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Biomedical subjects

D J Clarke

Publications and source records attributed to D J Clarke.

At least 37 records · Page 2Linked to original sources
PubMed2001

Bile bilirubin pigment analysis in disorders of bilirubin metabolism in early infancy.

BACKGROUND: Early and accurate diagnosis of Crigler-Najjar syndrome, which causes prolonged unconjugated hyperbilirubinaemia in infancy, is important, as orthotopic liver transplantation is the definitive treatment. AIM: To determine whether bilirubin pigment analysis of bile in infants with prolonged unconjugated hyperbilirubinaemia provides useful diagnostic information in the first 3 months of life. METHODS: Retrospective review of patients with prolonged unconjugated hyperbilirubinaemia referred to the liver unit, Birmingham Children's Hospital, for the diagnosis of Crigler-Najjar syndrome. Bile bilirubin pigment composition was determined by high performance liquid chromatography. Initial diagnoses were made based on the result of bile bilirubin pigment composition. Final diagnoses were made after reviewing the clinical course, response to phenobarbitone, repeat bile bilirubin pigment composition analysis, and genetic studies. RESULTS: Between 1992 and 1999, nine infants aged less than 3 months of age with prolonged hyperbilirubinaemia underwent bile bilirubin pigment analyses. Based on these, two children were diagnosed with Crigler-Najjar syndrome (CNS) type 1, six with CNS type 2, and one with Gilbert's syndrome. Five children whose initial diagnosis was CNS type 2 had resolution of jaundice and normalisation of serum bilirubin after discontinuing phenobarbitone, and these cases were thought to be normal or to have Gilbert's syndrome. One of the initial cases of CNS type 1 responded to phenobarbitone with an 80% reduction in serum bilirubin consistent with CNS type 2. In all, the diagnoses of six cases needed to be reviewed. CONCLUSIONS: Early bile pigment analysis, performed during the first 3 months of life, often shows high levels of unconjugated bilirubin or bilirubin monoconjugates, leading to the incorrect diagnosis of both type 1 and type 2 Crigler-Najjar syndrome.

Bile Pigments↗
PubMed2000

Coordinated spindle assembly and orientation requires Clb5p-dependent kinase in budding yeast.

The orientation of the mitotic spindle along a polarity axis is critical in asymmetric cell divisions. In the budding yeast, Saccharomyces cerevisiae, loss of the S-phase B-type cyclin Clb5p under conditions of limited cyclin-dependent kinase activity (cdc28-4 clb5Delta cells) causes a spindle positioning defect that results in an undivided nucleus entering the bud. Based on time-lapse digital imaging microscopy of microtubules labeled with green fluorescent protein fusions to either tubulin or dynein, we observed that the asymmetric behavior of the spindle pole bodies during spindle assembly was lost in the cdc28-4 clb5Delta cells. As soon as a spindle formed, both poles were equally likely to interact with the bud cell cortex. Persistent dynamic interactions with the bud ultimately led to spindle translocation across the bud neck. Thus, the mutant failed to assign one spindle pole body the task of organizing astral microtubules towards the mother cell. Our data suggest that Clb5p-associated kinase is required to confer mother-bound behavior to one pole in order to establish correct spindle polarity. In contrast, B-type cyclins, Clb3p and Clb4p, though partially redundant with Clb5p for an early role in spindle morphogenesis, preferentially promote spindle assembly.

CDC28 Protein Kinase, S cerevisiae↗
PubMed2000

Checkpoints controlling mitosis.

Each year many reviews deal with checkpoint control.((1-5)) Here we discuss checkpoint pathways that control mitosis. We address four checkpoint systems in depth: budding yeast DNA damage, the DNA replication checkpoint, the spindle assembly checkpoint and the mammalian G2 topoisomerase II-dependent checkpoint. A main focus of the review is the organization of these checkpoint pathways. Recent work has elucidated the order-of-function of several checkpoint components, and has revealed that the S phase, DNA damage and spindle assembly checkpoints each have at least two parallel branches. These steps forward have largely come from kinetic studies of checkpoint-defective mutants.

Adaptation, Physiological↗
PubMed2000

Real-time dynamics of dopamine released from neuronal transplants in experimental Parkinson's disease.

