Pentti Kuusela

Fibroblast surface antigen: A new serum protein

Abstract Material solubilized from the surface of cultured chick embryo fibroblasts contains a major antigen also present in normal chicken serum. Fluorescent antiserum stains the fibroblast membrane but not that of other cells. The antigen is synthesized by fibroblasts. [ 35 S]Methionine is incorporated into the molecule in cell cultures and the antigen is present in cells cultivated in heterologous serum for an extended period of time. In serum it has a high molecular weight and is an α-globulin. It seems to be a previously unrecognized serum protein. We suggest the name fibroblast surface antigen for it.

Hydrophobic domains affect the collagen‐binding specificity and surface polymerization as well as the virulence potential of the YadA protein of Yersinia enterocolitica

The YadA surface protein of enteropathogenic Yersinia species contains two highly hydrophobic regions: one close to the amino terminal, and the other at the carboxy‐terminal end of the YadA polypeptide. To study the role of these hydrophobic regions, we constructed 66 bp deletion mutants of the yadA genes of Yersinia enterocolitica serotype O:3 strain 6471/76 (YeO3) and of O:8 strain 8081 (YeO8). The mutant proteins, YadAYeO3‐Δ83–104 and YadAYeO8‐Δ80–101, lacked 22 amino acids from the amino‐terminal hydrophobic region, formed fibrillae and were expressed on the cell surface. Bacteria expressing the mutated protein lost their auto‐agglutination potential as well as their collagen‐binding property. Binding to fibronectin and laminin was affected differently in the YeO3 and the YeO8 constructs. The deletion did not influence YadA‐mediated complement inhibition. Loss of the collagen‐binding property was associated with loss of virulence in mice. We also constructed a number of YadAYeO3 deletion mutants lacking the hydrophobic carboxy‐terminal end of the protein. Deletions ranging from 19 to 79 amino acids from the carboxy terminus affected polymerization of the YadA subunits, and also resulted in the loss of the YadA expression on the cell surface. This suggests that the carboxy terminus of YadA is involved in transport of the protein to the bacterial outer surface.

The O75X adhesin of uropathogenic Escherichia coli is a type IV collagen-binding protein

Interaction of the basement‐membrane binding O75X adhesin of uropathogenic Escherichia coli with various extracellular matrix proteins was studied. The adhesin showed strong binding to type IV collagen immobilized on microtitre plates, whereas other collagens, laminin and fibronectin, were only weakly recognized. Similarly, specific binding of [125I]‐labelled type IV collagen to 075X‐positive bacteria was shown. Interaction of the two proteins was also demonstrated by affinity chromatography of the O75X adhesin on immobilized type IV collagen. The adhesin bound strongly to the immobilized N‐terminal 7S domain of type IV collagen, and the binding of [125I]‐labelled type IV collagen to O75X‐positive bacteria was inhibited by the soluble 7S domain. Binding of O75X to type IV collagen and to its 7S domain was specifically inhibited by chloramphenicol but was not affected by periodate or endoglycosidase‐H treatment of the glycoproteins. Our results show that the 7S domain of type IV collagen is the basement membrane receptor for the O75X adhesin and suggest an interaction based on protein‐protein recognition. Inhibition of the interaction by chloramphenicol favours the supposition that a modified tyrosine is involved in the binding site.

Immunological interspecies cross-reactions of fibroblast surface antigen (fibronectin).

Abstract The fibroblast surface antigen (SF antigen, fibronectin) a connective tissue component, is known to be present on the surface of fibroblasts and glial cells and to be shed to the surrounding medium. Anti-human fibronectin antibodies prepared in sheep reacted not only with human fibronectin but also with fibronectin in plasma and in fibroblasts of several other species including chicken. Sheep anti-chicken fibronectin similarly cross-reacted with some mammalian fibronectins. The cross-reactivity of these two fibronectins was destroyed by pronase, but not by periodate treatment. The cross-reaction between avian and human fibronectin suggests that fibronectin has remained highly conserved in evolution. This may be to do its functions as cell surface protein.

Genome Diversification in Staphylococcus aureus: Molecular Evolution of a Highly Variable Chromosomal Region Encoding the Staphylococcal Exotoxin-Like Family of Proteins

ABSTRACT Recent genomic studies have revealed extensive variation in natural populations of many pathogenic bacteria. However, the evolutionary processes which contribute to much of this variation remain unclear. A previous whole-genome DNA microarray study identified variation at a large chromosomal region (RD13) of Staphylococcus aureus which encodes a family of proteins with homology to staphylococcal and streptococcal superantigens, designated staphylococcal exotoxin-like (SET) proteins. In the present study, RD13 was found in all 63 S. aureus isolates of divergent clonal, geographic, and disease origins but contained a high level of variation in gene content in different strains. A central variable region which contained from 6 to 10 different set genes, depending on the strain, was identified, and DNA sequence analysis suggests that horizontal gene transfer and recombination have contributed to the diversification of RD13. Phylogenetic analysis based on the RD13 DNA sequence of 18 strains suggested that loss of various set genes has occurred independently several times, in separate lineages of pathogenic S. aureus, providing a model to explain the molecular variation of RD13 in extant strains. In spite of multiple episodes of set deletion, analysis of the ratio of silent substitutions in set genes to amino acid replacements in their products suggests that purifying selection (selective constraint) is acting to maintain SET function. Further, concurrent transcription in vitro of six of the seven set genes in strain COL was detected, indicating that the expression of set genes has been maintained in contemporary strains, and Western immunoblot analysis indicated that multiple SET proteins are expressed during the course of human infections. Overall, we have shown that the chromosomal region RD13 has diversified extensively through episodes of gene deletion and recombination. The coexpression of many set genes and the production of multiple SET proteins during human infection suggests an important role in host-pathogen interactions.

