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Biomedical subjects

J Parkinson

Publications and source records attributed to J Parkinson.

At least 19 recordsLinked to original sources
PubMed2001

Alphaherpesvirus proteins related to herpes simplex virus type 1 ICP0 induce the formation of colocalizing, conjugated ubiquitin.

Herpes simplex virus type 1 immediate early protein ICP0 influences virus infection by inducing the degradation of specific cellular proteins via a mechanism requiring its RING finger and the ubiquitin-proteasome pathway. Many RING finger proteins, by virtue of their RING finger domain, interact with E2 ubiquitin-conjugating enzymes and act as a component of an E3 ubiquitin ligase. We have recently shown that ICP0 induces the accumulation of colocalizing, conjugated ubiquitin, suggesting that ICP0 can act as or contribute to an E3 ubiquitin ligase. In this report we demonstrate that the ICP0-related RING finger proteins encoded by other alphaherpesviruses also induce colocalizing, conjugated ubiquitin, thereby suggesting that they act by similar biochemical mechanisms.

Cell Line↗
PubMed2000

A family of secreted mucins from the parasitic nematode Toxocara canis bears diverse mucin domains but shares similar flanking six-cysteine repeat motifs.

Infective larvae of the parasitic nematode Toxocara canis secrete a family of mucin-like glycoproteins, which are implicated in parasite immune evasion. Analysis of T. canis expressed sequence tags identified a family of four mRNAs encoding distinct apomucins (Tc-muc-1-4), one of which had been previously identified in the TES-120 family of glycoproteins secreted by this parasite. The protein products of all four cDNAs contain signal peptides, a repetitive serine/threonine-rich tract, and varying numbers of 36-amino acid six-cysteine (SXC) domains. SXC domains are found in many nematode proteins and show similarity to cnidarian (sea anemone) toxins. Antibodies to the SXC domains of Tc-MUC-1 and Tc-MUC-3 recognize differently migrating members of TES-120. TES-120 proteins separated by chromatographic methods showed distinct amino acid composition, mass, and sequence information by both Edman degradation and matrix-assisted laser desorption ionization/time of flight mass spectrometry on peptide fragments. Tc-MUC-1, -2, and -3 were shown to be secreted mucins with real masses of 39.7, 47.8, and 45.0 kDa in contrast to their predicted peptide masses of 15.7, 16.2, and 26.0 kDa, respectively. The presence of SXC domains in all mucin products supports the suggestion that the SXC motif is required for mucin assembly or export. Homology modeling indicates that the six-cysteine domains of the T. canis mucins adopt a similar fold to the sea anemone potassium channel-blocking toxin BgK, forming three disulfide bonds within each subunit.

Amino Acid Sequence↗
PubMed2000

Dissecting sites important for complement regulatory activity in membrane cofactor protein (MCP; CD46).

Membrane cofactor protein (MCP; CD46), a widely distributed regulator of complement activation, is a cofactor for the factor I-mediated degradation of C3b and C4b deposited on host cells. MCP possesses four extracellular, contiguous complement control protein modules (CCPs) important for this inhibitory activity. The goal of the present study was to delineate functional sites within these modules. We employed multiple approaches including mutagenesis, epitope mapping, and comparisons to primate MCP to make the following observations. First, functional sites were located to each of the four CCPs. Second, some residues were important for both C3b and C4b interactions while others were specific for one or the other. Third, while a reduction in ligand binding was invariably accompanied by a parallel reduction in cofactor activity (CA), other mutants lost or had reduced CA but retained ligand binding. Fourth, two C4b-regulatory domains overlapped measles virus interactive regions, indicating that the hemagglutinin docks to a site important for complement inhibition. Fifth, several MCP regulatory areas corresponded to functionally critical, homologous positions in other CCP-bearing C3b/C4b-binding proteins. Based on these data and the recently derived crystal structure of repeats one and two, computer modeling was employed to predict MCP structure and examine active sites.

Amino Acid Sequence↗
PubMed2000

Conserved surface-exposed K/R-X-K/R motifs and net positive charge on poxvirus complement control proteins serve as putative heparin binding sites and contribute to inhibition of molecular interactions with human endothelial cells: a novel mechanism for evasion of host defense.

