bluuebunny/crossref_metadata_2025 huggingface dataset:
Human Amyloid P Component: A Circulating Lectin that Modulates Immunological Responses
Authors: LI J. J. , PEREIRA M. E. A. , DELELLIS R. A. , McADAM K. P. W. J.
Published: August 2010

Amyloid P component (AP/SAP), a glycoprotein, precipitated with purified snail galactans from Helix pomatia and Ariania arbustorum in a dose‐dependent manner. Radiolabelled AP hinds to human peripheral blood mononuclear cells (PBMC), erythrocytes, and cells derived from human non‐T, non‐B acute lymphocytic leukaemia. The AP cell binding is specific in that it is dose‐dependent and can be blocked both by excess cold AP and by Helix promatia galactan. although it cannot be blocked by an equal amount of the monosaccharide galactose. In vitro studies of human PBMC immune responses demonstrated that AP inhibits PBMC proliferation responses to mycobacterial purified protein derivative and to phytohaemagglutinin and the humoral, antibody response to pokeweed mitogen. The AP‐induced suppression of non‐specific antibody production by human PBMC was dependent on the time at which AP was added to the culture. AP was suppressive if added in the first 48 h of the 7‐day culture, and the suppression could not be reversed by washing the cells after the exposure to AP. The mechanism of AP‐induced immunosuppression is still unclear, but human SAP circulates as a pair of pentameric rings, having ten identical subunits that bind to galactose polymers, and our present data suggest that AP affects the immune response through its properties as a lectin.
Distribution of Ig Classes and IgG Subclasses among Human B Cells Activated by Nocardia opaca‐Delipidated Cell Mitogen or by Pokeweed Mitogen
Authors: THI BICH‐THUY LÈ , CIORBARU‐BAROT R. , REVILLARD J.‐P.
Published: August 2010

The relative proportions of cells synthesizing the three major Ig classes or one of the four IgG subclasses in cultures stimulated with pokeweed mitogen (PWM) or Nocardia‐delipidated cell mitogen (NDCM) were investigated. In cultures of human peripheral Mood mononuclear cells (PB MNC) stimulated with PWM, the number of IgG‐comaining cells (CC) was higher than the number of IgM‐CC, and a substantial number of IgA‐CC was found. Conversely, in NDCM‐stimulated PB MNC cultures IgM‐CC outnumbered IgG‐CC and only few IgA‐CC were detected. In those cultures, the removal of T cells resulted in an increase in the number of IgM‐CC concomitant with a decrease in the number of IgG‐CC. A substantial number of cells containing simultaneously IgG or IgA in addition to IgM could be found in PWM‐stimulated cultures. These cells were virtually absent in NDCM‐stimulated cultures. The relative proportions of IgG subclass‐CC were IgG1‐CC>IgG2‐CC>IgG3‐CC≥IgG4‐CC in PWM‐stimulated and IgG2‐CC>IgG1‐CC>IgG3‐CC≥IgG4‐CC in NDCM‐stimulated cultures. The removal of T cells from NDCM‐stimulated cultures did not result in major alteration of this distribution. The role of T cells and of the genomic order of the Igh‐C genes in their phenotypic expression triggered in vitro by PBA is discussed.
Studies of Monoclonal Antibodies Specific for Major Histocompatibility Complex Products of the Rat
Authors: HEMMI S. , FENNER M. , BINDER C. , BINZ H. , WINTERHALTER K. , WIGZELL H.
Published: August 2010

Polyclonal anti‐idiotypic antibodies against monoclonal antibodies with specificity for RT1‐encoded antigens were induced in syngeneic, allogeneic, and xenogeneic hosts. The immune response to the idiotypic determinants on monoclonal antibodies was T‐cell‐dependent. The anti‐idiotypic antibodies, independent of whether they were induced in syngeneic, allogeneic, or xenogeneic (after proper absorption) hosts, showed an exquisite specificity for the monoclonal antibody used for the induction. No cross‐reactivity with other monoclonal antibodies could be observed.
Studies of Monoclonal Antibodies Specific for Major Histocompatibility Complex Products of the Rat
Authors: HEMMI S. , FENNER M. , BINZ H. , WINTERHALTER K. , WIGZELL H.
Published: August 2010

Sixteen monoclonal anti‐idiotypic antibodies were prepared against RTI antigen‐specific monoclonal antibodies by means of an inhibition assay. Three (110‐3, 112‐89, and 133‐4) were produced against antibody 3‐8C2 (19), three (178‐60, 181‐14, and 184‐38) against antibody 11/23/5, and three (144‐3, 144‐33, and 146‐31) against antibody 1‐5A4. One monoclonal antiidiotypic antibody (21–39) was directed against antibody 1‐8D2 and six (CE1‐3, CE2‐1, CE7‐2, CE8‐46, CE9‐35, and CE 10‐2) against antibody 3‐12A1. The anti‐idiotypic antibodies were all highly specific for the respective monoclonal antibody used for the induction of the antiidiotype and did not cross‐react with other monoclonal antibodies.
Mycoplasma Mimicry of Lymphokine Activity in T‐Cell Lines
Authors: RUUTH E. , RÅNBY M. , FRIEDRICH B. , PERSSON H. , GOUSTIN A. , LEANDERSON T. , COUTINHO A. , LUNDGREN E.
Published: August 2010

During screening of T‐T hybridomas for B‐cell‐stimulating factors (BSF) we found in the conditioned medium of such a line (TUH‐U) an activity that turned out to cosediment with a contaminating strain of Mycoplasma arginini on centrifugation. Since it only affected B blasts and only to a minor extent small cells and since it did not induce immunoglobulin secretion, the mycoplasma strain seemed to mimic a BSF. M. arginini was also found in two other T‐T hybridoma producer lines but not in a clone of the EL‐4 line. Three other M. arginini isolates were not stimulatory for B cells. The presented results point towards the importance of careful control for mycoplasma contamination in established cell lines.
Production of Specific Antibodies by Cerebrospinal Fluid Lymphocytes in Patients with Herpes Zoster, Mumps Meningitis and Herpes Simplex Virus Encephalitis
Authors: FORSBERG P. , KAM‐HANSEN S. , FRYDÉN A.
Published: August 2010