Intrastriatal transplantation of foetal midbrain dopamine (DA) neurons ameliorates the fundamental symptoms of dopaminergic denervation in clinical and experimental parkinsonism despite providing only restricted reinnervation. To understand how DA function is restored by these grafts we used fast-scan cyclic voltammetry at a carbon-fiber microelectrode in vitro to monitor directly and in "real time" the dynamics of graft-derived DA. Simulations of Michaelis-Menten kinetics were used to model the experimental observations. We show that the concentration of DA released by a single depolarizing pulse is significantly lower in grafted than intact striata. On the other hand, the extracellular lifetime of DA in grafts is extended due to a marked reduction in the rate maximum (V(max)) for DA reuptake by the DA transporter. Moreover, variations in V(max) and release occur in parallel: where DA release is lowest, V(max) is lowest and vice versa. The consequences of these dynamics are twofold. First, during repeated depolarization at a physiological firing frequency, when net extracellular concentrations reflect DA release versus uptake, ambient levels of extracellular DA within the graft are restored to normal. Second, the protracted extracellular lifetime of DA will increase the number and extracellular sphere of its postsynaptic actions. This effect will be most prominent where DA availability (and thus V(max)) is most restricted. Thus, these data demonstrate that dopaminergic grafts restore striatal dopaminergic function with extracellular dynamics of DA that are different from those of intact striatum but which can normalize ambient DA levels and permit transmission over an extended sphere.

Animals↗
PubMed2000

Premitotic chromosome individualization in mammalian cells depends on topoisomerase II activity.

When DNA topoisomerase II (topo II) activity is inhibited with a non-DNA-damaging topo II inhibitor (ICRF-193), mammalian cells become checkpoint arrested in G2-phase. In this study, we analyzed chromosome structure in cells that bypassed this checkpoint. We observed a novel type of chromosome aberration, which we call omega-figures. These are entangled chromosome regions that indicate the persistence of catenations between nonhomologous sequences. The number of omega-figures per cell increased sharply as cells evaded the transient block imposed by the topo II-dependent checkpoint, and the presence of caffeine (a checkpoint-evading agent) potentiated this increase. Thus, the removal of nonreplicative catenations, a process that promotes chromosome individualization in G2, may be monitored by the topo II-dependent checkpoint in mammals.

Animals↗
PubMed2000

Problem behaviors associated with 15q- Angelman syndrome.

Caregivers of persons with Angelman syndrome completed the Aberrant Behavior Checklist and Reiss Screen for Maladaptive Behavior. Seventy-three replies were received, and comparisons were made with other published data. Responses indicated that 15q- Angelman syndrome is associated with such problems as lack of speech, overactivity, restlessness, and eating and sleep problems. Episodes of inappropriate laughter were only reported for 57%, despite being considered a cardinal feature of the syndrome; eating problems (64%) and a fascination with water (68%) were reported more frequently. Overactivity was more of a problem for children; Aberrant Behavior Checklist Factor IV (Hyperactivity) was negatively correlated with age. Scores were mostly lower than for previously studied etiological groups. Therapeutic effort should be put into programs to address these problems.

Adolescent↗
PubMed1999

Cyclin-dependent kinase and Cks/Suc1 interact with the proteasome in yeast to control proteolysis of M-phase targets.

Cell cycle-specific proteolysis is critical for proper execution of mitosis in all eukaryotes. Ubiquitination and subsequent proteolysis of the mitotic regulators Clb2 and Pds1 depend on the cyclosome/APC and the 26S proteasome. We report here that components of the cell cycle machinery in yeast, specifically the cell cycle regulatory cyclin-dependent kinase Cdc28 and a conserved associated protein Cks1/Suc1, interact genetically, physically, and functionally with components of the 26S proteasome. A mutation in Cdc28 (cdc28-1N) that interferes with Cks1 binding, or inactivation of Cks1 itself, confers stabilization of Clb2, the principal mitotic B-type cyclin in budding yeast. Surprisingly, Clb2-ubiquitination in vivo and in vitro is not affected by mutations in cks1, indicating that Cks1 is not essential for cyclosome/APC activity. However, mutant Cks1 proteins no longer physically interact with the proteasome, suggesting that Cks1 is required for some aspect of proteasome function during M-phase-specific proteolysis. We further provide evidence that Cks1 function is required for degradation of the anaphase inhibitor Pds1. Stabilization of Pds1 is partially responsible for the metaphase arrest phenotype of cks1 mutants because deletion of PDS1 partially relieves the metaphase block in these mutants.

Adaptor Proteins, Signal Transducing↗
PubMed1999

The Pds1 anaphase inhibitor and Mec1 kinase define distinct checkpoints coupling S phase with mitosis in budding yeast.

In most eukaryotic cells, DNA replication is confined to S phase of the cell cycle [1]. During this interval, S-phase checkpoint controls restrain mitosis until replication is complete [2]. In budding yeast, the anaphase inhibitor Pds1p has been associated with the checkpoint arrest of mitosis when DNA is damaged or when mitotic spindles have formed aberrantly [3] [4], but not when DNA replication is blocked with hydroxyurea (HU). Previous studies have implicated the protein kinase Mec1p in S-phase checkpoint control [5]. Unlike mec1 mutants, pds1 mutants efficiently inhibit anaphase when replication is blocked. This does not, however, exclude an essential S-phase checkpoint function of Pds1 beyond the early S-phase arrest point of a HU block. Here, we show that Pds1p is an essential component of a previously unsuspected checkpoint control system that couples the completion of S phase with mitosis. Further, the S-phase checkpoint comprises at least two distinct pathways. A Mec1p-dependent pathway operates early in S phase, but a Pds1p-dependent pathway becomes essential part way through S phase.

