathway, were not associated to the phagocytes. Furthermore, agarose beads coated with human C3b and C3bi after incubation with isolated complement factors (C3, D, B), were associated to 30-40% of the phagocytes. About 80% of the cell-associated agarose beads was intracellularly located. Conversion by trypsin treatment (0.01%) of agarose bound C3bi to C3d, abolished the association of such beads to the macrophages. The results demonstrate that salmon macrophages possess complement receptors that bind human C3b and C3bi. Agarose beads coated with these ligands (C3b and C3bi) are attached and ingested by the phagocytes.

Animals↗
PubMed1979

Limited proteolysis of complement components C2 and factor B. Structural analogy and limited sequence homology.

A method is described for the simultaneous purification of milligram quantities of complement components C2 and Factor B. Both products are homogeneous by the criteria of polyacrylamide-gel electrophoresis and N-terminal sequence analysis. Component C2 is cleaved by serine proteinase C1s at an X-Lys bond to give fragment C2a (approx. mol.wt. 74000) and fragment C2b (approx. mol.wt. 34000). The two fragments can be separated by gel filtration without the need for reducing or denaturing agents. Fragment C2b represents the N-terminal end of the molecule. Similar results were seen on cleavage of Factor B by Factor D in the presence of component C3. Again two non-covalently linked fragments are formed. The smaller, fragment Ba (approx. mol.wt. 36,000),) has threonine as the N-terminal residue, as does Factor B; the larger, fragment Bb (approx. mol. wt. 58000), has lysine as the N-terminal residue. A similar cleavage pattern is obtained on limited proteolysis of Factor B by trypsin, suggesting an Arg-Lys-or Lys-Lys bond at the point of cleavage. Although component C2 and Factor B show no apparent N-terminal sequence homology, a limited degree of sequence homology is seen around the sites of proteolytic cleavage.

Amino Acid Sequence↗
PubMed1994

The endogenous vascular elastase that governs development and progression of monocrotaline-induced pulmonary hypertension in rats is a novel enzyme related to the serine proteinase adipsin.

We showed previously a cause and effect relationship between increased activity of an endogenous vascular elastase (EVE) and experimentally induced pulmonary hypertension in rats. We now report the isolation and characterization of EVE. Degenerate oligonucleotides synthesized to homologous sequences in serine elastases were used in a PCR with rat pulmonary artery (PA) cDNA. The PCR product hybridized to a 1.2-kb mRNA and the intensity of hybridization was threefold increased in RNA from rat hypertensive PA at a timepoint when EVE activity was increased. The PCR product was used to screen a cDNA library and sequences obtained encoded rat adipsin. We then used immunoaffinity to purify EVE. An antibody to the elastin-binding protein was used to remove this competitor of elastase from the PA extract and the elastolytic activity increased 100-fold. The enzyme was purified using an antibody that recognizes NH2-terminal sequences of serine proteinases and the eluate was further purified using an antibody raised against recombinant adipsin. A single band at 20 kD immunoreactive with the adipsin antibody was resolved as an active enzyme on an elastin substrate gel. Immunogold labeling with an antibody to an adipsin peptide sequence localized EVE to PA smooth muscle cells. This is the first isolation of EVE; it appears to be a novel enzyme related to the serine proteinase adipsin originally found in adipose tissue.

Amino Acid Sequence↗
PubMed1982

Functional active complement components secreted by guinea pig peritoneal macrophages.

In our studies on complement secretion functional C1, C4, C2, C3, P, D and B were clearly identified in the same cultures. Functional assays did not allow the detection of C5 to C9. Spontaneous C3 activation occurred at a very low level in culture supernatants. The responsible enzyme was identified as a metallo-enzyme. Upon addition of antibody-coated sheep erythrocytes (EA) to culture supernatant it was possible to induce C3 activation as indicated by the apparent formation of EAC1423. Zymosan was also able to activate C3 in culture supernatant after addition of purified functional factor B indicating efficient cooperation of factors of the alternative pathway. Thus in this in vitro system macrophages not only provide C3 but also all factors for spontaneous and induced C3 activation. If these secretory functions reflect in vivo properties of macrophages, our results may indicate that C3 and its activating systems are most relevant for local cooperation between macrophages and the complement system in inflammation and antimicrobial defense. Therefore availability of these essential factors at any time is secured by local production.

