Alpha Polypeptide Chains Research Articles

Upregulation of gamma‐2 laminin‐332 in the mouse ear vesicant wound model

Epithelial cell migration during wound healing is regulated in part by enzymatic processing of laminin-332 (formerly LN-5), a heterodimer formed from alpha, beta, and gamma polypeptide chains. Under static conditions, laminin-332 is secreted into the extracellular matrix as a proform and has two chains processed to smaller forms, allowing it to anchor epithelial cells to the basement membrane of the dermis. During incisional wounding, laminin gamma2 chains in particular are processed to smaller sizes and function to promote epithelial sheet migration over the wound bed. The present study examines whether this same function occurs following chemical injury. The mouse ear vesicant model (MEVM) was used to follow the pathology in the ear and test whether processed laminin-332 enhances epithelial cell migration. Skin biopsies of sulfur mustard (SM) exposed ears for several time points were analyzed by histology, immunohistochemistry, real-time PCR, and Western blot analysis. SM exposure greatly increased mRNA levels for laminin-gamma2 in comparison to the other two chains. Protein production of laminin-gamma2 was upregulated, and there was an increase in the processed forms. Protein production was in excess of the amount required to form heterotrimeric laminin-332 and was associated with the migrating epithelial sheet, suggesting a potential role in wound healing for monomeric laminin-gamma2.

Establishment of humanized anti-interleukin-5 receptor alpha chain monoclonal antibodies having a potent neutralizing activity

Human interleukin-5 is the key cytokine involved in regulating the production and function of human eosinophils. IL-5 binds to its specific receptor composed of two heterogeneous alpha and beta polypeptide chains (hIL-5Ralpha and betac) that are expressed on the cell surface. The hIL-5Ralpha specifically binds IL-5 without involvement of the betac. It has been suggested that neutralizing antibodies to hIL-5Ralpha could serve as a therapeutic agent in eosinophil-associated diseases. We describe here the creation and biologic activities of a mouse monoclonal antibody against hIL-5Ralpha that blocks the following IL-5 dependent activities (a) binding of the IL-5 ligand to its receptor, (b) IL-5 dependent growth of hIL-5R expressing cells, and (c) IL-5-induced adhesion of human eosinophils. We also describe the process for humanization of the mouse Mab towards development of a therapeutic MAb. The humanized version of the monoclonal antibody also displayed potent neutralizing activity against IL-5 dependent activities.

Covalent labeling of functional states of the acetylcholine receptor. Effects of antagonists on the receptor conformation.

Photoaffinity labeling of membrane-bound nicotinic acetylcholine receptor from Torpedo marmorata electric tissue with the ion-channel blocker [3H]TPMP+ reveals various functional states of the receptor protein if labeling is performed with ms time resolution. In the resting and in the activated state most of the label is incorporated into the alpha-polypeptide chains of the receptor complex. When equilibrated with agonists and antagonists, predominantly the delta-polypeptide chain (and to a lesser extent the beta-chain) reacts with the photolabel. Reactivity of the delta-chain increases after exposure to cholinergic effectors with a half-life slower than the kinetics of receptor activation or rapid desensitization. Agonists and antagonists stimulate photolabelling of the delta-chain with different kinetics. For acetylcholine, carbamoylcholine and suberyldicholine the half-life of the reactivity increases is 400 - 500 ms; for the antagonists hexamethonium, d-tubocurarine and flaxedil it is about 10 s. The latter slow kinetics are also observed when the receptor is preequilibrated with agonists or antagonists prior to mixing with [3H]TPMP+ and starting the photoreaction. We conclude that time-resolved photoaffinity labeling can convalently mark protein structures involved in receptor functions. Of special interest is the observation that antagonists also induce a conformational change in the receptor protein.

