Functional expression of CCR1, CCR3, CCR4, and CXCR4 chemokine receptors on human platelets

Platelets are known to contain platelet factor 4 and beta-thromboglobulin, alpha-chemokines containing the CXC motif, but recent studies extended the range to the beta-family characterized by the CC motif, including RANTES and Gro-alpha. There is also evidence for expression of chemokine receptors CCR4 and CXCR4 in platelets. This study shows that platelets have functional CCR1, CCR3, CCR4, and CXCR4 chemokine receptors. Polymerase chain reaction detected chemokine receptor messenger RNA in platelet RNA. CCR1, CCR3, and especially CCR4 gave strong signals; CXCR1 and CXCR4 were weakly positive. Flow cytometry with specific antibodies showed the presence of a clear signal for CXCR4 and weak signals for CCR1 and CCR3, whereas CXCR1, CXCR2, CXCR3, and CCR5 were all negative. Immunoprecipitation and Western blotting with polyclonal antibodies to cytoplasmic peptides clearly showed the presence of CCR1 and CCR4 in platelets in amounts comparable to monocytes and CCR4 transfected cells, respectively. Chemokines specific for these receptors, including monocyte chemotactic protein 1, macrophage inflammatory peptide 1alpha, eotaxin, RANTES, TARC, macrophage-derived chemokine, and stromal cell-derived factor 1, activate platelets to give Ca(++) signals, aggregation, and release of granule contents. Platelet aggregation was dependent on release of adenosine diphosphate (ADP) and its interaction with platelet ADP receptors. Part, but not all, of the Ca(++) signal was due to ADP release feeding back to its receptors. Platelet activation also involved heparan or chondroitin sulfate associated with the platelet surface and was inhibited by cleavage of these glycosaminoglycans or by heparin or low molecular weight heparin. These platelet receptors may be involved in inflammatory or allergic responses or in platelet activation in human immunodeficiency virus infection.

Aberrant low expression level of bovine beta-lactoglobulin is associated with a C to A transversion in the BLG promoter region

Beta-lactoglobulin (beta-LG) is the major whey protein in cow's milk. It is well established that the predominant 2 genetic variants, beta-LG A and B, are differentially expressed. Extensive investigation of the genetic variation in the promoter region of the BLG gene revealed the existence of specific haplotypes associated with the A and B variants, respectively. However, the genetic basis for the differential expression of BLG A and B alleles is still elusive. We have previously reported a quantitative beta-LG B variant, characterized by a very low beta-LG protein expression level. Here, we report that the corresponding BLG allele (BLG B*) shows a correspondingly low mRNA expression level. Comparative DNA sequencing of 7,670 bp of the BLG B* allele and the established BLG B allele revealed a unique difference of a C to A transversion at position 215 bp upstream of the translation initiation site (g.-215C>A). This mutation segregated perfectly with the differential phenotypic expression in a paternal half-sib family and could be confirmed in 2 independent cases. The sequence of the BLG B allele in the region of the mutation is highly conserved among 4 related ruminant species. The site of the mutation corresponds to a putative consensus-binding sequence for the transcription factors c-Rel and Elk-1 as predicted by searching the TRANSFAC database. The beta-LG B* site might be relevant in the natural production of milk of low beta-LG content.

Amino acid sequence of the alpha subunit and computer modelling of the alpha and beta subunits of echicetin from the venom of Echis carinatus (saw-scaled viper)

Echicetin, a heterodimeric protein from the venom of Echis carinatus, binds to platelet glycoprotein Ib (GPIb) and so inhibits platelet aggregation or agglutination induced by various platelet agonists acting via GPIb. The amino acid sequence of the beta subunit of echicetin has been reported and found to belong to the recently identified snake venom subclass of the C-type lectin protein family. Echicetin alpha and beta subunits were purified. N-terminal sequence analysis provided direct evidence that the protein purified was echicetin. The paper presents the complete amino acid sequence of the alpha subunit and computer models of the alpha and beta subunits. The sequence of alpha echicetin is highly similar to the alpha and beta chains of various heterodimeric and homodimeric C-type lectins. Neither of the fully reduced and alkylated alpha or beta subunits of echicetin inhibited the platelet agglutination induced by von Willebrand factor-ristocetin or alpha-thrombin. Earlier reports about the inhibitory activity of reduced and alkylated echicetin beta subunit might have been due to partial reduction of the protein.

