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R A Cox

Publications and source records attributed to R A Cox.

At least 91 records · Page 5Linked to original sources
PubMed1989

Recognition of specific Physarum alpha-tubulin isotypes by a monoclonal antibody. Sequence heterogeneity around the acetylation site at lysine 40.

The monoclonal antibody 6-11B-1 recognises specifically the acetylated form of alpha-tubulin. The acetylation event occurs on a unique lysine residue, lysine 40. Using 6-11B-1, acetylated alpha-tubulin was detected in myxamoebae but not plasmodia of Physarum polycephalum. Following chemical acetylation plasmodial alpha-tubulin was detected by 6-11B-1. The monoclonal antibody KMP-1 recognises certain Physarum alpha-tubulin isotypes but only in non-acetylated form. Whilst recognising all the non-acetylated fraction of myxamoebal alpha-tubulin only a proportion of plasmodial alpha-tubulin was recognised by KMP-1. Peptides were synthesised corresponding to the acetylation domains (containing lysine 40) of myxamoebal alpha-tubulin and the inferred acetylation domains of two plasmodial-specific alpha-tubulin isotypes. The only difference between the two peptides was at a single residue corresponding to amino acid 44 in the polypeptide. Tyrosine was present in myxamoebal alpha-tubulin and glycine was present in the plasmodial specific peptides; the peptides are referred to as the Tyr44 and Gly44 peptides respectively. Both peptides in acetylated form blocked 6-11B-1 reactivity towards acetylated myxamoebal alpha-tubulin. The Tyr44 but not the Gly44 peptide blocked KMP-1 reactivity towards non-acetylated myxamoebal alpha-tubulin. Tyrosine at position 44 is not found in any other known alpha-tubulin. Thus a unique antigenic determinant exists in certain Physarum alpha-tubulin isotypes, close to the acetylation site at lysine 40. This antigenic determinant forms part of the KMP-1 recognition epitope and explains the unique isotype selectivity of this monoclonal antibody.

Acetylation↗
PubMed1989

Determination and evolutionary significance of nucleotide sequences near to the 3'-end of 16S ribosomal RNA of mycobacteria.

The nucleotide sequence at the 3'-end of 16Sr-RNA (nucleotides 1305-1508) was determined, by the primer extension method, for Mycobacterium smegmatis, Mycobacterium tuberculosis and Mycobacterium vaccae, in addition to Mycobacterium leprae. No differences in nucleotide sequence were detected, indicating that this region of 16SrRNA is highly conserved among mycobacteria. The nucleotide sequence common to the four above-mentioned mycobacteria differs from that reported for species of other genera. For example, for helix 39 (nucleotides 1408-1491) the mycobacterial sequence has 58% similarity with the Escherichia coli sequence, 74% similarity with the Bacillus subtilis sequence and 93% similarity with the Streptomyces sequence. The observations support the assignment of M. leprae to the genus Mycobacterium.

Base Sequence↗
PubMed1989

Structure and expression of an alpha-tubulin gene of Physarum polycephalum.

Fragments of Physarum polycephalum DNA generated by partial digestion with Sau3A were cloned into phage-lambda EMBL4. A recombinant (phage-lambda E alpha Tu) containing an alpha-tubulin (E alpha-tubulin) gene was isolated. The E alpha-tubulin gene is part of the alt B locus. The gene was sequenced and was found to contain seven intervening sequences. The alpha-tubulin isotype (E alpha-tubulin) encoded by the gene has a methionine residue at the C-terminus. The E alpha-tubulin gene has much in common with the N alpha-tubulin gene cloned into phage-lambda NM1149 [M. J. Monteiro & R. A. Cox (1987) J. Mol. Biol. 193, 427-438]. However, the gene products E alpha-tubulin and N alpha-tubulin differ in amino acid sequence near to the C-terminus. E-peptide (corresponding to amino acids 440-448 of E alpha-tubulin) and N-peptide (corresponding to amino acids 437-445 of N alpha-tubulin) were synthesised and used to raise antibodies (E-antibodies and N-antibodies). The antibodies were used to show that on two-dimensional gel electrophoresis N alpha-tubulin travels to the alpha 1 position and E alpha-tubulin moves to the alpha 2 position. Gene-specific DNA probes were used to show that transcripts of the E alpha-tubulin gene were present in the plasmodial but not in the amoebal phase of the life cycle. The E- and N-antibodies detected E alpha- and N alpha-tubulins in plasmodia but not in amoebae, confirming that the expression of the E alpha- and N alpha-tubulin genes is regulated during development. E alpha- and N alpha-tubulin were shown to be components of spindle microtubules by indirect immunofluorescence microscopy.

