Similar Antigenic Properties Research Articles

In-silico formulation of a next-generation polyvalent vaccine against multiple strains of monkeypox virus and other related poxviruses.

Mpox (formerly known as monkeypox) virus and some related poxviruses including smallpox virus pose a significant threat to public health, and effective prevention and treatment strategies are needed. This study utilized a reverse vaccinology approach to retrieve conserved epitopes for monkeypox virus and construct a vaccine that could provide cross-protection against related viruses with similar antigenic properties. The selected virulent proteins of monkeypox virus, MPXVgp165, and Virion core protein P4a, were subjected to epitope mapping for vaccine construction. Two vaccines were constructed using selected T cell epitopes and B cell epitopes with PADRE and human beta-defensins adjuvants conjugated in the vaccine sequence. Both constructs were found to be highly antigenic, non-allergenic, nontoxic, and soluble, suggesting their potential to generate an adequate immune response and be safe for humans. Vaccine construct 1 was selected for molecular dynamic simulation studies. The simulation studies revealed that the TLR8-vaccine complex was more stable than the TLR3-vaccine complex. The lower RMSD and RMSF values of the TLR8 bound vaccine compared to the TLR3 bound vaccine suggested better stability and consistency of hydrogen bonds. The Rg values of the vaccine chain bound to TLR8 indicated overall stability, whereas the vaccine chain bound to TLR3 showed deviations throughout the simulation. These results suggest that the constructed vaccine could be a potential preventive measure against monkeypox and related viruses however, further experimental validation is required to confirm these findings.

Does the Inclusion of CD34+ B-cell Progenitors (Hematogones) in Stem Cell Enumeration of Apheresis Product Using ISHAGE Protocol Affect the Final Harvest dose Adequacy and the Outcome of Transplantation? A Single Institution Experience from Southern India.

Hematogones have similar antigenic and light scatter properties when compared to CD34+ hematopoietic stem cells (HSC) but they form a separate cluster with dimmer CD45 expression. These should be excluded while enumerating HSC, as their inclusion can overestimate and hence affect the final dose of HSC. However, their exact impact on the outcome of HSC transplant (HSCT) is not entirely known and hence this study was undertaken to address these issues, if any. This was a retrospective study in which patients undergoing HSCT were included, and flow cytometric enumeration was done on the apheresis product using single platform ISHAGE protocol. The gating of all plots was reviewed and carefully studied for hematogone population which would have otherwise been included in the original gating. Totally 78 patients underwent HSCT during the study period. On re-analysis, it was found that 10/78 (12.8%) cases had a separate hematogone population which was included in the HSC in the original analysis. Out of these 10 cases, 7/51 and 3/27 were in autologous and allogenic subgroup respectively. However, all the ten cases ultimately had adequate final stem cell dose and had successful engraftment. The inclusion of hematogones in CD34+ HSC enumeration of apheresis products did not yield any impact on neither the final dose nor the outcome of transplant in this study. However, it is recommended to exclude them from the final count when they are > 10% of the final HSC lest it overestimate the final harvest dose and outcome of HSCT.

Development of mutant human immunoreactive trypsinogen 1 (IRT1) and mutant human immunoreactive trypsinogen 2 (IRT2) for use in immunoassays

Immunoreactive Trypsinogen (IRT) is a protein-based pancreatic proenzyme that has an important role in protein digestion in humans. In human body, once IRT present in the small intestine, the proteolytic cleavage activates trypsinogen into trypsin. When IRT is in the active form, it is capable to cleave antibodies, other proteins and even itself while it is desired to use in immunoassays. According to the literature, there are three important IRT isoforms called Immunoreactive Trypsinogen 1 (IRT1), Immunoreactive Trypsinogen 2 (IRT2), and Immunoreactive Trypsinogen 3 (IRT3). However, trypsinogen 1 (cationic trypsinogen, IRT1) and trypsinogen 2 (anionic trypsinogen, IRT2) are the major isoforms in human pancreatic juice and used in the diagnosis of cystic fibrosis (CF). In this study, it is aimed to restrain its proteolytic activity with K23D mutation, which changes lysine (K) residue at the 23rd position to aspartic acid (D). Because we wanted to produce a hassle-free human recombinant immune reactive trypsinogen proenzyme which has similar antigenic properties with the native form. It is also aimed that the mutant IRTs do not exhibit proteolytic activity for the development of durable detection kits with a longer shelf life for both two isoforms. The innovation was actualized in order to use IRTs as a standard antigen in Immunoassays such as ELISA kits. The gene was synthesized as mutated and expressed in P. pastoris X-33 strain. The loss of proteolytic activity has been proven with the BAEE test. Antigenic properties of K23D IRTs and the effect of proteolytic inactivation on their performance in immunoassays were assessed with ELISA and Western Blot. In ELISA results K23D mutated IRTs showed higher signals than Wild-Type forms.

