Biomedical subjects

B M van den Berg

Publications and source records attributed to B M van den Berg.

At least 19 recordsLinked to original sources

PubMed2000

Protection and humoral immune responses against Bordetella pertussis infection in mice immunized with acellular or cellular pertussis immunogens.

In the present study, protection against Bordetella pertussis infection and humoral immunological responses in mice has been assessed upon immunization with custom-made acellular pertussis vaccines (ACVs) and whole-cell pertussis vaccine (WCV). Mice were immunized, next intranasally infected with B. pertussis and during 14 days the number of bacteria in the trachea and lungs and the level of serum antibodies were determined. ACV contained five immunogens, filamentous hemagglutinin, pertactin, fimbriae serotypes 2 and 3, and chemically detoxified pertussis toxin (PMC-5), or three immunogens, filamentous hemagglutinin, pertactin, and genetically detoxified (BC-3) or chemically detoxified pertussis toxin (SKB-3). Immunization with a high or low dose of ACV or WCV resulted in significant protection against B. pertussis, with differences in the degree of protection between the vaccines. The lowest protection was found with a low dose of SKB-3 and WCV. The pattern of cytokine production by spleen cells of immunized, non-infected, mice indicated that T-helper 1 cells are activated by vaccination with WCV, and T-helper 1 and T-helper 2 cells are involved in the immune response upon vaccination with ACVs. Each vaccine stimulated the production of IgG, but not IgA, antibodies. In mice immunized with ACV, elimination of B. pertussis from trachea and lungs correlated significantly with the titre of IgG1, but not IgG2a, antibodies.

Animals↗

PubMed2000

Binding of murine antibodies against whole-cell pertussis vaccine or filamentous haemagglutinin by Bordetella pertussis from patients with whooping cough.

In 1996 an unexpected rise in the incidence of whooping cough occurred in the Netherlands, and antigenic divergence between vaccine strains and clinical isolates has been suggested as a cause for this phenomenon. To investigate this assumption, the binding of murine antibodies against the whole-cell pertussis vaccine or filamentous haemagglutinin, an important protective antigen, to a limited number of Bordetella pertussis strains isolated during different time-periods (1991-92, 1994 and 1996) was assessed. The results showed that all strains were recognized equally well by these antisera, indicating that filamentous haemagglutinin was unchanged during the time-periods examined. Although over the years changes have occurred in at least two surface proteins of B. pertussis, these changes are too subtle to be recognized by the antibodies raised in mice. Further research is required to assess whether antigenic variation of B. pertussis has an effect on protective immunity.

Adhesins, Bacterial↗

PubMed1999

The light chain of factor VIII comprises a binding site for low density lipoprotein receptor-related protein.

In the present study, the interaction between the endocytic receptor low density lipoprotein receptor-related protein (LRP) and coagulation factor VIII (FVIII) was investigated. Using purified components, FVIII was found to bind to LRP in a reversible and dose-dependent manner (K(d) approximately 60 nM). The interaction appeared to be specific because the LRP antagonist receptor-associated protein readily inhibited binding of FVIII to LRP (IC(50) approximately 1 nM). In addition, a 12-fold molar excess of the physiological carrier of FVIII, i.e. von Willebrand factor (vWF), reduced the binding of FVIII to LRP by over 90%. Cellular degradation of (125)I-labeled FVIII by LRP-expressing cells ( approximately 8 fmol/10(5) cells after a 4.5-h incubation) was reduced by approximately 70% in the presence of receptor-associated protein. LRP-directed antibodies inhibited degradation to a similar extent, indicating that LRP indeed contributes to binding and transport of FVIII to the intracellular degradation pathway. Degradation of FVIII was completely inhibited by vWF. Because vWF binding by FVIII involves its light chain, LRP binding to this subunit was studied. In ligand blotting experiments, binding of FVIII light chain to LRP could be visualized. More detailed analysis revealed that FVIII light chain interacts with LRP with moderate affinity (k(on) approximately 5 x 10(4) M(-1) s(-1); k(off) approximately 2.5 x 10(-3) s(-1); K(d) approximately 50 nM). Furthermore, experiments using recombinant FVIII C2 domain showed that this domain contributes to the interaction with LRP. In contrast, no association of FVIII heavy chain to LRP could be detected under the same experimental conditions. Collectively, our data demonstrate that in vitro LRP is able to bind FVIII at the cell surface and to mediate its transport to the intracellular degradation pathway. FVIII-LRP interaction involves the FVIII light chain, and FVIII-vWF complex formation plays a regulatory role in LRP binding. Our findings may explain the beneficial effect of vWF on the in vivo survival of FVIII.