Animals↗
PubMed1988

A novel cleavage product of human complement component C3 with structural and functional properties of cobra venom factor.

The generation of two cleavage products of human C3, termed C3o and C3p, by incubation with a C3-cleaving protease isolated from cobra venom (Naja naja siamensis) is described. The venom protease removes the C3p fragment (Mr approximately 33,000) from the C3dg region of the C3 alpha-chain. The major cleavage fragment C3o (Mr approximately 140,000) contains the unaltered beta-chain of C3 and two alpha-chain-derived polypeptides of Mr approximately 29,000 and Mr approximately 38,000, respectively. Amino-terminal amino acids sequence analysis of C3p and the three chains of C3o allowed the identification of the exact location of the two alpha-chain-derived fragments of C3o and the three cleavage sites of the venom protease. The chain structure of C3o resembles those of C3c and cobra venom factor. In contrast to C3c but like cobra venom factor (and C3b), C3o was found to support the activation of the serine protease Factor B by cleavage in the presence of Factor D and Mg2+ into Bb and Ba, generating an enzymatically active complex that is able to cleave a fluorogenic peptide substrate for C3 convertases. Since the only stretch of amino acid residues of C3o not present in C3c is the carboxyl terminus of the Mr approximately 29,000 chain of C3o, it is suggested that this region is important for the interaction with Factor B and convertase formation.

Amino Acid Sequence↗
PubMed1987

Human complement proteins D, C2, and B. Active site mapping with peptide thioester substrates.

The specificity and reactivity of complement serine proteases D, B, Bb, C2, and C2a were determined using a series of peptide thioester substrates. The rates of thioester hydrolysis were measured using assay mixtures containing the thiol reagent 4,4'-dithiodipyridine at pH 7.5. Each substrate contained a P1 arginine residue, and the effect of various groups and amino acids in the P2, P3, P4, and P5 positions was determined using kcat/Km values to compare reactivities. Among peptide thioesters corresponding to the activation site sequence in B, dipeptide thioesters containing a P2 lysine residue were the best substrates for D. Extending the chain to include a P3 or P4 amino acid resulted in loss of activity, and neither the tripeptide nor the tetrapeptide containing the cleavage sequence of B was hydrolyzed. Overall, D cleaved fewer substrates and was 2-3 orders of magnitude less reactive than C1s against some thioester substrates. C2 and fragment C2a had comparable reactivities and hydrolyzed peptides containing Leu-Ala-Arg and Leu-Gly-Arg, which have the same sequence as the cleavage sites of C3 and C5, respectively. The best substrates for C2 and C2a were Z-Gly-Leu-Ala-Arg-SBzl and Z-Leu-Gly-Leu-Ala-Arg-SBzl, respectively, where Bzl is benzyl. B was the least reactive among these complement enzymes. The best substrate for B was Z-Lys-Arg-SBzl with a kcat/Km value of 1370 M-1 s-1. The catalytic fragment of B, Bb, had higher activity toward these peptide thioester substrates. The best substrate for Bb was Z-Gly-Leu-Ala-Arg-SBzl with a kcat/Km similar to C2a and 10 times higher than the value for B. Both C2a and Bb were considerably more reactive against C3-like than C5-like substrates. Bovine trypsin hydrolyzed thioester substrates with kcat/Km approximately 10(3) higher than the complement enzymes. These thioester substrates for D, B, and C2 should be quite useful in kinetic and active site studies of the purified enzymes.

Animals↗
PubMed1985

Serum concentration of cystatin C, factor D and beta 2-microglobulin as a measure of glomerular filtration rate.