Two alpha subunits and one beta subunit of meprin zinc-endopeptidases are differentially expressed in the zebrafish Danio rerio

Meprins are members of the astacin family of metalloproteases expressed in epithelial tissues, intestinal leukocytes and certain cancer cells. In mammals, there are two homologous subunits, which form complex glycosylated disulfide-bonded homo- and heterooligomers. Both human meprin alpha and meprin beta cleave several basement membrane components, suggesting a role in epithelial differentiation and cell migration. There is also evidence that meprin beta is involved in immune defence owing to its capability of activating interleukin-1beta and the diminished mobility of intestinal leukocytes in meprin beta-knockout mice. Here we show for the first time by reverse transcription PCR, immunoblotting and immunofluorescence analyses that meprins are expressed not only in mammals, but also in the zebrafish Danio rerio. In contrast to the human, mouse and rat enzymes, zebrafish meprins are encoded by three genes, corresponding to two homologous alpha subunits and one beta subunit. Observations at both the mRNA and protein level indicate a broad distribution of meprins in zebrafish. However, there are strikingly different expression patterns of the three subunits, which is consistent with meprin expression in mammals. Hence, D. rerio appears to be a suitable model to gain insight into the basic physiological functions of meprin metalloproteases.

Placental growth factor-1 attenuates vascular endothelial growth factor-A-dependent tumor angiogenesis during beta cell carcinogenesis

Members of the vascular endothelial growth factor (VEGF) family are critical players in angiogenesis and lymphangiogenesis. Although VEGF-A has been shown to exert fundamental functions in physiologic and pathologic angiogenesis, the exact role of the VEGF family member placental growth factor (PlGF) in tumor angiogenesis has remained controversial. To gain insight into PlGF function during tumor angiogenesis, we have generated transgenic mouse lines expressing human PlGF-1 in the beta cells of the pancreatic islets of Langerhans (Rip1PlGF-1). In single-transgenic Rip1PlGF-1 mice, intra-insular blood vessels are found highly dilated, whereas islet physiology is unaffected. Upon crossing of these mice with the Rip1Tag2 transgenic mouse model of pancreatic beta cell carcinogenesis, tumors of double-transgenic Rip1Tag2;Rip1PlGF-1 mice display reduced growth due to attenuated tumor angiogenesis. The coexpression of transgenic PlGF-1 and endogenous VEGF-A in the beta tumor cells of double-transgenic animals causes the formation of low-angiogenic hPlGF-1/mVEGF-A heterodimers at the expense of highly angiogenic mVEGF-A homodimers resulting in diminished tumor angiogenesis and reduced tumor infiltration by neutrophils, known to contribute to the angiogenic switch in Rip1Tag2 mice. The results indicate that the ratio between the expression levels of two members of the VEGF family of angiogenic factors, PlGF-1 and VEGF-A, determines the overall angiogenic activity and, thus, the extent of tumor angiogenesis and tumor growth.

Proapoptotic BH3-only protein Bid is essential for death receptor-induced apoptosis of pancreatic beta-cells