Animals↗

PubMed1999

Serial analysis of gene expression to assess the endothelial cell response to an atherogenic stimulus.

Activation of human, arterial endothelial cells (ECs) is an early event in the pathogenesis of atherosclerosis. To identify the repertoire of genes that are differentially expressed after activation, we used serial analysis of gene expression (SAGE) to compare the mRNA spectrum of quiescent ECs with that of ECs activated for 6h with a strong atherogenic stimulus. SAGE methodology generates concatenated 'tags' of 10bp that are derived from a specific mRNA. About 5% of over 12000 tags analyzed is derived from genes that are differentially expressed (at least 5-fold up- or downregulated). These transcript tags are derived from only 56 genes, close to 1% of the total number of analyzed genes. Among these 56 differentially expressed genes are 42 known genes, including the hallmark endothelial cell activation markers interleukin 8 (IL-8), monocyte chemoattractant protein 1 (MCP-1), vascular cell adhesion molecule 1 (VCAM-1), plasminogen activator inhibitor 1 (PAI-1), Gro-alpha, Gro-beta and E-selectin. Differential transcription of a selection of the upregulated genes was confirmed by Northern blot analysis. A novel observation is the upregulation of activin betaA mRNA, a member of the transforming growth factor beta family. Apparent discrepancies between this novel technology and conventional methods are discussed. In conclusion, we demonstrate that for the application of SAGE, a moderate number of analyzed transcript tags suffices to reveal the significant alterations of EC transcription that results from a strong atherogenic stimulus.

Activins↗

PubMed1999

Activation of complement receptor 3 on human monocytes by cross-linking of very-late antigen-5 is mediated via protein tyrosine kinases.

Bordetella pertussis interacts with very-late antigen-5 (VLA-5) receptors on the human monocyte resulting in cross-linking of these receptors followed by activation of complement receptor 3 (CR3) and firm adhesion of B. pertussis to these monocytes. In the present study we investigated whether protein tyrosine kinases are involved in the activation of CR3 on monocytes, which was assessed by the binding of C3bi-coated erythrocytes (EC3bi). Pre-incubation of monocytes with tyrphostin-A47, a specific protein tyrosine kinase inhibitor, before adherence of the cells to an anti-VLA-5 monoclonal antibody-coated surface, or addition of tyrphostin-A47 within 10 min of the adherence to such surface, reduced the binding of EC3bi to monocytes significantly. Pre-incubation of monocytes with tyrphostin-A47 reduced the binding of B. pertussis to such monocytes as well. Inhibitors of protein kinase A and/or C had no effect on EC3bi binding to monocytes. Cross-linking of VLA-5 on monocytes resulted in tyrosine phosphorylation of several proteins. Together, these results indicate that protein tyrosine kinases are involved in the VLA-5-induced activation of CR3 on human monocytes.

Antibodies, Monoclonal↗

PubMed1999

Role of antibodies against Bordetella pertussis virulence factors in adherence of Bordetella pertussis and Bordetella parapertussis to human bronchial epithelial cells.