Hemagglutinin-specific neutralization of subacute sclerosing panencephalitis viruses.

Subacute sclerosing panencephalitis (SSPE) is a progressive, lethal complication of measles caused by particular mutants of measles virus (MeV) that persist in the brain despite high levels of neutralizing antibodies. We addressed the hypothesis that antigenic drift is involved in the pathogenetic mechanism of SSPE by analyzing antigenic alterations in the MeV envelope hemagglutinin protein (MeV-H) found in patients with SSPE in relation to major circulating MeV genotypes. To this aim, we obtained cDNA for the MeV-H gene from tissue taken at brain autopsy from 3 deceased persons with SSPE who had short (3–4 months, SMa79), average (3.5 years, SMa84), and long (18 years, SMa94) disease courses. Recombinant MeVs with a substituted MeV-H gene were generated by a reverse genetic system. Virus neutralization assays with a panel of anti-MeV-H murine monoclonal antibodies (mAbs) or vaccine-immunized mouse anti-MeV-H polyclonal sera were performed to determine the antigenic relatedness. Functional and receptor-binding analysis of the SSPE MeV-H showed activity in a SLAM/nectin-4–dependent manner. Similar to our panel of wild-type viruses, our SSPE viruses showed an altered antigenic profile. Genotypes A, G3, and F (SSPE case SMa79) were the exception, with an intact antigenic structure. Genotypes D7 and F (SSPE SMa79) showed enhanced neutralization by mAbs targeting antigenic site IIa. Genotypes H1 and the recently reported D4.2 were the most antigenically altered genotypes. Epitope mapping of neutralizing mAbs BH015 and BH130 reveal a new antigenic site on MeV-H, which we designated Φ for its intermediate position between previously defined antigenic sites Ia and Ib. We conclude that SSPE-causing viruses show similar antigenic properties to currently circulating MeV genotypes. The absence of a direct correlation between antigenic changes and predisposition of a certain genotype to cause SSPE does not lend support to the proposed antigenic drift as a pathogenetic mechanism in SSPE.

Thyroid peroxidase (TPO) expressed in thyroid and breast tissues shows similar antigenic properties.

BackgroundThyroid peroxidase (TPO) is essential for physiological function of the thyroid gland. The high prevalence of thyroid peroxidase antibodies (TPOAbs) in patients with breast cancer and their protective role had previously been demonstrated, indicating a link between breast cancer and thyroid autoimmunity. Recently, TPO was shown to be present in breast cancer tissue samples but its antigenicity has not been analyzed.MethodsIn this study, we investigated TPO expression levels in a series of fifty-six breast cancer samples paired with normal (peri-tumoral) tissue and its antigenic activity using a panel of well-characterized murine anti-human TPOAbs.ResultsWe have shown that TPO transcripts were present in both normal and cancer tissue samples, although the amounts in the latter were reduced. Additionally, we observed that TPO levels are lower in more advanced cancers. TPO protein expression was confirmed in all tissue samples, both normal and cancerous. We also found that the antigenicity of the immunodominant regions (IDRs) in breast TPO resembles that of thyroid TPO, which is crucial for effective interactions with human TPOAbs.ConclusionsExpression of TPO in breast cancer together with its antigenic activity may have beneficial effects in TPOAb-positive breast cancer patients. However, further studies are needed to confirm the beneficial role of TPOAbs and to better understand the underlying mechanism.

