Biomedical subjects

D J Clarke

Publications and source records attributed to D J Clarke.

At least 19 recordsLinked to original sources

PubMed2006

Structural and functional studies of defensin-inspired peptides.

Mammals have evolved complex self-defence mechanisms to protect themselves from infection. This innate immune system comprises a large family of hundreds of peptides and proteins which have potent antibiotic activity at nanomolar concentrations. The defensins are a group of small cationic peptides which contain a high proportion of positively charged and hydrophobic amino acids. Their exact mechanism of antimicrobial action is unclear, but it is thought that the defensins bind to and disrupt the outer cell membrane which ultimately causes lysis and cell death. They are characterized by six conserved cysteine residues which oxidize to form three intramolecular disulphide (S-S) bonds. The human and mouse defensins have been subdivided into classes based on their sequence, site of expression and the S-S bond connectivity of the cysteine residues. Alpha-defensins are connected by cysteines 1 and 6, 2 and 4, and 3 and 5, whereas beta-defensins have a 1-5, 2-4 and 3-6 cysteine S-S connectivity. We present our structural and functional studies of a novel mouse beta-defensin-related peptide (Defr1) which contains only five cysteine residues. Synthetic Defr1 was more active than its six-cysteine analogue against a large panel of pathogens. High-resolution MS techniques revealed that Defr1 contains an unusual defensin structure. These studies have guided the design of novel peptides to explore the roles of defensins as antibiotics and as stimulants of the immune response.

Animals↗

PubMed2005

Clozapine treatment of psychosis associated with velo-cardio-facial syndrome: benefits and risks.

BACKGROUND: Clozapine is licensed for the treatment of psychotic illnesses resistant to other antipsychotic medications. Velo-cardio-facial syndrome (VCFS) is associated with a vulnerability to psychotic illness that may be resistant to treatment with conventional typical and atypical antipsychotics. PATIENTS AND METHODS: A 32-year-old man with intellectual disability (ID) and a long history of treatment-resistant psychosis was found to have VCFS. Treatment with typical antipsychotic drugs and with one atypical olanzapine produced no improvement. RESULTS: Treatment with clozapine produced an improvement in psychotic symptoms and associated behavioural abnormalities, but caused hypersalivation, constipation and a seizure disorder. The latter led to two fractures, one requiring surgery. The addition of sodium valproate stopped seizures. CONCLUSIONS: Clozapine may improve psychotic symptoms for people with ID associated with VCFS, but clinicians should be alert for potential adverse effects.

Abnormalities, Multiple↗

PubMed2005

The pbgPE operon in Photorhabdus luminescens is required for pathogenicity and symbiosis.

Photorhabdus is a genus of gram-negative Enterobacteriaceae that is pathogenic to insect larvae while also maintaining a mutualistic relationship with nematodes from the family Heterorhabditis, where the bacteria occupy the gut of the infective juvenile (IJ) stage of the nematode. In this study we describe the identification and characterization of a mutation in the pbgE1 gene of Photorhabdus luminescens TT01, predicted to be the fifth gene in the pbgPE operon. We show that this mutant, BMM305, is strongly attenuated in virulence against larvae of the greater wax moth, Galleria mellonella, and we report that BMM305 is more sensitive to the cationic antimicrobial peptide, polymyxin B, and growth in mildly acidic pH than the parental strain of P. luminescens. Moreover, we also show that the lipopolysaccharide (LPS) present on the surface of BMM305 does not appear to contain any O antigen. Complementation studies reveal that the increased sensitivity to polymyxin B and growth in mildly acidic pH can be rescued by the in trans expression of pbgE1, while the defects in O-antigen assembly and pathogenicity require the in trans expression of pbgE1 and the downstream genes pbgE2 and pbgE3. Finally, we show that BMM305 is defective in symbiosis as this mutant is unable to colonize the gut of the IJ stage of the nematode. Therefore, we conclude that the pbgPE operon is required for both pathogenicity and symbiosis in P. luminescens.