Vaccinia virus complement control protein (VCP) has been shown to possess the ability to inhibit both classical and alternative complement pathway activation. The newly found ability of this protein to bind to heparin has been shown in previous studies to result in uptake by mast cells, possibly promoting tissue persistence. It has also been shown to reduce chemotactic migration of leukocytes by blocking chemokine binding. In addition, this study shows that VCP-through its ability to bind to glycosaminoglycans (heparin-like molecules) on the surface of human endothelial cells-is able to block antibody binding to surface major histocompatibility complex class I molecules. Since heparin binding is critical for many functions of this protein, we have attempted to characterize the molecular basis for this interaction. Segments of this protein, generated by genetic engineering of the DNA encoding VCP into the Pichia pastoris expression system, were used to localize the regions with heparin binding activity. These regions were then analyzed to more specifically define their properties for binding. It was found that the number of putative binding sites (K/R-X-K/R), the overall positive charge, and the percentage of positively charged amino acids within the protein were responsible for this interaction.

Amino Acid Motifs↗
PubMed2000

Alphaherpesvirus proteins related to herpes simplex virus type 1 ICP0 affect cellular structures and proteins.

The herpes simplex virus type 1 (HSV-1) immediate-early protein ICP0 interacts with several cellular proteins and induces the proteasome-dependent degradation of others during infection. In this study we show that ICP0 is required for the proteasome-dependent degradation of the ND10 protein Sp100 and, as with the other target proteins, the ICP0 RING finger domain is essential. Further, comparison of the kinetics and ICP0 domain requirements for the degradation of PMI and Sp100 suggests that a common mechanism is involved. Homologues of ICP0 are encoded by other members of the alphaherpesvirus family. These proteins show strong sequence homology to ICP0 within the RING finger domain but limited similarity elsewhere. Using transfection assays, we have shown that all the ICP0 homologues that we tested have significant effects on the immunofluorescence staining character of at least one of the proteins destabilized by ICP0, and by using a recombinant virus, we found that the equine herpesvirus ICP0 homologue induced the proteasome-dependent degradation of endogenous CENP-C and modified forms of PML and Sp100. However, in contrast to ICP0, the homologue proteins had no effect on the distribution of the ubiquitin-specific protease USP7 within the cell, consistent with their lack of a USP7 binding domain. We also found that ICP0 by itself could induce the abrogation of SUMO-1 conjugation and then the proteasome-dependent degradation of unmodified exogenous PML in transfected cells, thus demonstrating that other HSV-1 proteins are not required. Surprisingly, the ICP0 homologues were unable to cause these effects. Overall, these data suggest that the members of the ICP0 family of proteins may act via a similar mechanism or pathway involving their RING finger domain but that their intrinsic activities and effects on endogenous and exogenous proteins differ in detail.

Alphaherpesvirinae↗
PubMed1999

Central modules of the vaccinia virus complement control protein are not in extensive contact.

The 28.6 kDa vaccinia virus complement control protein (VCP) is an inhibitor of the complement system and has therapeutic potential. It is composed of four domains or modules and is a homologue of complement receptor 1 (CR1) and other mammalian regulators of complement activation. A key aspect to structure-function relationships in these proteins is the extent of intramolecular module-module interactions, since these dictate the overall shape and flexibility of the molecules. A protein fragment (VCP approximately 2,3) encompassing modules 2 and 3 of VCP was over-expressed in Pichia pastoris. Ultracentrifugation showed that VCP approximately 2,3 is highly asymmetric with an axial ratio of 5.3:1, which is consistent with an end-to-end arrangement of the two modules. NMR spectroscopy, differential scanning calorimetry, CD and intrinsic tryptophan fluorescence were used to monitor unfolding of VCP approximately 2,3. Experiments performed over a range of temperatures and concentrations of guanidinium chloride revealed that module 2 unfolds under milder conditions than, and independently of, module 3. Unfolding of module 2 is not associated with extensive changes in amide (15)N and (1)H chemical shifts of module 3, implying that the modules do not form an extensive intermodular interface. Results obtained in this work for VCP approximately 2,3 are compared with those obtained in a study of CR1 modules 15-17 [Kirkitadze, Krych, Uhrin, Dryden, Smith, Cooper, Wang, Hauhart, Atkinson and Barlow (1999) Biochemistry 38, 7019-7031].