Serum concentrations of creatinine and of the three low molecular weight (LMW) proteins cystatin C, factor D of the complement system and beta 2-microglobulin were measured in 135 consecutive patients, whose glomerular filtration rates (GFR) were determined by Cr-EDTA. In the total patient series, the reciprocals of S-creatinine and S-cystatin C were numerically and, in males, significantly more closely correlated to GFR than the reciprocals of S-factor D. The reciprocals of beta 2-microglobulin showed a weaker correlation to GFR than those of the other three substances. The calculated glomerular elimination rates of creatinine, cystatin C and factor D were normally distributed, in contrast to those of beta 2-microglobulin. According to data presented so far, cystatin C seems to be the LMW protein of first choice when GFR is to be estimated by measuring the plasma concentration of a LMW protein.

Adolescent↗
PubMed1990

Adipogenesis in a murine bone marrow stromal cell line capable of supporting B lineage lymphocyte growth and proliferation: biochemical and molecular characterization.

Recent advances in long-term bone marrow (BM) culture techniques have allowed investigators to dissect cellular components responsible for lympho hematopoiesis. Consequently, a number of "stromal" cell clones have been developed which are capable of supporting B lineage lymphocyte growth and proliferation in vitro by direct cell-cell interactions and the release of cytokines. While much work has focused on the support function of these cells, questions remain regarding their own differentiation potential. We have examined adipogenesis in the cloned BM stromal cell, BMS2. The presence of hydrocortisone, methylisobutylxanthine, or 30% fetal calf serum each accelerated adipocyte differentiation. This process was accompanied by the accumulation of triglycerides and cholesterol esters along with the induction of adipocyte-specific enzymes. Likewise, the steady-state level of mRNA transcripts increased for genes related to lipid metabolism. However, the pattern of mRNA expression in BMS2 adipocytes differed from that of a well-established, pre-adipocyte cell line, 3T3-L1, with respect to the following genes: glycerol phosphate dehydrogenase, CAAT/enhancer binding protein and angiotensinogen. Adipocyte BMS2 cells retailed the ability to support stromal cell-dependent B lineage lymphocytes in methylcellulose assays. The adipocytes continued to express macrophage-colony-stimulating factor mRNA constitutively and interleukin 6 mRNA in an inducible manner, similar to the BMS2 pre-adipocytes. Together, these data document a close developmental relationship between a specialized fibroblasts and adipocytes in the BM and suggest that adipocyte stromal cells may play an active role in lympho-hematopoiesis.

Adipose Tissue↗
PubMed1990

Analysis of gene expression during adipogenesis in 3T3-F442A preadipocytes: insulin and dexamethasone control.

In the present study, we have investigated dexamethasone and insulin regulation of the expression of adipose-specific mRNA, namely, glycerophosphate dehydrogenase (G3PDH) and adipsin, at different stages of differentiation. During adipose conversion, insulin promotes an accumulation of G3PDH mRNA which is linked to cell differentiation; in fully differentiated cells, insulin is not required to maintain G3PDH gene expression. Differentiating cells in serum deprived medium already exhibit, at day 1, a maximal amount of mRNA encoding for adipsin, which is tenfold decreased by 10 nM of insulin; insulin also exerts a negative effect on the abundance of adipsin mRNA in mature cells. This result indicates that adipsin appears to be a very early marker of adipose conversion, the gene expression of which is down-regulated by the presence of insulin. Dexamethasone (DEX) decreases the G3PDH message at all stages of adipose conversion, while it promotes the accumulation of adipsin mRNA mainly in differentiating cells. In DEX-treated adipocytes, the transcription efficiency of the G3PDH gene is not altered, and reduction to 50% of the message is due essentially to an approximately twofold decrease in its half-life.

Adipose Tissue↗
PubMed1992

Adipogenesis in a myeloid supporting bone marrow stromal cell line.