We applied a new method consisting of short‐term culture (18 h) of lymphocytes from cerebrospinal fluid (CSF‐L) and peripheral blood (PBL) in viral antigen‐coaled ELISA plates and subsequent measurement of IgG and IgM antibodies bound to antigen. Utilizing mumps virus, herpes simplex virus (HSV), varicella zoster virus (VZV). and measles virus as antigens, we demonstrated production by CSF‐L. of antibodies against the aetiological agent only in all patients with mumps meningitis and HSV encephalitis and also in all patients with herpes zoster without central nervous system (CNS) symptoms. This might be considered as direct evidence that specific antibodies are produced within the CNS in inflammatory nervous system diseases. CSF‐L. usually produced higher amounts of antibodies than the corresponding number of PBL., In comparison with concentrations of free antibodies determined in parallel, our method had higher specificity and sensitivity and gave more precise information about the antibody response in infections of the nervous system.
Insignificant Immune Phagocytosis Inhibition and Cytotoxicity by the Murine Monoclonal HLA‐DP Antibody B7/21 Tested on Human Monocytes and Macrophages
Authors: NEPPERT J.
Published: August 2010

On the basis of fluorescence flow cytometry data with the monoclonal antibody (MoAb) B7/21, and of the differing cell sizes, the HLA‐DP antigen density on monocytes is estimated to be 25% of that on B lymphocytes. The remote positions of the antigens are assumed to be responsible for the markedly reduced inhibitory capacity of the MoAb B7/21 in immune phagocytosis by monocytes and for the absence of complement‐dependent cytotoxicity of this MoAb to monocytes. The findings from experiments on peritoneal macrophages are comparable. Therefore HLA‐DP may not be important for the functioning of the monocytes and macrophages.
The Attachment of Serum‐and Plasma‐Derived C3 to Solid‐Phase Immune Aggregates and its Relation to Complement‐Mediated Solubilization of Immune Complexes
Authors: BAATRUP G. , SVEHAG S.‐E. , JENSENIUS J. C.
Published: August 2010

The interaction between immune aggregates and complement (C) was investigated Solid‐phase immune aggregates were prepared by coating microwells with heat‐aggregated bovine serum albumin (BSA) followed by rabbit anti‐BSA antibody. The immune aggregates were reacted with human serum or citrated plasma al 370C. The binding of C3 components was investigated with biotinylated F(ab′)2 antibodies In C3c and C3d and avidin‐coupled alkaline phosphatase. The form of the incorporated C3. whether C3b‐iC3b or C3dg. can be deduced from the response with these two antibodies. The maximal binding of C3b‐iC3h to the immune aggregates was observed within 5 min of incubation with serum or citrated plasma The conversion to C3dg was evident by a decrease in bound anti‐C3c concomitant with increasing anti C3d reactivity within about 10min of incubation. When the classical C pathway activation was inhibited, the binding of C3b‐iC3b was delayed by 20–30 min. whereas stopping of the alternative pathway did not influence the initial kinetics of the reaction. The addition of human red blood cells had no measurable influence on the degradation of bound C3b‐iC3b.125I‐lebelled anti‐BSA antibody bound to the solid‐phase BSA was not released during the C3 incorporation. The incorporation of C3b into the immune aggregates was mediated equally well by serum and by citrated plasma. The incorporation of C3b‐iC3b into immune complexes (IC) is thought to be responsible for the C‐mediated solubilization (CMS) of IC. Citrated plasma, however, exerted no CMS capacity when measured by a radiometric assay, The CMS capacity of serum was inhibited by citrate, but could then be restored by adding Ca2+ and Mg2+, whereas no CMS could be demonstrated with citrated plasma to which divalent metal ions were added.
Monoclonal Antibodies against Pancreatic Islet‐Cell‐Surface Antigens Selected by Flow Cytofluorometry
Authors: VISSING H. , PAPADOPOULOS G. , LERNMARK Å.
Published: August 2010

BALB/c mice were immunized with human islets of Langerhans and spleen cells from two mice. found to develop cell‐surface antibodies against insulin‐producing rat islet tumour RIN‐5F cells, were fused with mouse myeloma cells. Antibody‐producing hybrids were cloned on the basis of their production of surface antibodies reactive with paraformaldehyde‐fixed RIN‐5F cells by indirect immunofluorescence analysis in the fluorescence‐activated cell sorter. Among 236 primary clones, eight stable cell lines producing islet‐cell‐surface antibodies were eventually cloned. Antibody 2G3 (IgM) reacted with viable normal rat islet cells and high insulin‐producing rat islet tumour RIN5‐A2 cells, while 3G3(IgM) only reacted with RIN5‐A2 cells. AntibodyβB1 (IgG1) reacted with all islet cells tested and detected an M121k component in immunoblotting experiments with RIN‐5AH cell plasma membrane proteins electrophoretically transferred to nitrocellulose filters. Antibody 7F6 (IgM) reacted with all islet and non‐islet cells tested and delected bands of M166k and 27k by immunoblotting. Antibodies γB3,γB6, γC2, and 6BI (all IgM) showed varying degrees of binding to different islet cells, but reacted only weakly with non‐islet human cells. It is concluded that monoclonal antibodies against pancreatic islet cells may define specific endocrine islet‐cell‐surface determinants.
Inhibition of in Vitro Alloreactivity by Cyclosporin A: Evidence for an Inter‐Individual Variation in Sensitivity
Authors: SANDER B. , BRIGATI C. , MÖLLER E.
Published: August 2010

