Search PubMed⌕ Search

Biomedical subjects

D Mills

Publications and source records attributed to D Mills.

At least 73 records · Page 4Linked to original sources
PubMed1990

Use of a synthetic dodecapeptide (malantide) to measure the cyclic AMP-dependent protein kinase activity ratio in a variety of tissues.

1. The cyclic AMP-dependent protein kinase activity-ratio assay was investigated by comparing histone and a synthetic peptide, malantide [Malencik & Anderson (1983) Anal. Biochem. 132, 32-40], as substrates. 2. In several tissues the activity ratio was higher when assayed with histone as the substrate; this result was obtained in control tissues and also in those incubated with agents known to increase cyclic AMP. The effect of these agents to increase the activity ratio was more clearly demonstrated with malantide. 3. The higher activity ratios observed with histone are due to: (a) measurement of phosphorylation not catalysed by cyclic AMP-dependent protein kinase; (b) activation of cyclic AMP-dependent protein kinase by histone during the assay. 4. When tissues were homogenized in buffers without NACl, lower activity ratios were found, owing to the catalytic subunit being artifactually removed from the supernatant. 5. We conclude that the measured activity ratio more faithfully reflects that in the tissue when NaCl is included in the homogenization buffer and malantide is used in the assay. This was confirmed in experiments where cyclic AMP-dependent protein kinase was added to the tissue before homogenization, and no dissociation of the exogenous enzyme was observed.

Amino Acid Sequence↗
PubMed1990

Electron microscopy of hybridoma cells with special regard to monoclonal antibody production.

Electron microscopy of mouse hybridoma cell lines shows that the major difference between non, low and high producer cell lines is the amount of endoplasmic reticulum. Vesicular-tubular or cavernous structures of endoplasmic reticulum, which can survive long after cell death, are particularly abundant in producer cell lines. Immunogold labelling with anti-mouse IgG reveals that antibodies are predominantly located in these structures. The cell membrane undergoes structural changes during the late stages of batch culture with the disappearance of microvilli and the appearance of blebs and deep indentations. Necrosis disrupts the cytoplasmic structures and the nucleus is last to degrade.

Animals↗
PubMed1990

Electrophoretic karyotyping without the need for generating protoplasts.

Chromosome samples for pulsed-field electrophoresis have been prepared without first generating protoplasts. The technique involves treatment of intact, agarose-solidified cell material with protease in the presence of EDTA and SDS. Saccharomyces cerevisiae, Ustilago hordei, Tilletia caries, and T. controversa karyotypes are clearly resolved with this technique. Colonies of U. hordei and S. cerevisiae removed from the surface of agar-solidified media and prepared for PFGE by this abbreviated method also yield well resolved karyotypes.

Chromosomes, Fungal↗
PubMed1990

Construction of a stable shuttle vector for high-frequency transformation in Pseudomonas syringae pv. syringae.

A cryptic 80.3-kilobase plasmid, pOSU900, in Pseudomonas syringae pv. syringae strain J900 could be cured by treatment with mitomycin without affecting the pathogenicity of J900 on the host, Phaseolus vulgaris L. The replication region of pOSU900 was identified, subcloned, and modified for construction of a high-copy cloning vector. This vector could be transformed into Pseudomonas strains with high efficiency (ca. 10(6) transformants per microgram of DNA) and was very stable during growth of the host bacteria in planta.

Cloning, Molecular↗
PubMed1988

A rapid procedure for isolating hemopoietic cell nuclei.

A new method for isolating cell nuclei is described which involves freezing and thawing cells in 2% Tween 40, then gentle homogenization to release nuclei, followed by immediate microcentrifugation through 50% sucrose. Purified nuclei were obtained in 3 min and yields of 78-95% were obtained from a variety of human hemopoietic cells. Electron microscope analysis of nuclei obtained from HL60 cells showed that 89% of the nuclei were intact and have an appropriate morphology. A low level of contamination with other organelles was revealed by electron microscopy and by using specific assays for plasma membrane, mitochondria, lysosomes, Golgi membrane, and endoplasmic reticulum (0.5-5.5%). The value of the technique is that nuclear proteins and small metabolites which might be lost by rapid leakage from isolated nuclei and the possibility of biochemical modification of cellular constituents are minimized by using a rapid isolation procedure.

Cell Fractionation↗
PubMed1988

Molecular analysis of the neurogenic locus mastermind of Drosophila melanogaster.