The bone marrow stroma contains pre-adipocyte cells which are part of the hemopoietic microenvironment. Cloned stromal cell lines differ both in their ability to support myeloid and lymphoid development and in their ability to undergo adipocyte differentiation in vitro. These processes have been examined in the +/+2.4 murine stromal cell line and compared to other stromal and pre-adipocyte cell lines. In long-term cultures, the +/+2.4 stromal cells support myeloid cell growth, consistent with their expression of macrophage-colony stimulating factor mRNA. However, despite the presence of mRNA for the lymphoid supportive cytokines interleukins 6 and 7, +/+2.4 cells failed to support stromal cell dependent B lineage lymphoid cells in vitro, suggesting that these stromal cells exhibit only a myelopoietic support function. The +/+2.4 cells differentiate into adipocytes spontaneously when cultured in 10% fetal bovine serum. The process of adipogenesis can be accelerated by a number of agonists based on morphologic and gene marker criteria. Following induction with hydrocortisone, methylisobutylxanthine, indomethacin, and insulin in combination, a time dependent increase in the steady state mRNA and enzyme activity levels of the following adipocyte specific genes was observed: adipocyte P2, adipsin, CAAT/enhancer binding protein, and lipoprotein lipase. In contrast, adipogenesis was accompanied by a slight decrease in the signal intensity of the macrophage-colony stimulating factor mRNA level, similar to that which has been reported in other bone marrow stromal cell lines. These data demonstrate that although the lympho-hematopoietic support function of pre-adipocyte bone marrow stromal cell lines is heterogeneous, they share a common mechanism of adipogenesis.

Adipose Tissue↗
PubMed1990

Participation of C3 and its ligands in complement activation.

C3, the most abundant complement protein in blood, plays a central role in the activation sequence of the complement system as well as in host defense. Expression of the multiple functions of C3 requires its cleavage by highly specific enzymes termed C3 convertases. C3 in a conformationally altered form, C3H2O, resulting from the slow spontaneous hydrolysis of the internal thioester bond of native C3, initiates the assembly of a C3 convertase which continuously cleaves C3 in the blood at slow rates generating a constant supply of small amounts of C3b. When an activator of the alternative complement pathway is present, C3b becomes covalently attached to its surface via an ester or amide bond. Activator surface-bound C3b initiates the assembly of an "amplification" C3 convertase, C3bBb(P), which can efficiently activate C3 and generate additional convertase complexes on the surface of the activator. C3b generated by an amplification or classical pathway C3 convertase can also bind covalently to the noncatalytic subunit, C3b or C4b, respectively, resulting in the generation of a C5 convertase, an enzyme catalyzing the cleavage/activation of C5. In terms of participation in host defense, several fragments of C3, including C3a, C3b, iC3b, and C3dg, mediate a number of important functions such as increased vascular permeability, enhancement of phagocytosis, elimination of immune complexes, and perhaps also proliferative responses and/or differentiation of B cells.

Complement Activation↗
PubMed1978

A radial hemolysis method in agarose for the functional assay of properdin.

A simple one-step radial hemolytic assay for properdin has been devised. In this assay, the test material is introduced into a well in an agarose plate containing optimal concentrations of normal human serum immunochemically depleted of properdin (RP), EGTA, magnesium ions and unsensitized guinea pig erythrocytes. Following radial diffusion, the area of the hemolytic zones resulting from the activation of the alternative complement pathway and bystander lysis of guinea pig erythrocytes was directly proportional to the concentration of properdin in the test material. The assay is specific reproducible and sensitive and the correlation with the radioimmunoassay for properdin is very good. The assay has been used to measure properdin activity in animal sera.

Animals↗
PubMed1989

Regulation of gene expression by insulin in adipose cells: opposite effects on adipsin and glycerophosphate dehydrogenase genes.