Altered specificity of single-chain antibody fragments bound to pandemic H1N1-2009 influenza virus after conversion of the phage-bound to the soluble form

BackgroundIn 2009, a novel influenza A/H1N1 virus (H1N1pdm) quickly spread worldwide and co-circulated with then-existing seasonal H1N1 virus (sH1N1). Distinguishing between these 2 viruses was necessary to better characterize the epidemiological properties of the emergent virus, including transmission patterns, pathogenesis, and anti-influenza drug resistance. This situation prompted us to develop a point-of-care virus differentiation system before entering the 2009–2010 influenza season. Aiming to establish H1N1pdm-specific detection tools rapidly, we employed phage display libraries to select H1N1pdm-specific single-chain variable fragments (scFvs).FindingsHuman single-fold scFv libraries (Tomlinson I + J) underwent selection for the ability to bind H1N1pdm virus particles. Three rounds of panning brought 1152 phage-bound scFvs, of which 58 clones reacted with H1N1pdm specifically or preferentially over sH1N1 in an enzyme-linked immunosorbent assay (ELISA). After conversion of the scFvs to soluble form, 7 clones demonstrating high/stable expression were finally obtained. However, all the soluble scFvs except No. 29 were found to have lost their specificity/preference for H1N1pdm in ELISA. The specificity/preference of No. 29 was also confirmed by immunofluorescence assay and immunoprecipitation, and the viral nucleoprotein was identified by ELISA as its target protein. The change in specificity associated with scFv conversion from phage-bound to soluble form could be due to loss of phage scaffold pIII protein, which likely provides structural support for the scFv antigen-binding site. It is also possible that the similar antigenic properties of H1N1pdm and sH1N1 led to the observed alterations in scFv specificity.DiscussionUsing a phage display library, we obtained 7 soluble scFv clones reactive against H1N1pdm; however, only 1 showed specificity/preference toward H1N1pdm. Our results confirmed that using phage display libraries was highly advantageous for the rapid development of molecules to detect target antigens. However, our results also indicated that this strategy might not have been effective for selecting H1N1pdm-specific antibodies during the 2009 pandemic, where the co-circulating sH1N1 virus shared similar antigenic properties. This suggests that it might be advisable to use a synthetic scFv phage display library by strategically considering the characteristics of target antigens and the potential situations.

Stonefish antivenom neutralises the inflammatory and cardiovascular effects induced by scorpionfish Scorpaena plumieri venom

Venomous fish are often involved in human accidents and symptoms of envenomation include local (intense pain and swelling) and systemic effects (cardiovascular and neurological disorders). However the only commercially available antivenom is against the Indo-Pacific stonefish Synanceja trachynisStonefish Antivenom (SFAV). The aim of the present study was to evaluate the potential of SFAV in neutralising the in vivo effects of some toxic activities of scorpionfish Scorpaena plumieri venom (SpV), and the in vitro immuno cross-reactivity. The SpV (7.5–100 μg/animal) caused nociceptive and dose-dependent edematogenic responses in the mice footpad. In rats SpV (300 μg/kg, i.v.) produced immediate and transient increase in arterial blood pressure and decrease in heart rate. Prior incubation of SpV with SFAV (1 μg SpV/1 U SFAV) abolished the inflammatory response, and significantly attenuated the cardiovascular effects induced by SPV. Western blotting analysis on two-dimensional SDS-PAGE of S plumieri venom proteins using SFAV proved that the epitopes recognized by SFAV are shared with the ∼98 kDa proteins. This is the first report of venom similarities between Indo-Pacific and Atlantic venomous fish, suggesting that the SpV compound responsible for inflammatory and cardiovascular effects possesses similar biochemical and antigenic properties to those found in stonefish venom.

Conversion of MDCK cell line to suspension culture by transfecting with human siat7e gene and its application for influenza virus production