Amino Acid Sequence↗
PubMed1989

The development of ciliated and mucus cells from basal cells in hamster tracheal epithelial cell cultures.

Hamster tracheal epithelia consist of three cell types: ciliated, mucus and basal cells. Autoradiographic data from several studies suggest that either basal or non-ciliated columnar cells may serve as stem cells for regeneration of lost or damaged ciliated and mucus cells. The objective of the present study was to examine the role of basal cells in the formation of ciliated and mucus cells in hamster tracheal epithelial (HTE) cell cultures via tritiated thymidine ([3H]-TdR) autoradiography. When 3 day cultures were pulsed with [3H]-TdR for 6 hr and incubated for 2 additional days in non-radioactive media (5 day total) label was present in the nuclei of basal and columnar epithelial cells suggesting that the labeled columnar cells may be derived from basal cells. However, the morphological reorganization occurring during this 2 day interval may create difficulties in this interpretation. Since these morphological changes are minimal during the 6 day to 8 day in vitro period, 6 day HTE cultures were pulsed with [3H]-TdR for 6 hr and incubated for 2 additional days in non-radioactive media (8 day total), and examined to further study the fate of labeled basal cells during this period. Analysis of these 8 day cultures revealed that labeled nuclei were present in both basal cells and adjacent ciliated and mucus cells. These results do not exclude the possibility of non-basal cell origin of ciliated and mucus cells in other systems but suggest that, at least in HTE cultures, undifferentiated basal cells have the ability to develop into ciliated and mucus cells.

Animals↗
PubMed1989

Development and characterization of a monoclonal antibody against the tube precipitin antigen of Coccidioides immitis.

Primary infection with Coccidioides immitis is commonly accompanied by the production of an immunoglobulin M precipitin antibody which is detected by the tube precipitin (TP) assay or by the immunodiffusion assay for TP antibody (IDTP assay). In the present investigation, spleen cells from spherulin-immunized BALB/c mice were fused with SP2/O Ag14 myeloma cells, and the resulting hybridomas were screened for antibody to the IDTP antigen by using an enzyme-linked immunosorbent assay. Positive hybridomas were cloned by limiting dilution and injected into pristane-primed mice for ascites production. Characterization of antibody reactivity was accomplished with the IDTP assay, two-dimensional immunoelectrophoresis, and immunoblotting. An immunoglobulin G1 monoclonal antibody which reacts with the IDTP antigen of C. immitis is described. The epitope that is recognized by the monoclonal antibody is also present, but to a lesser extent, on a second coccidioidal antigen which has been designated antigen 2. The monoclonal antibody was not reactive in immunoblots of histoplasmin or blastomycin, indicating that the epitope recognized by this antibody may be specific for C. immitis.

Animals↗
PubMed1989

Induction of tumor necrosis factor alpha by spherules of Coccidioides immitis.

The cytokine tumor necrosis factor alpha (TNF-alpha) functions as an immunomodulatory protein and as a mediator of cachexia. We report that viable or Formalin-killed spherules of Coccidioides immitis induced the secretion of TNF-alpha by peritoneal-exudate cells from BALB/c mice. The identification of the cytokine as TNF-alpha was based on its lytic activity against the TNF-alpha-sensitive LS murine fibrosarcoma cell line but not the TNF-alpha-resistant LR cell line, its neutralization by rabbit anti-TNF-alpha, and its secretion by peritoneal cells having characteristics of macrophages. The induction of TNF-alpha was to spherules and not to contaminating lipopolysaccharide (endotoxin), as evidenced by the finding that polymyxin B, a reagent that blocks the TNF-alpha-inducing component of lipopolysaccharide, did not negate the production of TNF-alpha in response to spherules, whereas pretreatment of spherules with hyperimmune goat antiserum to spherulin neutralized the induction of TNF-alpha by these cells. The demonstration that C. immitis activates macrophages to secrete TNF-alpha in vitro is a new finding and warrants studies to determine whether this cytokine is produced during active coccidioidomycosis.

