Summative assessment in North Thames (West)
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Education, Medical, Graduate↗
Biomedical subjects
P Pietroni
Publications and source records attributed to P Pietroni.
Structural analyses of gp45 sliding clamp interactions during assembly of the bacteriophage T4 DNA polymerase holoenzyme. I. Conformational changes within the gp44/62-gp45-ATP complex during clamp loading.
A multisubunit ring-shaped protein complex is used to tether the polymerase to the DNA at the primer-template junction in most DNA replication systems. This "sliding clamp" interacts with the polymerase, completely encircles the DNA duplex, and is assembled onto the DNA by a specific clamp loading complex in an ATP-driven process. Site-specific mutagenesis has been used to introduce single cysteine residues as reactive sites for adduct formation within each of the three subunits of the bacteriophage T4-coded sliding clamp complex (gp45). Two such mutants, gp45S19C and gp45K81C, are reacted with the cysteine-specific photoactivable cross-linker TFPAM-3 and used to track the changes in the relative positioning of the gp45 subunits with one another and with the other components of the clamp loading complex (gp44/62) in the various stages of the loading process. Cross-linking interactions performed in the presence of nucleotide cofactors show that ATP binding and hydrolysis, interaction with primer-template DNA, and release of ADP all result in significant conformational changes within the clamp loading cycle. A structural model is presented to account for the observed rearrangements of intersubunit contacts within the complex during the loading process.
Adenosine Triphosphate↗
Structural analyses of gp45 sliding clamp interactions during assembly of the bacteriophage T4 DNA polymerase holoenzyme. II. The Gp44/62 clamp loader interacts with a single defined face of the sliding clamp ring.
The phage T4 gp45 sliding clamp is a ring-shaped replication accessory protein that is mounted onto DNA in an ATP-dependent manner by the gp44/62 clamp loader. In the preceding paper (Pietroni, P., Young, M. C., Latham, G. J., and von Hippel, P. H. (1997) J. Biol. Chem. 272, 31666-31676), two gp45 mutants were exploited to probe interactions of the sliding clamp ring with the gp44/62 loading machinery at various steps during the clamp loading process. In this report, these studies are extended to examine the polarity of the binding interaction between gp45 and gp44/62. Three different gp45 mutants containing a single cysteine in three topographically distinct positions were used. Several different reporter groups, including extrinsic fluorophores, a photo-cross-linker, and a biotin linker for use in a novel "streptavidin interference assay," were covalently attached to these cysteine residues. Since gp45 is a trimeric protein, these three different mutations allowed us to probe up to nine distinct local environments along the surface of the sliding clamp in the presence and absence of other replication proteins. The results show that the gp44/62-ATP clamp loader complex binds exclusively to the C-terminal (S19C) face of the gp45 ring.
Adenosine Triphosphate↗
Structural analyses of gp45 sliding clamp interactions during assembly of the bacteriophage T4 DNA polymerase holoenzyme. III. The Gp43 DNA polymerase binds to the same face of the sliding clamp as the clamp loader.
In the preceding paper (Latham, G. J., Bacheller, D. J., Pietroni, P. , and von Hippel, P. H. (1997) J. Biol. Chem. 272, 31677-31684), we demonstrated that the T4 gp44/62-ATP clamp loader binds to the C-terminal face of the gp45 sliding clamp. Here we extend these results by exploring the structural relationship between the gp43 polymerase and the gp45 sliding clamp. Using fluorescence intensity and polarization techniques, as well as photo-cross-linking methods, we present evidence that gp43, like gp44/62, binds to the C-terminal face of gp45. In addition, we show that g43 binds to the gp45 clamp in two distinct interaction modes, depending on the presence or absence of template-primer DNA. When template-primer DNA is present, gp43 binds tightly to gp45 to form the highly processive DNA polymerase holoenzyme. Gp43 also binds to gp45 in the absence of template-primer DNA, but this interaction is more than 100 times weaker than gp43-gp45 binding on DNA. Specific interactions between gp43 and the C-terminal face of gp45 are maintained in both modes of binding. These results underscore the pivotal role of template-primer DNA in modulating the strength of protein-protein interactions during DNA synthesis and provide additional insight into the structural requirements of the replication process.
