Characterisation of the 5 kDa growth hormone isoform

Objectives: The 5 kDa N-terminal fragment of 43 amino acids of human growth hormone (GH) shows a specific and significant in-vivo insulin-like activity. This isoform can be easily obtained by solid phase synthesis methods. Our objective in this study is to describe this procedure in detail and to provide structural information of the protein.

Methods: Solid phase synthesis was employed for the synthesis of the 5 kDa GH isoform. Circular dichroism and limited proteolysis have been carried out to provide structural information about the folded state of the protein in solution. Surface plasmon resonance was used to compare the structural equivalence between the synthetic protein and a proteolytic homologue at an antibody binding level. For this purpose, a murine monoclonal antibody specific for the 5 kDa isoform was generated and characterised employing this and several other GH isoforms.

Results: Circular dichroism and proteolysis results suggested that the C-terminal segment of the 5 kDa protein folds in an α-helix. The comparison of the synthetic product to its proteolytic homologue at an antibody binding level suggested structural equivalency. A highly specific antibody against the 5 kDa GH isoform was generated with null cross-reactivity for 17, 20 and 22 kDa isoforms. Kinetic data on the interaction with the synthetic 5 kDa GH was obtained.

Conclusions: The structure of the protein appears to be different in comparison to when it is included within the 22 kDa GH isoform. Finally, a highly specific antibody has been generated. The possible significance of the 5 kDa protein as a potential agent for obesity-related diseases is discussed.     

Strong association of HLA-B27 heavy chain with β2-microglobulin

Monoclonal antibody TG1 recognizes specifically antigens HLA-B27, B7, B22 and B17 on cell surface in cytotoxicity and cytofluorometry tests. When cell detergent extracts were subjected to SDS PAGE under mild conditions (no heating and no reduction of the sample) followed by Western blotting, TG1 detected exclusively a complex of B27 heavy chains with β₂-microglobulin (as a 50 kDa band) whereas the other B-locus antigens (B7, B22, B17) were detected as free 43 kDa heavy chains under the same conditions. When the samples were boiled prior to SDS PAGE, TG1 detected again the 43 kDa free heavy chains of B7, B22 and B17 but no zone corresponding to B27 could be detected indicating that the epitope in free B27 chains is more sensitive to denaturation by SDS. Thus, our main finding is that the interaction of HLA-B27 heavy chain with β₂-microglobulin appears to be stronger than that of the other HLA-B chains. The resistance of the HLA-B27/β₂-microglobulin complex to the SDS dissociation is strikingly similar to the behavior of MHC class II molecules under similar conditions. Thus, it may be speculated that HLA-B27 complexes can be also more stable than other MHC class I molecules under more physiological dissociative conditions (e.g. in endosomal compartments). This feature might potentially influence antigen presentation by HLA-B27 and contribute to the well known disease linkage of HLA-B27.     

A component of an antigenic rhoptry complex of Plasmodium falciparum is modified after merozoite invasion

Human antibodies affinity purified on an adsorbent prepared from a cDNA clone (Ag44) expressing a portion of a rhoptry antigen were used to characterize the synthesis and fate of the antigen in the asexual blood stages of Plasmodium falcipartum. The rhoptry antigen is synthesized in the mature trophozoite-stage parasites as a 103 kDa polypeptide, is present in the schizonts and merozoites as a 105 kDa polypeptide, is discharged from the rhoptries and found in the newly invaded red cells as a 110 kDa polypeptide. Anti-Ag44 antibodies immunoprecipitate the antigen and two additional polypeptides of 135 and 150 kDa from lysates of infected cells and from culture supernatants. The three polypeptides are associated in a non-covalent complex that persists in the newly invaded red cells. All the components of the high molecular weight rhoptry complex are antigenic and can be precipitated with immune human serum. The 135 kDa polypeptide is identical to a 140 kDa rhoptry antigen previously identified by a monoclonal antibody.     

