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Biomedical subjects

C S Jones

Publications and source records attributed to C S Jones.

At least 55 records · Page 3Linked to original sources
PubMed1998

Molecular characterisation of intermediate snail hosts and the search for resistance genes.

The relationship between schistosomes and their intermediate hosts is an extremely intricate one with strains and species of the parasite depending on particular species of snail, which in turn may vary in their susceptibility to the parasites. In order to gain a better understanding of the epidemiology of the disease we have been investigating the use of molecular markers for snail identification and for studying host-parasite relationships. In this paper we will draw on examples concerning schistosomiasis in West and East Africa to illustrate how a molecular analysis can be used as part of a "total evidence" approach to characterisation of Bulinus species and provide insights into parasite transmission. Particular emphasis is given to ribosomal RNA genes (rRNA), random amplified polymorphic DNA (RAPDs) and the mitochondrial gene cytochrome oxidase I (COI). Snails resistant to infection occur naturally and there is a genetic basis for this resistance. In Biomphalaria glabrata resistance to Schistosoma mansoni is known to be a polygenic trait and we have initiated a preliminary search for snail genomic regions linked to, or involved in, resistance by using a RAPD based approach in conjunction with progeny pooling methods. We are currently characterising a variety of STSs, (sequence tagged sites) associated with resistance. These can be used for local linkage and interval mapping to define genomic regions associated with the resistance trait. The development of such markers into simple dot-blot or specific PCR-based assays may have a direct and practical application for the identification of resistant snails in natural populations.

Animals↗
PubMed1997

Retinoic acid inhibition of cell cycle progression in MCF-7 human breast cancer cells.

Cell cycle analysis indicates that retinoic acid (RA) inhibition of MCF-7 cell growth occurs through induction of G1 arrest with a concomitant reduction in the proportion of cells in S and G2 + M phases. RA did not affect cyclins D1, A, and E and cyclin-dependent kinase 2 (CDK2) expression, but significantly reduced cyclin D3 and CDK4 expression after 24 h. RA also inhibited cyclin B1 and CDC2 expression, possibly responsible for the reduction of the proportion of cells in G2 + M and S phases. RA did not induce p16 and p27 expression, but obviously reduced p21 level in MCF-7 cells. The retinoid markedly reduced pRB protein level and abrogated pRB phosphorylation after 48 h; it also reduced transcription factor E2F1 expression at both the mRNA and protein levels. E2F1 promoter activity was reduced by 60%, which is probably responsible, at least in part, for the reduction of E2F1 expression in RA-treated MCF-7 cells. These observations demonstrate a marked effect of RA on some of the key cell cycle regulatory proteins in MCF-7 cells. Cyclin D3 and CDK4 are likely the early targets of RA, followed by reduced pRB expression and phosphorylation, as well as by the inhibition of the E2F1 transcription factor which controls progression from G1 to S phase. Most of these events precede the observed reduction in MCF-7 cell growth, which begins at Day 3 of RA treatment.

Breast Neoplasms↗
PubMed1997

Autocatalytic activation of human cathepsin K.

The in vitro activation of the recombinant purified human cathepsin K (EC 3.4.22.38) was examined by mutagenesis. Cathepsin K was expressed as a secreted proenzyme using baculovirus-infected Sf21 insect cells. Spontaneous in vitro activation of procathepsin K occurred at pH 4 and was catalyzed by exogenous mature cathepsin K. Three intermediates were identified as resulting from cleavages after Glu19, Ser98, and Glu110. The mature enzyme was composed of mixture of enzymes with N termini of Gly113, Arg114, and Ala115 with varying ratios depending on the preparation. Molecular weight determinations were consistent with the absence of carbohydrate in the mature protein, while electrospray mass spectroscopy indicated that six of the eight cysteine residues were in disulfide linkage, and that the protein had Met329 as the C-terminal residue. A mutant was constructed in which the active site Cys139 was changed to Ser. [Ser139,Ala163]Procathepsin K (containing mutation C139S,S163A) failed to spontaneously process and was only partially processed in the presence of 1% exogenous wild-type mature cathepsin K forming intermediates, which were identical to those observed in the activation of wild-type. [Ser139,Ala163]Procathepsin K could be fully processed to mature enzyme by including one equivalent of wild-type procathepsin K in the activation mixture. These results indicated that in vitro activation of the procathepsin K was an autocatalytic process.