OBJECTIVE: Apoptosis of pancreatic beta-cells is critical in both diabetes development and failure of islet transplantation. The role in these processes of pro- and antiapoptotic Bcl-2 family proteins, which regulate apoptosis by controlling mitochondrial integrity, remains poorly understood. We investigated the role of the BH3-only protein Bid and the multi-BH domain proapoptotic Bax and Bak, as well as prosurvival Bcl-2, in beta-cell apoptosis. RESEARCH DESIGN AND METHODS: We isolated islets from mice lacking Bid, Bax, or Bak and those overexpressing Bcl-2 and exposed them to Fas ligand, tumor necrosis factor (TNF)-alpha, and proinflammatory cytokines or cytotoxic stimuli that activate the mitochondrial apoptotic pathway (staurosporine, etoposide, gamma-radiation, tunicamycin, and thapsigargin). Nuclear fragmentation was measured by flow cytometry. RESULTS: Development and function of islets were not affected by loss of Bid, and Bid-deficient islets were as susceptible as wild-type islets to cytotoxic stimuli that cause apoptosis via the mitochondrial pathway. In contrast, Bid-deficient islets and those overexpressing antiapoptotic Bcl-2 were protected from Fas ligand-induced apoptosis. Bid-deficient islets were also resistant to apoptosis induced by TNF-alpha plus cycloheximide and were partially resistant to proinflammatory cytokine-induced death. Loss of the multi-BH domain proapoptotic Bax or Bak protected islets partially from death receptor-induced apoptosis. CONCLUSIONS: These results demonstrate that Bid is essential for death receptor-induced apoptosis of islets, similar to its demonstrated role in hepatocytes. This indicates that blocking Bid activity may be useful for protection of islets from immune-mediated attack and possibly also in other pathological states in which beta-cells are destroyed.

Projection structure of DtpD (YbgH), a prokaryotic member of the peptide transporter family

Cellular uptake of di- and tripeptides has been characterized in numerous organisms, and various transporters have been identified. In contrast, structural information on peptide transporters is very sparse. Here, we have cloned, overexpressed, purified, and biochemically characterized DtpD (YbgH) from Escherichia coli, a prokaryotic member of the peptide transporter family. Its homologues in mammals, PEPT1 (SLC15A1) and PEPT2 (SLC15A2), not only transport peptides but also are of relevance for uptake of drugs as they accept a large spectrum of peptidomimetics such as beta-lactam antibiotics, antivirals, peptidase inhibitors, and others as substrates. Uptake experiments indicated that DtpD functions as a canonical peptide transporter and is, therefore, a valid model for structural studies of this family of proteins. Blue native polyacrylamide gel electrophoresis, gel filtration, and transmission electron microscopy of single-DtpD particles suggest that the transporter exists in a monomeric form when solubilized in detergent. Two-dimensional crystallization of DtpD yielded first tubular crystals that allowed the determination of a projection structure at better than 19 A resolution. This structure of DtpD represents the first structural view of a member of the peptide transporter family.

Routine phenotypic identification of bacterial species of the family Pasteurellaceae isolated from animals

Pasteurellaceae are bacteria with an important role as primary or opportunistic, mainly respiratory, pathogens in domestic and wild animals. Some species of Pasteurellaceae cause severe diseases with high economic losses in commercial animal husbandry and are of great diagnostic concern. Because of new data on the phylogeny of Pasteurellaceae, their taxonomy has recently been revised profoundly, thus requiring an improved phenotypic differentiation procedure to identify the individual species of this family. A new and simplified procedure to identify species of Actinobacillus, Avibacterium, Gallibacterium, Haemophilus, Mannheimia, Nicoletella, and Pasteurella, which are most commonly isolated from clinical samples of diseased animals in veterinary diagnostic laboratories, is presented in the current study. The identification procedure was evaluated with 40 type and reference strains and with 267 strains from routine diagnostic analysis of various animal species, including 28 different bacterial species. Type, reference, and field strains were analyzed by 16S ribosomal RNA (rrs) and rpoB gene sequencing for unambiguous species determination as a basis to evaluate the phenotypic differentiation schema. Primary phenotypic differentiation is based on beta-nicotinamide adenine dinucleotide (beta-NAD) dependence and hemolysis, which are readily determined on the isolation medium. The procedure divides the 28 species into 4 groups for which particular biochemical reactions were chosen to identify the bacterial species. The phenotypic identification procedure allowed researchers to determine the species of 240 out of 267 field strains. The procedure is an easy and cost-effective system for the rapid identification of species of the Pasteurellaceae family isolated from clinical specimens of animals.