MDCK cells are currently being considered as an alternative to embryonated eggs for influenza virus propagation and hemagglutinin (HA) production intended for vaccine manufacturing. MDCK cells were found suitable for the virus production but their inability to grow in suspension burdens the process of scale up and hence their production capability. Anchorage-dependent MDCK cells were converted to anchorage-independent cells, capable of growing in suspension as a result of transfection with the human siat7e gene (ST6GalNac V). This gene was previously identified as having an important role in cellular adhesion when the transcriptions of genes from anchorage-dependent and anchorage-independent HeLa cells were compared. Unlike the parental MDCK cells, the siat7e-expressing cells were capable of growing in shake flasks as suspension cultures, achieving maximum concentration of 7 x 10(5) cells/mL while keeping close to 100% viability throughout the growth phase. In production experiments, the siat7e-expressing cells were infected with the Influenza B/Victoria/504/2000 strain. It was determined that the cell-derived viruses retained similar antigenic properties as those obtained from egg-derived viruses and their nucleotide sequences were identical. The specific production of hemagglutinin (expressed in hemagglutination units per 10(6) cells) from the siat7e-expressing cells was approximately 20 times higher than the specific production from the parental MDCK cells. If this suspension process scales up, the production potential of HA from 10 L of siat7e-expressing cells at a concentration of 10(6) cells/mL would be equivalent to the amount of HA obtained from 10,000 embryonated eggs.

Expression of HPV-11 L1 protein in transgenic Arabidopsis thaliana and Nicotiana tabacum

BackgroundWe have investigated the possibility and feasibility of producing the HPV-11 L1 major capsid protein in transgenic Arabidopsis thaliana ecotype Columbia and Nicotiana tabacum cv. Xanthi as potential sources for an inexpensive subunit vaccine.ResultsTransformation of plants was only achieved with the HPV-11 L1 gene with the C-terminal nuclear localization signal (NLS-) encoding region removed, and not with the full-length gene. The HPV-11 L1 NLS- gene was stably integrated and inherited through several generations of transgenic plants. Plant-derived HPV-11 L1 protein was capable of assembling into virus-like particles (VLPs), although resulting particles displayed a pleomorphic phenotype. Neutralising monoclonal antibodies binding both surface-linear and conformation-specific epitopes bound the A. thaliana-derived particles and – to a lesser degree – the N. tabacum-derived particles, suggesting that plant-derived and insect cell-derived VLPs displayed similar antigenic properties. Yields of up to 12 μg/g of HPV-11 L1 NLS- protein were harvested from transgenic A. thaliana plants, and 2 μg/g from N. tabacum plants – a significant increase over previous efforts. Immunization of New Zealand white rabbits with ~50 μg of plant-derived HPV-11 L1 NLS- protein induced an antibody response that predominantly recognized insect cell-produced HPV-11 L1 NLS- and not NLS+ VLPs. Evaluation of the same sera concluded that none of them were able to neutralise pseudovirion in vitro.ConclusionWe expressed the wild-type HPV-11 L1 NLS- gene in two different plant species and increased yields of HPV-11 L1 protein by between 500 and 1000-fold compared to previous reports. Inoculation of rabbits with extracts from both plant types resulted in a weak immune response, and antisera neither reacted with native HPV-11 L1 VLPs, nor did they neutralise HPV-11 pseudovirion infectivity. This has important and potentially negative implications for the production of HPV-11 vaccines in plants.

Detection and Identification of Salmonella Typhimurium in Bovine Diarrhoeic Fecal Samples by Immunomagnetic Separation and Multiplex PCR Assay

The aim of this study was to use the immunomagnetic separation (IMS) test plus a multiplex polymerase chain reaction (m-PCR) assay to detect Salmonella at genus level and also for the identification of Salmonella enterica serovar Typhimurium in bovine diarrhoeic fecal samples. In all, 400 bovine diarrhoeic fecal specimens were examined by conventional bacterial culture, IMS, and m-PCR. For m-PCR assay, four set primers were selected: 139-141, specific for inv-A gene of Salmonella spp and the RfbJ, FliC and FljB, specific for the rfbJ, FliC and fljB genes of Salmonella Typhimurium or other Salmonella serovars with similar antigenic properties. Thirty-three (8.25%) out of the 400 fecal samples were culture positive for Salmonella serovars. Of these, 66.7% (22 of 33) were Salmonella enterica serovar Typhimurium, and 9.1% (three of 33) were serovar Dublin. In the IMS + m-PCR, four amplified product (663, 526, 284 and 183 bp) were found in all specimens that had serovar Typhimurium (4,5,12:i:1,2), they corresponded, respectively, to the rfbJ, fljB, inv-A and Flic genes of this serovar. In serovar Dublin (1,9,12:g,p:-), Georgia (6,7:b:e,n,z(15)) and, Enteritidis (1,9,12;g,m:-) only one PCR product (284 bp) was amplified from the inv-A gene. In serovars Augustenborg (6,7:i:1,2) and Lindenburg (6,8:i:1,2) three positive bands (526, 284 and 183 bp) were amplified corresponding to the fljB, inv-A and Flic genes, respectively. In serovar Virchow (6,7:r:1,2) two amplified products (284 and 526 bp) from the inv-A and FliC genes were observed. In serovar Gloucster (1,4,12(27):i:1,w) three fragments (183, 284 and 663) from the FliC, inv-A and, rfbJ genes respectively, were observed. In the positive control as expected, four PCR products were amplified corresponding to the FliC, inv-A, fljB and rfbJ genes, respectively. In conclusion, the results of this study showed that detection of Salmonella at genus level with universal ST139-141 primers and identification of Salmonella Typhimurium by using specific primers of O4, H(2):1, 2 and H(1) antigens can potentially permit to more readily evaluate fecal and other types of samples for the presence of these organisms. Compared to bacteriological culture the combination of IMS and m-PCR resulted a faster method for the detection and identification of Salmonella at genus and serovar level by using of universal and specific primers.