Amino Acid Sequence↗
PubMed1997

The isolation of RNA from raspberry (Rubus idaeus) fruit.

Previous attempts to extract high-quality, total RNA from raspberry (Rubus idaeus) fruits using published protocols have proven to be unsuccessful. Even the use of protocols developed for the extraction of RNA from other fruit tissue has resulted in low yields (1) or the isolation of degraded RNA (2). Here, we report on the development of a quick and simple method of extracting total RNA from raspberry fruit. Using this method, high yields of good quality, undegraded RNA were obtained from fruit at all stages of ripening. The RNA is of sufficient quality for northern analysis and cDNA library construction.

Fruit↗
PubMed1996

Temporal patterns of clonality and parasitism in a population of freshwater bryozoans.

Clonal reproduction is commonly incorporated into the life cycles of many metazoans. However, whether and how such highly clonal animals persist in the face of natural enemies remains poorly understood. Here we report the first temporal genetic study of a clonal population, the freshwater bryozoan Cristatella mucedo, and the associated prevalence of a myxozoan parasite. High levels of both clonality and parasitism persisted over a 3 year period. Random amplified polymorphic DNA markers revealed four distinct clones of C. mucedo. The two most common clones varied in abundance with the significantly more common clone in the first year becoming the significantly less common by the third year. There was no evidence that the most common clone was disproportionately infected. These results are discussed in relation to predictions of the Red Queen and the metapopulation dynamics of clonal organisms.

Animals↗
PubMed1996

Dietary retinoic acid inhibits mouse skin carcinogenesis irrespective of age at initiation.

In the two-stage protocol of skin carcinogenesis, the carcinogen 7,12-dimethylbenz[a]anthracene (DMBA) is applied to the skin of mice at around seven weeks of age. We previously performed DMBA initiation at three weeks of age to study the effect of pharmacological (30 micrograms/g diet) dietary retinoic acid (RA) on skin carcinogenesis. In this study we asked whether dietary pharmacological RA is equally effective against skin carcinogenesis when mice are initiated with (DMBA) at 7 weeks of age and then subjected to weekly applications of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) or mezerein (MEZ) for 20 weeks. Similar to the three-week initiation protocol, high dietary RA inhibited papilloma incidence and yield in MEZ- but not in TPA-promoted female SENCAR mice. In addition, carcinoma incidence and yield were decreased by high dietary RA in TPA- as well as MEZ-treated mice. These data demonstrate that the high dietary RA diet is as effective in inhibiting papilloma and carcinoma formation when the DMBA is applied at seven weeks of age as at three weeks.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1995

The H-ras oncogene interferes with retinoic acid signaling and metabolism in NIH3T3 cells.

We have previously shown that retinoic acid (RA) fails to induce transglutaminase C in H-ras transformed NIH-3T3 cells. Therefore, we investigated the effect of the H-ras oncogene on the metabolism of RA and on the expression of the cellular RA-binding protein I mRNA. HPLC analysis of the media and cell extracts demonstrated that H-ras-transformed cells metabolize RA to a much lesser extent than control cells, resulting in a higher concentration of RA in H-ras cells. Although inactive in endogenous transglutaminase induction, H-ras cell-associated RA was shown to be biologically available to induce activation of a reporter construct containing a retinoid response element and in stimulating transglutaminase activity in nontransfected cells. Cellular RA-binding protein I mRNA, supposedly involved in RA storage, was significantly increased in the H-ras-transformed cells. These data demonstrate that, even though H-ras-transformed cells accumulate up to 20 fold the concentration of RA as NIH-3T3 cells, they fail to show transglutaminase induction, suggesting that H-ras interferes with signal transduction by RA.