Cyclosporin A (CyA) is the immunosuppressive treatment of choice in preventing allograft rejection and graf‐versus‐host disease. However, clinical trials indicate that there may exist inter‐individual variations in sensitivity to the drug. We have approached this issue by studying CyA‐mediated inhibition of in vitro alloreactions. as measured by mixed lymphocyte reactions (MLR) and cell‐mediated lympholysis (CML), using mouse and human normal spleen cells. The differences between individuals in the CyA concentration required 50% inhibition of the human alloreactions were twentyfold for the MLR and fortyfold for the CML. Moreover, the CML appeared to be inhibited, at lower doses of the drug. No correlation was found between inhibitory doses and variables such as the magnitude of the response, age, or human leukocyte antigen phenotype. When the same experiments were performed with mouse spleen cells, no differences were observed among eight inbred strains. The lack of demonstrable individual sensitivity of mice in relation to humans is discussed.
The Role of the CD8‐Positive Subset of T Cells in Proliferative Responses to Soluble Antigens
Authors: SOLLID L. , BRUSERUD Ø. , GAUDERNACK G. , THORSBY E.
Published: August 2010

Using magnetic monosized polymer particles (M 450) coated with a monoclonal mouse IgM anti‐CD8 (ITI 5C2) antibody, we were able to selectively remove and isolate functionally active CD8+ T cells from human peripheral blood mononuclear cells. Isolated CD8+ cells did not respond by proliferation lo soluble antigens, but proliferated in response to phytohaemag‐glutinin. However, in the presence of CD4+ T cells. CD8+ cells were able to mount a substantial proliferation when stimulated with soluble antigens. Depletion of CD8+ cells decreased rather than increased the T‐cell responses to the antigens glyc‐gli, Coxsackie B4, and mumps in healthy individuals. We therefore found no indication of involvement of functionally‐active CD8+‐ suppressor cells in vitro, The T‐cell responsiveness to these antigens has previously been shown to be influenced by HLA‐DR‐associated restriction elements, but the tendency for decreased responsiveness to these antigens by CD8 depletion seemed independent of the DR type of the cell donors. As in healthy subjects. CD8 depletion resulted in a decreased responsiveness to the gluten antigen glyc‐gli in untreated and treated coeliac disease patients and to Coxsackic B4 and mumps antigens in Type 1 diabetics.
The Role of the CD8‐Positive Subset of T Cells in Proliferative Responses to Soluble Antigens
Authors: BRUSERUD Ø. , SOLLID L. , GAUDERNACK G. , THORSBY E.
Published: August 2010

The role of CD8 (T8, Leu 2)‐positive T lymphocytes in the proliferative T‐lymphocyte response to mumps and Coxsackie B4 viral antigens in vitro was investigated. The frequency among enriched T‐lymphocyte blasts of antigen‐reactive T lymphocytes (ARTL) restricted by different DR‐associated elements was investigated, using antigenic restimulation with allogeneic antigen‐presenting cells in a limiting dilution assay. A decreased frequency of DR3‐restricted and an increased frequency of DR4‐restricted mumps and Coxsackie B4 ARTL were seen in the limiting dilution assay, whether or not HLA class I determinants were shared in the antigenic restimulation. Removal of CD8‐positive cells did not increase the primary in vitro responsiveness to mumps and Coxsackie B4 viral antigens, and did not change the DR‐associated differences in the frequency of ARTL seen in the limiting dilution assay.
Quantitative Evidence Against Inactivation of Self‐Reactive B‐Cell Clones
Authors: KARRAY S. , LYMBERI P. , AVRAMEAS S. , COUTINHO A.
Published: August 2010

The frequencies of murine B‐cell precursors developing into clones secreting antibodies which hind to autologous (mouse) or heterologous (rabbit or human) forms of the same protein antigen (myosin and albumin) were determined in an attempt to directly lest the hypothesis of higher decay rates of B lymphocytes exposed to self‐antigens. The results exclude, on a quantitative basis, any form of inactivation or deletion of such cells.
Further Characterization of Internal Image‐Bearing Anti‐Idiotypic Antibodies: Specific Binding to Immunoglobulin Receptors on Murine Hybridoma Cells Secreting Antibodies to Hepatitis B Surface Antigen.
Authors: KENNEDY R. C. , HENKEL R. D. , DREESMAN G. R.
Published: August 2010

The further characterization of internal image anti‐idiotypic antibodies (anti‐Id) that represent a potential alternative vaccine candidate for type B viral hepatitis is described. The anti‐Id preparation contains an internal image component or related epitope that mimics hepatitis B surface antigen (HBsAg) and binds to murine hybridoma cells that secrete antibodies to HBsAg (anti‐HBs). This binding to anti‐HBs‐secreting hybridomas was partially inhibited by intact HBsAg particles and was associated with the expression of an interspecies idiotype. Immunoprecipitation studies demonstrated that the anti‐Id bound to immunoglobulin molecules expressed on the surface of the hybridoma cells. These data suggest that internal image anti‐Id, which induces an in vivo antibody response by antigenic mimicry in the absence of HBsAg, binds to anti‐HBs molecules on the surface of cells actively secreting anti‐HBs. The possible mechanism for internal image anti‐Id‐based antibody vaccines that mimic the overall conformation of antigens associated with infectious agents is discussed.
Characterization of Human Mononuclear Cells after Positive Selection with Immunomagnetic Particles
Authors: LEA T. , SMELAND E. , FUNDERUD S. , VARTDAL F. , DAVIES C. , BEISKE K. , UGELSTAD J.
Published: August 2010

We have investigated the possibility to employing magnetic monodisperse polymer particles for positive selection of human peripheral blood mononuclear cell populations. By carefully titrating the ratio between particles and cells we succeeded in isolating a number of cell populations that could be cultivated subsequently in vitro for functional studies. The success of the procedure is partly dependent on the properties of the monoclonal antibodies used to sensitize the cells. Provided these antibodies do not read with membrane structures involved in the transduction of activating signals, highly purified, quiescent cell populations can be recovered in a single fractionation step. In most instances panicles will detach from the isolated cells by overnight culture, and the particles can then be removed from the system by a suitable magnet. T lymphocytes, subpopulations of I lymphocytes, and B lymphocytes have been isolated in this way and studied in a variety of functional assay systems. Comparison with cells obtained after negative selection clearly demonstates the usefulness of this technique, especially if the membrane marker selected for it is not directly engaged in the activation processes.
Increase in T‐Cell Subsets of Oral Mucosa: a Late Immune Response in Patients with Treated Coeliac Disease?*
Authors: Lähteenoja H. , Toivanen A. , Viander M. , Räihä I. , Rantala I. , Syrjänen S. , Mäki M.
Published: August 2010