Immunization with whole-cell pertussis vaccines (WCV) containing heat-killed Bordetella pertussis cells and with acellular vaccines containing genetically or chemically detoxified pertussis toxin (PT) in combination with filamentous hemagglutinin (FHA), pertactin (Prn), or fimbriae confers protection in humans and animals against B. pertussis infection. In an earlier study we demonstrated that FHA is involved in the adherence of these bacteria to human bronchial epithelial cells. In the present study we investigated whether mouse antibodies directed against B. pertussis FHA, PTg, Prn, and fimbriae, or against two other surface molecules, lipopolysaccharide (LPS) and the 40-kDa outer membrane porin protein (OMP), that are not involved in bacterial adherence, were able to block adherence of B. pertussis and B. parapertussis to human bronchial epithelial cells. All antibodies studied inhibited the adherence of B. pertussis to these epithelial cells and were equally effective in this respect. Only antibodies against LPS and 40-kDa OMP affected the adherence of B. parapertussis to epithelial cells. We conclude that antibodies which recognize surface structures on B. pertussis or on B. parapertussis can inhibit adherence of the bacteria to bronchial epithelial cells, irrespective whether these structures play a role in adherence of the bacteria to these cells.

Adhesins, Bacterial↗

PubMed1999

Role of Bordetella pertussis virulence factors in adherence to epithelial cell lines derived from the human respiratory tract.

During colonization of the respiratory tract by Bordetella pertussis, virulence factors contribute to adherence of the bacterium to the respiratory tract epithelium. In the present study, we examined the roles of the virulence factors filamentous hemagglutinin (FHA), fimbriae, pertactin (Prn), and pertussis toxin (PT) in the adherence of B. pertussis to cells of the human bronchial epithelial cell line NCI-H292 and of the laryngeal epithelial cell line HEp-2. Using B. pertussis mutant strains and purified FHA, fimbriae, Prn, and PT, we demonstrated that both fimbriae and FHA are involved in the adhesion of B. pertussis to laryngeal epithelial cells, whereas only FHA is involved in the adherence to bronchial epithelial cells. For PT and Prn, no role as adhesion factor was found. However, purified PT bound to both bronchial and laryngeal cells and as such reduced the adherence of B. pertussis to these cells. These data may imply that fimbriae play a role in infection of only the laryngeal mucosa, while FHA is the major factor in colonization of the entire respiratory tract.

Adhesins, Bacterial↗

PubMed1998

Isoelectric focusing in the vegetable seed industry.

Different types of electrophoretic procedures were developed for hybrid purity testing based on starch gel electrophoresis (SGE), vertical polyacrylamide gel electrophoresis (PAGE), and isoelectric focusing (IEF). For the most important vegetables these methods are much faster than plant grow-outs and relatively inexpensive. Compared to SGE and PAGE methods, horizontal IEF proved to be more efficient for large-scale hybrid purity testing. These developments were made possible by the basic work of Harry Rilbe and improvements that were initiated as a result of Rilbe's work. The present paper describes a number of milestones during this developmental period, starting with the isoelectric focusing concept of Harry Rilbe up to the large-scale application of IEF. Further, a comparison of IEF with DNA fingerprinting methods along with the future of both techniques is discussed with respect to hybrid purity testing in the vegetable seed industry. When it comes to a choice between the use of either IEF or a DNA-based method, efficiency and efficacy determines the method which is best suited for hybrid purity testing. It is also concluded that in the future we will see an increased use of both IEF as well as DNA-based methods for hybrid purity testing because expectations of growers has increased; consequently they will accept fewer inbreds in a hybrid variety, especially when growing in a greenhouse.

Industry↗

PubMed1998

Plasminogen activator inhibitor 1 contains a cryptic high affinity receptor binding site that is exposed upon complex formation with tissue-type plasminogen activator.