Animals↗

PubMed2003

The transmembrane domain of the DnaJ-like protein DjlA is a dimerisation domain.

DjlA is a bitopic inner membrane protein, which belongs to the DnaJ co-chaperone family in Escherichia coli. Overproduction of DjlA leads to the synthesis of colanic acid, resulting in mucoidy, via the activation of the two-component regulatory system RcsC/B that controls the cps (capsular polysaccharide) operon. This induction requires both the co-chaperone activity of DjlA, in cooperation with DnaK and GrpE, and its unique transmembrane (TM) domain. Here, we show that the TM segment of DjlA acts as a dimerisation domain: when fused to the N-terminal DNA-binding domain of the lambda cI repressor protein, it can substitute for the native C-terminal dimerisation domain of cI, thus generating an active cI repressor. Replacing the TM domain of DjlA by other TM domains, with or without dimerising capacity, revealed that dimerisation is not sufficient for the induction of cps expression, indicating an additional sequence- or structurally specific role for the TM domain. Finally, the conserved glycines present in the TM domain of DjlA are essential for the induction of mucoidy, but not for dimerisation.

Amino Acid Sequence↗

PubMed2002

Behavioural aspects of Pollitt syndrome: a 32-year follow-up of a case described by R. J. Pollitt and colleagues in 1968.

In 1968, R. J. Pollitt and colleagues described a syndrome characterized by abnormally brittle, sulphur-deficient hair (trichothiodystrophy), intellectual disability (ID) and growth retardation. One of the two siblings originally described by the above authors has recently been re-assessed by the present authors following a referral for advice about ritualistic behaviours. Her current clinical features are described, and the literature concerning trichothiodystrophy and ID is summarized.

Abnormalities, Multiple↗

PubMed2002

Genetic analysis of the RcsC sensor kinase from Escherichia coli K-12.

The Rcs two-component pathway is involved in the regulation of capsule production in Escherichia coli. RcsC is predicted to be the sensor component of this two-component pathway, and in this study we present the first genetic data that support the role of RcsC as a hybrid sensor kinase.

Amino Acid Sequence↗

PubMed2002

Prader-Willi syndrome, compulsive and ritualistic behaviours: the first population-based survey.

BACKGROUND: Obsessive-compulsive disorder has been reported in association with Prader-Willi syndrome. AIMS: To report the nature and prevalence of compulsive and similar symptoms associated with Prader-Willi syndrome in a population ascertained as completely as possible. METHOD: Attempted complete ascertainment of people with Prader-Willi syndrome in eight English counties. Administration of standardised rating scales and a structured interview. Comparison with people with learning disability and high body mass indices. RESULTS: Prader-Willi syndrome was associated with high rates of ritualistic behaviours, such as the need to ask or to tell something, insistence on routines, hoarding and ordering objects and repetitive actions and speech, compared with the control group, and was negatively correlated with IQ and socialisation age. Typical obsessive-compulsive symptoms, such as checking, counting and cleaning compulsions or obsessional thoughts, were not found. CONCLUSIONS: Ritualistic and compulsive behaviours occur more frequently in association with Prader-Willi syndrome than among people with intellectual disability and significant obesity.

Adolescent↗

PubMed2002

Effects of antioxidant pretreatment on the survival of embryonic dopaminergic neurons in vitro and following grafting in an animal model of Parkinson's disease.