Anaphase↗
PubMed1999

Effects of anaesthetics and lazaroid U-83836E on survival of transplanted rat dopaminergic neurones.

We investigated whether different methods of anaesthesia, used on the pregnant rat when collecting embryonic donor tissue, and a lipid peroxidation inhibitor (lazaroid U-83836E) affect the survival of grafted embryonic dopaminergic neurones in hemiparkinsonian rats. There was no difference in either functional recovery or survival of dopaminergic neurones between the different euthanasia groups: (a) isoflurane sedation followed by cervical dislocation, (b) equithesin- or (c) euthatal-anaesthesia. However, lazaroid-treatment enhanced both behavioural recovery and transplant survival.

Anesthetics, Combined↗
PubMed1999

Apoptosis in primary cultures of E14 rat ventral mesencephala: time course of dopaminergic cell death and implications for neural transplantation.

Transplantation using fetal nigral grafts has been performed by various groups worldwide in over 200 Parkinson's disease (PD) patients in an attempt to restore dopaminergic (DA) input to the striatum. However, the proportion of the implanted DA neurons that survives, whether using suspension, partially dissociated, or solid grafts, is small, often as low as 5 to 10%, which is insufficient to allow a full functional recovery. A significant proportion of the transplanted neurons in animal models of PD has been shown to die via apoptosis, but the reason for this is unclear. Since the methods used to prepare donor tissue for neural transplantation and in vitro culture are identical, we have looked at the time course of DA neuron loss following cell suspension preparation using an in vitro assay system and considered whether the procedures used may, in part, be responsible for the poor DA neuron survival. Primary dissociated cultures of E14 rat ventral mesencephala were incubated for different periods in serum-containing and serum-free media. After fixation, the TUNEL method, as well as ethidium bromide and acridine orange, were used to detect apoptosis, and DA neurons were localized immunocytochemically. Results showed that most apoptosis occurred during the first 24 h and that 50% of the DA neurons were lost in the first 8 h. Double-immunofluorescent labeling confirmed the presence of TUNEL+ve nuclei within DA neurons. There was no difference in either the extent or rate of loss between the serum-containing and serum-free medium during the first 32 h. We suggest, therefore, that existing methods used to prepare cell suspensions probably induce apoptosis and may need to be modified in order to increase the survival of DA neurons.

Anesthetics↗
PubMed1999

Competence for assembly of sister chromatid cores is progressively acquired during S phase in mammalian cells.

Condensed sister chromatids possess a protein scaffold or axial core to which loops of chromatin are attached. The sister cores are believed to be dynamic frameworks that function in the organization and condensation of chromatids. Chromosome structural proteins are implicated in the establishment of sister chromatid cohesion and in the maintenance of epigenetic phenomena. Both processes of templating are tightly linked to DNA replication itself. It is a question whether the structural basis of sister chromatid cores is templated during S phase. As cells proceed through the cell cycle, chromatid cores undergo changes in their protein composition. Cytologically, cores are first visualized at the start of prometaphase. Still, core assembly can be induced in G1 and G2 when interphase cells are fused with mitotic cells. In this study, we asked if chromatid cores are similarly able to assemble in S-phase cells. We find that the ability to assemble cores is transiently lost during local replication, then regained in chromosome regions shortly after they have been replicated. We propose that core templating occurs coincident with DNA replication and that the competence for the assembly of the sister chromatid cores is acquired shortly after passage of replication forks.

Animals↗
PubMed1999

Nonepitopic antibody binding sequence: implications in screening and development of peptide vaccines.

We describe the interaction of a nonepitopic synthetic decapeptide sequence comprising, GQVLQGAIKG, derived from a random sequence with polyclonal IgGs from various animal sources. GQVLQGAIKG was screened for antibody binding activity using ELISA techniques. The peptide showed similar binding characteristics to the IgGs tested. The results were similar whether we used peptide acid or amide. MAP (multiple antigen peptide)-type construct of the peptide was synthesised and employed as an approach to enhance peptide-IgG interaction. The construct, (GQVLQGAIKG)(4)-K(2)-K, showed significant antibody binding activity relative to its monomeric form. These results show that nonepitopic sequences may contribute to binding activity observed in peptide library screening and development of peptide based vaccines. As a cautionary point the measure of antibody binding cannot alone be used to classify peptide as an antigen.

Antigen-Antibody Reactions↗
PubMed1999

Utility of modified DCR-10 criteria in the diagnosis of depression associated with intellectual disability.