The low density lipoprotein receptor-related protein (LRP), a multi-functional endocytic receptor, mediates the cellular internalization of tissue-type (t-PA) and urokinase-type (u-PA) plasminogen activator and their complexes with plasminogen activator inhibitor type 1 (PAI-1). LRP preferentially binds the complexed forms, exemplified by equilibrium dissociation constants (KD) that are at least an order of magnitude lower than those of the free components. To understand the molecular interactions, underlying the preference of the receptor for complexes rather than for the free components, we have performed a detailed analysis of the affinity and kinetics of the binding of PAI-1 and t-PA:PAI-1 complexes to the receptor, using surface plasmon resonance. To assess the involvement of the heparin-binding domain of PAI-1 for the interaction with LRP, we determined the equilibrium dissociation constants for the binding to LRP of a panel of PAI-1 mutants with single- and multiple amino-acid substitutions of the basic residues that constitute the heparin binding site of PAI-1 (K65, K69, R76, K80 and K88). The binding of these PAI-1 mutants was partially reduced with a 2 to 4 fold increase in KD values for single (K80, K88) and combined (K80, 88) substitution mutant proteins respectively. LRP binding of complexes, composed of t-PA with either wild type PAI-1 or any one of the single PAI-1 mutants indicated a major role of lysine 69 (K69) for the binding of t-PA:PAI-1 complexes to LRP (KD values of 6.1, 3.7. 75.4, 5.4, 12.5 and 8.1 nM for wild type, K65A, K69A, R76A, K80A and K88A complexes, respectively). Since the KD for the binding of free t-PA to LRP is 158 nM, we conclude that the PAI-1 moiety harbors the major determinant for t-PA:PAI-1 complex binding to LRP. The in vitro binding studies were extended by binding and clearance studies with COS-1 cells. Degradation of both 125I-t-PA:PAI-1 K69A and 125I-t-PA:PAI-1 K69A K80A K88A complexes after 2 h of incubation was reduced compared to the degradation of 125I-t-PA:PAI-1 complexes. We conclude that PAI-1 contains a cryptic binding site (lysine 69) for LRP, that is specifically expressed upon t-PA:PAI-1 complex formation.

Amino Acid Substitution↗

PubMed1997

Molecular analysis of ligand binding to the second cluster of complement-type repeats of the low density lipoprotein receptor-related protein. Evidence for an allosteric component in receptor-associated protein-mediated inhibition of ligand binding.

The low density lipoprotein receptor-related protein (LRP), a member of the low density lipoprotein receptor gene family, mediates the cellular uptake of a diversity of ligands. A folding chaperone, the 39-kDa receptor-associated protein (RAP) that resides in the early compartments of the secretory pathway inhibits the binding of all ligands to the receptor and may serve to prevent premature binding of ligands to the receptor during the trafficking to the cell surface. To elucidate the molecular interactions that underlie the interplay between the receptor, RAP, and the ligands, we have analyzed and delineated the binding sites of plasminogen activator inhibitor-1 (PAI-1), tissue-type plasminogen activator (t-PA).PAI-1 complexes, RAP, and the anti-LRP Fab fragment Fab A8. To that end, we have generated a series of soluble recombinant fragments spanning the second cluster of complement-type repeats (C3-C10) and the amino-terminal flanking epidermal growth factor repeat (E4) of LRP (E4-C10; amino acids 787-1165). All fragments were expressed by stably transfected baby hamster kidney cells and purified by affinity chromatography. A detailed study of ligand binding to the fragments using surface plasmon resonance revealed the presence of three distinct, Ca2+-dependent ligand binding sites in the cluster II domain (Cl-II) of LRP. t-PA.PAI-1 complexes as well as PAI-1 bind to a domain located in the amino-terminal portion of Cl-II, spanning repeats E4-C3-C7. Adjacent to this site and partially overlapping is a high affinity RAP-binding site located on repeats C5-C7. Fab A8, a pseudo-ligand of the receptor, binds to a third Ca2+-dependent binding site on repeats C8-C10 at the carboxyl-terminal end of Cl-II. Next, we studied the RAP-mediated inhibition of ligand binding to LRP and to Cl-II. As expected, we observed a strong inhibition of t-PA.PAI-1 complex and Fab A8 binding to LRP by RAP (IC50 congruent with 0.3 nM), whereas in the reverse experiment, competition of t-PA. PAI-1 complexes and Fab A8 for RAP binding to LRP could only be shown at high concentrations of competitors (>/=1 microM). Interestingly, even though the equilibrium dissociation constants for the binding of RAP to LRP and to Cl-II are similar, the binding of the ligands to Cl-II is only prevented by RAP at concentrations that are at least 2 orders of magnitude higher than those required for inhibition of ligand binding to LRP. Our results favor models that propose RAP-induced allosteric inhibition of ligand binding to LRP that may require LRP moieties that are located outside Cl-II of the receptor.