The effect of pretreating cell suspensions of embryonic rat ventral mesencephala (VM) with antioxidant combinations on the survival of dopaminergic (DA) neurons was studied in vitro and following transplantation into the unilateral 6-hydroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease. The in vitro experiments examined the effects of two thiol antioxidants, N-acetyl-L-cysteine (NAC) and reduced glutathione (GSH), and a member of the lazaroid family of 21-aminosteroids, U-83836E, singly and in combination, on survival of DA neurons derived from dissociated E14 rat VM tissue. For in vivo studies, cell suspensions were pretreated with combinations of NAC, GSH, and U-83836E prior to transplanting into 6-OHDA-lesioned rats to investigate whether DA neuron survival could be further improved. NAC, GSH, and U-83836E individually increased DA neuron survival in vitro and a combination of all three resulted in the greatest survival. In vivo, pretreatment with U-83836E alone resulted in a significantly greater reduction in amphetamine-induced rotation 6 weeks postgrafting compared with a control group receiving nontreated graft tissue. This functional effect correlated with a significant improvement in DA neuron survival 6 weeks postgrafting. The thiol combination pretreatment of NAC and GSH, and the triple combination of NAC, GSH, and U-83836E, however, failed to improve both functional recovery and DA neuron survival when compared with the nontreated control grafts.

Acetylcysteine↗

PubMed2001

Neuronal nitric oxide synthase immunohistochemistry and 4,5-diaminofluorescein diacetate: tools for nitric oxide research.

Nitric oxide plays an important role in many biological processes including the cardiovascular, immune, reproductive and nervous systems. There is much interest surrounding this molecule and the enzyme responsible for its synthesis, nitric oxide synthase. In order to investigate the role of nitric oxide in various biological processes it is necessary to be able to identify the released molecule itself as well as nitric oxide synthase. Detection of nitric oxide synthase was achieved by optimisation of the immunohistochemical localisation of the neuronal isoform of the enzyme in primary cultures of rat ventral mesencephala and in fixed adult rat brain sections. Furthermore, using 4,5-diaminofluorescein diacetate, we have directly visualised endogenously produced nitric oxide in mesencephalic cultures and demonstrated the potential use of this indicator for visualising nitric oxide produced in vivo.

Animals↗

PubMed2001

UBA domains mediate protein-protein interactions between two DNA damage-inducible proteins.

The Saccharomyces cerevisiae genes RAD23 and DDI1 were identified in a screen for multicopy suppressors of the temperature-sensitivity of a mutant allele of S. cerevisiae PDS1. Pds1 is a regulator of anaphase that needs to accumulate and then be degraded by the ubiquitin-proteasome pathway at the metaphase-anaphase transition for cells to progress normally through mitosis. Both the Rad23 and Ddi1 pds1 suppression phenotypes depend on a shared motif known as a UBA domain found in a variety of proteins associated with ubiquitin metabolism. UBA domains were found to be essential for homodimerization of Rad23 and heterodimerization between Rad23 and Ddi1, but not for homodimerization of Ddi1. This observation, coupled with the findings that Rad23 and Ddi1 UBA domains bind ubiquitin and that dimerization of Rad23 blocks ubiquitin binding, suggests a possible mechanism for regulating Rad23 and Ddi1 function.

Amino Acid Sequence↗

PubMed2001

A novel role of the budding yeast separin Esp1 in anaphase spindle elongation: evidence that proper spindle association of Esp1 is regulated by Pds1.

In Saccharomyces cerevisiae, the metaphase-anaphase transition is initiated by the anaphase-promoting complex-dependent degradation of Pds1, whereby Esp1 is activated to promote sister chromatid separation. Although this is a fundamental step in the cell cycle, little is known about the regulation of Esp1 and how loss of cohesion is coordinated with movement of the anaphase spindle. Here, we show that Esp1 has a novel role in promoting anaphase spindle elongation. The localization of Esp1 to the spindle apparatus, analyzed by live cell imaging, is regulated in a manner consistent with a function during anaphase B. The protein accumulates in the nucleus in G2 and is mobilized onto the spindle pole bodies and spindle midzone at anaphase onset, where it persists into midanaphase. Association with Pds1 occurs during S phase and is required for efficient nuclear targeting of Esp1. Spindle association is not fully restored in pds1 mutants expressing an Esp1-nuclear localization sequence fusion protein, suggesting that Pds1 is also required to promote Esp1 spindle binding. In agreement, Pds1 interacts with the spindle at the metaphase-anaphase transition and a fraction remains at the spindle pole bodies and the spindle midzone in anaphase cells. Finally, mutational analysis reveals that the conserved COOH-terminal region of Esp1 is important for spindle interaction.