Background and aims: In coeliac disease, the gut involvement is gluten‐dependent. Following the introduction of a gluten‐free diet, inflammatory cell infiltration decreases in the small intestinal mucosa. Our hypothesis was that the oral mucosa might mirror the changes found in coeliac disease similarly to the mucosa of the small intestine. Thus, the number of inflammatory cells in the oral mucosa would decrease in patients with coeliac disease on a gluten‐free diet. Methods: The distribution CD45RO+ and CD3+ T cells, T‐cell subpopulations (CD4+ , CD8+ , T‐cell receptor (TCR)αβ+ and TCRγδ+ cells) and HLA DR expression were studied in the buccal mucosa of 15 untreated and 44 gluten‐free diet treated coeliac disease patients, and of 19 controls. All 15 patients with untreated coeliac disease were immunglobulin (Ig)A endomysial antibody positive and all 44 patients on gluten‐free diet except one were endomysial antibody negative, as were all control subjects. Results: Untreated coeliac disease patients did not differ from controls in the densities of CD45RO+ cells, CD3+ cells or of T‐cell subsets. In contrast, in treated coeliac disease patients, a significant increase in the numbers of mast cells, CD3+ and CD4+ lymphocytes was found in the lamina propria of oral mucosa as compared with patients with untreated coeliac disease and controls. The increase in CD3+ T cells was in part owing to an increase in lymphocytes expressing no TCR. No differences were found in the expression of human leucocyte antigen (HLA) DR in the epithelium or in the lamina propria in the patient groups studied or in the controls. In treated coeliac disease patients only a few TCRγδ+ T cells were found intraepithelially and in the lamina propria, but these cells were not detected in the lamina propria of oral mucosa of patients with untreated coeliac disease or in the controls. Conclusions: The infiltration of T cells into oral mucosa was increased in treated coeliac disease patients in spite of adherence to a gluten‐free diet. Because the CD3+ T cell count was higher than those of the TCRαβ+ and TCRγδ+ T cells, there must be other cells involved, probably natural killer (NK) cells. The increase in T‐cell subsets in the treated coeliac disease patients seems not to result from poor dietary compliance, but might occur as a late immune response in coeliac disease and reflect chronic immunologic stimulation followed by regeneration of memory T cells.
Expression Pattern of Immune Suppressive Cytokines and Growth Factors in Oesophageal Adenocarcinoma Reveal a Tumour Immune Escape‐promoting Microenvironment
Authors: Milano F. , Jorritsma T. , Rygiel A. M. , Bergman J. J. , Sondermeijer C. , Ten Brinke A. , vanHam S. M. , Krishnadath K. K.
Published: August 2010

Immunotherapy for solid cancers, such as oesophageal adenocarcinoma (OAC), is generally hampered by an unfavourable immunological tumour microenvironment. This prompted us to investigate the nature of the OAC environment. Biopsies of tumour and normal control tissues were collected from 17 OAC patients, and investigated using fluorescent immunohistochemistry (IHC) for the expression of cyclooxygenase‐2 (COX‐2), vascular endothelial growth factor (VEGF), transforming growth factor‐β, indoleamine 2–3 dioxygenase, CXCL3 and CXCR1, and for measuring a panel of cytokines by cytometric bead array (CBA), and for Granzyme B (GrB), Perforin and PI9 detection by semi‐quantitative PCR (QPCR). IHC showed that expression of all the above‐mentioned factors is upregulated in 80–93% of the tumours. By QPCR, the cytokine interleukin (IL)‐8 was significantly upregulated in tumour samples (P < 0.05). IL‐6, IL‐10, GrB and Perforin did not show any significant difference between normal and tumour samples, whereas PI9 levels were significantly higher in normal when compared with the tumour samples. CBA confirmed upregulation of IL‐8 and show upregulation of IL‐1β in the tumours (P < 0.05). Regarding IL‐6 and interferon (IFN)‐γ, no significant differences were observed between normal and tumour tissues. The OAC microenvironment is characterized by a lack of cytokines and factors that normally would enhance anti‐cancer responses, such as IFN‐γ and GrB, and by a high expression of several immuno‐suppressive factors, such as COX‐2, VEGF and IL‐8. For future improvement of treatment efficacy of OAC patients, it will be of importance to combine immunotherapy with immune‐modulating agents.
Solution conformation of N‐terminal fragments of trichosanthin small domain (TCS 182‐200)
Authors: HU HONG Y. , LU ZI X. , DU YU C.
Published: August 2010

Three peptides, T14, T18 and TDK, derived from the N‐terminus of trichosanthin small domain (TCS 182‐200) have been investigated by circular dichroism. Secondary structure and structural transitions of the above peptides under different conditions were studied. Alcohol prompts a transition of the T18 peptide from a β‐sheet to an α‐helical structure. It also increases the α‐helicities of T14 and TDK. The β‐sheet of T18 peptide appears more hydrophobic than the α‐helix of T14 or TDK. The effects of polypeptide sequence and solvent on secondary structure formation of these model peptides are discussed.
Differential involvement of disulfide bridges on the folding of a scorpion toxin
Authors: CALABRO V. , SABATIER J.M. , BLANC E. , LECOMTE C. , RIETSCHOTEN J. VAN , DARBON H.
Published: August 2010