Animals↗

PubMed1997

Horizontal ultrathin-layer multi-zonal electrophoresis of DNA: an efficient tool for large-scale polymerase chain reaction (PCR) fragment analysis.

A rapid and efficient procedure for large-scale analysis of polymerase chain reaction (PCR) fragments in the range of 200-3000 base pairs is presented. The procedure is based on horizontal ultrathin-layer multi-zonal (HUME) electrophoresis of PCR fragments in polyacrylamide gels followed by silver staining. HUME gels can be prepared rapidly using a simple procedure called the flap technique. The electrophoretic set-up allows the use of multi-channel pipettes for sample loading. Separation and detection of the PCR fragments from sample preparation to silver staining can be carried out in 2 h. Using four electrophoresis units, one technician can analyze 400 PCR fragment samples in 2 h.

DNA Fragmentation↗

PubMed1996

State of the art of large-scale genetic purity testing of hybrid vegetable seeds using isoelectric focusing at PetoSluis.

During the past ten years we have been engaged in developing and applying isoelectric focusing techniques to test the genetic quality of vegetable seeds. We started with isoelectric focusing using carrier ampholytes (IEF-CA), and continued research with isoelectric focusing in immobilized pH gradients (IEF-IPG) and two-dimensional electrophoresis (IPG-DALT). In addition, we have developed equipment and procedures for large-scale seed and seedling homogenization, sample preparation and semi-automatic gel staining. Moreover, we have optimized the sample application and gel running setup for large-scale analysis. We have developed hybrid purity (inbred) testing methods for all important vegetables, e.g., melon, cole crops, tomato, pepper, watermelon, squash, cucumber, radish etc. using either IEF-CA or IEF-IPG of seed or seedling proteins, followed by specific or general protein staining. To indicate the efficiency of the equipment and procedures developed we present results of two of our hybrid purity test methods, namely for brassica using polymorphism for phosphoglucomutase (PGM) from dry seeds, and for tomato using polymorphism for alcohol dehydrogenase (ADH) from imbibed seeds. We show that one person can routinely analyze 1536 individual seeds per day at a cost of about US +0.11 per seed for chemicals, materials, and electrophoresis equipment.

Alcohol Dehydrogenase↗

PubMed1995

Identification of functional interaction sites on proteins using bacteriophage-displayed random epitope libraries.

We describe a phage-display-based method to identify epitopes or interaction sites on proteins. DNA encoding the protein of interest is partially degraded with DNase I to generate random fragments of 50-200 bp. These fragments are then cloned into a phagemid vector that has been modified to allow the expression of the random fragments and the construction of a (bacterio)phage-displayed random epitope library. Phages displaying functional epitopes can be selected from these libraries by affinity selection or panning. To test this method we have constructed a random-epitope library for human plasminogen-activator inhibitor 1 and used this library to map the epitope of a monoclonal antibody (mAb) directed against this protein. By alignment of the selected overlapping epitope-containing fragments, we were able to locate the epitope of the mAb on a stretch of 39 amino acids spanning from E128 to V166. The approach may also be applied to more complex systems than single-protein genes, such as viral genomes or complete cDNA libraries.

Amino Acid Sequence↗

PubMed1995

Binding of FimD on Bordetella pertussis to very late antigen-5 on monocytes activates complement receptor type 3 via protein tyrosine kinases.