Anaphase↗

PubMed2001

The Ras pathway and spindle assembly collide?

Although alterations in Ras signalling are found in about 30% of human cancers, the transforming activity of oncogenic Ras is not fully understood. In a recent paper, a putative Ras1 effector in S. pombe, named Scd1, was reported to localize to mitotic spindles. Scd1 physically associates with Moe1, a factor that may contribute to the inherent instability of microtubules (MTs) and appears to be needed for proper spindle function. Altered MT dynamics within the spindle are likely to affect spindle assembly and chromosome capture, processes that need to be delicately controlled if cells are to guard against genome instability and transformation. BioEssays 23:307-310, 2001.

Animals↗

PubMed2001

Synchronous nuclear-envelope breakdown and anaphase onset in plant multinucleate cells.

Multinucleate plant cells with genetically balanced nuclei can be generated by inhibiting cytokinesis in sequential telophases. These cells can be used to relate the effect of changes in the distribution of nuclei in the cytoplasm to the control of the timing of cell cycle transitions. Which mitotic cell cycle events are sensitive to differences in the amount of cytoplasm surrounding each chromosomal complement has not been determined. To address this, we maximized the cell size by transiently inhibiting replication, while cell growth was not affected. The nuclei of 93% of the elongated cells reached prophase asynchronously compared to 46% of normal-sized multinucleate cells. The asynchronous prophases of normal-sized cells became synchronous at the time of nuclear-envelope breakdown, and the ensuing metaphase plate formation and anaphase onset and progression occurred synchronously. The elongated multinucleate cells were also very efficient in synchronizing the prophases at nuclear-envelope breakdown, in the prophase-to-prometaphase transition. However, 2.4% of these cells broke down the nuclear envelope asynchronously, though they became synchronous at the metaphase-to-anaphase transition. The kinetochore-microtubular cycle, responsible for coordinating the metaphase-to-anaphase transition and for the rate of sister segregation to opposite spindle poles during anaphase, remained strictly controlled and synchronous in the different mitoses of a single cell, independently of differences in the amount of cytoplasm surrounding each mitosis or its ploidy. Moreover, the degree of chromosome condensation varied considerably within the different mitotic spindles, being higher in the mitoses with the largest surrounding cytoplasm.

Allium↗

PubMed2001

A festival of cell-cycle controls.

The second biennial Salk Cell Cycle meeting convened on 22 June 2001 in San Diego, California. Organized by Tony Hunter and Susan Forsburg of the Salk Institute, the five-day conference was highlighted by enlightening science and plenty of San Diego sunshine. Presentations covered a broad range of contemporary cell-cycle topics, ranging from regulation of DNA replication and mitosis to DNA damage recognition and checkpoint control.

Animals↗

PubMed2001

Mec1p regulates Pds1p levels in S phase: complex coordination of DNA replication and mitosis.

Genetic evidence suggests that the securin Pds1p is the target of a late-S-phase checkpoint control. Here we show that Pds1p becomes essential once two-thirds of the genome has been replicated and that the coupling of the completion of genome replication with mitosis relies on the regulation of Pds1p levels. Mec1p is needed to maintain Pds1p levels under S-phase checkpoint conditions. In contrast, Rad53p and Chk1p, needed for the stabilization of Pds1p in the context of the G2 DNA-damage checkpoint pathway, are dispensable. Thus, the Pds1p-dependent late-S-phase checkpoint pathway couples replication with mitosis but is mechanistically distinct from the G2 DNA-damage checkpoint. Finally, we show that the inhibition of spindle elongation in early S phase, controlled by the Mec1p/Rad53p branch, is not regulated via Pds1p/Esp1p. This can mechanistically explain the need for branched S-phase checkpoint controls.