Phylogeny of the family Pasteurellaceae based on rpoB sequences

Sequences of the gene encoding the beta-subunit of the RNA polymerase (rpoB) were used to delineate the phylogeny of the family Pasteurellaceae. A total of 72 strains, including the type strains of the major described species as well as selected field isolates, were included in the study. Selection of universal rpoB-derived primers for the family allowed straightforward amplification and sequencing of a 560 bp fragment of the rpoB gene. In parallel, 16S rDNA was sequenced from all strains. The phylogenetic tree obtained with the rpoB sequences reflected the major branches of the tree obtained with the 16S rDNA, especially at the genus level. Only a few discrepancies between the trees were observed. In certain cases the rpoB phylogeny was in better agreement with DNA-DNA hybridization studies than the phylogeny derived from 16S rDNA. The rpoB gene is strongly conserved within the various species of the family of Pasteurellaceae. Hence, rpoB gene sequence analysis in conjunction with 16S rDNA sequencing is a valuable tool for phylogenetic studies of the Pasteurellaceae and may also prove useful for reorganizing the current taxonomy of this bacterial family.

The AtProT family. Compatible solute transporters with similar substrate specificity but differential expression patterns

Proline transporters (ProTs) mediate transport of the compatible solutes Pro, glycine betaine, and the stress-induced compound gamma-aminobutyric acid. A new member of this gene family, AtProT3, was isolated from Arabidopsis (Arabidopsis thaliana), and its properties were compared to AtProT1 and AtProT2. Transient expression of fusions of AtProT and the green fluorescent protein in tobacco (Nicotiana tabacum) protoplasts revealed that all three AtProTs were localized at the plasma membrane. Expression in a yeast (Saccharomyces cerevisiae) mutant demonstrated that the affinity of all three AtProTs was highest for glycine betaine (K-m = 0.1-0.3 mM), lower for Pro (K-m = 0.4-1 mM), and lowest for gamma-aminobutyric acid (K-m = 4-5 mM). Relative quantification of the mRNA level using real-time PCR and analyses of transgenic plants expressing the beta-glucuronidase (uidA) gene under control of individual AtProT promoters showed that the expression pattern of AtProTs are complementary. AtProT1 expression was found in the phloem or phloem parenchyma cells throughout the whole plant, indicative of a role in long-distance transport of compatible solutes. beta-Glucuronidase activity under the control of the AtProT2 promoter was restricted to the epidermis and the cortex cells in roots, whereas in leaves, staining could be demonstrated only after wounding. In contrast, AtProT3 expression was restricted to the above-ground parts of the plant and could be localized to the epidermal cells in leaves. These results showed that, although intracellular localization, substrate specificity, and affinity are very similar, the transporters fulfill different roles in planta.

Genomic cloning and expression of three murine UDP-galactose: beta-N-acetylglucosamine beta1,3-galactosyltransferase genes.

Based on the detection of expressed sequence tags that are similar to known galactosyltransferase sequences, we have isolated three novel UDP-galactose:beta-N-acetylglucosamine beta1, 3-galactosyltransferase (beta3GalT) genes from a mouse genomic library. The three genes, named beta3GalT-I, -II, and -III, encode type II transmembrane proteins of 326, 422, and 331 amino acids, respectively. The three proteins constitute a distinct subfamily as they do not share any sequence identity with other eucaryotic galactosyltransferases. Also, the entire protein-coding region of the three beta3GalT genes was contained in a single exon, which contrasts with the genomic organization of the beta1,4- and alpha1, 3-galactosyltransferase genes. The three beta3GalT genes were mainly expressed in brain tissue. The expression of the full-length murine genes as recombinant baculoviruses in insect cells revealed that the beta3GalT enzymes share the same acceptor specificity for beta-linked GlcNAc, although they differ in their Km for this acceptor and the donor UDP-Gal. The identification of beta3GalT genes emphasizes the structural diversity present in the galactosyltransferase gene family.