The pluripotent cytokine pleiotrophin is induced by wounding in human mesangial cells

Mesangial re-modeling and mesangial cell (MC) migration are features of several glomerular diseases including mesangiocapillary glomerulonephritis. In vitro investigations have recently identified ADAM-15, a multidomain adamalysin, as central to the migration of MC. The current study used array technology to investigate the expression of other genes in migrating cells and identified pleiotrophin (PTN), platelet-derived growth factor alpha polypeptide chain, colony stimulating factor, and four members of the tumor necrosis factor-alpha superfamily as major genes that were upregulated. Transcriptional induction of PTN was confirmed by reverse transcription-polymerase chain reaction and Northern blotting and induction of the protein by Western blotting and immunohistochemical localization. PTN was observed associated with mesangial 'hillocks' in confluent MC cultures. In contrast, in models of migration, migrating cells had the highest expression of cell-associated PTN. PTN protein was less evident, however, in the conditioned medium of MCs. Treatment of MC with heparanase removed PTN from the cells suggesting that its localization was owing to an association with heparan sulfates on the cell surface or in the extracellular matrix. This is the first description of the expression of PTN by human MCs and the data suggest that it is rapidly induced in cells that are triggered to migrate. The result of this induction is currently under investigation.

The DY genes of the cattle MHC: expression and comparative analysis of an unusual class II MHC gene pair.

The major histocompatibility complex of cattle (BoLA) contains the class II genes DYA and DIB which are transcribed with a dendritic cell restricted distribution. As part of the process to determine whether these genes have any functional significance, we demonstrate that they form a closely linked pair characteristic of other expressed class II MHC molecules. Accepted nomenclature convention suggests that BoLA-DIB should therefore be renamed BoLA-DYB. Analysis of the first full-length DYA and DYB transcripts revealed open reading frames with potential to translate 253 and 259 amino acid proteins, respectively. Comparative sequence analysis between the DY polypeptides and classical cattle, human and mouse class II MHC alpha and beta polypeptide chains revealed 16 unique amino acid residues at positions predicted to form and line the putative peptide-binding region. Expression of tagged constructs demonstrates for the first time that the DY genes of cattle are capable of translating distinctive class II MHC alpha and beta polypeptide chains.

Sequence analysis and expressional regulation of messenger RNAs encoding beta subunits of follicle-stimulating hormone and luteinizing hormone in the red-bellied newt, Cynops pyrrhogaster.

Two distinct cDNAs encoding beta subunits of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were cloned from the cDNA library constructed for the pituitary of the red-bellied newt, Cynops pyrrhogaster, and sequenced. The newt FSHbeta and LHbeta cDNAs encode polypeptides of 129 and 131 amino acids, including signal peptides of 20 and 19 amino acids, respectively. The number and position of cysteine and N-glycosylation in each of the beta subunits of FSH and LH, which are considered essential for assembly of the alpha subunit, are well conserved between the newt and other tetrapods. The high homology (41.6%) between the beta subunits of newt FSH and LH imply less specificity of FSH and LH in gonadal function. One cDNA encoding the common polypeptide chain alpha subunit of FSH and LH was also isolated from the newt pituitary gland. The mRNAs of FSHbeta, LHbeta, and the alpha subunit were expressed only in the pituitary gland among various newt tissues. Double-staining with in situ hybridization and immunohistochemistry revealed coexpression of FSHbeta and LHbeta in the same newt pituitary cells. Ovariectomy induced a significant increase in FSHbeta mRNA levels, but there was no significant change in LHbeta or alpha subunit mRNA levels compared with those in control animals. Taken together, these data suggest that two kinds of gonadotropins, namely FSH and LH, are expressed in the same gonadotropin-producing cells in the pars distalis of the newt as well as in other tetrapods and that the expression of FSHbeta is negatively regulated by the ovaries.

Human CD8 beta, but not mouse CD8 beta, can be expressed in the absence of CD8 alpha as a beta beta homodimer.