Amino Acid Sequence↗
PubMed1999

Centric diatom morphogenesis: a model based on a DLA algorithm investigating the potential role of microtubules.

Diatoms are single-celled algae which possess characteristic rigid cell walls (frustules) composed of amorphous silica. Frustule formation occurs within a specialised organelle termed the silica deposition vesicle (SDV). During diatom morphogenesis, silica particles are transported to the SDV by silica transport vesicles. Once released within the SDV, the particles are then thought to diffuse until they encounter part of the growing aggregate upon which they adhere. The particles may then undergo a further period of surface relocalisation (sintering) which leads to a smoothing of the surface. A number of computer simulations based on a modified diffusion-limited aggregation (DLA) algorithm, have been undertaken to investigate the potential role of microtubules (which are known to be associated with the periphery of the SDV) in localising deposition of new siliceous material. Based on our findings, we present a new model of diatom morphogenesis which is able to account for many morphological features of diatoms including the influence of environmental effects such as changes in pH and salinity, and the formation of a regular branched pattern.

Algorithms↗
PubMed1999

Beyond micromachining: the potential of diatoms.

Diatoms are microscopic, single-celled algae that possess rigid cell walls (frustules) composed of amorphous silica. Depending on the species of diatom and the growth conditions, these frustules can display a wide range of different morphologies. It is possible to design and produce specific frustule morphologies that have potential applications in nanotechnology.

Biosensing Techniques↗
PubMed1999

Team-based planning: new tools for new times.

The collaborative process of team-based planning draws upon the strengths of a team and develops the skills that healthcare providers need to build consensus during this time of significant change. This article describes three examples of team-based planning and shows how applying an integrated set of tools can help formulate plans and create new options to meet the challenges facing today's healthcare organizations.

Conflict, Psychological↗
PubMed1999

Perinatal outcome after in-vitro fertilization-surrogacy.

The perinatal outcome of pregnancies (both single and multiple) established after in-vitro fertilization (IVF)-surrogacy was evaluated and compared to the outcome of pregnancies that resulted from standard IVF. Analysis of medical records and a telephone interview with physicians, IVF-surrogates, and commissioning mothers were conducted to assess prenatal follow up and delivery care in several hospitals. 95 IVF-surrogates delivered 128 liveborn (65 singletons, 27 sets of twins and two sets of triplets). The commissioning mothers and the IVF-surrogates average ages were 37.7 +/- 5.0 and 30.4 +/- 4.7 years old respectively. IVF-surrogates carrying twin and triplet gestations delivered substantially earlier than those who gestated singleton pregnancies (36.2 +/- 0.4 versus 35.5 versus 38.7 +/- 0.3 weeks gestation respectively; P < 0.001). Twin newborns were significantly lighter than singleton infants born through IVF-surrogacy (2.7 +/- 0.06 versus 3.5 +/- 0.07 kg; P < 0.001). The incidence of low birth weight infants rose from 3.3% in the single births to 29.6% (P < 0.01) in the twins and to 33.3% in the triplets born through IVF-surrogacy. The incidence of prematurity was significantly greater in both twins delivered by IVF-surrogates (20.4%) and infertile IVF patients (58%). The occurrence of pregnancy-induced hypertension and bleeding in the third trimester was four to five times lower in the IVF-surrogates, independently of whether they were carrying multiples. The incidence of Caesarean section was 21.3% for singleton gestations, while two times higher in the IVF-surrogates carrying multiples (56.3%). Postpartum complications occurred in 6.3% of patients and the incidence of malformation was similar to those reported for the general population. The results provide general reassurance regarding perinatal outcome to couples who wish to pursue IVF-surrogacy.

Adult↗
PubMed1999

Herpes simplex virus type 1 immediate-early protein vmw110 induces the proteasome-dependent degradation of the catalytic subunit of DNA-dependent protein kinase.