The prognostic value of preoperative serum levels of CA 19-9 and CEA in patients with pancreatic cancer

The prognostic value of preoperative serum levels of CA 19-9 and CEA was evaluated in 160 patients with pancreatic cancer. The survival of patients whose tumour marker value was below a certain cut-off level was compared with the survival of those with a higher value using the log-rank test. The lowest cut-off level dividing patients into groups with significant difference in survival (P < 0.05) was determined by graphical analysis of chi-square values at different cut-off levels. If stage of disease was not taken into account, there was a significant difference in survival between patients with low vs high preoperative CA 19-9 and CEA levels. When patients were classified according to stage, a difference was found for CA 19-9 in stage II-III patients. Patients with preoperative CA 19-9 below 370 U ml-1 had a significantly better prognosis than those with a higher level (P < 0.05). In stage I and stage IV patients, no significant difference was found between the groups at any cut-off level. The analysis of CEA showed a significant difference in survival only in stage IV patients, with CEA above 15 ng ml-1 being associated with shorter survival. In conclusion, in patients with stage II-III disease, particularly in patients with a non-resectable tumour, in whom the exact spread of the disease may be difficult to evaluate even at operation, the preoperative CA 19-9 level seems to have a prognostic value.

Comparison of CA 19-9 and carcinoembryonic antigen (CEA) levels in the serum of patients with colorectal diseases

The serum levels of CA 19-9 and carcinoembryonic antigen (CEA) were determined in 37 patients with benign colorectal diseases and in 111 patients with newly discovered colorectal carcinomas or clinically verified relapses. In cancer patients, the CA 19-9 level ranged from normal (0-37 U ml-1) to 77,500 U ml-1 whereas all samples but one from patients with benign colorectal diseases had a normal value. CA 19-9 was increased in 46% and 45% of patients with an advanced (Dukes C or D) carcinoma or a verified recidive, respectively. Only one out of 26 patients (4%) with a localized (Dukes A or B) carcinoma displayed an elevated CA 19-9 level (greater than 37 U ml-1). No clear correlation was found between the CA 19-9 and CEA levels. The sensitivity of the CA 19-9 test (36%) was poorer than that of the CEA assay (69%), but the new test was markedly more specific (97% vs 70%) than the CEA assay.

Type V collagen as the target for type-3 fimbriae, enterobacterial adherence organelles

Tissue‐binding specificity of the type‐3 fimbriae of pathogenic enteric bacteria was determined using frozen sections of human kidney. A wild‐type Kleb‐siella sp. strain and the recombinant strain Escherichia coli HB101(pFK12), both expressing type‐3 fimbriae, as well as the purified type‐3 fimbriae effectively bound to sites at or adjacent to tubular basement membranes, Bowmans capsule, arterial walls, and the interstitial connective tissue. Bacterial adherence to kidney was decreased after collagenase treatment of the tissue sections. Recombinant strains expressing type‐3 fimbriae specifically adhered to type V collagen immobilized on glass slides, whereas other collagens, fibronectin or laminin did not support bacterial adherence. In accordance with these findings, specific binding of purified type‐3 fimbriae to immobilized type V collagen was demonstrated. Specific adhesion to type V collagen was also seen with the recombinant strain HB101(pFK52/pDC17), which expresses the mrkD gene of the type‐3 fimbrial gene cluster in association with the pap‐encoded fimbrial filament of E. coli, showing that the observed binding was mediated by the minor lectin (MrkD) protein of the type‐3 fimbrial filament. The interaction is highly dependent on the conformation of type V collagen molecules since type V collagen in solution did not react with the fimbriae. Specific binding to type V collagen was also exhibited by type‐3 fimbriae strains of Yeisinia and Salmonella, showing that the ability to use type collagen as tissue target is widespread among enteric bacteria.

Antibacterial activities of temporin A analogs

Temporin A (TA) is a small, basic, highly hydrophobic, antimicrobial peptide amide (FLPLIGRVLSGIL‐NH2) found in the skin of the European red frog, Rana temporaria. It has variable antibiotic activities against a broad spectrum of microorganisms, including clinically important methicillin‐sensitive and ‐resistant Staphylococcus aureus as well as vancomycin‐resistant Enterococcus faecium strains. In this investigation the antimicrobial activity and structural characteristics of TA synthetic analogs were studied. For antibacterial activity against S. aureus and enterococcal strains, the hydrophobicity of the N‐terminal amino acid of TA was found to be important as well as a positive charge at amino acid position 7, and bulky hydrophobic side chains at positions 5 and 12. Replacing isoleucine with leucine at amino acid positions 5 and 12 resulted in the greatest enhancement of antibacterial activity. In addition, there was little difference between the activities of TA and its all‐D enantiomer, indicating that the peptide probably exerts its effect on bacteria via non‐chiral interactions with membrane lipids.

Evaluation of CA 19-9 as a serum tumour marker in pancreatic cancer.

Serum concentrations of the CA 19-9 antigen were determined in 91 patients with pancreatic cancer and in 111 patients with benign pancreatic, biliary and hepatocellular diseases. The CA 19-9 concentration was above the cut-off limit (37 U ml-1) in 78% of the patients with pancreatic cancer and high levels (greater than 500 U ml-1) were seen in 56% of these patients. Elevated levels were also seen in benign diseases (22%), especially in patients with extrahepatic cholestasis (up to 440 U ml-1). Hepatocellular jaundice and pancreatitis were associated with normal values (84% of the patients), or with only slightly elevated CA 19-9 levels (up to 88 U ml-1). The CA 19-9 test can be useful as an additional diagnostic tool for the detection of pancreatic cancer. Preliminary results suggest that the CA 19-9 assay can be used in the monitoring of surgically treated patients.