The T cell coreceptor CD8 exists on mature T cells as disulfide-linked homodimers of CD8 alpha polypeptide chains and heterodimers of CD8 alpha- and CD8 beta-chains. The function of the CD8 alpha-chain for binding to MHC class I and associating with the tyrosine kinase p56lck was demonstrated with CD8 alpha alpha homodimers. CD8 alpha beta functions as a better coreceptor, but the actual function of CD8 beta is less clear. Addressing this issue has been hampered by the apparent inability of CD8 beta to be expressed without CD8 alpha. This study demonstrates that human, but not mouse, CD8 beta can be expressed on the cell surface without CD8 alpha in both transfected COS-7 cells and murine lymphocytes. By creating chimeric proteins, we show that the murine Ig domain of CD8 beta is responsible for the lack of expression of murine CD8 beta beta dimers. In contrast to CD8 alpha alpha, CD8 beta beta is unable to bind MHC class I in a cell-cell adhesion assay. Detection of this form of CD8 should facilitate studies on the function of the CD8 beta-chain and indicates that caution should be used when interpreting studies on CD8 function using chimeric protein with the murine CD8 beta beta Ig domain. In addition, we demonstrate that the Ig domains of CD8 alpha are also involved in controlling the ability of CD8 to be expressed. Mutation of B- and F-strand cysteine residues in CD8 alpha reduced the ability of the protein to fold properly and, therefore, to be expressed.

Monoclonal antibodies as an instrument to study fibrin polymerization

Examples of using monoclonal antibodies (MAb) for studying the fibrin polymerization mechanism are considered. MAb with epitopes situated in the fibrin polymerization sites or in the recognition sites of enzymes thrombin, plasminogen, and factor XIII, which are the functional partners of fibrin, are primarily discussed. The MAb to epitopes in various regions of A alpha, B beta, and gamma polypeptide chains of the functionally important E, D, and alpha C domains of fibrin are successively described.

Epitope mapping of a monoclonal antibody directed against the alpha-subunit of phosphofructokinase-1 from Saccharomyces cerevisiae by screening phage display libraries.

Phosphofructokinase-1 from Saccharomyces cerevisiae is composed of two types of subunits, alpha and beta. Subunit-specific monoclonal antibodies were raised to elucidate structural and functional properties of both subunits. One monoclonal antibody, alpha-F3, binds to an epitope either at the C-terminal or at the N-terminal part of the alpha-polypeptide chain. By screening a heptapeptide library with this monoclonal antibody, a set of heptapeptides was selected, which contained the consensus sequences D-A-F and D-S-F. Two heptapeptides with these motifs were synthesized in order assess their capacity to inhibit the binding of antibody alpha-F3 to native phosphofructokinase-1. The peptide G-I-K-D-A-F-L inhibited the binding more strongly (IC50 = 1.5 microM) than the peptide A-P-W-H-D-S-F (IC50 = 33.3 microM). Sequence matching revealed the presence of the D-A-F motif in the polypeptide chain of phosphofructokinase-1 at amino acid position 172-174. As a control, the nonapeptide A-P-T-S-K-D-A-F-L which corresponds to the sequence of the putative epitope was tested in the inhibition assay. In view of the high inhibitory capacity (IC50 = 0.3 microM) it was concluded that this nonapeptide represents the continuous epitope of phosphofructokinase-1 that is recognized by antibody alpha-F3.

Molecular cloning and expression in Escherichia coli of a gene coding for bovine S100A1 protein and its Glu32-->Gln and Glu73-->Gln mutants.

Calcium binding S100A1 protein consists of two S100 alpha subunits. On the basis of sequence homology to other S100 proteins it is believed that the binding loops are formed by amino-acid residues 19-32 and 62-73 of S100 alpha polypeptide chain. In the oxidized form of the protein the subunits are linked covalently with each other by a disulphide bond between their Cys85 residues. A synthetic gene coding for bovine S100 alpha subunit was constructed and cloned into a derivative of pAED4 plasmid. The gene was expressed in Escherichia coli utilizing the T7 expression system. The expression products were purified and identified using mass spectrometry and by sequencing of their N- and C-termini. Three different forms (a, b, and c) of S100 alpha were produced: with the native sequence, with the initiator methionine at the N-terminus, and with an additional alanine at the C-terminus as well as with the initiator methionine. The material was partly oxidized. Interestingly, only the homodimers of a, b, and c species were formed. The total yield of the protein was about 50 mg/l of culture. Genes coding for Glu32-->Gln and Glu73-->Gln mutants of S100 alpha were obtained by site-directed mutagenesis and expressed in the same system. In both cases similar mixtures of oxidized and reduced a, b, and c species have been obtained. The total yield of E73Q mutant is similar to that of the native protein and that of E32Q lower by about a half. As expected, the mutants of S100 alpha subunits bind only one calcium ion.