Cell Cycle Proteins↗

PubMed2001

UBA domains of DNA damage-inducible proteins interact with ubiquitin.

Rad23 is a highly conserved protein involved in nucleotide excision repair (NER) that associates with the proteasome via its N-terminus. Its C-terminal ubiquitin-associated (UBA) domain is evolutionarily conserved from yeast to humans. However, the cellular function of UBA domains is not completely understood. Recently, RAD23 and DDI1, both DNA damage-inducible genes encoding proteins with UBA domains, were implicated genetically in Pds1-dependent mitotic control in yeast. The UBA domains of RAD23 and DDI1 are required for these interactions. Timely degradation of Pds1 via the ubiquitin/proteasome pathway allows anaphase onset and is crucial for chromosome maintenance. Here, we show that Rad23 and Ddi1 interact directly with ubiquitin and that this interaction is dependent on their UBA domains, providing a possible mechanism for UBA-dependent cell cycle control. Moreover, we show that a hydrophobic surface on the UBA domain, which from structural work had been predicted to be a protein-protein interaction interface, is indeed required for ubiquitin binding. By demonstrating that UBA domains interact with ubiquitin, we have provided the first indication of a cellular function for the UBA domain.

Amino Acid Sequence↗

PubMed2001

Philosophy: a key to open the door to critical thinking.

It has been suggested that philosophy is a bit like manure: pile it high and it rots and stinks, but spread it around and it becomes surprisingly useful. Philosophical enquiry, used sparingly, can provide a means to develop critical and analytical ability in nurses. A key principle of philosophical enquiry is the development of skills to rigorously examine and debate ideas or explanations about, for example, the nature of being human. In recent years nurses have been exhorted to critically examine or critically analyse nursing knowledge and nursing theory. It is contended that nurses have rarely been formally prepared during their education to develop the necessary critical thinking skills to examine some of the central questions about the nature of their role, and the knowledge needed to be effective in that role. This paper will discuss the development and experience of delivering a module called 'Nursing: Art and Science' within a post-registration degree programme. It is argued that philosophical enquiry offers one means of equipping nurses with the critical capacity to examine the issues which may shape the future of nursing practice.

Education, Nursing, Graduate↗

PubMed2001

Dosage suppressors of pds1 implicate ubiquitin-associated domains in checkpoint control.

In budding yeast, anaphase initiation is controlled by ubiquitin-dependent degradation of Pds1p. Analysis of pds1 mutants implicated Pds1p in the DNA damage, spindle assembly, and S-phase checkpoints. Though some components of these pathways are known, others remain to be identified. Moreover, the essential function of Pds1p, independent of its role in checkpoint control, has not been elucidated. To identify loci that genetically interact with PDS1, we screened for dosage suppressors of a temperature-sensitive pds1 allele, pds1-128, defective for checkpoint control at the permissive temperature and essential for viability at 37 degrees C. Genetic and functional interactions of two suppressors are described. RAD23 and DDI1 suppress the temperature and hydroxyurea, but not radiation or nocodazole, sensitivity of pds1-128. rad23 and ddi1 mutants are partially defective in S-phase checkpoint control but are proficient in DNA damage and spindle assembly checkpoints. Therefore, Rad23p and Ddi1p participate in a subset of Pds1p-dependent cell cycle controls. Both Rad23p and Ddi1p contain ubiquitin-associated (UBA) domains which are required for dosage suppression of pds1-128. UBA domains are found in several proteins involved in ubiquitin-dependent proteolysis, though no function has been assigned to them. Deletion of the UBA domains of Rad23p and Ddi1p renders cells defective in S-phase checkpoint control, implicating UBA domains in checkpoint signaling. Since Pds1p destruction, and thus checkpoint regulation of mitosis, depends on ubiquitin-dependent proteolysis, we propose that the UBA domains functionally interact with the ubiquitin system to control Pds1p degradation in response to checkpoint activation.

Amino Acid Sequence↗