Herpes simplex virus type 1 (HSV-1) infection causes the active degradation of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), and this process is reliant on the expression of the HSV-1 immediate-early protein Vmw110. In this study we investigated in more detail the mechanism by which the degradation occurs, the domains of Vmw110 which are required, and whether Vmw110 is by itself sufficient for the effect. We found that proteasome inhibitors prevented the degradation of DNA-PKcs, indicating the involvement of a proteasome pathway. Furthermore, the continued activity of DNA-PK during infection in the presence of these inhibitors indicated that Vmw110 does not directly alter the enzyme activity of DNA-PKcs prior to its degradation in a normal infection. Indeed, Vmw110 was found to bind to neither the catalytic nor Ku subunits of DNA-PK. Using mutant Vmw110 viruses we show that the RING finger domain of Vmw110 is essential for the induced degradation of DNA-PKcs but that the ability of Vmw110 to bind to a cellular ubiquitin-specific protease (HAUSP) is not required. When expressed in the absence of other viral proteins, Vmw110 was sufficient to cause the degradation of DNA-PKcs, indicating that the effect on the stability of DNA-PKcs was a direct consequence of Vmw110 activity and not an indirect Vmw110-dependent effect of virus infection. Finally, the Vmw110-induced degradation of DNA-PKcs and loss in DNA-PK activity appears to be beneficial to HSV-1 infection, as virus replication was more efficient in cells lacking DNA-PKcs, especially at low multiplicities of infection.

Binding Sites↗
PubMed1998

High resolution 1H NMR spectroscopic studies on dynamic biochemical processes in incubated human seminal fluid samples.

High resolution 600 MHz 1H NMR spectroscopy was used to investigate the changes in biochemical composition of whole human seminal fluid (SF) and an artificial mixture of prostatic (PF) and seminal vesicle fluid (SVF). A variety of time-related biochemical changes were monitored simultaneously and non-invasively in SF, including enzymatic hydrolysis of phosphorylcholine to choline and polypeptides to amino acids. The fastest NMR-observable reactions in SF were the conversion of phosphorylcholine to choline (t1/2 approximately equal to 9 min) and uridine-5'-monophosphate (UMP) to uridine (t1/2 < 2 min). UMP has not previously been detected in SF because of its rapid hydrolysis. Artificial mixtures of separately obtained prostatic and SVF showed very similar biochemical changes to those observed in whole SF. Addition of EDTA to SF incubated for 2 min post ejaculation strongly inhibited peptide hydrolysis. Zn2+, present in whole SF was shown to be non EDTA-chelatable 2 min after ejaculation, whereas after 7 min, a singlet signal from the ethylenic protons of the Zn-EDTA2- complex was clearly observed which remained constant after 7 min. This indicates that soon after ejaculation (< 5 min) Zn2+ is immobilised in a macromolecular complex which is rapidly broken down by proteolytic enzymes, the released Zn2+ then being free to react with EDTA. Mg- and Ca-EDTA2- complexes were observed at 2 min and remained constant (at 1.4 and 2.1 mM, respectively) throughout the entire time course of the experiment. These studies cast new light on the time-related biochemical changes occurring in the post-ejaculatory SF which may have an important role in reproductive function.

Chelating Agents↗
PubMed1998

The disruption of ND10 during herpes simplex virus infection correlates with the Vmw110- and proteasome-dependent loss of several PML isoforms.

The small nuclear structures known as ND10 or PML nuclear bodies have been implicated in a variety of cellular processes including response to stress and interferons, oncogenesis, and viral infection, but little is known about their biochemical properties. Recently, a ubiquitin-specific protease enzyme (named HAUSP) and a ubiquitin-homology family protein (PIC1) have been found associated with ND10. HAUSP binds strongly to Vmw110, a herpesvirus regulatory protein which has the ability to disrupt ND10, while PIC1 was identified as a protein which interacts with PML, the prototype ND10 protein. We have investigated the role of ubiquitin-related pathways in the mechanism of ND10 disruption by Vmw110 and the effect of virus infection on PML stability. The results show that the disruption of ND10 during virus infection correlates with the loss of several PML isoforms and this process is dependent on active proteasomes. The PML isoforms that are most sensitive to virus infection correspond closely to those which have recently been identified as being covalently conjugated to PIC1. In addition, a large number of PIC1-protein conjugates can be detected following transfection of a PIC1 expression plasmid, and many of these are also eliminated in a Vmw110-dependent manner during virus infection. These observations provide a biochemical mechanism to explain the observed effects of Vmw110 on ND10 and suggest a simple yet powerful mechanism by which Vmw110 might function during virus infection.

Animals↗