Partial identification of the epitope on D-dimer for monoclonal antibody, DD-3B6/22

This study provides new information about the recognition site on fibrin for DD-3B6/22, a monoclonal antibody used in the diagnosis of thrombotic disease and proposed as a delivery agent for radioisotopes to visualize thrombi in situ. Preliminary chemical modification and proteolytic digestion of D-dimer revealed key residues in the interaction of D-dimer with its diagnostic antibody DD-3B6/22. Following the initial survey, thermolysin was used to produce digestion fragments for more specific sequence analysis. The gamma-polypeptide chain of D-dimer was confirmed as a recognition site for the antibody, with the residues S86RK88 being implicated in binding, and a second, novel site of antibody interaction was identified on the alpha-polypeptide as D105NTYNR110. Based on evidence from this study and from a previous investigation of the DD-3B6/22 binding site on D-dimer (Devine and Greenberg), a model for the DD-3B6/22 binding site is proposed to comprise regions from parts, at or near the N-terminus, of at least two different subunits: one region on the alpha-polypeptide chain of one D monomer and another region on the gamma-polypeptide chain of an adjacent D monomer. The model is discussed in the context of the predicted secondary and tertiary structure of D-dimer.

Antigen-specific apoptosis in immortalized T cells by soluble MHC class II-peptide complexes.

The recognition of T cell receptors (TCR) by purified major histocompatibility complex (MHC) class II-peptide complexes in the absence of costimulatory signals leads to the induction of T cell non-responsiveness or anergy. In a recent study using human T cell clones, it was observed that prolonged incubation of resting T cells with soluble MHC II-peptide complexes appears to result in T cell apoptosis. The present study shows that the engagement of TCR by soluble MHC II-peptide complexes also results in antigen-specific apoptosis in immortalized T cells. Apoptosis was demonstrated in a herpes saimiri virus (HSV) transformed human T cell clone (SS8T) restricted for HLA-DR2 in association with an epitope from the myelin basic protein [MBP(84-102)]. A dose- and time-dependent T cell death was observed upon incubation of SS8T cloned T cells with purified complexes of native human HLA-DR2 and MBP(83-102)Y83 peptide. The specificity of T cell apoptosis was demonstrated by exposing SS8T cells with DR2 alone and DR2 bound to another high affinity epitope [MBP(124-143)] from the same MBP. Recently, we have shown that the complexes of HLA-DR2 and [MBP(83-102)Y83] can be reconstituted by refolding Escherichia coli expressed individual DR2 alpha and beta (B5*0101) polypeptide chains lacking the transmembrane region. When SS8T cloned T cells were exposed to purified reconstituted rDR2.MBP(83-102)Y83 complexes, similar apoptosis of T cells was observed. Agarose gel analysis of T cells incubated with complexes showed a degradation of celluar deoxyribonucleic acid (DNA) to oligonucleosomal bands, a characteristic of apoptosis. The quantitative detection of DNA strand breaks was performed by pulsing T cells with 5-bromo-2'-deoxyuridine (BrdU) followed by the detection of BrdU-labelled DNA fragments using an antibody sandwich enzyme-linked immuno assay (ELISA). The fragmentation of DNA was also measured by double fluorescence flow cytometry by 3' end labelling of fragmented DNA with biotinylated-deoxyuridine triphosphate (dUTP) in the presence of terminal deoxynucleotide transferase (TdT) enzyme. The expression of the bcl-2 protein in SS8T cells following TCR engagement by soluble MHC II-peptide complexes was monitored by chemiluminescence blot analysis using anti-bcl-2 monoclonal antibody. Finally, the nucleosomal condensation of T cells following complex treatment, characteristics of typical apoptosis, was demonstrated by transmission electron microscopy. These results suggest that the binding of soluble MHC class II-peptide complexes to TCR induces antigen-specific apoptosis in transformed CD4 positive T cells in vitro. Such induction of apoptosis by soluble MHC II-peptide complexes may provide a novel therapeutic strategy to delete autoreactive T cells in various autoimmune diseases.