Nonopsonized Bordetella pertussis bind to human monocytes by means of the virulence factors filamentous hemagglutinin (FHA), pertactin, and the minor fimbrial subunit FimD. Receptors on monocytes that mediate binding of B. pertussis to these cells include complement receptor type 3 (CR3), which binds to FHA of B. pertussis, and very late antigen-5 (VLA-5), which binds to an, as yet, unknown ligand on these bacteria. In the present study, the possibility that FimD acts as a ligand for VLA-5 was investigated. Soluble fibronectin, which is the natural ligand for VLA-5, or mAbs against VLA-5 inhibited binding to monocytes of B. pertussis strains that express FimD but not of mutant strains that lack FimD. Beads that were coated with the fusion protein maltose-binding protein-FimD bound to adherent monocytes, and this binding was inhibited by soluble fibronectin or mAb against the alpha- or beta-chain of VLA-5, while soluble collagen or mAb against VLA-4, VLA-6, CR3, or HLA class II had no effect. Down-modulation of VLA-5 on the apical surface of monocytes by plating the cells onto surfaces precoated with anti-VLA-5 mAb also inhibited binding of beads coated with maltose-binding protein-FimD to monocytes, while precoating of the surfaces with mAb against VLA-6 or CR3 had no effect. These results indicate that VLA-5 on monocytes serves as a receptor for FimD on B. pertussis. Binding of C3bi-coated erythrocytes to monocytes, which is a measure of the binding activity of CR3, was enhanced when monocytes were adhered onto plates precoated with purified fimbriae of B. pertussis, while precoating with fimbriae lacking FimD had no effect. Precoating of the plates with FimD-containing fimbriae also enhanced binding of B. pertussis, which express FHA, but not of strains that lack FHA, to monocytes. The enhanced binding of C3bi-coated erythrocytes and B. pertussis to monocytes could be markedly inhibited by tyrphostin-47, a protein tyrosine kinase inhibitor. These results demonstrate that interaction of FimD of B. pertussis with VLA-5 on monocytes activates CR3, which requires protein tyrosine kinases and results in enhanced binding of B. pertussis to the latter receptor via FHA.

Bacterial Adhesion↗

PubMed1995

Analysis of the binding of pro-urokinase and urokinase-plasminogen activator inhibitor-1 complex to the low density lipoprotein receptor-related protein using a Fab fragment selected from a phage-displayed Fab library.

The low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor (LRP) mediates endocytosis of a number of structurally unrelated ligands, including complexes of plasminogen activator inhibitor type 1 (PAI-1) and tissue-type plasminogen activator (t-PA) or urokinase plasminogen activator (u-PA), free t-PA, single-chain urokinase (pro-u-PA), alpha 2-macroglobulin-protease complexes, and lipoprotein lipase. So far, all ligands have in common the fact that they bind to the receptor in a Ca(2+)-dependent way and the fact that binding to the receptor can be inhibited by a 39-40-kDa protein, termed the receptor-associated protein. To obtain inhibitory antibodies for the analysis of the structure and function of the receptor we applied the combinatorial immunoglobulin repertoire cloning technique in order to specifically select monoclonal Fab fragments directed against Ca(2+)-dependent epitopes. In this report we describe the isolation of a Fab fragment (Fab A8) showing a high relative affinity for the receptor (0.5 nM). The binding of this Fab fragment to purified LRP is inhibited in the presence of 5 mM EDTA, receptor-associated protein, and lipoprotein lipase (IC50 values of 1.4 and 31 nM, respectively). By immunoblotting of CNBr-digested LRP it is shown that Fab A8 binds to a fragment that harbors the second cluster of cysteine-rich complement-type repeats flanked by epidermal growth factor repeats. Binding studies using 125I-labeled ligands and immobilized receptor show that Fab A8 partially inhibits the binding of [125I]u-PA.PAI-1 complexes (IC50 = 1.1 nM) and completely inhibits the binding of [125I]pro-u-PA to the receptor (IC50 = 2.2 nM). No inhibition was observed for the binding of 125I-labeled methylamine-activated alpha 2-macroglobulin or [125I]t-PA.PAI-1 to LRP. Degradation of [125I]u-PA.PAI-1 complexes by COS-1 cells was also partially (43%) inhibited by Fab A8. Our results provide evidence for the presence of an interaction site for pro-u-PA localized in the second cluster of cysteine-rich repeats that is unrelated to the t-PA.PAI-1 or methylamine-activated alpha 2-macroglobulin interaction sites.