Animals↗
PubMed1989

The role of basal cells in attachment of columnar cells to the basal lamina of the trachea.

The mechanism by which basal cells play a role in attachment of airway epithelium to the basal lamina has not been determined. Our hypothesis is that basal cells form a structural bridge between columnar cells and the basal lamina via hemidesmosomes, the cytoskeleton, and desmosomes. To evaluate this hypothesis, we determined the percentage of the columnar cell surface area associated with attachment to the basal lamina and the basal cell in tracheal epithelia of different heights. Tracheas from mice, hamsters, rats, bonnet monkeys, cats, and sheep were prepared for electron microscopy by standard techniques. The height of the epithelia ranged from 8.6 microns in the hamster to 56.8 microns in the sheep. The number of basal cells/100 microns ranged from 3.4 in the hamster to 21.4 in the sheep. The percentage of the basal lamina covered by basal cells increased from 32.6 in the hamster to 94.7 in the sheep. In the shorter epithelia of the hamster, 32% of the columnar cell attachment to the basal lamina was indirect through basal cells, and in the taller epithelia of the sheep, 92% of the columnar cell attachment was by this means. Conversely, the percentage of columnar cell surface in contact with the basal lamina decreased from 67.4% in the hamster to 5.3% in the sheep. These data demonstrate that basal cells play a role in attachment of columnar epithelium to the basal lamina by presenting a surface area for cell-to-cell attachment, thus acting as a bridge between columnar cells and the basal lamina.

Animals↗
PubMed1989

Antigenic structure of Coccidioides immitis.

Coccidioides immitis is a unique fungal pathogen, characterized by a saprobic mycelial phase that gives rise to infectious arthroconidia which in host tissue, convert into a morphologically distinct spherule-endospore phase. This multiphasic cycle presents a formidable challenge to the host. The cell wall of C. immitis is a major reservoir of immunoreactive antigens and thus figures prominently in host-parasite interaction in this disease. Extracts of cell walls have yielded antigens reactive with the TP and CF antibodies, antigens that elicit in vivo and in vitro T-cell responses, and antigens that augment or suppress host response. By using techniques of 2D-IEP and Western blotting, investigators have provided evidence that the TP antigen has a predominant polysaccharide composition, with a molecular weight range of 130-330 kD. This antigen may have tolerogenic activity, as suggested by the induction of IgM antibody early during the disease, which then wanes and is followed by a modest or nondemonstrable IgG antibody response. In contrast, the CF antigen is highly immunogenic, inducing IgG levels within 1 to 3 months of clinical illness, which then persist throughout the course of disease. Compositional analyses of the CF antigen indicate that this heat-labile precipitinogen is a glycolipoprotein complex with a molecular weight range of 43-48 kD. The skin test active component(s) of C. immitis has not been purified, but preliminary evidence suggests that it may be a polymeric antigen designated as Ag2. This polymeric antigen is susceptible to enzymatic treatment with mannosidase and protease, indicating a mannoprotein composition, although other constituents may be present as well. Despite differences in the chemical composition of Ag2 and the TP antigen, these two components coelute on molecular sieve, ion-exchange, and lectin affinity columns and both antigens are solubilized from cell walls with alkali. These similar chromatographic profiles suggest that Ag2 and the TP antigen are intimately associated and perhaps present as a complex. Comparative analyses of morphological forms of C. immitis have provided evidence that extracts from spherules are more potent than those from the mycelial phase in eliciting immune responses. These differences may, however, be attributed to quantitative rather than qualitative differences in the antigenic composition of these cell types. In summary, many advances have been made in the isolation and characterization of antigens from this pathogenic fungus. However, additional work is required to fully delineate their production and expression during the morphogenetic phases of C. immitis and to assess their role in the pathophysiology of coccidioidomycosis.