Domain structure of phage P4 alpha protein deduced by mutational analysis

Bacteriophage P4 DNA replication depends on the product of the alpha gene, which has origin recognition ability, DNA helicase activity, and DNA primase activity. One temperature-sensitive and four amber mutations that eliminate DNA replication in vivo were sequenced and located in the alpha gene. Sequence analysis of the entire gene predicted a domain structure for the alpha polypeptide chain (777 amino acid residues, M(r) 84,900), with the N terminus providing the catalytic activity for the primase and the middle part providing that for the helicase/nucleoside triphosphatase. This model was confirmed experimentally in vivo and in vitro. In addition, the ori DNA recognition ability was found to be associated with the C-terminal third of the alpha polypeptide chain. The type A nucleotide-binding site is required for P4 replication in vivo, as shown for alpha mutations at G-506 and K-507. In the absence of an active DnaG protein, the primase function is also essential for P4 replication. Primase-null and helicase-null mutants retain the two remaining activities functionally in vitro and in vivo. The latter was demonstrated by trans complementation studies, indicating the assembly of active P4 replisomes by a primase-null and a helicase-null mutant.

Phosphorylation of α-crystallin in rat lenses is stimulated by H 2O 2 but phosphorylation has no effect on chaperone activity

Alpha crystallin (alpha), a phosphorylated structural protein of the lens, has been shown to be a chaperone preventing other lens proteins from aggregating. It is now demonstrated that with oxidative stress imposed on cultured rat lenses, the incorporation of labeled phosphate into the alpha polypeptide chains increased by two to four times over a 90-min period in comparison to control experiments. The phosphorylation rate of the B chain, alpha B, was twice that of the A chain, alpha A. However, phosphorylation of the alpha chains has an insignificant effect on the chaperone activity of alpha or the individual alpha A and alpha B chains as measured by suppressing the thermally induced aggregation of beta low or gamma crystallins. It was also found that the alpha A aggregates are more effective chaperones than the alpha B aggregates. The size of the macromolecules resulting from reaggregation of the isolated non-phosphorylated or phosphorylated alpha B chains are not markedly effected by phosphorylation. However, phosphorylation of the alpha A chain leads to a heterogeneous population with two major species, one similar in size to alpha A and another approximately twice as large. It is concluded that the phosphorylation of alpha is associated with some other function of the protein than that of chaperone activity and that this function may be linked to a protective response to oxidative stress.

Chloroplasts Can Accommodate Inclusion Bodies: EVIDENCE FROM A MUTANT OF CHLAMYDOMONAS REINHARDTII DEFECTIVE IN THE ASSEMBLY OF THE CHLOROPLAST ATP SYNTHASE

We identified two neighboring missense mutations in the chloroplast atpA gene which are responsible for the defect of ATP synthase assembly in the FUD16 mutant from Chlamydomonas reinhardtii. The two corresponding amino acid substitutions, Ile184-->Asn and Asn186-->Tyr, occurred at strictly conserved sites among the alpha and beta subunits of (C)F1 complexes from bacteria, mitochondria, and chloroplasts. The altered region in the alpha polypeptide chain is located 7 amino acids downstream of the P-loop, which forms most of the conserved nucleotide binding site. Although the resulting chloroplast mutant fails to accumulate most of the ATP synthase subunits, it displays an increased intracellular content in both the alpha and beta subunits. We demonstrate that the two subunits do not bind to the thylakoid membranes but associate and overaccumulate in the chloroplast stroma as inclusion bodies. Increased rates of synthesis of the two subunits in the mutant point to an early interaction between the two subunits during their biogenesis.