The neurogenic loci comprise a small group of genes which are required for proper division between the neural and epidermal pathways of differentiation within the neuroectoderm. Loss of neurogenic gene function results in the misrouting of prospective epidermal cells into neuroblasts. A molecular analysis of the neurogenic locus mastermind (mam) has been initiated through transposon tagging with P elements. Employing the Harwich strain as the source of P in a hybrid dysgenesis screen, 6000 chromosomes were tested for the production of lethal mam alleles and eight mutations were isolated. The mam region is the site of residence of a P element in Harwich which forms the focus of a chromosome breakage hotspot. Hybrid dysgenic induced mam alleles elicit cuticular and neural abnormalities typical of the neurogenic phenotype, and in five of the eight cases the mutants appear to retain a P element in the cytogenetic region (50CD) of mam. Utilizing P element sequence as probe, mam region genomic DNA was cloned and used to initiate a chromosome walk extending over 120 kb. The physical breakpoints associated with the hybrid dysgenic alleles fall within a 60-kb genomic segment, predicting this as the minimal size of the mam locus barring position effects. The locus contains a high density of repeated elements of two classes; opa (CAX)n and (dC-dA)n.(dG-dT)n. A preliminary study of the transcriptional activity of the mam region is presented.

Alleles↗
PubMed1988

Molecular analysis of a pathogenicity locus in Pseudomonas syringae pv. syringae.

One of the chromosomal regions of Pseudomonas syringae pv. syringae encoding pathogenicity factors had been mapped into a 3.9-kilobase-pair fragment in previous studies. Promoter probe analysis indicated the existence of a promoter near one end of the fragment. DNA sequencing of this fragment revealed the existence of a consensus promoter sequence in the region of the promoter activity and two open reading frames (ORFs) downstream. These ORFs, ORF1 and ORF2, encoded putative polypeptides of 40 and 83 kilodaltons, respectively. All ORF1::Tn5 as well as ORF2::Tn5 mutant strains were nonpathogenic on susceptible host bean plants and were unable to elicit hypersensitive reactions on nonhost tobacco plants. The deduced amino acid sequence of the 83-kilodalton polypeptide contained features characteristic of known integral membrane proteins. Fusion of the lacZ gene to ORF2 led to the expression of a hybrid protein inducible in Escherichia coli. The functions of the putative proteins encoded by ORF1 and ORF2 are unknown at present.

Amino Acid Sequence↗
PubMed1988

Characterization of a novel nuclear envelope protein restricted to certain cell types.

The monoclonal antibody AGF2.3 identifies a nuclear envelope protein that is restricted to certain cell types. In particular, this antigen shows a reduced level of expression during haemopoietic cell maturation. In this study, we have examined the relationship of this protein to known nuclear envelope proteins that have a similar molecular mass. Antigen extraction and immunoelectron microscope studies revealed that the AGF2.3 protein is an integral membrane protein present at both the inner and outer aspects of the nuclear envelope. The protein is not associated with nuclear pores and therefore is distinct from pore complex proteins. The AGF2.3 protein does not have ATPase activity. Therefore, this protein is also distinct from a myosin heavy chain-like ATPase that is associated with the nuclear envelope. The AGF2.3 antibody identifies a novel nuclear envelope protein. Further studies of the biochemical nature of the AGF2.3 protein should provide insight into novel cellular processes at the nuclear envelope relating to the lineage or maturation status of cells.

Antibodies, Monoclonal↗
PubMed1987

Identification and mapping of regions that confer plasmid functions and of sites for excisive recombination of plasmid pMMC7105.

Strain PP808 of Pseudomonas syringae pv. phaseolicola contains pEXC8080 (34.6 kb), the smallest of several plasmids that originated by partial excision of the cryptic plasmid, pMMC7105 (150 kb), from the host chromosome. This excision plasmid is derived entirely of sequences from pMMC7105 and contains a 24 kb region referred to as common DNA, which is present in each of the other excision plasmids. A six enzyme restriction endonuclease map was constructed of pEXC8080. The replication region was mapped by identifying small restriction fragments that conferred replication properties to pMB1 plasmids that otherwise fail to replicate in Pseudomonas. This region is located within the common DNA and is 0.8-3.8 kb in size. Sequences from pEXC8080 failed to stabilize pMB1 derivatives in Pseudomonas in the absence of antibiotic selection, but stability functions were mapped to a region of pMMC7105 that presumably remains integrated in the chromosome of strain PP808. An incompatibility region was mapped to a 7.3 kb region on pEXC8080 that is closely linked to, but not included within, the replication region. The recombination site was mapped to a 1.2 kb region of the fusion fragment that was formed upon excision of pEXC8080. RS-I, a repetitive sequence found on pMMC7105 was present in the fusion fragment at the site of recombination. RS-I was also mapped to BamHI fragments that recombined upon excision of pEXC8080 and suggest that it provides sites for homologous recombination.