Analysis of the serotype and genotype correlation of VP1 and the 5' noncoding region in an epidemiological survey of the human enterovirus B species.

The sequence identity of the enterovirus VP1 gene has been shown to correlate with the serotype concept. Enterovirus molecular typing methods are therefore often based on sequencing of the VP1 genomic region and monophyletic clustering of VP1 sequences of a homologous serotype. For epidemiological surveillance, 342 enterovirus samples obtained from patients with aseptic meningitis in Belgium from 1999 to 2002 were first diagnosed as being enterovirus positive by amplification of the 5' noncoding region (5'NCR) by reverse transcription (RT)-PCR. Subsequently, samples were molecularly typed by RT-nested PCR amplification and sequencing of a portion of the VP1 gene. Phylogenetic analyses were performed to investigate enteroviral evolution and to examine the serotype and genotype correlation of the two genomic regions. Our typing results demonstrated echovirus 30, echovirus 13, echovirus 18, and echovirus 6 to be the most predominant types. Echoviruses 13 and 18 were considered to be emerging human serotypes since 2000 and 2001, respectively, as they had been rarely reported before. Several serotypes existed as multiple genotypes (subtypes) from 1999 to 2002, but genomic differences mainly resided at synonymous sites; these results strongly suggest that the subtypes exhibit similar antigenic properties. Phylogenetic analyses confirmed that VP1 is an adequate region for molecular typing. Serotype-specific clusters are not observed commonly in phylogenetic trees based on the 5'NCR, and the phylogenetic signal in the 5'NCR was found to be particularly low. However, some substructure in the 5'NCR tree made a tentative prediction of the enterovirus type possible and was therefore helpful in PCR strategies for VP1 (e.g., primer choice), provided some background knowledge on the local spectrum of enteroviruses already exists.

Isolation and Characterization of Glycophorin from Nucleated (Chicken) Erythrocytes

A sialoglycoprotein fraction was isolated from chicken erythrocytes by two methods based on the phenol extraction or chloroform/2-propanol extraction of differently prepared erythrocyte membranes. Both preparations gave in SDS–PAGE two major PAS-stained bands (GP2 and GP3), which migrated as 60- and 33-kDa species, respectively, compared to reference proteins, or as 44- and 23-kDa molecules, compared to human glycophorins. Some less abundant slower migrating PAS-stained components, antigenically related to GP2 and GP3, also were detected. No evidence for the presence of antigenically distinct glycoproteins of leukosialin type was obtained. Interconversion in SDS–PAGE, similar carbohydrate composition, and similar antigenic properties of GP2 and GP3 indicated that they are a dimer and monomer, respectively, of the same glycoprotein which shows properties that allow it to be classified as a glycophorin. Lectin binding studies and methylation analysis of β-elimination products of chicken glycophorin preparation showed the presence of O-glycans and N-glycans. The major O-glycans include sialylated Galβ1–3GalNAc units and more complex GlcNAc-containing chains. Among the N-glycans, there are complex-type biantennary structures with a bisecting GlcNAc residue, accompanied by chains with additional antennas linked to α-mannose residues. A characteristic feature of the chicken glycophorin is a relatively high proportion of N-glycans to O-glycans, compared to the glycophorin A from human erythrocytes.