Refolding and Reconstitution of Functionally Active Complexes of Human Leukocyte Antigen DR2 and Myelin Basic Protein Peptide from Recombinant α and β Polypeptide Chains

Major histocompatibility complex (MHC) class II molecules are cell surface heterodimeric glycoproteins consisting of one alpha and one beta polypeptide chain of similar size. These molecules play a critical role in immune recognition by displaying processed antigens to CD4-positive T helper cells. Several attempts to express the MHC class II molecules by recombinant methods in various systems resulted in either failure or poor recovery of the intact heterodimer. The present study describes our successful effort to refold and reconstitute HLA DR2 heterodimer from individually expressed alpha and beta polypeptide chains lacking the transmembrane hydrophobic regions in Escherichia coli, in the presence of an immunodominant epitope analog from human myelin basic protein (b-MBP(83-102)Y83). The reconstituted DR2 heterodimer complex was selectively purified from unfolded alpha and beta chains using heterodimer-specific monoclonal antibody (L243) coupled to a solid support. The detection of two polypeptide chains in the purified refolded DR2-peptide complex preparations was accomplished by Western blot analysis and enzyme-linked immunosorbent assay using heterodimer- and chain-specific polyclonal antibodies, and the presence of equimolar amounts of both alpha chain and beta chain in the reconstituted complex preparation was confirmed by a double label experiment. The quantitation of the bound peptide in complex preparation was measured by incubating two chains in the presence of 125I-labeled peptide. An increase in the yield of refolded and reconstituted DR2-peptide complexes was observed with increasing peptide concentration in the reaction mixture. Finally, the functional activity of the reconstituted DR2 complexes was measured by their ability to stimulate gamma-interferon production by SS8T cloned T cells in an antigen-specific and dose-dependent manner. These results demonstrate that biologically active complexes of human DR2.b-MBP (83-102)Y83 can be prepared by proper folding of human leukocyte antigen DR2 alpha and beta chains in the presence of antigenic peptide. The yield of such DR2 heterodimers with bound peptide is several thousand-fold higher over native DR2 purified from transformed B cells. Since purified MHC class II-peptide complexes have been shown to prevent autoimmune diseases in various animal models, reconstituted heterodimer complexes may have significant clinical relevance in antigen-specific treatment of various autoimmune diseases. In addition, such complexes with increased yield will provide better understanding of the trimolecular interactions between MHC-peptide and T cell receptor.

Intramolecular charge heterogeneity in purified major histocompatibility class II alpha and beta polypeptide chains

Major histocompatibility (MHC) class II antigens are heterodimeric cell surface glycoproteins consisting of an alpha and a beta chain. Although one-dimensional SDS-polyacrylamide gel electrophoresis analysis of purified MHC class II antigens shows a single diffuse band for each chain, multiple spots of identical molecular size were observed for each chain when analyzed by two-dimensional electrophoresis. The basis of this heterogeneity has not been clearly defined and has been predicted partially to be due to glycosylation and/or phosphorylation of the mature protein. To investigate the role of the three N-linked oligosaccharides of the alpha and beta chains in determining the isoelectric point of each chain, affinity-purified MHC class II antigens from human and rat sources were deglycosylated using asparagine amidase. The complete enzymatic removal of all three N-linked oligosaccharides was confirmed by SDS-polyacrylamide gel electrophoresis as well as by four different lectin-linked Western blot analyses. Two-dimensional gel analysis of the deglycosylated molecules shows no significant difference from the fully glycosylated chains. We have expressed truncated forms of the HLA DR2 chains which lack the transmembrane and cytoplasmically exposed regions in Escherichia coli. Two-dimensional electrophoresis of these single chains also reveal multiple banding patterns. The two-dimensional banding patterns described are unaffected by exposure to acidic or basic conditions, increased gel running time in the first dimension, treatment of the proteins with alkaline phosphatase to remove any potential phosphorylation, or preincubation in the presence of iodoacetamide. Multiple forms of recombinant alpha and beta chains were also observed in Tris-glycine-urea gels which merged into a single band in the presence of SDS. In addition, partially fractionated bands from preparative isoelectric focusing gels, when refocused, showed an identical number of multiple spots spanning the same range of isoelectric points. These results together suggest that each polypeptide chain of MHC class II antigens may exist in multiconformational forms, and the observed charge heterogeneity is independent of glycosylation and phosphorylation of the proteins.