Characterization of IgE-binding epitopes on Candida albicans enolase

Candida albicans enolase is one of the important allergens in Candida allergy. We isolated and purified 46 kDa C. albicans enolase (CAE) from C. albicans and characterized epitopes for IgE antibody by lectin-blotting and enzymatic digestion followed by sodium dodecyl sulfale polyacrylamide gel electrophoresis (SDS-PAGE) and immunobiotting. Lectin blotting and deglycozilation indicated that this protein did not contain polysaccharide side chains. The purified CAE and recombinant fusion protein produced from CAE gene possessed common epitopes for IgE antibody. We estimated IgE binding epitopes on the basis of reported amino acid sequences from the analysis of cDNA encoding CAE. V8 protease digestion of CAE gave six polypeptide fragments (A-F). The N-termini of each fragment were confirmed by amino acid sequence and the C-termini were estimated by molecular weights of each fragment and the specific cutting site of V8 protease. Fragment C (25.0 kDa; F-171-I-399) reacted to 90% IgE antibodies examined, whereas fragments D (21.0 kDa; F-171-1-360), E (16.2kDa: F-171-D-317) and F (13.0kDa; A-47-E-170) showed no IgE binding. Our results suggest that epitopes for IgE antibodies exist near the C-terminal of the protein.     

Immune Response to Natural and Recombinant Antigens of Helicobacter pylori in Patients with Dyspeptic Complaints

 Sera of 223 dyspeptic patients with endoscopic findings of nonulcer dyspepsia (72%), gastric ulcer (15%) and duodenal ulcer (13%) were tested for antibodies against Helicobacter pylori with an enzyme immunoassay and an immunoblot technique using lysates of Helicobacter pylori cells as antigen source. One hundred and fifty-one (68%) sera were found to be positive for Helicobacter pylori IgG with both methods; 5% of the positive results in the enzyme immunoassay were false-positive due to cross-reactions mainly of proteins with a molecular mass of 43–66 kDa. Since cross-reactivity not only reduces the diagnostic value of the immunoassay but also complicates evaluation of the immunoblot results, an attempt was made to overcome these problems by using specific purified recombinant proteins instead of the crude cell preparations as antigens. Of the commonly recognised immunogens of Helicobacter pylori, antibodies against a cell surface protein of 26 kDa, the small urease subunit (29 kDa) and the cytotoxin-associated protein (130 kDa) were identified as highly sensitive serological markers for inclusion in a recombinant antigen mixture for Helicobacter pylori screening. Only the cytotoxinassociated protein was confirmed to be an indicator immunogen for ulcerogenic strains. To assess the reliability of recombinant fragments of this protein in serological screening, the reactivity of antibody to purified fragments of the cytotoxin-associated protein was compared with that to the natural protein. A C-terminal recombinant fragment of 58 kDa showed results identical to those obtained with the natural protein and was thus considered to be an appropriate component of an antigen mixture for serological detection of Helicobacter pylori.     

Preparation of F(ab′)2 fragments from monoclonal mouse IgG1 suitable for use in radioimaging

Monoclonal antibodies of IgG1 subclass raised against purified human prostate-specific acid phosphatase were subjected to different procedures to produce F(ab′)₂ fragments suitable for radioimaging of prostatic cancer, following derivatization and labeling with radionuclides. The main aim was to obtain highly purified fragments with preserved immunological activity. Optimized pepsin digestion led to the formation of mainly F(ab′)₂ and Fab fragments, and, following Sephacryl S-200 gel filtration, the yield of pure F(ab′)₂ fragments was 24 ± 11% of the theoretical maximum. After papain digestion in the absence of thiols, no formation of Fab fragments was observed, and the F(ab′)₂ fragments formed could be efficiently separated from Fc fragments by chromatofocusing or ion exchange chromatography. The yield of F(ab′)₂ fragments from papain digestion was 50 ± 5% of the theoretical maximum. Both the above procedures gave F(ab′)₂ fragments with immunoreactivity and affinity identical to those of the precursor IgG1, despite the fact that isoelectric focusing profiles of the two F(ab′)₂ preparations differed, suggesting different digestion sites.     

