The Role of Glycosaminoglycan Binding of Staphylococci in Attachment to Eukaryotic Host Cells

Attachment of microorganisms to host cells is believed to be a critical early step in microbial pathogenesis. The aim of the study was to determine the role of the known glycosaminoglycan (GAG) binding activity of Staphylococcus aureus and coagulase-negative staphylococci (CoNS) in their attachment to six different eukaryotic cell lines. Three staphylococcal species expressing GAG binding capacity—S. aureus, S. epidermidis, and S. hemolyticus—were chosen for investigation. Six different eukaryotic cell lines, endothelial HUVEC and EA. hy 926 cells, epithelial A549 and HeLa S3 cells, fibroblasts HEL Sp 12 and macrophages J774.A1, were included. A modified ELISA with biotinylated bacteria was used for estimating the adhesion of staphylococci to each of the cell lines. Our results showed that staphylococci adhered to each of the cell lines studied, although the binding of CoNS strains to epithelial cells was lower than to the other cells. The attachment to all cell types could be partially decreased by pretreatment of the bacteria with various polysulfated agents (highest inhibition was 60%), as well as by chlorate and heparitinase treatment of the cells. These observations may suggest that at least one mode of staphylococcal attachment utilizes GAG chains present on the surface of virtually all adherent cells. Received: 6 September 2000 / Accepted: 29 December 2000     

The recombinant congenic strains—a novel genetic tool applied to the study of colon tumor development in the mouse

The development of tumors in mice is under multigenic control, but, in spite of considerable efforts, the identification of the genes involved has so far been unsuccessful, because of the insufficient resolution power of the available genetic tools. Therefore, a novel genetic tool, the RC (Recombinant Congenic) strains system, was designed. In this system, a series of RC strains is produced from two inbred strains, a background strain and a donor strain. Each RC strain contains a different small subset of genes from the donor strain and the majority of genes from the background strain. As a consequence, the individual genes of the donor strain which are involved in the genetic control of a multigenic trait, become separated into different RC strains, where they can be identified and studied individually. One of the RC strains series which we produced is made from the parental strains BALB/cHeA (background strain) and STS/A (donor strain). We describe the genetic composition of this BALB/cHeA-C-STS/A (CcS/Dem) series and show, using 45 genetic autosomal markers, that it does not deviate from the theoretical expectation. We studied the usefulness of the CcS/Dem RC strains for analysis of the genetics of colon tumor development. The two parental strains, BALB/cHeA and STS/A, are relatively resistant and highly susceptible, respectively, to the induction of colon tumors by 1,2-dimethylhydrazine (DMH). The individual RC strains differ widely in colon tumor development after DMH treatment; some are highly susceptible, while others are very resistant. This indicates that a limited number of genes with a major effect are responsible for the high susceptibility of the STS strain. Consequently, these genes can be mapped by further analysis of the susceptible RC strains. The differences between the RC strains were not limited to the number of tumors, but the RC strains differed also in size of the tumors and the relative susceptibility of the two sexes. Our data indicate that the number of tumors and the size of tumors are not controlled by the same genes. The genetics of these different aspects of colon tumorigenesis can also be studied by the RC strains. The DMH-treated mice of the parental strains and the RC strains also developed anal tumors and haemangiomas in varying numbers. The strain distribution pattern (SDP) of susceptibility for each of the three types of tumors induced by DMH is different, indicating that development of these tumors is under control of different, largely non-overlapping, sets of genes. Thus, with a single series of RC strains, genes involved in tumorigenesis in various organs and tissues can be studied separately. These results indicate that the novel genetic tool, the RC strain system, offers new possibilities for analysis of the multigenic control of tumor development.     

Comparison of early passage, senescent and hTERT immortalized endothelial cells

The need for standardized experimental conditions to gain relevant and reproducible results has increased the demand for well characterized continuously growing cell lines that exhibit the characteristics of their normal counterparts. Immortalization of normal human cells by ectopic expression of the catalytic subunit of human telomerase (hTERT) has shown to result in highly differentiated cell lines. However, the influence of the increased telomerase activity on the protein expression profile was not investigated so far. Therefore, we have immortalized human umbilical vein endothelial cells (HUVECs) by hTERT overexpression and compared them to their normal early passage and senescent counterparts. This study, including a proteomic approach, shows that ectopic hTERT expression leads to a stable growing cell line. Although these cells are highly differentiated, the protein expression profile of the cell line is different to that of normal early passage and senescent cells.     

