Tenascin-C expression by angiogenic vessels in human astrocytomas and by human brain endothelial cells in vitro.

The expression of the extracellular matrix glycoprotein tenascin-C (TN) is enhanced in human astrocytomas and correlates with angiogenesis. To determine whether vascular cells are able to synthesize TN, we investigated the expression of TN protein and mRNA in nine astrocytomas. Immunogold electron microscopy in two glioblastomas multiforme detected the presence of TN in an extracellular perivascular location and to a lesser extent among tumor cells, confirming light microscopy immunohistochemical findings. In situ hybridization of astrocytomas using a digoxigenin-labeled antisense riboprobe detected strong staining for TN mRNA in vascular cells, especially in hyperplastic vessels, including those at the invasive edge of the tumors but not in vessels of normal brains. We observed weaker staining in tumor cells indicating a higher level of TN mRNA in vascular than in tumor cells. No staining was detected with the sense probe. Moreover, we investigated the ability of human brain microvessel endothelial cells (HBMECs) in primary culture to synthesize TN in vitro. Western blot analysis of the culture supernatants from HBMECs detected large amounts of TN. Immunogold silver staining demonstrated the presence of TN on the surface of HBMECs and in the subendothelial matrix. The distribution of TN mRNA in vascular cells of astrocytomas and the ability of HBMECs to synthesize TN in vitro demonstrate that vascular cells, including endothelial cells, are a major source of TN associated with angiogenesis. Furthermore, our results suggest that TN expression may be associated with endothelial cell activation and may play an important role in angiogenesis.

Physical mapping of the mec region of an Australian methicillin-resistant Staphylococcus aureus lineage and a closely related American strain.

Methicillin-resistant (Mcr) staphylococci contain chromosomal DNA that is absent from Mcs cells. This extra DNA harbours the methicillin resistance determinant mec and often other resistance determinants. The mec region can differ substantially in structure among different isolates. We present studies on the mec region of a group of Staphylococcus aureus isolates prevalent in Australia and London. Southern hybridization analyses of a prototype Australian isolate, ANS46, and an isogenic Mcs deletion mutant, ANS62, allowed the physical map of the region to be extended to 55 kb. The DNA corresponding to the deletion, which includes mec and resistance determinants for mercury, cadmium (Cd) and tetracycline, amounted to 41 kb. It was bounded precisely at one end by the macrolides-lincosamides-streptogramin B (MLS)-resistance transposon, Tn554. Near the other end was an element with homology to Tn554, psi Tn554, which carried the Cdr determinant. The mec region of an American Mcr isolate, R35, was found to be virtually the same as that of ANS46, except that it lacked Tn554. Another class of American Mcr isolates, prevalent since 1987, differs markedly from ANS46 in mec region organization. However, this other American class also contains an insertion of Tn554 in the mec region, and the attachment site for this insertion was found to have significant homology to attachment sites for the Tn554 and psi Tn554 insertions in the mec region of the Australian strain. These results suggest possible roles of Tn554 and Tn554-like elements in the evolutionary variation of the mec region.

Physical mapping of the mec region of an American methicillin-resistant Staphylococcus aureus strain.

We mapped part of the mec region of a locally prevalent strain of Staphylococcus aureus. The mec region was found to harbor an insert of the transposon Tn554, which encodes spectinomycin and macrolide-lincosamide-streptogramin B resistance, and a 4.6-kb segment of DNA that contains the kanamycin resistance gene aadD. This 4.6-kb segment appears to be an integrated form of a previously described plasmid, pUB110, and is flanked by copies of the insertion sequence IS257. The integration event may be an example of processes that have led to accretion of resistance determinants in the mec region of S. aureus.

Tn554 inserts in methicillin-resistant Staphylococcus aureus from Australia and England: comparison with an American methicillin-resistant group.

We have compared methicillin-resistant (Mcr) Staphylococcus aureus isolates from Australia, the UK and the USA with regard to chromosomal inserts of the macrolides-lincosamides-streptogramin B (MLS)-resistance transposon Tn554. The American isolates were known to have a distinctive Tn554 insert, designated insert 6, which was closely associated epidemiologically with the methicillin-resistance phenotype. Southern blots of DNA from Australian and London, UK Mcr isolates were hybridized with a range of probes related to Tn554. The isolates had similar or identical Tn554 inserts, and we consider them to be a single group, designated 'Australondon'. Australondon isolates were compared in detail with a deletion mutant, ANS62, that had lost the methicillin-resistance determinant mec, plus other resistance determinants resident in the mec region of the chromosome, and with an American Mcr isolate containing Tn554 insert 6. The Australondon isolates had three Tn554 inserts. Sequence analysis with the polymerase chain reaction showed that all of these inserts differed from classical Tn554 in that the 3'-terminal residues of the transposons were reverse complements of the usual GATGTA. One of the Australondon inserts, designated 6B, closely resembled Tn554 insert 6 in the sequence of its left flanking chromosomal DNA. This insert was found to abut the deletion from the mec region which results in strain ANS62. We infer that Tn554 insert 6B is part of the mec region of the chromosome in Australondon isolates, supporting the idea that insert 6 of the American isolates is also part of this chromosomal region.

IS257 and small plasmid insertions in the mec region of the chromosome of Staphylococcus aureus.

Four copies of the insertion sequence IS257 are found in the mec region of the chromosome of the Australian methicillin resistant Staphylococcus aureus (MRSA) strain ANS46, two flanking a merAmerB sequence (encoding resistance to mercurial compounds), the other two flanking an integrated copy of the plasmid pT181 (tetracycline resistance). The termini of the integrated copy of the plasmid pT181 carry a direct repeat of 8 bp of plasmid sequence, but otherwise there are no similarities in the 8 bp sequences flanking the four copies of IS257 in this strain. Integrated copies of pT181 in strains R35 (a New Jersey MRSA) and GH32 (MRSA of Greek origin) have the same terminal repeat as in ANS46, suggesting either a specific site of insertion of IS257 into the free plasmid before integration into the chromosome, or a common evolutionary lineage for these geographically diverse isolates. A different 8 bp terminal repeat of plasmid sequence is found in the chromosomally integrated copy of pUB110 (flanked by a pair of IS257s) in R155, another New Jersey MRSA. This 8 bp repeat differs from that reported previously for pUB110/IS257 inserted into the plasmid pSK41, indicating insertion of IS257 into different sites of pUB110 before integration into the chromosome or into pSK41. In the plasmid pSK1, the two outer copies of IS257 of the three associated with Tn4003 (trimethoprim resistance) are also flanked by 8 bp repeats.(ABSTRACT TRUNCATED AT 250 WORDS)