Adenosine Triphosphate↗
Is complementary medicine holistic?
The term 'holism' is explored using both Smuts' original definition and that developed by the Gestalt theoreticians. It is compared to reductionism and the author posits that holism must encompass reductionalism if it is to study the relationship between parts and whole.
Complementary Therapies↗
Fluorescence monitoring of T4 polymerase holoenzyme accessory protein interactions during loading of the sliding clamp onto the template-primer junction.
Assembly of the T4 polymerase holoenzyme requires coordinated interactions among the core polymerase and the clamp loader (gp44/62) and sliding clamp (gp45) accessory proteins. Here we describe the creation of a mutant of gp45 that can be uniquely modified by fluorescent probes within each protein monomer at a site-specific cysteine residue. The fluorescently labeled gp45 was shown to have the same biological activity as the wild-type protein. These strike "labeled" gp45 adducts were then used in steady-state and fluorescence polarization studies to monitor the interaction of gp45 with the gp44/62 clamp-loading complex and template-primer DNA in the presence and absence of ATP, and of the non-hydrolyzable analog, adenosine 5'-O-(3-thiotriphosphate). We find that a complex of ATP-activated gp44/62 with appropriately labeled gp45 shows significant fluorescent enhancement (and an increase in fluorescent anisotropy), that can be partially reversed by interaction with template-primer DNA. Fluorescence-monitored binding curves between gp45 and ATP-activated gp44/62 reveal that the two protein complexes bind with a 1:1 stoichiometry. Analysis shows that these methods can be used to follow the ATP-driven loading of gp45 onto the template-primer by the gp44/62 clamp-loading complex, and in combination with the kinetic data presented in the companion article, provide insight into the rate-limiting steps during clamp assembly on template-primer DNA. A reaction pathway for this processivity clamp-loading process is proposed.
Adenosine Triphosphatases↗
Post-transcriptional regulation of CspA expression in Escherichia coli.
The Escherichia coli cspA gene, encoding the major cold-shock protein CspA, was deprived of its natural promoter and placed in an expression vector under the control of the inducible lambda PL promoter. After induction of transcription by thermal inactivation of the lambda ts repressor, abundant expression of the product (CspA) was obtained if the cells were subsequently incubated at 10 degrees C, but poor expression was obtained if the cells were incubated at 37 degrees C or 30 degrees C. The reason for this differential temperature-dependent expression was investigated and it was found that: (i) the CspA content of the cells decreased more rapidly at 37 degrees C compared to 10 degrees C, regardless of whether transcription was turned off by addition of rifampicin; (ii) both the chemical and functional half-lives of the cspA transcript were substantially longer at 10 degrees C compared to 37 degrees C; (iii) S30 extracts as well as 70S ribosomes prepared from cold-shocked cells translated CspA mRNA (but not phage MS2 RNA) more efficiently than equivalent extracts or ribosomes obtained from control cells grown at 37 degrees C; and (iv) purified CspA stimulated CspA mRNA translation. Overall, these results indicate that a selective modification of the cold-shocked translational apparatus favouring translation of CspA mRNA, and an increased stability of this mRNA at low temperature, may play an important role in the induction of cspA expression during cold shock.
Bacterial Proteins↗
Counselling in an inner city general practice: analysis of its use and uptake.
BACKGROUND: In recognition of the emotional problems which frequently underlie somatic complaints, practices increasingly offer counselling as part of their services to patients. In an inner city practice, a combination of short term counselling, volunteer befriending, community outreach and social work services is offered as a means of responding to the full range of patients' counselling needs. AIM: This study set out to establish the use and uptake of these services. METHOD: A retrospective analysis of patients referred for counselling over one year was carried out. RESULTS: The analysis identified a broad range of emotional problems among referred patients as well as problems of a practical nature. A quarter of the patients referred failed to keep their initial appointments or to complete their contracts. One fifth of the patients were referred on for longer term counselling and/or psychotherapy. Subsequent feedback revealed that preparation of a patient before referral was an important factor affecting uptake of counselling. CONCLUSION: Early assessment of the use and uptake of such services is essential if they are to be integrated successfully and a counsellor's individual skills employed effectively.
Adolescent↗
Musculoskeletal clinic in general practice: study of one year's referrals.