Effect of synthetic peptide fragments of β2-glycoprotein-I on the binding of antiphospholipid antibodies to cardiolipin-glycoprotein-I on the binding of antiphospholipid antibodies to cardiolipin

The synthetic fragment of β₂-glycoprotein-I peptide P5 (Phe280-Ser289) maximally inhibits the binding of anticardiolipin antibodies to cardiolipin. This β₂-glycoprotein-I sequence probably reacts with negatively charged phospholipids and antibodies. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 127, No. 1, pp. 50–52, January, 1999     

Comparison of the ability of wild type and stabilized human IgG4 to undergo Fab arm exchange with endogenous IgG4 in vitro and in vivo

Fab arm exchange by a stabilized anti-IL-31 IgG₄S228P monoclonal antibody (mAb) was studied using physiologically relevant antibody concentrations and thiol exchange conditions, and directly compared to that of matched wild type IgG₄ (IgG₄wt) and IgG₁ control antibodies. In vitro arm exchange between the test mAbs and a purified IgG₄wt exchange partner was monitored using capillary isoelectric focusing and a size-exclusion peak shift assay. Arm exchange between the test mAbs and IgG exchange partners with unknown specificity was monitored using only the shift assay. Studies were performed using single isotype human and mouse mAbs, unfractionated human, mouse, and cynomolgus monkey IgG, and human serum as the sources of the exchange partners. In vitro studies using human serum demonstrated that anti-IL-31 IgG₄S228P did not undergo significant Fab arm exchange with endogenous human IgG₄ whereas anti-IL-31 IgG₄wt underwent rapid and extensive Fab arm exchange. The in vitro results were corroborated by in vivo studies in which mice were injected with a mixture of either form of the test mAb and an excess of non-specific human IgG₄ exchange partner.     

Production of the matrix protein of Nipah virus in Escherichia coli: Virus-like particles and possible application for diagnosis

The broad species tropism of Nipah virus (NiV) coupled with its high pathogenicity demand a rapid search for a new biomarker candidate for diagnosis. The matrix (M) protein was expressed in Escherichia coli and purified using a Ni-NTA affinity column chromatography and sucrose density gradient centrifugation. The recombinant M protein with the molecular mass (M_r) of about 43 kDa was detected by anti-NiV serum and anti-myc antibody. About 50% of the M protein was found to be soluble and localized in cytoplasm when the cells were grown at 30 °C. Electron microscopic analysis showed that the purified M protein assembled into spherical particles of different sizes with diameters ranging from 20 to 50 nm. The purified M protein showed significant reactivity with the swine sera collected during the NiV outbreak, demonstrating its potential as a diagnostic reagent.     