Black Fire Ant ( Solenopsis Richteri ) Sting Producing Anaphylaxis: A Report Of 10 Cases from Najran

Black Fire Ant ( <i>Solenopsis Richteri</i> ) Sting Producing Anaphylaxis: A Report Of 10 Cases from Najran

Detection of an antibothropic fraction in opossum ( Didelphis marsupialis) milk that neutralizes Bothrops jararaca venom

An antibothropic fraction (ABF) from Didelphis marsupialis (opossum) serum, which is responsible for the neutralization of Bothrops jararaca venom was isolated by Perales et al. [Perales, J., Moussatché, H., Marangoni, S., Oliveira, B. and Domont, G. B. (1994). Isolation and partial characterization of an antibothropic complex from the serum of South American Didelphidae. Toxicon 32, 1237–1249]. The aim of this work was to verify the presence of this factor in opossum's milk, which could represent an additional protection for the neonatal opossum against bothropic venoms. An active milk fraction was isolated and showed similar physicochemical, structural, antigenic and biological properties when compared to ABF, indicating that they are probably the same protein.

Isolation and Comparison of Natural and Recombinant Human CENP-A Autoantigen

Anticentromere antibodies (ACA) are associated with systemic sclerosis (scleroderma) patients exhibiting the more benign or so called limited manifestation of the disease (lSSc). ACA reactivity is directed against multiple polypeptide targets, the smallest of which is designated CENP-A. CENP-A is not an abundant cellular constituent; therefore to maximize recovery, we developed a protocol with a minimum of steps to isolate CENP-A from a human cell line. The trace cellular amount of this protein clearly dictated the production of its recombinant counterpart to facilitate determination of the role of the CENP-A antigen in scleroderma pathogenesis. Here we describe the eukaryotic expression of CENP-A cDNA using baculovirus-mediated infection of insect cells. The non-fusion recombinant protein spans the natural residues of the human CENP-A protein and rCENP-A followed the same chromotographic sequence for purification as did the natural source. The availability of thebona fideantigen provided a critical standard upon which to document authenticity of the recombinant polypeptide. The two forms of this antigen have been compared and shown to exhibit similar physical and antigenic properties.

Human Phospholipase D1 Can Be Tyrosine-phosphorylated in HL-60 Granulocytes

The human phospholipase D1 (hPLD1) has recently been cloned. Although recent data have implicated PLD in receptor-stimulated secretion, the regulation of the activity of PLD enzymes remains to be clarified. Purified hPLD1 is activated by several cytosolic cofactors among which are protein kinase Calpha, ARF, and RhoA. In human granulocytes, a strong correlation between tyrosine phosphorylation of proteins and PLD activity has been established. In this study, the presence of hPLD1 in HL-60 granulocytes and its phosphorylation on tyrosine residues have been studied. We generated antipeptide antibodies (Abs) specific for hPLD1 but not PLD2 as shown by Western blotting (WB) of recombinant PLD1 and PLD2. These Abs identified the presence of hPLD1 in HL-60 cells with the bulk of it being detected in the membranes and only a minor fraction in the cytosol. The hPLD1 Abs detected a major band at 120 kDa (PLD1a) and a minor band at 115 kDa (PLD1b). The specificity of the Abs was confirmed using PLD antisera neutralized with the immunizing peptides. The two forms of hPLD1 were consistently detected by immunoprecipitation under nondenaturing and denaturing conditions following a WB analysis with hPLD1 Abs. Following exposure of HL-60 cells to peroxides of vanadate (V4+-OOH), an inhibitor of tyrosine phosphatases, hPLD1 was immunoprecipitated under nondenaturing conditions from HL-60 cell lysates and assayed for tyrosine phosphorylation by WB. hPLD1 comigrated with a 120-kDa tyrosine phosphorylated protein by gel electrophoresis. Other tyrosine-phosphorylated peptides of 160, 140, 135, 90, and 75-80 kDa were also detected in hPLD1 immune complexes. hPLD1 and the associated tyrosine-phosphorylated proteins were not immunoprecipitated by neutralized hPLD1 Abs. Using denaturing conditions, the PLD immunoprecipitates were sequentially immunoblotted with anti-PLD1 and anti-phosphotyrosine Abs. PLD1a and PLD1b were detected, and the major PLD1a protein was superimposable with a major tyrosine-phosphorylated protein detected at 120 kDa. Conversely, PLD1a and PLD1b were recovered, at least in part, in the anti-phosphotyrosine immunoprecipitates. These results provide evidence that two PLD1 forms are expressed in human granulocytes. Furthermore, in response to stimulation by V4+-OOH, PLD1 was tyrosine-phosphorylated and associated with several, presently undefined, tyrosine-phosphorylated proteins.

