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R W Williams

Publications and source records attributed to R W Williams.

At least 19 recordsLinked to original sources
PubMed1995

Hepatitis core antigen produced in Escherichia coli: subunit composition, conformational analysis, and in vitro capsid assembly.

The production and biochemical and physiocochemical analysis are described of recombinant-produced hepatitis B virus core antigen (HBcAg capsid) and the corresponding particle produced by a deletion mutant missing the C-terminal 39 residues (HBeAg). Conditions for producing HBeAg from HBcAg capsids by in vitro proteolysis are also described. The morphology and masses of these capsids were determined by scanning transmission electron microscopy. Both HBcAg and HBeAg capsids comprise two size classes that correspond to icosahedral lattices with triangulation numbers (T) of 3 and 4, containing 180 and 240 subunits per capsid, respectively. This dimorphism was confirmed by sedimentation equilibrium and sedimentation velocity measurements on a Beckman Optima XL-A analytical ultracentrifuge. More than 60% of HBcAg capsids were T = 4, whereas only 15-20% of HBeAg capsids were of this size class: the remainder, in each case, were T = 3. Circular dichroism and Raman spectroscopy were used to determine the overall secondary structures of HBcAg and HBeAg capsids. Both have high alpha-helical contents, implying that this capsid protein does not conform to the canonical beta-barrel motif seen for all plant and animal icosahedral viral capsids solved to date. We suggest that the C-terminal domain of HBcAg has a random coil conformation. In vitro dissociation of HBeAg capsids under relatively mild conditions yielded stable dimers. The reassociation of HBeAg dimers into capsids appears to be driven by hydrophobic processes at neutral pH. Capsid assembly is accompanied by little change in subunit conformation as judged by circular dichroism and fluorescence spectroscopy. The thermal stability of HBcAg capsids was compared calorimetrically with that of in vitro assembled HBeAg capsids. Both have melting temperatures > 90 degrees C, implying that the C-terminal region makes little difference to the thermal stability of HBcAg: nevertheless, we discuss its possible role in facilitating disassembly and the release of viral nucleic acid.

Calorimetry, Differential Scanning↗
PubMed1995

Genetic control of retinal projections in inbred strains of albino mice.

Mutations in the tyrosinase gene are often associated with a misrouting of retinal ganglion cell axons at the optic chiasm. In albinos, tyrosinase activity is lost and some ganglion cell axons that would normally project into the ipsilateral optic tract instead cross midline and project into the contralateral tract. The developmental mechanisms that cause this modification in neuronal connectivity are unknown. In this study, we screened six diverse strains of albino mice (strains 129, A, AKR, BALB/c, C57BL/6-c/c, and CD-1) to discover genetically determined variations and possible gene loci that might affect the severity of the albino decussation abnormality. Ganglion cells were retrogradely labeled with horseradish peroxidase, and the ipsilaterally and contralaterally projecting cells were counted. The average number of ipsilaterally projecting ganglion cells in the six albino strains varies from 1,000 to 1,300. Despite this variation, 1.8-1.9% of the total population projects ipsilaterally in each strain. In comparison, 2.8% project ipsilaterally in the pigmented strain, C57BL/6(-)+/+. However, the percentage of displaced, ipsilaterally projecting cells varies substantially among albino strains--from a low of 4% in strain CD-1 to a high of nearly 10% in C57BL/6-c/c. We conclude that even with large differences in genetic background and in absolute numbers of ganglion cells, there is no appreciable variation in the magnitude of decussation error among albino mice. The consistent effect of null alleles at tyrosinase suggests a comparably tight linkage between the biochemical activity of this enzyme and the mechanisms that control decussation phenotype.

Alleles↗
PubMed1995

Analysis of the retinas and optic nerves of achiasmatic Belgian sheepdogs.