T cell recognition pattern of bovine milk αS1-casein and its peptides

T cell recognition patterns of CAS1_Bovin, its limited hydrolysis, oxidized, reduced/alkylated, cyanogen bromide cleavage fractions and synthetic peptides were examined. Thirteen overlapping peptides covering the intact molecule, with chain lengths varied between 17 and 20 AA, were prepared by f-moc SPPS. In addition, six CNBr-cleavage fragments were obtained and extensively purified using RP/HPLC. Likewise, chemically modified derivatives and limited pepsin hydrolysate, were performed and the specificities were confirmed. Stimulation of PBMC and TCL cultures by the intact CAS1_Bovin molecule, synthetic peptides and modified derivatives were screened by [methyl-³H] thymidine incorporations. PBMC phenotype was performed by flow cytometry and the mean CD4⁺/CD8⁺ ratio of freshly prepared PBMC was compared with the ratio following specific CAS1_Bovin stimulation. CD4⁺ phenotypes (T_H1/, T_H2 and T_H0) were assigned by assay of four marker cytokines IL-4, IL-6, IL-10 and IFN-γ.Five CNBr fragments and seven of the thirteen tested peptides were recognized by specific TCL. The most reactive epitopes of CAS1_Bovin comprised seven motifs namely: peptides Cas 1–18, Cas 16–35, Cas 67–85, Cas 91–110, Cas 136–155, Cas 152–169 and Cas 166–183. The stimulation range for the seven peptides was 1058–2383 cpm. Stimulation for the CNBr fragments were, respectively, 8670, 5808, 3324, 5465, 2255 and 321 cpm. Cytokine assay showed that CD4⁺ T_H2 phenotype was dominant for half the number of patients, while T_H1 solely or combined T_H0 were represented in the other four cell culture filtrates.The T cell reactive epitopes described and their antibodies will be useful tools for methods in progress for the detection of masked casein epitopes encompassed in processed food.In conclusion, T cell recognition pattern of CAS1_Bovin was examined using extensively purified synthetic peptides and CNBr fragments. Five large and seven small peptides were clearly recognized. Peptides of chain length less than six AA were left unrecognised. CD4⁺ T_H2 phenotype was the most dominant TCL subpopulations found in atopic patients while CD4⁺ T_H1 was representative in the non-IgE mediated type IV hypersensitivity.     

Sensitive ELISA for Determination of Serum E2 Using a New Tracer E2-Biotin

Abstract

A new tracer conjugate of E₂-Biotin, with different spacers, was synthesized at position 3 in the estradiol molecule for first time. Immunoreactivity of the tracer was determined by reacting with the anti-E₂ monoclonal antibody. The monoclonal antibodies raised against E₂ were characterized for its use in ELISA detection systems of serum E₂. The purified antibody has a high affinity and specificity for E₂. The antibody and tracer were used for establishing a competitive ELISA for estradiol (E₂). The experimental results showed that the dose-response curve of the assay covered a range of 33–20,000 pg/mL (n = 8). The detection limit is 28.3 pg/mL (S/N = 3). The intra- and inter-assay coefficients of variation for the assay of serum samples ranged from 5.7 to 13.2% and from 5.3 to 10.6%, respectively. Precoated microtiter plates were dried at 4°C and they were stable for up to 3 months.     

Effect of hypothermia on renal sodium reabsorption

Summary The relationship between sodium reabsorption and oxygen consumption was studied in an isolated rabbit kidney preparation perfused with blood at 37, 28 and 19° C. When the temperature was lowered from 37° C to 28° C and to 19°C the rate of oxygen consumption and of the maximal P.A.H. excretion (Tm P.A.H.) decreased more than that of sodium reabsorption.TheQ ₁₀ for sodium reabsorption is about 1.8, while that for maximal P.A.H. excretion is 2.5. Some hypothesis on the possible mechanisms of the lowQ ₁₀ of the Na⁺ reabsorption are forwarded.Preliminary reports have been published [Boll. Soc. Ital. Biol. Sper.43, 1019–1023 (1966) and44, 1784–1787 (1967);45, 860–862 (1969) and45, 863–865 (1969)].     

Niflumic acid reduces the hyperpolarization-activated current (Ih) in rod photoreceptor cells

We examined the effects of niflumic acid (NFA), a chloride channel blocker, on the hyperpolarization-activated current (I_h) in newt rod photoreceptors. At 100 μM, NFA delayed the activation of I_h induced by hyperpolarizing voltage pulses to −83 mV from a holding potential of −43 mV, and reduced the steady-state current. However, reduction by NFA was weakened when I_h was activated by hyperpolarizing steps to −123 mV, suggesting that these effects were voltage-dependent. The suppressive effects of NFA on I_h were accompanied by a negative shift in activation voltage. NFA also delayed the relaxation of I_h tail currents, showing that this drug also inhibited deactivation of the current. The reversal potential and the fully activated conductance were not affected. These observations suggest that NFA reduces I_h by modifying the gating kinetics of the underlying channels. The suppressive actions of NFA remained when intracellular Ca²⁺ was strongly chelated, and the failure of suppression by NFA in inside-out patches suggests that the agent may act on the I_h channel from the extracellular side. These results, obtained in rod photoreceptors, are consistent with similar effects of NFA on I_f in cardiac myocytes, suggesting that both currents share similar pharmacological properties.     