Leiurotoxin I is a neurotoxin, blocker of Ca2+‐activated apamin‐sensitive K+ channel, purified from the venom of the scorpion Leiurus quinquestriatus hebraeus. It is a 31‐residue polypeptide reticulated by three disulfide bridges, i.e. Cys3‐Cys21, Cys8‐Cys26 and Cys12‐Cys28. To investigate the role of these disulfide bridges in the folding of this toxin, analogs lacking one disulfide bridge were synthesized. The structures of two analogs in which two half‐cystines were replaced by α‐aminobutyrate residues to suppress one disulfide bridge, were analyzed by 1H NMR. The NMR studies reveal a three‐dimensional structure identical with the native toxin for the analog lacking disulfide bridge Cys3‐Cys21 and a loss of organized structure for another analog lacking disulfide bridge Cys12‐Cys28. These analogs are, respectively, fully active and only weakly active (2% of the residual activity) when tested in vitro for their ability to interact with their receptor channel and in vivo for their neurotoxic activity in mice. This suggests that disulfide bridge Cys12‐Cys28 is essential for the folding process. In contrast, the lack of disulfide bridge Cys3‐Cys21 does not affect the folding and the maintenance of bioactive conformation of Leiurotoxin I.
Conformational study of vasoactive intestinal peptide by computational methods
Authors: FILIZOLA MARTA , CARTENÌ‐FARINA MARIA , PEREZ JUAN J.
Published: August 2010

The conformational profile of vasoactive intestinal peptide (VIP) was characterized using computational methods. The strategy devised included a close examination of the conformational profile of the first 11 residues fragment followed by a study that considered the compatibility of the different conformations found with a continuation of the polypeptide chain in a‐helical conformation. Accordingly, a detailed analysis of the conformational preferences of the N‐terminal fragment of VIP(1–11) was carried out within the framework of the molecular mechanics approach, using simulated annealing in an iterative fashion as the sampling technique. In a second step, low‐energy structures of the fragment were fused to the remainder of the VIP chain in the form of two noninteracting α‐helices, according to a model of the structure of the peptide proposed from NMR studies. After investigation for compatibility of each of the low‐energy structures of VIP(1–11) with the two helical regions by energy minimization, only 5 of 35 structures were discarded. Analysis of the structures characterized indicates that most of the conformations of VIP(1–11), including the global minimum, can be described as bent conformations. Conformations exhibiting α‐turns and ß‐turns, previously proposed by NMR studies were also characterized. The conformational analysis also suggests that the common structural features found in VIP(1–11) should also be present in VIP. Finally, because of the sequence homology between VIP and Peptide T, and the fact that both are ligands of the CD4 receptor, both sets of low‐energy conformations were compared for similarity. The relevance of these results as guidance of the design of new peptide analogs targeted to the CD4 receptor is also discussed.
Random coil conformation for extended polyglutamine stretches in aqueous soluble monomeric peptides
Authors: ALTSCHULER ERIC LEWIN , HUD NICHOLAS V. , MAZRIMAS JOSEPH A. , RUPP BERNHARD
Published: August 2010

Several neurodegenerative diseases have been found to be strongly associated with proteins containing a polyglutamine stretch which is greatly expanded from approximately 20 glutamines in normal individuals to more than 40 in affected individuals. A conformational change in the expanded polyglutamine stretch has been suggested to form the molecular basis for disease onset. Model peptides containing polyglutamine tend to aggregate and become insoluble. We have synthesized readily water‐soluble monomeric peptides by flanking polyglutamine stretches with sequences rich in alanine and lysine. Circular dichroism measurements show that polyglutamine stretches of length 9 or 17 adopt a random coil configuration in aqueous solution. We think that in the disease‐associated peptides for normal individuals the stretches of ∼20 glutamines are in a random coil conformation, whereas in affected individuals the polyglutamine stretch may be in some other conformation. Our method to design soluble monomeric peptides containing extended polyglutamine stretches may be generally useful in studying other highly aggregating peptides.
Size‐exclusion chromatography of protected synthetic polypeptide tandems in an organic solvent
Authors: KARNOUP ANTON S. , SHIRYAEV VIACHESLAV M. , MEDVEDKIN VIACHESLAV N. , MITIN YURY V.
Published: August 2010

A novel method for analytical and preparative size exclusion chromatography of large water‐insoluble protected peptides in an organic solvent was developed. This method was applied to analysis and separation of protected synthetic peptide tandem repeats and to a control of the reaction of peptide fragment coupling. Columns containing Toyopearl HW‐40, HW‐50, HW‐55 and HW‐60 gels of Fine grade were used, and the selectivity of each sorbent, as well as the Chromatographie behaviour of the peptides on them, were examined. Separation ranges of these gels applied to separation of protected peptides in DMF were shown to be much smaller (ca. 400–14000 Da) than those of the same gels applied to protein separations in water buffers (100–1000000 Da). An initial evaluation of efficiency of Toyopearl HW Superfine grade gels with respect to similar separations was performed. © Munksgaard 1997.
Crystal structure of a dipeptide Boc‐Aib‐Phe‐OMe
Authors: BALAKRISHNAN R. , PARTHASARATHY R. , RAMASUBBU N.
Published: August 2010

In order to understand the effect of the restrictions posed by the Aib residue on peptide conformation we studied the crystal structure of a dipeptide tBoc‐Aib‐Phe‐OMe. Crystals of this compound are triclinic, space group PI with a= 9.600(1) Å, b=10.262(1) Å, c= 10.799(1) Å, α= 98.43°(1), β=99.18°(1), °=98.87°(1), V= 1021.69(18) Å3 and Z=2. The structure was solved by direct methods and refined to an R‐factor of 4.98%. The backbone conformational angles for the Aib residue in molecule A are in the left‐handed helical region, while in molecule B they are in the right‐handed helical region. The Phe residue in molecule A is in the right‐handed helical conformation, while in molecule B it is in the β‐region. The peptide units are trans and show significant deviation from planarity [(ω1= 166.67(5)° and ω2=–177.9(5)]. © Munksgaard 1997.
Conformational consequences of i, i + 3 cystine linkages: nucleation for α‐helicity?
Authors: PELLEGRINI MARIA , ROYO MIRIAM , CHOREV MICHAEL , MIERKE DALE F.
Published: August 2010