Increased gamma/delta-positive T-cells in blood and bronchoalveolar lavage of patients with sarcoidosis and hypersensitivity pneumonitis

A small population of T-cells does not express the conventional T-cell receptor (TCR), characterized by the alpha and beta polypeptide chains (alpha/beta TCR) but two polypeptides termed gamma and delta (gamma/delta TCR). Changes in gamma/delta TCR expression may be relevant as the cause or consequence of several diseases. Our study was undertaken to determine and compare the distribution of T-cells expressing gamma/delta TCR in blood and bronchoalveolar lavage (BAL) of patients with sarcoidosis, hypersensitivity pneumonitis (HP), idiopathic pulmonary fibrosis (IPF), and of healthy controls. In addition, the association between gamma/delta TCR of blood T-lymphocytes and accessory molecules was analysed. Using direct immunofluorescence with the anti-gamma/delta TCR and anti-CD3 monoclonal antibodies (MoAbs) followed by flow cytometric analysis, the blood of patients with pulmonary sarcoidosis, HP, IPF and of healthy controls was analysed. To reveal the association between gamma/delta TCR of blood T-lymphocytes and the accessory molecules, expression of CD4, CD8 and CD25 were determined. Calculating the percentage and the total number of CD3+ gamma/delta TCR cells in blood, the data indicated a significant increase of gamma/delta T-cells in individuals with pulmonary sarcoidosis and HP, compared to healthy controls and IPF patients. In sarcoidosis patients with elevated CD3+ gamma/delta TCR levels, significantly lower CD4/CD8 ratios were observed. In addition, our data demonstrate a correlation between the decrease of CD4+ cells in blood and the amplified appearance of gamma/delta TCR expression in sarcoidosis patients, but not in HP patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Selected physico-chemical properties of succinylated legumin from pea (Pisum sativum L.).

Selected physico-chemical properties of pea legumin before and after succinylation have been investigated using isoelectric focusing, PAGE, SDS-PAGE, hydrophobicity measurements, SE-HPLC and RP-HPLC. Exhaustive succinylation shifted the I.P. of legumin from 4.75 to 3.5. The stepwise dissociation of legumin by increasing succinylation has been confirmed both by means of PAGE in a nondenaturing system, and by SE-HPLC. The results of SDS-PAGE provided evidence for the exposure of alpha-polypeptide chains in the native legumin. High succinylation resulted in a decrease of the surface hydrophobicity (S0) measured by both fluorescence probes (cis-parinaric acid and anilino-naphthalene sulfonic acid). RP-HPLC gave a response both to conformational changes and the introduced succinyl residues.

Der Haptoglobin-Polymorphismus - nicht nur eine genetische Markierungshilfe

The biological activities of the haptoglobin polymorphism are controlled by continuous DNA sequences coding for the HP alpha and Hp beta polypeptide chains and forming with a linked Hp related gene the haptoglobin gene complex on chromosome 16. Probably, this DNA domain originates from the gene family of the serine proteases after having lost the informations for the proteolytic functions. Instead of this, the haptoglobins have acquired other qualities, among them the hemoglobin binding capacity, inserted into the Hp beta chain. The Hp polymorphism is constituted by the evolutionary progressive DNA sequences for the Hp alpha chains, which probably have activation functions. The haptoglobins display immunoregulative abilities, which can be immunosuppressive by inhibition of the lymphocyte reactivity or immunoinductive by influencing the IgM biosynthesis, adapted to the functional requirements. In this field, Hp 2-2 has a stronger effect than the two other Hp types. Moreover, the haptoglobins inhibit the prostaglandin synthesis and protect against harmful oxidation processes. These qualities are based on the hemoglobin binding ability and can be realized by Hp 1-1 with the comparatively highest efficacy. Further on, the haptoglobins are protease inhibitors. Finally, Hp 2-2 is associated with higher albumin and ceruloplasmin serum levels than Hp 2-1 and Hp 1-1. Evidently, the haptoglobins are inserted into a widely ramified network of biological functions. The selective advantages and disadvantages of the Hp polymorphism are noticeable under pathological conditions in case of malignant tumors, inflammations, autoimmune diseases, allergic illness, affective psychoses and affective lability favouring addiction.