Polyclonal anti-histamine H<Subscript>2</Subscript> receptor antibodies detect differential expression of H<Subscript>2</Subscript> receptor protein in primary vascular cell types

Objective and Design:One of the factors defining cellular response might be the distribution and density of receptor subtypes on cell membranes. It was our aim to quantify and compare histamine H₂ receptor expression in primary vascular cell types. We have therefore generated antibodies directed against the second extra-cellular loop of the H₂ receptor.Methods:The specificity of polyclonal anti-H₂ receptor antibodies designed for this purpose was examined by Western blot analysis and immunohistochemistry. H₂ receptor expression was quantified by ELISA. Regulation of H₂ receptor gene expression was analyzed by competitive RT-PCR.Results:Our results indicate that the polyclonal antibodies specifically interact with the histamine H₂ receptor. Furthermore, utilizing these antibodies we were able to show significant differences in H₂ receptor levels in human umbilical arterial and vein endothelial cells as well as smooth muscle cells.Conclusions:We conclude that the antibodies generated against the extra-cellular domain of the H₂ receptor are specific and can be utilized to detect and quantify H₂ receptor expression. Furthermore, the significant differences in H₂ receptor expression in different vascular cell types might play a critical role in defining histamine induced cellular responses during physiological or pathophysiological processes.Received 25 August 2003; returned for revision 10 October 2003; accepted by A. Falus 22 December 2003     

Vλ-light chain genes reconstitute immune responses to defined carbohydrate antigens or haptens by utilizing different VH genes

The contribution of the λ-light chain to the development of peripheral B cell repertoire and generation of specific antibodies to haptens and polysaccharide antigens was studied in genetically manipulated kappa-deficient and λ₂-transgenic mice. The results clearly demonstrate a non-stoichiometric V_H gene family expression in the absence of k-light chain and suggest a non-stochastic pairing between V_H and V_λ genes, expressed in the peripheral B cell repertoire. A shift in V_H gene utilization in the case of Vl_λ⁺ antibodies was evident in response to β2–6 fructosan and TNP hapten. These observations demonstrate the availability of compensatory mechanisms in the absence of V_K genes and are consistent with the hypothesis that V_H gene family expression is controlled by genetic factors from inside the V_H locus. Furthermore, genetic factors from outside the V_H locus, namely restricted available light chain diversity, may lead to a shift in V_H gene utilization in the peripheral B cell repertoire.     

Significant association of interleukin 8 −251T/A polymorphism with smoking behavior in a Japanese population

Accumulating evidence indicates that the genotype may impact on smoking behavior and a deeper understanding of the molecular basis could lead to more effective strategies for preventing initiation of the habit and to help smokers to quit. Since individual variation in airway responsiveness to cigarette smoke might have an important influence, we have focused on associations between smoking behavior and polymorphisms affecting the inflammatory cytokine, IL-8. In the present study, 453 Japanese non-cancer outpatients (191 males and 262 females) who visited Aichi Cancer Center Hospital were genotyped for the IL8 –251T/A polymorphism, and age- and sex-adjusted odds ratios (aORs) for smoking were estimated using a logistic regression model. The aORs for IL8 251-TA and AA combined, genotypes associated with high production of IL-8, were 0.52 (95% CI 0.33–0.82, P=0.004) for ever having smoked and 0.55 (0.33–0.92, P=0.023) for being a current smoker. Our results suggest that the inflammatory-prone genotype of IL8 may act to deter initiation or characteristics of the smoking habit.     