Methods to introduce specific secondary structural elements into peptides and proteins are vital for the rational design of peptide and non‐peptide drug candidates as well as in the de novo design of proteins. Here the incorporation of a disulfide linkage between cysteine residues spaced three amino acids apart (i, i+3) as a method to induce helicity is examined. Two dodecamer peptides, A‐V‐S‐E‐C‐Q‐L‐C‐H‐D‐K‐G‐NH2, differing in the chirality of the cysteine at the fifth position (the i position), have been synthesized and conformationally studied both in the linear and cyclized form. This peptide sequence, derived from the N‐terminal sequence of parathyroid hormone related protein, does not form helices, even as part of the 1‐34 fully active domain of the protein. The four analogs (two cyclic and two linear) were analyzed both in aqueous solution and in the presence of sodium dodecyl sulfate micelles. In aqueous solution the linear peptides display no evidence for secondary structure, while the cyclization induces a turn centered about the cysteine residues. In the presence of micelles the linear form of the peptides adopts bent conformations, containing turns, but results from both NMR and CD provide no evidence of helices. The oxidized l, l‐peptide in the micellar solution does not present a well defined conformation, although the presence of one helical turn is evident. The cyclic d, l analog adopts a helical structure (not an α‐helix) extending from residue 2 to 9, with non‐standard φ, ψ values caused by the presence of the d‐amino acid. These results clearly illustrate that the ability of d‐Cys(i), Cys(i+ 3) cyclization to initiate helix formation depends greatly on the solvent used. Therefore, any drug‐design principle utilizing this modification for helix nucleation must keep the environment in which the peptide is biologically active in mind. © Munksgaard 1997.
NMR and circular dichroism studies on the conformation of a 44‐mer peptide from a CD4‐binding domain of human immunodeficiency virus envelope glycoprotein
Authors: CHANG DING‐KWO , CHIEN WEI‐JYUN , CHENG SHU‐FANG , CHEN SHUI‐TEIN
Published: August 2010

Two‐dimensional NMR, circular dichroism (CD) experiments and molecular modeling were performed to study the secondary structure of a 44‐mer peptide fragment derived from the C4 region of gp120 of human immunodeficiency virus in aqueous solution. It was found a nascent helical structure exists following a type I turn near the N‐terminus of the peptide. The proline residue in the turn appears to serve as a helix initiator. The helical structure was in fast dynamic equilibrium with β‐ or random coil form on the NMR scale. A reverse turn was identified at a section containing two consecutive proline residues. A nascent helical structure has been detected for the region near the C‐terminus of the 44‐mer peptide. Higher helical content for the peptide is also indicated by CD studies on TFE titration. Thus it is proposed that, in more apolar medium, the Pro‐Pro turn and the segment amino‐terminal to it, spanning about 20 amino acids, may be converted into helix structure. Moreover, the region near the C‐terminus of the peptide may also be induced into helix, so that a helix‐turn‐helix structure may be formed in the C4 domain of gp120. A helical wheel representation of this stretch shows amphipathicity of the helix. The biological implication of the conformational adaptibility of the peptide was discussed. © Munksgaard 1997.
A systematic approach to the solid‐phase synthesis of linear and cyclic pseudopeptide libraries containing ψ[CH2NH] amide bond surrogates
Authors: WEN JAMES J. , SPATOLA ARNO F.
Published: August 2010

A systematic approach has been adopted for the synthesis and characterization of a series of linear and cyclic pseudopeptide mixtures containing the ψ[CH2NH] amide replacement. The parent structures were based on biologically relevant compounds including an enkephalin analog, H‐Tyr‐d‐Ala‐Gly‐Phe‐Leu‐OH, and an Arg‐Gly‐Asp peptide sequence. The linear mixtures containing 4 and 64 pseudopeptide components with 1, 2 or 3 amide bond surrogates were synthesized using Boc‐SPPS. The amount of desired linear pseudopeptides in the mixtures ranged from 67 to 90% as determined by integration of HPLC peak areas. Comparative studies indicated: (i) racemization is not a problem in the synthesis of pseudopeptide mixtures containing the ψ[CH2NH] surrogate; and (ii) protection of the ψ[CH2NH] surrogate with a benzyloxycarbonyl group during the synthesis is beneficial. Cyclic mixtures containing 4 and 256 cyclic pseudopeptide components with a single amide bond surrogate were synthesized using a resin‐bound cyclization approach featuring side‐chain attachment of Boc‐Asp‐OFm to the solid support. Cyclization kinetic studies revealed that the newly developed HATU coupling reagent provided a fast cyclization rate for a pseudopeptide mixture and that the position of the reduced peptide bond within a peptide mixture had only a small effect on the cyclization rates of the mixture. Pseudopeptide libraries permit the more efficient bioassay of complex structures and can also be used to reveal more rapidly trends in physicochemical variables. For example, we observed that the expected increase in hydrophilicity with ψ[CH2NH] substitutions during RP‐HPLC analysis did not continue with several such replacements. © Munksgaard 1997.
New fluorogenic substrates for the study of secondary specificity of prolyl oligopeptidase
Authors: NOULA CATHERINE , KOKOTOS GEORGE , BARTH TOMISLAV , TZOUGRAKI CHRYSSA
Published: August 2010