Animals↗
PubMed1989

Death caused by a constricting snake--an infant death.

A 21-month-old male was found dead in his crib. An escaped pet python snake (Python reticulatus) was found near the child on a cribside shelf. Autopsy findings included petechiae associated with asphyxial death plus puncture wounds consistent with the reptile's teeth. This report details this 1982 death of a child in Reno, Nevada, and includes illustration of the reptile's bite marks plus description of snake jaw mechanics in swallowing.

Animals↗
PubMed1988

Identification of EcoRV fragments spanning the N alpha-tubulin gene of Physarum.

The N alpha-tubulin gene of Physarum polycephalum has an EcoRV site at codons 252/253. EcoRV digestion of physarum DNA generated two EcoRV fragments per gene copy comprising both coding and flanking sequences. Hybridisation probes which included coding sequences upstream from the central EcoRV site cross-hybridised with another alpha-tubulin gene. Probes derived from either 5'- or 3'-flanking regions were gene-specific. These probes identified two EcoRV fragments in the haploid strain CLdAXE viz 5.4 kb (5'-fragment) and 6.2 kb (3'-fragment). The same two fragments were identified in EcoRV digests of DNA of the diploid strain M3CVIII, and a second form of the gene was also identified comprising two fragments viz 5.0 kb (5'-end) and 5.5 kb (3'-end). Both forms gave rise to an identical 4.65 kb HindIII fragment as judged by restriction mapping.

Deoxyribonucleases, Type II Site-Specific↗
PubMed1988

Structure and expression of an actin gene of Physarum polycephalum.

Physarum polycephalum (strain M3CVIII) contains four unlinked actin gene loci, each with two alleles (ardA1, ardA2, ardB1, ardB2, ardC1, ardC2, ardD1 and ardD2). The 4800 base HindIII fragment of the ardC2 allele was previously isolated as a recombinant phage lambda. We now report the structure of the actin gene sequences (C-actin gene). The gene, which contains four intervening sequences, codes for the principal actin isotype of plasmodia and it is expressed in both the haploid myxamoebal and diploid plasmodial phases of the life cycle. The C-actin isotype is closely related to actins of Dictyostelium, Acanthamoebae, Drosophila, sea urchin and mammalian cytoplasmic actin, and more distantly related to actins of yeast, Entamoebae and Tetrahymena. The ardC1 and ardC2 alleles differ by a 700(+/- 100) base-pair insertion/deletion in the vicinity of the 3' end of the transcribed region of the gene.

Actins↗
PubMed1988

Immunogenetic analysis of human tuberculosis.

An investigation was undertaken to determine if susceptibility and/or immune response to Mycobacterium tuberculosis was associated with histocompatible leukocyte antigen (HLA) phenotype frequencies of class I or II antigens. Comparisons of the HLA phenotypes of 51 Mexican-American patients with tuberculosis and 54 healthy subjects who differed in their skin-test reactivity to purified protein derivative (PPD) revealed that HLA-DR3 was significantly decreased in patients with tuberculosis, compared with healthy persons who were tuberculin skin-test positive. Although no association was observed between HLA phenotype and skin test reactivity to PPD in tuberculous patients, we did observe an increase in the HLA-A9-B40 phenotype in patients who manifested a strong in vitro proliferative response to PPD, whereas the HLA-B14-DR1 phenotype was increased in patients who exhibited a low proliferative response to this antigen.

Adult↗
PubMed1988

Partial nucleotide sequence of 16S ribosomal RNA isolated from armadillo-grown Mycobacterium leprae.

Ribosomal RNA (rRNA) was isolated from Mycobacterium leprae recovered from infected tissue of the Nine-banded Armadillo, and nucleotide sequences near the 3' end of the 16S species were determined by primer extension in the presence of dideoxynucleotides. Previously published data for bacterial 16S rRNAs show a pattern of conserved and non-conserved sequences that fit a common secondary structure. Our data for M. leprae fits this general pattern.

Animals↗