An autosomal recessive mutation carried in a family of black Belgian sheepdogs eliminates the optic chiasm--all retinal ganglion cell axons extend directly into the ipsilateral optic tract. One key issue we are trying to resolve is whether the retina or the chiasm is the principal site of mutant gene action. In this study, we have examined retinas of mutants to discover any associated changes in retinal structure. Retinas of mutant animals are relatively normal. Inner and outer nuclear layers are qualitatively indistinguishable from those of normal dogs. The principal difference is that the area centralis of mutants is smaller and had a lower peak ganglion cell density than that of normal dogs (8,100 vs 10,500/mm2, P < 0.05). This mutant phenotype is similar to that seen in retinas of Siamese cats and albino ferrets. Beyond area centralis, the central-to-peripheral gradient in ganglion cell density is normal in mutants. The size of the optic nerves, density of axons, and total number of axons do not differ between mutant and normal dogs. One of three mutant dogs had a small abnormal optic chiasm. Retrograde labeling of ganglion cells demonstrated that the residual crossed projection originated from cells in a widespread region in nasal retina and not solely from the peripheral nasal region, as might be expected of an anti-albino. Although our analysis does not rule out the retina as a site of mutant gene action, the modest differences between mutant and normal retinas suggest that the mutation either acts outside the retina or exerts a highly specific effect on ganglion cell trajectories alone.

Animals↗
PubMed1995

Atrazine treatment potentiates excretion of mutagenic urine in 2,6-dinitrotoluene-treated Fischer 344 rats.

Atrazine (ATZ), an s-triazine herbicide, is a widespread environmental contaminant. The hepatocarcinogenic component of technical grade dinitrotoluene, 2,6-dinitrotoluene (2,6-DNT, 19.5%), is a byproduct of trinitrotoluene synthesis and is found at production sites. This study explores the effect of ATZ treatment on the bioactivation of the promutagen, 2,6-DNT. Male Fischer 344 rats (5 weeks old) were administered 50 mg/kg of ATZ by gavage for 5 weeks. At 1, 3, and 5 weeks, both DMSO-control and ATZ-pretreated rats were treated p.o. with 75 mg/kg of 2,6-DNT and were housed in metabolism cages for urine collection. Sulfatase- and beta-glucuronidase-treated, concentrated urine was bioassayed for urinary mutagens in a microsuspension modification of the Salmonella assay with and without metabolic activation. No significant change in mutagen excretion was observed in ATZ-treated rats; however, an elevation in direct-acting urine mutagens from rats receiving ATZ and 2,6-DNT at weeks 1 (359 +/- 68 vs. 621 +/- 96 revertants/ml) and 5 (278 +/- 46 vs. 667 +/- 109 revertants/ml) of treatment was observed. The increase in production of urinary mutagens was accompanied by an elevation in small intestinal nitroreductase activity. Increases in large intestinal nitroreductase and beta-glucuronidase were observed after 5 weeks. There was no apparent effect of ATZ following 5 weeks of treatment on the production of 2,6-DNT-derived hepatic DNA adducts. ATZ treatment modifies intestinal enzymes responsible for promutagen bioactivation, and potentiates the excretion of mutagenic urine in 2,6-DNT-treated animals.

Animals↗
PubMed1995

CDR molecular localization of possible anti-idiotypic anti-DNA antibodies in normal subjects, patients with SLE, and SLE first-degree relatives.

Patients with active systemic lupus erythematosus (SLE) with disease worsening or severe flares frequently show very low levels of serum immunoglobulin G (IgG) anti-F(ab')2 antibody. Anti-F(ab')2 antibody probably represents a polyclonal collection of generic anti-idiotypic antibodies involved in immune homeostasis. We synthesized the entire variable regions of the heavy and light chains (VH and VL) of two monoclonal anti-DNA antibodies, V88 and 2A4, as overlapping 7-mers on small polypropylene pins and tested these linear segments of anti-DNA V-regions for reactivity against serum samples from 10 normal subjects with high serum IgG anti-F(ab')2, 11 normal subjects with low anti-F(ab')2, 5 patients with SLE with active uncontrolled disease, 3 patients with SLE in remission, and 8 unaffected normal first-degree SLE relatives. VH and VL regions of a human monoclonal IgG anti-rabies antibody were also tested as a control. Concordant IgG antibody reacting with the same complementarity-determining regions (CDRs) was arbitrarily scored as indicative of the presence of anti-idiotypic antibody in test serum samples. Among normal subjects with either high or low serum anti-F(ab')2 levels, 10% to 21% showed strong concordant anti-CDR reactions with either the monoclonal anti-DNA or the control monoclonal anti-rabies V-region sequences. However, all patients with active SLE showed no detectable anti-CDR-reactive antibody. Patients with SLE in remission often showed return of strong concordant anti-CDR antibody. Normal unaffected SLE relatives also showed high levels of anti-CDR reactivity for both monoclonal anti-DNA and anti-rabies antibody sequences.

Adolescent↗
PubMed1992

Structure of clonal and polyclonal cell arrays in chimeric mouse retina.

