Biomedical subjects
A P Jackson
Publications and source records attributed to A P Jackson.
Endocytosis in the brain: the role of clathrin light-chains.
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Preliminary crystallographic analysis of the ATP-hydrolysing domain of the Escherichia coli DNA gyrase B protein.
The 43 kDa N-terminal ATPase domain of the Escherichia coli DNA gyrase B protein has been purified from an over-expressing strain. This protein has been crystallized in two crystal forms, both in the presence of the non-hydrolysable ATP analogue 5'-adenylyl-beta,gamma-imidodiphosphate. The first crystal form is monoclinic P2(1), with cell dimensions a = 76 A, b = 88 A, c = 82 A, beta = 105.5 degrees, and diffracts to at least 2.7 A resolution using synchrotron radiation. Crystal density measurements suggest that there are two molecules in the asymmetric unit (Vm = 3.08 A3/Da). The second crystal form is orthorhombic C222(1), with cell dimensions a = 89.2 A, b = 143.1 A and c = 79.8 A. The crystals diffract to beyond 3 A and are stable for at least 100 hours when exposed to X-rays from a rotating anode source. The asymmetric unit of this crystal form appears to contain one molecule (Vm = 2.96 A3/Da). Data have already been collected to 5 A resolution from native crystals of this second form, and to 6 A resolution from three heavy-atom derivatives. Electron density maps calculated using phases obtained from these derivatives show features consistent with secondary structural elements, and have allowed the molecular boundary to be determined. Higher resolution native and derivative data are being collected.
Conservation of resources in community intervention.
Evaluated the applications of principles of resource conservation and management in community interventions and compared these to a more individual, perception-based psychological perspective. Conservation of resource theory suggests that promotion of well-being and prevention of disease depend on the availability and successful management of resources. When resources are lacking, lost, or when invested without consequent gain, people become vulnerable to psychological and physical disorder and debilitated functioning. Resources, in this context, are defined as those things people value or those things that may help people obtain that which they value. Conservation of resource theory further suggests that resources are to a large extent common to all people, more common within a given culture and time in the developmental cycle, and only to a small extent idiographic. Further, conservation of resource theory posits that resources operate within an ecological context where feedback, sharing, and exchange operate between the individual, social context, and environment. Given these principles, it is reasoned that community interventions must acknowledge the solid base of most problems and accept that interventions must target resources and be intensive enough to change the ecology in which resources operate.
The behaviour of heavy metals in sewage sludge-amended soils.
Soils amended with sewage sludges generally contain elevated concentrations of a wide range of heavy metals and are therefore of interest with regard to their potential impact on human health. This review considers the concentrations of heavy metals in sewage sludges and in the soils amended with them. The effects of sludge amendments on soil properties, the speciation of heavy metals and their bioavailability are reviewed. Variations in heavy metal accumulation between crop species are considered, together with the effects of sludge-borne heavy metals on soil microorganism activity. Perhaps the most important questions to be addressed are the changes in the bioavailability of the heavy metals and their distribution in the soil profile during the residual period. The consequences of the application of sewage sludges to agricultural soils, with regard to the long-term bioavailability and movement of metals in soil profiles, are incompletely understood.
Somatic cell mapping of bovine clathrin light chain genes: identification of a new bovine syntenic group.
Two bovine DNA probes (LCa and LCb) complementary to the clathrin light chain genes were hybridized to DNAs from bovine hamster hybrid somatic cell lines retaining different combinations of bovine chromosomes. Concordancy of retention of the clathrin genes was compared to existing syntenic data for the domestic cow. LCb identified a single locus. CLTB, concordant with the genes encoding bovine anti-Müllerian hormone (AMH) and bovine osteonectin from bovine syntenic group U22. LCa recognized two loci. CLTAL1 from a previously unidentified bovine syntenic group. U25, and CLTAL2 which is concordant with GGTB2, a gene marker for bovine syntenic group U18.
Conservation and diversity in families of coated vesicle adaptins.
The complete sequence of the beta adaptin subunit of the plasma membrane adaptor complex from coated vesicles has been elucidated. Complementary cDNA clones from human fibroblasts, rat lymphocytes, and bovine lymphocytes have been isolated, sequenced, and compared with each other and with beta adaptin sequences from rat brain (Kirchhausen, T., Nathanson, K.L., Matsui, W., Vaisberg, A., Chow, E.P., Burne, C., Keen, J.H., and Davis, A.E. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 2612-2616). Surprisingly, the 937-amino acid beta adaptin polypeptide is totally conserved between species. This remarkable homology contrasts with the absence of significant sequence similarity between the alpha (Robinson, M.S. (1989) J. Cell Biol. 108, 833-842) and beta adaptins of the plasma membrane adaptor complex. Diversity within each adaptin family is created by the expression of different genes and by tissue-specific differential splicing. The structures of the beta and alpha adaptins can both be divided into two globular domains interconnected by a variable and potentially flexible stalk domain.
The accumulation of cadmium by vegetables grown on soils contaminated from a variety of sources.