3T3 Cells↗
PubMed1995

Conversion of murine Fabs isolated from a combinatorial phage display library to full length immunoglobulins.

The use of combinatorial Ig libraries displayed on the surface of bacteriophage has advantages over traditional hybridoma techniques for the generation of mAbs but in many instances full length Igs may be more desirable than Fab fragments. Two murine Fabs reactive with the human complement component C5a, recovered from a combinatorial library, were converted to full length IgG2a mAbs. The VH and VL domains of these antibodies were removed from the bacterial expression vector used for the combinatorial library construction, and subcloned into individual mammalian expression vectors containing the corresponding Ig heavy and light chain constant regions. The subcloning relied on 5' restriction endonuclease sites encoded by the oligonucleotide primers originally used to amplify the Ig cDNAs and 3' sites conserved in CH1 and C kappa. These vectors were co-transfected into COS cells yielding full length IgG2a versions of the anti-C5a antibodies. The mAbs, purified from the culture supernatant, retained the full activity of the Fabs, binding specifically to and neutralizing human recombinant C5a. Refined versions of the mammalian expression vectors have been constructed for single step conversion of murine recombinant Fabs, recovered from combinatorial libraries, to IgG2a mAbs.

Animals↗
PubMed1995

Retinoic acid and methotrexate specifically increase PHA-E-lectin binding to a 67-kDa glycoprotein in LA-N-1 human neuroblastoma cells.

Retinoic acid (RA) decreased growth and increased morphologic differentiation of human neuroblastoma LA-N-1 cells. These phenomena correlated with a specific enhancement of PHA-E lectin binding to a glycoprotein of MW 67 kDa (gp67). Gp67 was found susceptible to N-glycanase and displayed BSA binding by affinity chromatography analysis. The chemotherapeutic agent methotrexate (MTX) also reduced growth and induced differentiation of LA-N-1 cells. In addition, the cells responded to MTX as well as to doxorubicin by a marked increase in PHA-E binding to gp67. We conclude that reduced growth and induction of morphological differentiation of LA-N-1 cells correlates with increased binding of PHA-E to gp67.

Amidohydrolases↗
PubMed1995

Differential male genetic success determines gene flow in an experimentally manipulated mouse population.

Sexual selection arises when genetically different males show heritable differences in reproductive success. Mouse mating behaviour involves both male competition and female choice. In this paper we show that introduced Y-linked DNA markers spread more extensively through a natural population than do genes inherited matrilineally. Differences in mating success between the sexes and among individual males may alter the pattern and rate of gene flow in natural populations. Another interesting possibility is that the success of the introduced Y chromosome may be attributable to so-called 'selfish' traits, such as sex-linked meiotic drive or intra-uterine competition. However, this study provides little unequivocal evidence to support this view. Differential success of introduced versus resident males may have implications for the reintroduction of endangered mammals into residual wild populations.

Animals↗
PubMed1995

Neutralizing murine monoclonal antibodies to human IL-5 isolated from hybridomas and a filamentous phage Fab display library.