One of the most striking results of recent cell-lineage studies of vertebrate retina is the marked variability in the size and types of clones marked by retroviral transfection and dye injection of embryonic progenitor cells. Is this variability due to microenvironmental modulation of cell determination, to lineage restriction, or to experimental perturbation of the progenitor cells? We have taken advantage of species-specific DNA probes to mark groups of lineage-related cells in experimental mouse chimeras. This method of marking cells has two distinct advantages over previous methods: direct manipulation of progenitor cells is avoided, and clones are established at an earlier stage of retinal development. The most notable feature of retinal cohorts in chimeras is their structural uniformity--each is a solid radial array that contains the same ratio of major cell types as the retina itself. This is true even of the smallest monoclonal cohorts, which contain fewer than 200 cells. Our results provides compelling empirical support for the hypothesis that the murine retina is made up of hundreds of relatively homogeneous radial units, each derived from single retinal precursor cells. This finding is inconsistent with micro-environmental modulation of clone structure early in development. We raise the possibility that the heterogeneity among clones marked by dye injection and transfection is due to progressive lineage restriction or to experimental perturbation of the retinal progenitor cells.

Animals↗
PubMed1992

Lineage versus environment in embryonic retina: a revisionist perspective.

The idea that microenvironmental cues act alone late in development to determine a cell's phenotype has dominated recent discussion of, retinal development, and has successfully displaced the notion of any role for cell lineage in the process of cell determination. We argue that there is, in fact, evidence favoring a degree of lineage restriction during the development of the vertebrate retina. We propose that environmental factors modulate a process of progressive lineage restriction. In this model, progenitor cells are viewed as having unequal potential, and their progeny are viewed as being committed to one of the major retinal cell classes before the stage at which they become postmitotic.

Animals↗
PubMed1992

Development of source testing, analytical, and mutagenicity bioassay procedures for evaluating emissions from municipal and hospital waste combustors.

Incineration is currently being used for disposal of about 10% of the solid waste generated in the United States, and this percentage will likely increase as land disposal declines. Siting new incinerators, however, is often controversial because of concerns related to the possibility of adverse health effects and environmental contamination from long-term exposure to stack emissions. Specific concerns relate to the adequacies of a) stack emission testing protocols, b) existing regulations, and c) compliance monitoring and enforcement of regulations. U.S. Environmental Protection Agency laboratories are cooperatively conducting research aimed at developing new testing equipment and procedures that will allow a more comprehensive assessment of the complex mixture of organics that is present in stack emissions. These efforts are directed specifically toward developing source testing equipment and procedures, analytical procedures, and bioassay procedures. The objectives of this study were to field test two types of high-volume source dilution samplers, collect stack samples for use in developing analytical and mutagenicity bioassay procedures, and determine mutagenicity of organics associated with emission particles from two municipal waste combustors and a hospital waste combustor. Data are presented for particle concentrations and emission rates, extractable organic concentrations and emission rates, and Salmonella (Ames) mutagenic potency and emission rates. The mutagenic emission rates and emission factors are compared to other incinerators and combustion sources.

Air Pollutants↗
PubMed1991

The structure of human acidic fibroblast growth factor and its interaction with heparin.

The secondary and tertiary structure of recombinant human acidic fibroblast growth factor (aFGF) has been characterized by a variety of spectroscopic methods. Native aFGF consists of ca. 55% beta-sheet, 20% turn, 10% alpha-helix, and 15% disordered polypeptide as determined by laser Raman, circular dichroism, and Fourier transform infrared spectroscopy; the experimentally determined secondary structure content is in agreement with that calculated by the semi-empirical methods of Chou and Fasman (Chou, P. Y., and Fasman, G. C., 1974, Biochemistry 13, 222-244) and Garnier et al. (Garnier, J. O., et al., 1978, J. Mol. Biol. 120, 97-120). Using the Garnier et al. algorithm, the major secondary structure components of aFGF have been assigned to specific regions of the polypeptide chain. The fluorescence spectrum of native aFGF is unusual in that it is dominated by tyrosine fluorescence despite the presence of a tryptophan residue in the protein. However, tryptophan fluorescence is resolved upon excitation above 295 nm. The degree of tyrosine and tryptophan solvent exposure has been assessed by a combination of ultraviolet absorption, laser Raman, and fluorescence spectroscopy; the results suggest that seven of the eight tyrosine residues are solvent exposed while the single tryptophan is partially inaccessible to solvent in native aFGF, consistent with recent crystallographic data. Denaturation of aFGF by extremes of temperature or pH leads to spectroscopically distinct conformational states in which contributions of tyrosine and tryptophan to the fluorescence spectrum of the protein vary. The protein is unstable at physiological temperatures. Addition of heparin or other sulfated polysaccharides does not affect the spectroscopic characteristics of native aFGF. These polymers do, however, dramatically stabilize the native protein against thermal and acid denaturation as determined by differential scanning calorimetry, circular dichroism, and fluorescence spectroscopy. The interaction of aFGF with such polyanions may play a role in controlling the activity of this growth factor in vivo.