Genes and enzymes involved in caffeic acid biosynthesis in the actinomycete Saccharothrix espanaensis

The saccharomicins A and B, produced by the actinomycete Saccharothrix espanaensis, are oligosaccharide antibiotics. They consist of 17 monosaccharide units and the unique aglycon N-(m,p-dihydroxycinnamoyl)taurine. To investigate candidate genes responsible for the formation of trans-m,p-dihydroxycinnamic acid (caffeic acid) as part of the saccharomicin aglycon, gene expression experiments were carried out in Streptomyces fradiae XKS. It is shown that the biosynthetic pathway for trans-caffeic acid proceeds from L-tyrosine via trans-p-coumaric acid directly to trans-caffeic acid, since heterologous expression of sam8, encoding a tyrosine ammonia-lyase, led to the production of trans-p-hydroxycinnamic acid (coumaric acid), and coexpression of sam8 and sam5, the latter encoding a 4-coumarate 3-hydroxylase, led to the production of trans-m,p-dihydroxycinnamic acid. This is not in accordance with the general phenylpropanoid pathway in plants, where trans-p-coumaric acid is first activated before the 3-hydroxylation of its ring takes place.     

Role of Heme–Hemopexin in Human T-Lymphocyte Proliferation

Heme–hemopexin supports and stimulates proliferation of human acute T-lymphoblastic (MOLT-3) cells, suggesting the participation of heme in cell growth and division. MOLT-3 cells express approximately 58,000 hemopexin receptors per cell (apparent K_d20 nM), of which about 20% are on the cell surface. Binding is dose- and temperature-dependent, and growth in serum-free IMDM medium is stimulated by 100–1000 nMheme–hemopexin, consistent with the high affinity of the receptor for hemopexin, and maximal growth is seen in response to 500 nMcomplex. Growth was similar in defined minimal medium supplemented with either low concentrations of heme–hemopexin or iron-transferrin, and either of these complexes were about 80% as effective as a serum supplement. Heme–hemopexin, but not apo–hemopexin, reversed the growth inhibition caused by desferrioxamine showing that heme–iron derived from heme catabolism is used for cell growth. Cobalt-protoporphyrin (CoPP)–hemopexin, which binds to the receptor but is not transported intracellularly [Smithet al.,(1993)J. Biol. Chem.268, 7365], also stimulated cell proliferation in serum-free IMDM but did not “rescue” the cells from desferrioxamine. Furthermore, CoPP–hemopexin effectively competed for the hemopexin receptor with heme–hemopexin and diminished its growth stimulatory effects. In addition, protein kinase C (PKC) is translocated to the plasma membrane within 5 min after heme–hemopexin is added to the medium, reaches maximum activity within 5–10 min, and declines to unstimulated levels by 30 min. Heme–hemopexin and CoPP-hemopexin both augmented MOLT-3 cell growth stimulated by serum. Thus, heme–hemopexin not only functions as an iron source for T-cells but occupancy of the hemopexin receptor itself triggers signaling pathway(s) involved in the regulation of cell growth. The stimulation of growth of human T-lymphocytes by heme–hemopexin is likely to be a physiologically relevant mechanism at sites of injury, infection, and inflammation.     

Characterization of a Spodoptera frugiperda multiple nucleopolyhedrovirus isolate that does not liquefy the integument of infected larvae

Here we report the characterization of a Spodoptera frugiperda multiple nucleopolyhedrovirus isolate, named SfMNPV-6nd, that does not cause the liquefaction of the host integument. The sequencing of the chitinase A (v-chiA) gene from SfMNPV-6nd revealed that it had a frameshift mutation that greatly reduced size of the putative enzyme. In order to evaluate the suitability of SfMPNV-6nd as a biopesticide, this isolate was compared with the highly virulent SfMNPV-19. Our results showed that the LC(50) of the two isolates were not significantly different, but that SfMNPV-6nd took a longer period of time to kill second instar S. frugiperda than SfMNPV-19.