Conventional hybridomas and combinatorial Ab libraries were used to develop neutralizing murine mAbs to human IL-5. Mice were immunized with rIL-5. Spleens from two mice were used to generate hybridomas. Spleens from an additional three mice were used to construct a combinatorial library. In both instances, Abs were identified and selected by ELISA using 96-well plates coated with rIL-5. These Abs were tested for the ability to block binding of iodinated rIL-5 to the alpha-chain of the human IL-5 receptor (IL-5R alpha) and to inhibit proliferation of IL-5-dependent cells. By hybridoma technology, 16 mAbs were obtained, 11 of which blocked binding to IL-5R alpha, including three that inhibited proliferation. Quantitative binding assays and sequence analysis revealed that these latter three mAbs were closely related. Combinatorial cloning and selection by phage display was used to isolate 24 bacterial colonies secreting Fabs that bound to 125I-rIL-5 and to rIL-5-coated plates. Sequencing of 10 of the Fabs indicated that four unique Abs were obtained, comprising one predominant VH paired with one of two different VL. The sequence of the Fabs was distinct from the sequences of the neutralizing mAbs. In contrast to the mAbs, none of the Fabs blocked binding of 125I-IL-5 to IL-5R alpha or neutralized the biologic activity of IL-5. The inability to identify neutralizing Fabs was shown not to result from their monovalency, because a Fab derived from one of the neutralizing mAbs, by cloning and expression of its Fd and kappa light chains, retained neutralizing activity. By chain shuffling, pairing of the Fd fragment of the heavy chain of one of the neutralizing mAbs (2B6), with the light chain library derived from the IL-5-immunized mice, neutralizing Fabs were obtained. These Fabs contained light chain sequences closely related to the original light chain of 2B6. Hence, chain shuffling allowed detection of a light chain sequence that was not evident upon two-chain combinatorial selection. The results reveal differences in the Abs obtained from a combinatorial library vs hybridomas and demonstrate how these approaches can be used in concert to select mAbs with neutralizing activity.

Amino Acid Sequence↗
PubMed1995

Molecular characterization of trimethoprim resistance in salmonellas isolated in Sicily, 1985-1988.

The occurrence of trimethoprim (Tp) resistance in salmonellas isolated from humans and water samples in Sicily between 1985 and 1988 has been investigated and the Tp resistance mechanisms have been further characterized on the basis of hybridization with probes for the dihydrofolate reductase (DHFR) genes types I, II, IV and V. Of 765 strains examined, high level (> 1000 mg/l) resistance to Tp was identified in 23 strains (3%). In 22 of these strains, such resistance was associated with resistance to sulphonamides. Six serovars with Tp-resistant strains were identified, Salmonella typhimurium (14 strains), S. enteridis (2), S. agona (2), S. mbandaka (2), S. virchow (2), S. indiana (1). In all strains with high level Tp resistance, resistance to this antimicrobial was plasmid-encoded, in most strains by plasmids with MWs ranging from 70-100 MDa. On the basis of restriction endonuclease analysis, four different categories of Tp resistance plasmids were identified in Tp-resistant strains of S. typhimurium. Hybridization with the DHFR I probe was observed in three strains of Tp-resistant S. typhimurium and two strains of Tp-resistant S. enteritidis; in contrast, in none of the strains tested was there any detectable hybridization with the probes for DHFR types II, IV and V. It is concluded that the DHFR type I resistance mechanism, common in Tp-resistant enterobacteria in many European countries, is relatively uncommon in Tp-resistant salmonellas isolated in Sicily. Furthermore, the DHFR V resistance mechanism, previously identified in strains of Shigella sonnei isolated in Sicily and associated with travellers from Sri Lanka, has not yet appeared in salmonellas in Sicily.

Acute Disease↗
PubMed1994

Isolation of neutralizing anti-C5a monoclonal antibodies from a filamentous phage monovalent Fab display library.

A panel of mAbs against the activated complement component C5a was obtained from a filamentous phage M13-Fab display library generated from mice immunized with human rC5a. Fabs isolated from the library after iterative selection vs rC5a bound to both rC5a and purified C5. To isolate Fabs specific for neoepitopes expressed on C5a but not on the native complement component C5 the library was rescreened in a competitive manner. The phage Fab library was first incubated with immobilized C5 to deplete C5 reactive Fabs. The C5 nonadherent phage were then incubated with immobilized rC5a in the presence of soluble C5. Bound phage were eluted and subjected to two additional cycles of subtraction with immobilized C5 and selection with immobilized rC5a in the presence of soluble C5. After three cycles of this competitive biopanning four Fabs reactive with rC5a were isolated. Two bound preferentially with and neutralized C5a. Competitive biopanning of phage display libraries may increase the probability of identification of Abs of the desired specificity.

Amino Acid Sequence↗