Anti-Bacterial Agents↗
PubMed1987

Differentiation of newborn rat adipocyte precursors in defined serum-free medium.

Newborn rat adipocyte precursors, isolated from inguinal fat pads of 2 day-old NBR rats proliferate and undergo adipose differentiation in defined medium in the absence of serum when cultivated on polylysine coated dishes in DME-F12 medium supplemented with fibronectin, insulin, transferrin and FGF. After 7 days in culture in these conditions, 90% of the cells have undergone differentiation as measured by the increase of G3PDH specific activity and by the accumulation of triglycerides in their cytoplasm. In contrast, the cells cultivated in the presence of 10% fetal bovine serum, have a limited ability to differentiate. These results indicate that newborn rat adipocyte precursors from inguinal fat pads do not require the presence of an undefined adipogenic factor in order to differentiate in culture. In contrast, proliferation and differentiation are dependent on the presence of insulin in the culture medium. Moreover, the data presented in this paper show that the rat adipocyte precursor culture represents a rapid and reproducible system for investigating the processes of adipose tissue development and for studying the negative and positive regulators of the adipose differentiation in a controlled environment.

Adipose Tissue↗
PubMed1987

Design and synthesis of a theophylline bonded-phase column for HPLC--application to separation of aromatic carboxylic acids.

A stationary phase has been designed and synthesized in which theophylline residues are covalently bonded to a silica support through an eight carbon hydrocarbon linkage. The phase offers improved resolution in the separation of aromatic carboxylic acids over that available with conventional reversed phase supports. The column is relatively stable. Retention can be modified by adjusting mobile phase composition with respect to pH, electrolyte type and concentration, and organic modifier as well as by manipulating the temperature at which chromatography is carried out. The capacity factors, k', for a series of ring substituted benzoic acids were correlated with the complexation constants previously reported for these compounds with theophylline in bulk phase solution.

Journal Article↗
PubMed1985

The effects of binocular and monocular occlusion on the number of optic nerve axons containing degenerative organelles.

In this study chicks were incubated in the dark and then reared for 2 days with either none, one or both eyes occluded. Cross-sections of the optic nerves were examined for profiles of axons containing accumulations of degenerative organelles. There were significantly more organelle-containing axons in the non-occluded and monocularly occluded birds compared with the binocularly occluded birds. There was no significant difference between the non-occluded and monocular group. Furthermore, there was no significant difference between the occluded and non-occluded eyes of monocular birds. We propose that the rise in organelle-containing axons during development reflects a phenomenon involved in the linking of the eyes, and that visual input to a single eye is sufficient to hasten this process.

Animals↗
PubMed1985

Myelogenesis and estimation of the number of axons in the anterior commissure of the chick (Gallus gallus).

By use of light- and electron microscopy the anterior commissure of the chick was studied at different times during development. Between the 19th day of incubation and the 35th day after hatching the cross-sectional area of the anterior commissure, as determined from mid-sagittal sections, undergoes a 6-fold increase in size. Thereafter the area remains fairly constant. The total number of fibres in the anterior commissure was estimated to be 89000. The full complement of fibres is already present by the 19th day of incubation. Myelogenesis occurs mainly between the 19th day of incubation and the 35th day after hatching, concomitant with the increase in cross-sectional area. From the 35th day after hatching, myelinated fibres comprise approximately 40% of the total number of fibres. The median diameter of unmyelinated fibres is about 0.35-0.40 micron. The median diameters of myelinated axons and fibres are 0.8-1.0 micron and 1.1-1.3 micron, respectively.

Animals↗
PubMed1985

Evidence for self-absorption of terminals by developing axons of retinal ganglion cells in the chick.

The appearance of membrane-bound degenerative organelles in chick optic nerve axons was studied at the electron microscopic level. A semiquantitative analysis revealed a sharp increase in the number of axons containing accumulations of such organelles during the second day after hatching. In dark-reared chicks this increase was retarded, suggesting the presence of a light-influenced event in the early post-hatch period.

Animals↗