Topology of Kell blood group protein and the expression of multiple antigens by transfected cells

Kell is one of the major blood group systems in human red blood cells (RBCs). The Kell antigens are carried on a 731 amino acid glycoprotein that is thought to span the erythrocyte membrane once. Rabbit antibodies to three synthetic peptides, derived from different parts of the Kell protein, were used to determine the topology of Kell protein on the RBC. Antibodies to a C-terminal peptide and to a peptide derived from amino acid residues 410 to 439 reacted with RBCs treated with 0.2 mol/L dithiothreitol. An antibody to the N-terminal peptide reacted with inside-out RBC vesicles but not with right-side-out vesicles nor with intact RBCs, showing that Kell is a type II membrane protein and that the extracellular portion of the protein is folded by disulfide bonds. By transfection, Kell protein was expressed on the cell surface of surrogate cells, and the transfected cells expressed similar antigenic properties as native RBCs. Kell protein was expressed in COS- 1 and K562 cells and in Sf9 cells infected by the Baculovirus system. Transfected K562 cells expressed several high-incidence antigens but not the low-incidence antigen K1.

Topology of Kell Blood Group Protein and the Expression of Multiple Antigens by Transfected Cells

Kell is one of the major blood group systems in human red blood cells (RBCs). The Kell antigens are carried on a 731 amino acid glycoprotein that is thought to span the erythrocyte membrane once. Rabbit antibodies to three synthetic peptides, derived from different parts of the Kell protein, were used to determine the topology of Kell protein on the RBC. Antibodies to a C-terminal peptide and to a peptide derived from amino acid residues 410 to 439 reacted with RBCs treated with 0.2 mol/L dithiothreitol. An antibody to the N-terminal peptide reacted with inside-out RBC vesicles but not with right-side-out vesicles nor with intact RBCs, showing that Kell is a type II membrane protein and that the extracellular portion of the protein is folded by disulfide bonds. By transfection, Kell protein was expressed on the cell surface of surrogate cells, and the transfected cells expressed similar antigenic properties as native RBCs. Kell protein was expressed in COS-1 and K562 cells and in Sf9 cells infected by the Baculovirus system. Transfected K562 cells expressed several high-incidence antigens but not the low-incidence antigen K1.

The mitochondrial ATP / ADP carrier: Interaction with detergents and purification by a novel procedure

The interaction of several classes of detergents with mitochondrial ATP / ADP carrier (AAC) was studied. The detergents that were best suited for solubilization of active AAC differed in several physico-chemical properties, but contained relatively rigid or planar hydrophobic (sub)moieties. Based on specific binding of AAC to Blue Sepharose, a novel method for the purification of the AAC was developed. The new method gave AAC samples which were devoid of non-essential lipids and allowed to purify AAC isoenzymes from several species and tissues to a significantly higher degree of purity than that achieved up to now. Western blot analysis of purified AACs with an antiserum against chicken heart AAC confirmed that immunological variability is more important between tissues than between species. In contrast to liver and kidney AACs, brain AAC displayed similar antigenic properties to heart AAC.

Canine-coronavirus (CCV) characterization in Spain. Epidemiological aspects.

In this paper the characterization of a canine-coronavirus (CCV) strain isolated in Spain is reported. The CCV cellular-infection cycle on A-72 cells was studied using electron-microscopy techniques. The isolate was found to have similar electrophoretic profile and antigenic properties to an American reference strain. Sera samples were obtained from several canine populations and tested for viral antibodies using two immunoenzymatic methods. Sera specificity was confirmed using immunoblot analysis. The agreement between both ELISA tests produced adequate results (kappa value = 0.64).