The secondary specificity of prolyl oligopeptidase (POP) has been studied by using a series of fluorogenic substrates containing the highly fluorescent 7‐amino‐4‐methyl‐2‐quinolinone (AMeq) marker. The substrates were dipeptides of the general formula Z‐X‐Pro‐NH‐Meq, bearing amino acid residues with variable functional groups [Met, Lys(Boc). Lys. His, Ser, Leu, Glu(OMe), Glu, Cys(Bzl)] at the P2 position, and the tripeptide Z‐Asn‐Cys(Bzi)‐Pro‐NH‐Meq. The kinetic parameters for their hydrolysis by porcine kidney POP were determined at λex= 360 nm and λem= 430 nm. All the dipeptide substrates showed a high affinity to the enzyme and could be used for its fluorometric determination. The S2 binding subsite of POP can accomodate amino acid residues with a bulky side group, while it prefers a positively charged group (free Lys) instead of a negatively charged one (free Glu). © Munksgaard 1997.
HIV‐1 protease inhibitors containing a novel C2 symmetrical hydroxyalkylgem‐diamino core structure
Authors: MARASTONI MAURO , SALVADORI SEVERO , BORTOLOTTI FABRIZIO , TOMATIS ROBERTO
Published: August 2010

Two series of peptidomimetics containing a novel C2 symmetrical hydroxyalkylgem‐diamino core structure were prepared, from amino acid starting materials, and evaluated as inhibitors of HIV‐1 protease (HIV‐1 Pr). 1, 1‐Diamino‐3‐hydroxypropane (gHse) derivatives showed weak inhibitory potency (IC50> 10 μM). In the 1, 1‐diamino‐2‐hydroxyethane (gSer) series, a compound containing P1/P1’benzyl and P2/P2’Fmoc substitueras, displayed a significant HIV‐1 Pr inhibition (IC50= 440 nM). © Munksgaard 1997.
Preparation and properties of Nα‐Bpoc‐amino acid pentafluorophenyl esters
Authors: CAREY ROBERT I. , BORDAS LESLIE W. , SLAUGHTER REED A. , MEADOWS BRIAN C. , WADSWORTH JAMES L. , HUANG HAIHONG , SMITH JESSE J. , FURUSJö ERIK
Published: August 2010

The preparation and properties are reported of several Nx‐Bpoc ‐amino acid pentafluorophenyl esters, including those bearing tert‐butyl‐, allyl‐ and trityl‐based protecting groups. These derivatives have been used in the solid‐phase peptide synthesis of sevral short peptides.
in vitro reactivity of the antineoplastic drug carmustin and acrolein with model peptides
Authors: CARBONE VIRGINIA , SALZANO ANNAMARIA , PUCCI PIERO , FIUME IMMA , POCSFALVI GABRIELLA , SANNOLO NICOLA , LANDA GIUSEPPE DI , MALORNI ANTONIO
Published: August 2010

The in vitro interaction of the antineoplastic drug 1, 3‐bis‐(2‐chloroethyl)‐1‐nitrosourea (BCNU) and acrolein with model peptides has been investigated in order to provide a detailed description of their electrophilic reactivity towards biological macromolecules. Following incubation with these substances, the modified species were separated by HPLC and identified by fast atom bombardment mass spectrometry, whereas the reactive amino acids within the peptide structure were assigned by tandem mass spectrometry. Incubation with BCNU led essentially to the formation of an N‐terminal carbamoyl derivative that slowly decomposed to form three isomeric structures and a very minor ethylated adduct. Alkylation with acrolein gave rise to a mixture of different adducts due to the reaction of both the double bond and the carbonyl group. Two species containing intramolecular cross‐links were also observed. These results constitute the prerequisite for in vitro and in vivo studies on the modification of haemoglobin in patients following treatment with antineoplastic drugs. © Munksgaard 1997.
X‐ray studies on crystalline complexes involving amino acids and peptides. XXXII. Effect of chirality on ionisation state, stoichiometry and aggregation in the complexes of oxalic acid with dl‐ and l‐lysine
Authors: VENKATRAMAN JANANI , PRABU MOSES M. , VIJAYAN M.
Published: August 2010

Crystals of the oxalic acid complex of dl‐lysine (triclinic Pl; a= 5.540(1), b= 10.764(2), c= 12.056(2) Å, α= 77.8(1), β= 80.6(1), γ= 75.6(1)°; R = 4.7% for 2023 observed reflections) contain lysine and semioxalate ions in the 1:1 ratio, whereas the ratio of lysine and semioxalate/oxalate ions is 2:3 in the crystals of the l‐lysine complex (monoclinic P21; a= 4.906(1), b= 20.145(4), c= 12.455(1) Å, β= 92.5(1)°; R = 4.4% for 1494 observed reflections). The amino acid molecule in the l‐lysine complex has an unusual ionisation state with positively charged α‐ and side‐chain amino groups and a neutral carboxyl group. The unlike molecules aggregate into separate alternating layers in the dl‐lysine complex in a manner similar to that observed in several of the amino acid complexes. The l‐lysine complex exhibits a new aggregation pattern which cannot be easily explained in terms of planar features, thus emphasizing the fundamental dependence of aggregation on molecular characteristics. Despite the differences in stoichiometry, ionisation state and long‐range aggregation patterns, the basic element of aggregation in the two complexes exhibits considerable similarity.
Destruxin analogs: variations of the α‐hydroxy acid side chain
Authors: CAVELIER FLORINE , JACQUIER ROBERT , MERCADIER JEAN‐LUC , VERDUCCI JEAN , TRARIS MICHEL , VEY ALAIN
Published: August 2010

This work describes the synthesis of three destruxin E cyclodepsipeptidic analogs. These compounds have an identical amino acid sequence but differ by the nature of the hydroxy acid residue which is 2‐hydroxy‐3‐phenylpropionic (Hpp), 2‐hydroxy‐5‐trimethylsilyl‐4‐pentynoic (Hpy‐TMS) and 2‐hydroxy‐4‐pentynoic (Hpy) acid, respectively. The insecticidal properties on the Galleria mellonella larvae (paralysis and lethal effect) of these analogs are presented in comparison with the natural destruxin E. All these compounds have toxic effects, the most potent being Hpy that induces the same effect as destruxin E.
Oxidized and phosphorylated synthetic peptides corresponding to the second and third tubulin‐binding repeats of the τ protein reveal structural features of paired helical filament assembly
Authors: HOFFMANN RALF , DAWSON NICOLA F. , WADE JOHN D. , OTVöS LASZLO
Published: August 2010