Insulin is known to play the role of a positive effector both in vitro on the adipose conversion process and in vivo on the fatty acid synthesis and esterification processes in adipose tissue. The effects of insulin on the expression of two genes activated during adipose conversion, glycerol-3-phosphate dehydrogenase (GPDH) and adipsin genes, have been investigated in 3T3 F442A adipose cells. Within a physiological range of concentrations, insulin exerts opposite effects on the levels of GPDH (EC50 approximately 0.2 nM) and adipsin (EC50 approximately 1 nM) mRNAs. Its negative effect on the abundance of adipsin mRNA involves primarily a rapid inhibition of the transcriptional rate (less than 2 h). Its positive effect on the abundance of GPDH mRNA is due to a stimulation of the transcriptional rate accompanied by a delayed stabilization of GPDH mRNA. In addition, insulin exerts a specific effect on the length of the poly(A) tract of the adipsin mRNA. These results show that a single mechanism for the regulation of adipose-related genes by insulin can be excluded but rather suggest a complex phenomenon in which various levels of regulation take place.

Adipose Tissue↗
PubMed1989

The antiglucocorticoid RU38486 is a potent accelerator of adipose conversion of 3T3-F442A cells.

We examined the effects of RU38486, a potent glucocorticoid and progestin antagonist, upon several aspects of 3T3-F442A adipocyte differentiation. RU38486 accelerated the onset of differentiation, as monitored by cell morphological changes, accumulation of lipid droplets and widespread increases in the rate of expression of several enzyme adipose markers and specific mRNAs. RU38486, at a maximal concentration of 1 microM, dramatically hastened the emergence of both fatty-acid synthetase (FAS) and glycerol-3-phosphate dehydrogenase (G3PDH) enzyme activities (550% and 450% above control values 4 days after confluence, respectively). RU38486 induction of G3PDH-specific activity ran parallel to an increase in G3PDH mRNA content (2.4-fold the control content 4 days after confluence). Moreover, RU38486-treated cells exhibited enhancement of adenylate cyclase sensitivity to both isoproterenol and ACTH (160% and 350% above control activities 8 days after confluence, respectively). While the level of expression of lipogenic markers reached similar values at the mature stage, RU38486 enabled cells to acquire hypersensitivity in terms of ACTH-stimulated adenylate cyclase activity. Similarly, adipsin gene expression was highly potentiated by the drug at day 15 post-confluence (5-fold the control value). RU38486 responsiveness observed in differentiating 3T3-F442A cells is dependent upon their prior developmental activation; none of the studied markers could be induced by the drug in the undifferentiating 3T3-C2 cell subclone. Finally, this antiglucocorticoid appears to be a useful tool for studies on adipose conversion in vitro; it could permit a re-evaluation of the role of glucocorticoids in the understanding of adipocyte development.

Adenylyl Cyclases↗
PubMed1991

Beta-adrenergic-cyclic AMP signalling pathway modulates cell function at the transcriptional level in 3T3-F442A adipocytes.

We studied the role of cAMP in the regulation of the expression of the adipsin gene and of some other adipose-specific genes including lipoprotein lipase (LPL), glycerophosphate dehydrogenase (G3PDH), and adipocyte P2 (aP2) in 3T3-F442A adipocytes. Northern blot analysis of isoproterenol (10(-6) M)-, forskolin (10(-5) M)- or 8-bromo-cAMP (10(-3) M)-treated adipocytes showed that the steady-state levels of adipsin mRNA were strongly reduced in a time-dependent and reversible manner. The concentration of isoproterenol giving a half-maximal effect in the down-regulation of the adipsin message was approximately 5 x 10(-8) M. Similarly, cell treatment by forskolin elicited a down-regulation of LPL and G3PDH mRNA levels but did not alter aP2 mRNA level. As determined by nuclear run-on assays, the rate of transcription of adipsin, LPL and G3PDH in isoproterenol-treated adipocytes was respectively 3, 3, and 2 times lower than in control adipocytes. These results indicate (1) that cAMP plays a dominant antilipogenic role in the fat cell through the transcriptional down-regulation of the expression of two major genes involved in triglyceride biosynthesis; (2) that cAMP does not reverse the adipocyte character; (3) hence, that cAMP suppresses adipsin expression at the transcriptional level, providing additional support for the role of adipsin protein in adipocyte metabolism.

3T3 Cells↗