The accumulation of cadmium by four crops (cabbage, carrot, lettuce and radish) grown on soils contaminated from a variety of sources was investigated in greenhouse pot experiments. Stepwise multiple regression analyses of the data revealed that, out of the 23 soil variables determined, only eight were significantly related to cadmium accumulation in the edible plant tissues. The most frequently occurring soil parameter was total cadmium, which was inversely related to plant cadmium accumulation (CdPlant tissue/CdSoil). This implies that, for the heterogeneous group of soils used, as the concentration of cadmium in the soil increases the proportion available to the plant decreases. This may be due to the presence of metallic ore particles and/or the high sorptive capacity of the most contaminated soils. When the data were divided into two groups: sewage sludge amended soils and inorganically contaminated soils, the R-squared values were usually enhanced and some differences occurred in the variables included in the multiple regression equations; this may be indicative of the differences in speciation. Cadmium accumulation by plants grown on sewage sludge amended soils was lower than that for the inorganically contaminated soils. The heterogeneity of the inorganically contaminated group of soils resulted in lower R-squared values for the multiple regression equations; this group of soils exhibited a wide range of soil variables, such as pH, and had been contaminated by a variety of means, such as atmospheric deposition from metal smelters and the dumping of mine wastes.
Measurement of the fracture toughness of some contact lens hydrogels.
The fracture toughness of three polymethacrylate contact lens hydrogel materials was measured using the method of Generalized Fracture Mechanics. One hydrogel (SNOFLEX S68) had a significantly higher fracture energy per unit area (Jc) than the others, probably because it undergoes plastic deformation at high strains. Differences in toughness imply differences in the mechanical durability of the contact lenses in normal use, which could not be so readily deduced from the results of tensile strength tests.
A cost-effective method for producing custom dental teaching aids using RTV silicone.
A method for making low-cost custom dental teaching aids, using components of standard models and a silicone replication technique, is described. The process is quick, and is suitable for both small and large numbers of individual model types.
Structure of human clathrin light chains. Conservation of light chain polymorphism in three mammalian species.
Complementary DNAs (cDNA) encoding the brain and non-brain forms of the human clathrin light chains LCa and LCb have been isolated, sequenced, and compared with their homologues in cow and rat. The significant differences that distinguish LCa from LCb and the brain from non-brain forms show remarkable preservation in all three species. These features include the position and sequence of the brain-specific inserts, a totally conserved region of 22 residues near the amino terminus, the LCb-specific phosphorylation site, the heavy chain binding site, and a distinctive pattern of cysteine residues near the carboxyl terminus. Unorthodox sequences for translation initiation and polyadenylation are found for LCb contrasting with LCa which exhibits orthodox regulatory sequences. Small insertions in human LCa revealed a duplicated sequence of 13 residues that flank the 22-residue conserved region. Only the carboxyl-terminal copy of this sequence is present in LCb. All sequences are consistent with the heavy chain binding site comprising an alpha-helical central region of the light chains. The hydrophobic face of this helix, which is presumed to interact with the heavy chain, is highly conserved between LCa and LCb, whereas the hydrophilic face shows considerable divergence. To help define the carboxyl-terminal limit of the heavy chain binding region, the epitope recognized by the CVC.6 monoclonal antibody was localized to residues 192-208 of LCa with glutamic acid 198 being of most importance. The faithful preservation of clathrin light chain polymorphism in three mammalian species provides evidence supporting a functional diversification of the brain and non-brain forms of LCa and LCb.
HLA-A and B polymorphisms predate the divergence of humans and chimpanzees.
Major histocompatibility complex (MHC) glycoproteins bind processed fragments of proteins and present them to the receptors of T lymphocytes. The extraordinary polymorphism of class I MHC molecules in man (HLA-A, B and C) and mouse (H-2 K, D and L) poses many questions concerning their diversification and evolution. Comparison of allelic sequences within a species suggests diversity is generated by the assortment of point mutations into varied combinations by mechanisms of recombination and gene conversion. We have now compared class I MHC alleles in two closely related species: humans (Homo sapiens) and chimpanzees (Pan troglodytes). Chimpanzee homologues of HLA-A, HLA-B and a non-classical gene have been identified. No features distinguishing human and chimpanzee alleles could be found. Individual HLA-A or B alleles are more closely related to individual chimpanzee alleles than to other HLA-A or B alleles. These results show that a considerable proportion of contemporary HLA-A and B polymorphism existed before divergence of the chimpanzee and human lines. The stability of the polymorphism indicates that hyper-mutational mechanisms are not necessary to account for HLA-A, B and C diversity.
Active site trapping of nucleotide by smooth and non-muscle myosins.