Algorithms↗
PubMed1991

Designed coiled-coil proteins: synthesis and spectroscopy of two 78-residue alpha-helical dimers.

Receptor-adhesive modular proteins are nongenetic proteins designed to contain ligand, spacer, coil, and linker modules and to interact strongly with integrins or other types of cell-surface receptors. We have designed, chemically synthesized, and characterized a 39-residue peptide chain having a 6-residue ligand module (Gly-Arg-Gly-Asp-Ser-Pro-) for adherence to Arg-Gly-Asp-binding integrin receptors, a 3-residue spacer module (-Gly-Tyr-Gly-) for flexibility, and a 30-residue coil module [-(Arg-Ile-Glu-Ala-Ile-Glu-Ala) 4-Arg-Cys-NH2] containing four 7-residue repeats for dimerization. This chain was designed to form a 78-residue noncovalent dimer (P39) by folding the coils of two chains into an alpha-helical coiled coil through hydrophobic interaction of eight pairs of Ile residues. Air oxidation of P39 gave P78, a 78-residue covalent dimer having a disulfide bridge linking its C termini. Raman spectroscopy indicated that both synthetic proteins have high alpha-helical content. Ultraviolet circular dichroic spectroscopy indicated that both dimers contain stable alpha-helical coiled coils. Its C-terminal disulfide bridge renders P78 significantly more stable than P39 to thermal denaturation or denaturation by urea. The coiled coil of P39 was 30% unfolded near 55 degrees C and half-unfolded in 8 M urea, while that of P78 was 30% unfolded only near 85 degrees C. These studies have demonstrated the feasibility of using these ligand, spacer, and coil modules to construct the designed coiled-coil proteins P39 and P78, a stage in the nanometric engineering of receptor-adhesive modular proteins.

Amino Acid Sequence↗
PubMed1991

A novel cytoarchitectonic area induced experimentally within the primate visual cortex.

The cerebral cortex is divisible into a number of cytoarchitectonic areas, but developmental mechanisms that regulate their number and size remain unknown. Here we provide evidence that reducing the population of selected thalamic fibers projecting into the primary visual cortex (area 17) of monkeys during midgestation induces the formation of a novel cytoarchitectonic area situated along the border of and embedded within area 17. This region, termed area X, differs cytoarchitectonically from both area 17 and the adjacent secondary visual cortex (area 18). We propose that an aberrant combination of thalamic and cortical connections acting on a portion of prospective area 17 deprived of its normal thalamic input may result in formation of a hybrid cortex. Our results support the protomap hypothesis of cortical parcellation and suggest how during evolution new cytoarchitectonic regions may arise by cell-cell interactions that depend on a unique combination of intrinsic properties of cortical neurons and afferent fibers.

Animals↗
PubMed1991

The human retina has a cone-enriched rim.

Video-enhanced imaging of retinal wholemounts reveals an abrupt change in the composition of the photoreceptor mosaic at the edge of the human retina. Cone densities rise threefold and rod densities fall tenfold in a 1-mm-wide peripheral band. Antibodies directed against cones confirm the identification of the major subtypes of photoreceptors within this peripheral band. The cone-enriched rim is most highly developed along the nasal retinal margin, an area where the extreme lateral periphery of the visual field is imaged. This rim of cones may function as part of a rapid-acting alert mechanism under conditions of moderate and bright illumination.

Adult↗
PubMed1991

Microdiskectomy--myth, mania, or milestone? An 18-year surgical adventure.

The term "microdiskectomy" in the medical literature defines absolutely no surgical parameters or specific techniques for the treatment of the herniated nucleus pulposus. I attempt to clarify frequently used slang and to design a logical mental and microsurgical approach to the problem of lumbar disk herniations. The rigid surgical technique of microlumbar diskectomy is closely examined.

Humans↗