A retrospective study of 11 inpatients with intellectual disability who received antidepressant treatment over a 12-month period indicated the utility of minor modifications to DCR-10 criteria in the diagnosis of depressive illness. The modifications were made by adding items found in previous research to be behavioural equivalents of depression in people with severe intellectual disability to DCR-10 criteria. The time course of symptom response to antidepressant treatment was documented. All 11 patients had a remission of depressive symptoms, as described by the modified criteria, within 5 weeks of starting antidepressant treatment

Adult↗
PubMed1998

Clb5-associated kinase activity is required early in the spindle pathway for correct preanaphase nuclear positioning in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, a single cyclin-dependent kinase, Cdc28, regulates both G1/S and G2/M phase transitions by associating with stage-specific cyclins. During progression through S phase and G2/M, Cdc28 is activated by the B-type cyclins Clb1-6. Because of functional redundancy, specific roles for individual Clbs have been difficult to assign. To help genetically define such roles, strains carrying a cdc28(ts) allele, combined with single CLB deletions were studied. We assumed that by limiting the activity of the kinase, these strains would be rendered more sensitive to loss of individual Clbs. By this approach, a novel phenotype associated with CLB5 mutation was observed. Homozygous cdc28-4(ts) clb5 diploids were inviable at room temperature. Cells were defective in spindle positioning, leading to migration of undivided nuclei into the bud. Occasionally, misplaced spindles were observed in cdc28-4 clb5 haploids; additional deletion of CLB6 caused full penetrance. Thus, CLB5 effects proper preanaphase spindle positioning, yet the requirement differs in haploids and diploids. The execution point for the defect corresponded to the time of Clb5-dependent kinase activation. Nevertheless, lethality of cdc28-4 clb5 diploids was not rescued by CLB2 or CLB4 overexpression, indicating a specificity of Clb5 function beyond temporality of expression.

Anaphase↗
PubMed1998

cAMP included during cell suspension preparation improves survival of dopaminergic neurons in vitro.

The physical process of cell suspension preparation from embryonic ventral mesencephala (VM) may be responsible for the low numbers of dopaminergic (DA) neurons that survive following neural transplantation or in vitro culture. In particular, the disruption of cell to extracellular matrix attachment may result in cell death through deactivation of a cAMP-dependent protein kinase involved in cell survival signalling. In an attempt to reduce this death, dibutyryl cAMP was included in all solutions from explant collection to final dissociation. Pretreatment with 700 microM dibutyryl cAMP resulted in 90% survival of the DA neurons originally plated, compared with only 40% in the untreated cultures, after 5 days in vitro. Treatment of VM explants in this manner may result in major improvements in neural transplantation as a technique for the treatment of Parkinson's disease.

Animals↗
PubMed1998

Creation of monosomic derivatives of human cultured cell lines.

Monosomic mammalian cell lines would be ideal for studying gene dosage effects, including gene imprinting, and for systematic isolation of recessive somatic mutants parallel to the invaluable mutants derived from haploid yeast. But autosomal monosomies are lethal in early development; although monosomies appear in tumors, deriving cell lines from these tumors is difficult and cannot provide several syngenic lines. We have developed a strategy for generating stable monosomic human cells, based on random autosomal integration of the gpt plasmid, partial inhibition of DNA topoisomerase II during mitosis to promote chromatid nondisjunction, and selection against retention of gpt. These are likely to be valuable as a source of otherwise inaccessible mutants. The strategy can also be used to generate partial mammalian monosomies, which are desirable as a source of information on recessive genes and gene imprinting.

Anaphase↗
PubMed1998

Increased sensitivity of E. coli to novobiocin, EDTA and the anticalmodulin drug W7 following overproduction of DjlA requires a functional transmembrane domain.

In earlier studies we found that E. coli is sensitive to anticalmodulin drugs such as W7. Mutants that are resistant to this drug were isolated, including wseA1. In an attempt to clone the wseA gene, we isolated a clone that restored sensitivity to the drug in the mutant. We found that this clone in fact suppresses W7 resistance through expression of djlA, which encodes a novel DnaJ-like protein. It was found previously that overproduction of DjlA could induce capsule synthesis via activation of the two-component regulatory pathway RcsC/B. In addition to suppression of wseA1, djlA overexpression increases the sensitivity of cells to EDTA and novobiocin, but not to other drugs tested. Although overexpression of a form of the protein carrying a mutation in, or lacking, the J-region of DjlA also led to increased sensitivity, indicating that the chaperone activity of this protein was not strictly required. the full-length, wild-type protein had a more pronounced effect. In contrast, a point mutation which affects the function of the transmembrane domain but not the localisation or stability of DjlA abolished the effects of DjlA overproduction.

Amino Acid Sequence↗