BACKGROUND: A musculoskeletal clinic, staffed by a general practitioner trained in osteopathy, medical acupuncture and intralesional injections, was set up in an inner London general practice in 1987. AIM: A retrospective study was undertaken of one year's referrals to the clinic in 1989-90 to determine how general practitioners were using the clinic in terms of problems referred; consultation patterns of patients attending the clinic and 12 months after initially being seen; and how access to the clinic influenced referrals to relevant hospital departments. METHOD: Day sheets were studied which recorded information on demographic characteristics of patients referred to the clinic and their problems, diagnoses made, duration of symptoms, number and range of treatments given, and recurrence of problems. Use of secondary referral sources was also examined. RESULTS: During the study year 154 of 3264 practice patients were referred to the musculoskeletal clinic, and attended a mean of 3.5 times each. Of all the attenders 64% were women and 52% were 30-54 years old. Eighty one patients (53%) presented with neck, back or sciatic pain. A specific traumatic, inflammatory or other pathological process could be ascribed to only 19% of patients. Regarding treatment, 88% of patients received osteopathic manual treatment or acupuncture, or a combination of these treatments and 4% received intralesional injections. Nine patients from the clinic (6%) were referred to an orthopaedic specialist during the year, two with acute back pain. Referrals to orthopaedic specialists by the practice as a whole were not significantly lower than the national average, although the practice made fewer referrals to physiotherapy and rheumatology departments than national figures would have predicted. Seventeen patients (11%) returned to the clinic with a recurrence of their main complaint within a year of their initial appointment; second courses of treatment were usually brief. CONCLUSION: The clinic encouraged a relatively low referral rate to musculoskeletal specialists outside the practice. However, a need was identified for better communication about the potential of the approaches used in order that referrals to secondary specialists, particularly orthopaedic specialists, could be further reduced.
Acupuncture Therapy↗
Partners or partisans? Patient participation at Marylebone Health Centre.
This paper outlines some of the issues which arose for patients and professionals involved in patient participation projects at the Marylebone health centre in London. It describes the projects undertaken and focuses on the practical implications of working with rather than for patients. Dilemmas surrounding patient participation are discussed, including the ways volunteers are rewarded, how doctors and patients can share knowledge, how participation is affected by professional boundaries, and why a regular group meeting may not necessarily be the best way to involve patients in decision making. The successes of patient participation are also highlighted.
Community Health Centers↗
Self care--who does best?
A clinically evaluated study to assess the benefits of a self care programme in general practice is described. There was significant improvement in psychiatric morbidity at the 6-month follow up, which was maintained at one year. The capacity of Multidimensional Health Locus of Control scales to predict long term clinical improvement was demonstrated, suggesting that a predominantly internal control of reinforcement appears to be crucial to the successful adoption of self care practices. The clinical and educational benefits of such programmes in dealing with a wide range of psychophysical problems in primary care is discussed.
Adult↗
Holistic medicine: new lessons to be learned.
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Complementary Therapies↗
Research trials in general practice: towards a focus on clinical practice.
With current moves towards an emphasis on the 'whole' patient rather than fragmenting the person into organ systems research methods need to be developed which reflect that emphasis and direct us in our endeavour as clinicians. It is possible to have a descriptive science of human behaviour which can be based upon clinical consultations. In this way the clinician is required to act as a clinical anthropologist as well as a clinical epidemiologist.
Clinical Competence↗
A multi-sample microdialysis apparatus for proteins and nucleic acids.
A multiposition microdialysis system suitable for simultaneous microsample applications (between 10 microL and 500 microL), has been developed. Each sample, contained in a specially designed microfuge dialysis tube (mDT), is dialysed independently from the other samples. Each mDT has its own membrane, and this feature allows the use of different membranes and dialysis times for different samples. The microdialysis apparatus is kept at constant temperature by an external thermostat, avoiding the use of a cold box. The dialysis release time for small ions, a parameter used for quantitation of microdialysis efficiency, decreases from 22.9 min (for a 200 microL sample) to 7 min (for a 50 microL sample). The sample is efficiently recovered by centrifugation. Quantitative recoveries (90%) of different proteins and DNA were achieved after microdialysis by mDT.
Microdialysis↗