The folded 10 S monomer conformation of smooth muscle myosin traps the hydrolysis products ADP and Pi in its active sites. To test the significance of this, we have searched for equivalent trapping in other conformational and assembly states of avian gizzard and brush border myosins, using formycin triphosphate (FTP) as an ATP analogue. When myosin monomers were in the straight-tail 6 S conformation, the hydrolysis products were released at about 0.03 s-1. Adoption of the folded 10 S monomer conformation reduced this rate by more than 100-fold, effectively trapping the products FDP and Pi in the active sites. This profound inhibition of product release occurred only on formation of the looped tail monomer conformation. In vitro-assembled myosin filaments released products at a comparable rate to free straight-tail 6 S monomers, and smooth muscle heavy meromyosin, which lacks the C-terminal two-thirds of the myosin tail, also did not trap the products in this way. Phosphorylation of the myosin regulatory light chain had no effect on the rate of product release from straight-tail 6 S myosin monomers or from myosin filaments. Rather, it allowed actin to accelerate product release. Phosphorylation acted also to destabilize the folded monomer conformation, causing the recruitment of molecules from the pool of folded monomers into the myosin filaments. The two processes of contraction and filament assembly are thus both controlled in vitro by light-chain phosphorylation. A similar linked control in vivo would allow the organization of myosin in the cell to adapt itself continuously to the pattern of contractile activity.
Molecular cloning of bovine class I MHC cDNA.
Two cDNA cloned from a Hereford cow B cell line (BL-3) have allowed the determination of the complete coding region for two class I molecules encoded by the bovine MHC (BoLA). The predicted protein sequences have all the features expected of expressed class I molecules that present peptide Ag to cytotoxic T cells. Comparison with class I molecules from other species strongly suggests these cDNA are derived from different genes and provides evidence for the existence of a second expressed class I BoLA locus. The BoLA proteins show greater similarity to HLA than to H-2 molecules, correlating with the cross-reactions of W6/32 and other murine anti-HLA-A,B,C mAb with BoLA molecules. The basis for the W6/32 epitope and the preferential association of H-2 class I H chains with bovine beta 2-m is examined.
Transient-kinetic studies of the adenosine triphosphatase activity of scallop heavy meromyosin.
Fluorescence stopped-flow experiments were performed to elucidate the elementary steps of the ATPase mechanism of scallop heavy meromyosin in the presence and in the absence of Ca2+. ATP binding and hydrolysis, as monitored by the change in tryptophan fluorescence, appear to be Ca2+-insensitive, whereas both Pi release and ADP release are markedly suppressed in the absence of Ca2+. Rate constants for Pi release are 0.2 s-1 and 0.002 s-1 and for ADP release are 6 s-1 and 0.01 s-1 in the presence and in the absence of Ca2+ respectively. Ca2+ binding to the specific site of the regulatory domain is rapid and its release occurs at 25 s-1, consistent with the time scale of a twitch of the striated adductor muscle. Nucleotide binding is a multi-step process requiring a minimum of three states. In such a model Ca2+ controls the rate of conformational changes at the active site in both the forward and the reverse direction, leading to a large dependence of the rate of nucleotide release, but a lesser effect on the overall equilibrium position. The kinetic trapping of nucleotides and Pi at the active site, in the absence of Ca2+, appears to be a fundamental step in suppressing the interaction of the myosin head with the thin filaments in relaxed molluscan muscle.
Kinetic trapping of intermediates of the scallop heavy meromyosin adenosine triphosphatase reaction revealed by formycin nucleotides.
The kinetics of interaction of formycin nucleotides with scallop myosin subfragments were investigated by exploiting the fluorescence signal of the ligand. Formycin triphosphate gives a 5-fold enhancement of the emission intensity on binding to heavy meromyosin, and the profile indicates that the kinetics of binding are Ca2+-insensitive. In contrast, the subsequent product-release steps show a marked degree of regulation by Ca2+. In the absence of Ca2+ formycin triphosphate turnover by the unregulated and the regulated heavy meromyosin fractions are clearly resolved, the latter showing a fluorescence decay rate of 0.002 s-1, corresponding to the Pi-release step. In the presence of Ca2+ this step is activated 50-fold. Formycin diphosphate release is also regulated by Ca2+, being activated from 0.008 s-1 to 5 s-1. In contrast with protein tryptophan fluorescence [Jackson & Bagshaw (1988) Biochem. J. 251, 515-526], formycin fluorescence is sensitive to conformational changes that occur subsequent to the binding step and demonstrate, directly, an effect of Ca2+ on both forward and reverse rate constants. Apart from a decrease in the apparent second-order association rate constants, formycin derivatives appear to mimic adenosine nucleotides closely in their interaction with scallop heavy meromyosin and provide a spectroscopic handle on steps that are optically silent with respect to protein fluorescence. A novel mechanism is discussed in which regulation of the formycin triphosphate activity by Ca2+ involves kinetic trapping of product complexes.
Porcelain facings in dentistry and the importance of clinical illustrations to the dentist and dental ceramist.
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The kinetics of calcium binding to fura-2 and indo-1.
The kinetics of Ca2+ dissociation from fura-2 and indo-1 were measured using a stopped-flow spectrofluorimeter. The dissociation rate constants were 84 s-1 and 130 s-1, respectively, in 0.1 M KC1 at 20 degrees C. The rate constants were insensitive to pH over the range 7.0 to 8.0. The second order association rate constants were estimated indirectly to be in the region of 5 X 10(8) M-1 X s-1 and thus approach the diffusion-controlled limit. The results demonstrate that these new generation indicators are well-suited to measure rapid changes in concentration of intracellular Ca2+.