Differential distribution of γ-aminobutyric acidA, receptor subunit (α1, α2, α3, α5 and β2+3) immunoreactivity in the medial prefrontal cortex of the rat

A detailed mapping of the γ-aminobutyric acid (GABA)_A receptor subunits (α₁, α₂, α₃ and β₂₊₃) in the infralimbic/ventral prelimbic region (IL/vPL) of the rat frontal cortex was carried out using subunit-specific antibodies. The α₁ and β₂₊₃ subunit antibodies immunostained all layers of the IL/vPL region. Layers II and III displayed immunostaining of cell bodies whereas I, V and VI showed predominantly neuropil staining. The size of the α₁-positive cell bodies corresponded to that of small interneurons (range, 20–55 μm²; mean ± SEM, 37 ± 5.5 μm²) as well as pyramidal cells or large interneurons (range, 87–135 μm²; mean ± SEM, 103.4 ± 9.7 μm²). However, β₂₊₃ antibody immunostained only small cell bodies. Immunoreactivity for α₂ was restricted to layers I and II, whereas α₃ and α₅ subunit expression was seen only in layer VI. The antibody to the α₂ subunit immunostained small cell bodies (range, 29–63 μm²; mean ± SEM, 32 ± 4.5 μm²) in layer II, resembling interneurons. Conversely, both α₃ and α₅ antibodies immunostained large cell bodies (range, 94–151 gmm²; mean ± SEM, 115.7 ± 13.4 μm²), consistent with pyramidal cell labelling in layer VI.     

Positive regulator for the expression of Mba protein of the virulence plasmid, pKDSC50, of Salmonella choleraesuis

A positive regulator was identified within a 2.3 kb fragment of the 6.4 kb mouse bacteremia region (mba region) of the virulence pKDSC50 plasmid of Salmonella choleraesuis. Sodium dodecylsulphate polyacrylamide gel electrophoresis showed that Escherichia coli K-12 carrying the recombinant plasmids of the 2.3 kb fragment produced Mba1 protein with a molecular mass of 32 kDa. The recombinant plasmids carrying a 4.1 kb fragment, the other part of 6.4 kb region, produced Mba2 (32 kDa), Mba3 (70 kDa) and Mba4 (29 kDa) proteins. All three proteins were expressed by using the lacZ promoter under isopropyl thiogalactoside induction. In contrast to this, Mba3 protein was overexpressed independently of the lacZ promoter when the 2.3 kb fragment coexisted either in cis or trans. These results suggest that Mba1 is a trans-acting positive regulator for the expression of the Mba3 protein of mba region of pKDSC50.     

Kinetic analysis of a human chorionic gonadotropin-β epitope–paratope interaction

Kinetics of protein–protein or ligand–ligate interaction has predominantly been studied by optical spectroscopy (particularly fluorescence) and surface plasmon resonance biosensors. Almost all such studies are based on association kinetics between ligand–ligate and suffer from certain methodological and interpretational limitations. Therefore, kinetic analyses of dissociation data of such interactions become indispensable. In the present investigation, the radiolabeled human chorionic gonadotropin-β (¹²⁵IhCGβ) was employed as a probe and nitrocellulose (NC) as a solid support to immobilize monoclonal antibody (MAb) G₁G₁₀.1. The NC–G₁G₁₀.1–¹²⁵IhCGβ complex (NC_com) was prepared and the dissociation of radiolabeled hCGβ was carried out in the presence of excess unlabeled ligate. From the experimental dissociation data under varying ionic strength, dissociation constants (k_{? 1}), association constants (k₊₁) and affinity constants (k_a) were calculated. The values obtained were utilized in exploring the amino acid residues constituting an epitopic region of hCGβ involved in interaction with the complementary paratope on MAb G₁G₁₀.1. Kinetic data of the present study supported our recently published findings [using single step-solid phase radioimmunoassay (SS-SPRIA)] that the core region of hCGβ epitope consists of Arg (94,95) and Asp (99) while a Lys (104) and a His (106) are in proximity to the core epitopic region. Based on the results of present investigation, we conclude that dissociation kinetics coupled with SS-SPRIA unequivocally provides considerable insight into the study of ligand–ligate interactions and epitope analysis.