The microtubule‐associated protein τ of normal brains is attached to tubulin through its 18‐amino‐acid repeat units. In the paired helical filaments (PHF) of Alzheimer's disease, however, τ is oligomerized in an abnormally hyperphosphorylated form (PHF‐τ). τ contains two cysteine residues in repeat units 2 and 3, but only the R3‐R3 homodimer is present in PHF‐τ. A serine residue two amino acids downstream of the R3 cysteine is a major phosphate acceptor site for protein kinase C. In the work reported here, we used synthetic peptides corresponding to R2, R3 and phosphorylated R3 to determine the binding of the τ repeat peptides to a peptide fragment corresponding to the C‐terminal domain of ß‐tubulin and to study the kinetics of homo‐ and heterodimer formation. Additionally, we studied two major biochemical properties of the peptides that distinguish between normal τ and PHF‐τ: conformation and metabolic stability. All R2 and R3 peptides bound specifically to the tubulin peptide regardless of the state of phosphorylation or dimerization. The reverse‐turn conformation of the τ repeat peptides in the presence of the tubulin peptide remained unaffected. Phosphorylation slightly loosened the turn structure of the monomeric and dimeric peptides, and did not univocally affect the serum stability of the peptides or the ability of the peptides to form dimers. The isolated R2 and R3 units formed homodimers approximately in the same rate. When the two peptides were mixed, however, the R2‐R3 heterodimer was formed preferentially over the homodimers. The dimers were generally more stable in human serum than the monomers. Our results with the synthetic peptide fragments of τ indicate that neither oxidation nor phosphorylation of the repeat units is able to generate extended structure such as that found in PHF‐τ. Additionally, phosphorylation of Ser324 does not appear to modulate the kinetics of oligomerization of τ, and in general biochemistry terms, does not affect disulfide bridge formation nearby. In agreement with studies at the full‐protein level, the formation of homodimers of the peptides, a model of the self‐association of τ, is not preferred. If the dimers are formed, however, their clearance is considerably slower than that of the monomers, explaining the remarkable protease resistance of PHF‐τ in the affected brains. © Munksgaard 1997.
Adrenocorticotropic hormone‐releasing activity of urotensin I and its fragments in vitro
Authors: OHTA NAOSHI , MOCHIZUKI TOHRU , HOSHINO MINORU , JUN LI , KOBAYASHI HIDESHI , YANAIHARA NOBORU
Published: August 2010

Adrenocorticotropic hormone (ACTH)‐releasing activity of synthetic carp Urotensin I (UI) and its ten synthetic fragments were examined using cultured rat pituitary cells. Both UI(1‐41) and rat CRF (rCRF) increased ACTH release in a similar fashion at a concentration range from 10 pm to 100 nm. The potency of UI(1‐41) was about one seventh that of rCRF on a molar basis. Four often UI fragments, UI(1‐36), UI(4‐36), UI(6‐36) and UI(1‐19) showed relatively strong ACTH‐releasing activity, whereas both UI(9–36) and UI(17–36) showed extremely weak ACTH‐releasing activity. However, all these fragments showed the activity in a dose‐dependent manner parallel with that of UI(1‐41). The activity of Ul(1‐36) was weaker than Ul(1–41), suggesting that the C‐terminal 37‐41 sequence is required to express the full ACTH‐release activity, although each of four C‐terminal fragments, UI(24‐36), UI(24‐41), UI(29‐36) and UI(29‐41), exhibited no activity. In summary, the 4‐19 amino acid sequence of UI(is important to exhibit ACTH‐releasing activity and the C‐terminal 37–41 sequence will be necessary to express the full activity.
Aza‐peptides. III. Experimental structural analysis of aza‐alanme and aza‐asparagine‐containing peptides
Authors: ANDRÉ FRÉDÉRIC , VICHERAT ANDRÉ , BOUSSARD GUY , AUBRY ANDRÉ , MARRAUD MICHEL
Published: August 2010

To determine the structural perturbations induced by the CαH→Nα exchange in aza‐peptides, we have examined by H NMR and IR spectroscopy various derivatives of the aza‐analogues of alanine, aspartic acid and asparagine in different organic solvents with increasing polarity. Their general formulas are: R'‐AzXaa‐NR2R3, R'‐Pro‐AzXaa‐NR2R3 and R‐AzXaa‐Pro‐NR2R3 (where AzXaa denotes the aza‐analogue of the amino acid residue Xaa = Ala, Asp, Asn; R = Boc, Z; R2, R3= H, Me, iPr). The aza‐analogue of an amino acid residue appears to be a strong p‐turn‐inducing motif, and the AzAsn carboxamide side‐chain is capable of interacting, as a proton donor, with the preceding peptide carbonyl group.
Esterification of 9‐Fluorenylmethoxycarbonyl‐glycosylated serine and cysteine derivatives with an hydroxymethyl resin
Authors: HARTH‐FRITSCHY ESTELLE , CANTACUZÈNE DANIÈLE
Published: August 2010

Esterification of glycosylated serine and cysteine derivatives with a 4‐alkoxybenzyl alcohol (Wang) resin is described. The classical methods of ester bond formation (symmetrical anhydride, 2‐(1H‐benzotriazol‐1‐yl)‐1,1,3,3‐tetramethyluronium tetrafluoroborate [TBTU]. /4‐dimethylaminopyridine [DMAP]. with or without 1‐hydroxybenzotriazole [HOBT]., pentafluorophenyl [Pfp]. esters) gave high percentages of racemization of the glycosylated serine or cysteine residues. To reduce the d‐amino acid content, we found that the best results were obtained with the highly efficient MSNT reagent (2,4,6‐mesitylenesulfonyl‐3‐nitro‐1,2,4‐triazolide), which gave a high yield of substitution of the resin and the lowest percentage of racemization. A difference in behavior was observed between the two amino acids. The glycosylated cysteine derivative always gave lower racemization than the analogous glycosylated serine.