Patient perceptions of osteomyelitis, septic arthritis and prosthetic joint infection: the psychological influence of methicillin-resistant Staphylococcus aureus.

INTRODUCTION: Patients form their own representations of their illness, which can be important determinants of their coping and influence outcome. Our aims were to (i) assess patient perceptions of osteomyelitis, septic arthritis and prosthetic joint infection, (ii) compare perceptions of methicillin-resistant Staphylococcus aureus (MRSA) with non-MRSA infection and (iii) investigate the emotional aspects of these infections. METHODS: A questionnaire was developed from the 'Illness Perception Questionnaire' of Weinman et al.with additional questions assessing emotional response. This was offered to all patients with osteomyelitis, septic arthritis and prosthetic joint infection attending the Liverpool Hospital Infectious Diseases Outpatient Clinic during a 3-month period. RESULTS: There were 91 respondents--25 with MRSA infection, 14 with MRSA colonization and 52 without MRSA. Seventy-nine per cent of all respondents felt that their infection was very serious and 76% felt their infection had had major consequences on their life. On multivariate analysis MRSA was associated with a greater emotional effect; the consequences and emotional effects of infection were greater in younger people and prosthetic joint infection was associated with less sense of control or cure. CONCLUSION: Osteomyelitis, septic arthritis and prosthetic joint infection have a significant effect on an individual. Ongoing support and education are important, particularly for the young, those with prosthetic joint infection and patients with MRSA.

DNA replication: stable driving prevents fatal smashes.

Cells respond to DNA damage during S phase by slowing chromosome replication. Recent results have shed light on the mechanism by which this 'intra-S phase' checkpoint is implemented.

The yeast mitotic cyclin Clb2 cannot substitute for S phase cyclins in replication origin firing.

Cyclin-dependent kinases (CDKs) drive the cell cycle, central to which is the accurate control of chromosome replication. In Saccharomyces cerevisiae, six closely related B-type cyclins (Clb1-6) drive the events of S phase and mitosis. Either Clb5 or Clb6 can activate early-firing replication origins, whereas only Clb5 can activate late origins. Clb1-4 are expressed later in the cell cycle. Whether Clb cyclins differ only in timing of expression, or else impart different kinase specificities is under ongoing investigation. This study shows that the expression of Clb2 during S phase in cells lacking Clb5 failed to rescue late origin activation. Early expression of Clb2 in cells lacking both Clb5 and Clb6 did not activate early origins on schedule to restore the correct S phase entry time. Therefore, Clb2 cannot drive timely activation of either early or late replication origins, demonstrating that Clb2-directed CDK has a specificity distinct from that driven by Clb5 and Clb6.

The regulation of replication origin activation.

At the start of the cell-division programme, proteins must be assembled onto replication origins to establish competence for initiation of DNA synthesis. At the correct moment, other effectors must then coordinate appropriate firing of the various origins to control entry into and progress through S phase. These processes are key targets of cell-cycle control, and understanding their regulation will provide a deeper knowledge of the mechanisms controlling cell proliferation.

CLB5-dependent activation of late replication origins in S. cerevisiae.

Replication origins in chromosomes are activated at specific times during the S phase. We show that the B-type cyclins are required for proper execution of this temporal program. clb5 cells activate early origins but not late origins, explaining the previously described long clb5 S phase. Origin firing appears normal in cIb6 mutants. In clb5 clb6 double mutant cells, the late origin firing defect is suppressed, accounting for the normal duration of the phase despite its delayed onset. Therefore, Clb5p promotes the timely activation of early and late origins, but Clb6p can activate only early origins. In clb5 clb6 mutants, the other B-type cyclins (Clb1-4p) promote an S phase during which both early and late replication origins fire.

Cdc7 is required throughout the yeast S phase to activate replication origins.

The long-standing conclusion that the Cdc7 kinase of Saccharomyces cerevisiae is required only to trigger S phase has been challenged by recent data that suggests it acts directly on individual replication origins. We tested the possibility that early- and late-activated origins have different requirements for Cdc7 activity. Cells carrying a cdc7(ts) allele were first arrested in G1 at the cdc7 block by incubation at 37 degrees C, and then were allowed to enter S phase by brief incubation at 23 degrees C. During the S phase, after return to 37 degrees C, early-firing replication origins were activated, but late origins failed to fire. Similarly, a plasmid with a late-activated origin was defective in replication. As a consequence of the origin activation defect, duplication of chromosomal sequences that are normally replicated from late origins was greatly delayed. Early-replicating regions of the genome duplicated at approximately their normal time. The requirements of early and late origins for Cdc7 appear to be temporally rather than quantitatively different, as reducing overall levels of Cdc7 by growth at semi-permissive temperature reduced activation at early and late origins approximately equally. Our results show that Cdc7 activates early and late origins separately, with late origins requiring the activity later in S phase to permit replication initiation.

Spc42p: a phosphorylated component of the S. cerevisiae spindle pole body (SPD) with an essential function during SPB duplication.

The 42-kD component of the S. cerevisiae spindle pole body (SPB) localizes to the electron-dense central plaque of the SPB. We have cloned the corresponding gene SPC42 (spindle pole component) and show that it is essential. Seven temperature-sensitive (ts) mutants in SPC42 were prepared by error-prone PCR. We found that a change to a proline residue in a potential coiled-coil region of Spc42p was responsible for the ts phenotype in at least three alleles, suggesting that formation of the coiled-coil is essential in normal function. The mutant cells showed a phenotype of predominantly single or bilobed SPBs often with an accumulation of unstructured electron-dense material associated with the bridge structure adjacent to the SPB. This phenotype suggests a defect in SPB duplication. This was confirmed by examining synchronized mutant cells that lose viability when SPB duplication is attempted. Spc42p is a phosphoprotein which shows some cell cycle-regulated phosphorylation. Overexpression of Spc42p causes the formation of a disc- or dome-shaped polymer composed of phosphorylated Spc42p, which is attached to the central plaque and associated with the outer nuclear membrane. Taken together, these data suggest that Spc42p forms a polymeric layer at the periphery of the SPB central plaque which has an essential function during SPB duplication and may facilitate attachment of the SPB to the nuclear membrane.

Expression in Escherichia coli of human lamins A and C: influence of head and tail domains on assembly properties and paracrystal formation.

We have expressed in Escherichia coli cDNA corresponding to human lamins A and C, together with a number of fragments produced using site-specific mutagenesis. The proteins produced in this way were characterised both biochemically and ultrastructurally, and appeared to retain their native conformation. Crosslinking showed that all fragments formed 4-chain molecular dimers ('tetramers') analogous to those formed by intact intermediate filament proteins. Shadowed preparations showed the presence of rod-like particles that closely resembled those observed for other intermediate filament proteins and their proteolytically prepared rod domains. Moreover, the expressed lamins and a series of fragments in which different domains had been deleted formed paracrystals similar to those observed with native material. Deletion of either the N- or C-terminal non-helical domains altered the solubility and aggregation properties of the expressed protein, indicating that both domains have a role in lamin assembly.