Amino Acid Sequence↗

PubMed1995

Association of protein polymorphism among pepper (Capsicum annuum L.) inbred lines with agronomic performance of their crosses.

The genetic variation of 10 morphologically similar pepper (Capsicum annuum L.) inbred lines has been analyzed by two-dimensional electrophoresis with immobilized pH gradient (IPG-DALT) of seed proteins. For all pairs of inbred lines genetic distance indices were calculated on the basis of the IPG-DALT analysis and a genetic tree was constructed. The protein polymorphism data of the 10 inbred lines were integrated into a pepper breeding program to validate the assumption that there is a higher chance of achieving better hybrid performance when the genetic distance between the parents is as great as possible. Field trials were performed in Turkey and consisted of a total of 27 crossings based on 9 inbred lines. Fifteen hybrids exceeded the crop yield of the better parent and 8 additional hybrids exceeded the mean crop yield of both parents. The genetic distance indices of the parental inbred lines based on protein polymorphism data were not significantly correlated to heterosis performance of the experimental hybrids (r = 0.20), indicating that protein polymorphism data add little to the prediction of single cross hybrid performance. Nevertheless, protein polymorphism data support the breeder in being more effective in finding out the best inbred combinations, because most of the less promising crossings can be omitted from the expensive and time-consuming performance tests. Those hybrids whose distance indices exceeded the mean of all 27 distance values (0.52) were superior to the mean value of all heterosis performances by 19%.(ABSTRACT TRUNCATED AT 250 WORDS)

Capsicum↗

PubMed1995

Genetic variability of carrot seed proteins analyzed by one- and two-dimensional electrophoresis with immobilized pH gradients.

The genetic variability of eight carrot populations representing commercially important morphological types was analyzed for seed protein polymorphism by one- and two-dimensional electrophoresis using immobilized pH gradients (IPG) in order to assess the potential of both techniques in carrot hybrid breeding as well as genetic quality testing of carrot trade seeds. The digitized silver-stained one- and two-dimensional gels of the urea/detergent-soluble seed proteins were subjected to computer analysis to estimate both the inter- and intrapopulation genetic variability. The results indicated that isoelectric focusing (IEF-IPG) of seed proteins is a powerful tool for quick screening of the genetic variability present between and within carrot populations, while two-dimensional polyacrylamide gel electrophoresis (IPG-DALT) can be employed to establish detailed genetic relationships of carrot populations selected by initial IEF-IPG screening in order to use genetic distant and homogeneous populations for successful carrot hybrid breeding. However, the applicability of IEF-IPG in genetic quality testing of carrot seeds seems to be restricted to hybrid purity testing. IPG-DALT is a far more powerful tool to profile unambiguously all carrot varieties commercially used in order to test the genetic quality of trade seeds, because this technique exhibits protein spots or spot combinations exclusively found in each of the eight varieties investigated.

Daucus carota↗

PubMed1995

Bordetella pertussis fimbriae bind to human monocytes via the minor fimbrial subunit FimD.

Nonopsonized Bordetella pertussis can bind to and become ingested by human monocytes. Previous studies demonstrated that mutant B. pertussis strains that lack fimbriae express a reduced adherence to monocytes. The present study was undertaken to investigate the involvement of the minor fimbrial subunit FimD in the adherence of B. pertussis to human monocytes using purified fimbriae, FimD, and strains with mutations in fimbrial genes. Flow cytometry demonstrated that purified B. pertussis fimbriae avidly bind to monocytes in a dose-dependent manner but bind less to neutrophils and hardly to lymphocytes; FimD-lacking fimbriae did not bind to monocytes. Purified fimbriae or FimD inhibited the binding of wild-type B. pertussis to monocytes in a dose-dependent manner and similarly inhibited the binding of a mutant defective in the major fimbrial subunits. It did not affect the binding of strains defective in FimD. These results prove that B. pertussis bind to human monocytes via the fimbrial minor subunit FimD.

Bacterial Adhesion↗