It has recently been shown using genetic markers that Ascaris in humans and pigs in Central America comprise reproductively isolated populations. We present a similar analysis for a region of China in which close association between pigs and humans has been the norm for thousands of years, and agricultural practices will result in frequent exposure to eggs from both sources. DNA fragments from selected regions of mitochondrial and ribosomal DNA were amplified by PCR and allelic forms identified following digestion with a panel of restriction enzymes, using DNA from a total of 115 individual worms from both people and pigs from 2 neighbouring villages. Significant frequency differences in both mtDNA haplotypes and the rDNA spacer were found between the 2 host-associated populations, indicating that they represented reproductively isolated populations. Mitochondrial haplotype frequencies were different from those observed in Guatemala and also from other Asian Ascaris populations, suggesting low levels of gene flow between populations. However, we found no evidence for significant heterogeneity in the genetic composition of Ascaris infrapopulations in either humans or pigs, possibly indicative of agricultural practices in China which have resulted in a random distribution of alleles within the parasite populations.

Animals↗

PubMed1998

Effects of transforming growth factor-beta and platelet-derived growth factor on human gingival fibroblasts grown in serum-containing and serum-free medium.

Both transforming growth factor-beta (TGF-beta) and platelet-derived growth factor (PDGF) have been shown to affect cell proliferation in vitro. The hypothesis being tested was that the effects of the 2 cytokines would be modulated by the presence of serum in the medium. Gingival fibroblasts, obtained from periodontally healthy patients, were maintained in primary culture. Dose response experiments were performed for each growth factor in serum-free medium and in medium containing natural or heat-inactivated fetal bovine serum (10% FBS). Changes in cell numbers were quantified by crystal violet staining. The optimal concentrations of the individual factors (10 ng/ml TGF-beta1, 20 ng/ ml PDGF-BB) were then used when the 2 factors were tested in various sequences. In serum-free medium or in medium with 10% natural serum, the response to PDGF-BB was dose-dependent up to 40 ng/ml; however, with 10% heat-inactivated serum, the maximal response was seen at 20 ng/ml. The largest increase in cell numbers was produced by the simultaneous exposure to the two cytokines, rather than a sequential presentation. The findings suggest that the 48-h growth response of human gingival fibroblasts to 10 ng/ml TGF-beta1 or 20 ng/ ml PDGF-BB in serum-free medium was equivalent to growth obtained in medium containing heat-inactivated 10% FBS without added growth factors.

Adult↗

PubMed1998

Characterization of the carbohydrate moiety of intestinal mucin-type sialoglycoprotein receptors for the K88ac fimbrial adhesin of Escherichia coli.

We have previously identified two mucin-type sialoglycoproteins from porcine intestinal epithelial cells with approximate molecular masses of 210 (intestinal mucin-type glycoprotein IMTGP-1) and 240 kDa (IMTGP-2) as receptors for the K88ab and K88ac fimbrial adhesins of Escherichia coli. These receptors are detected in intestinal brush border membrane preparations from pigs with adhesive phenotypes but not from pigs with nonadhesive phenotypes and are postulated to be important determinants of the susceptibility of pigs to K88ab+ and K88ac+ enterotoxigenic E. coli infections. Using exoglycosidase digestion studies, we have now determined that beta-linked galactose is an important component in the recognition of IMTGP-1 and IMTGP-2 by the K88ac adhesin. In addition, we observed a differential distribution of the K88ac adhesin binding activity of IMTGP-1 and IMTGP-2 along the crypt-villus axis, suggesting that receptor activity is dependent on the maturation state of the intestinal epithelial cells. Brush borders from immature intestinal epithelial cells possessed the highest concentrations of IMTGP-1 and IMTGP-2 receptor activity, with a progressive decrease in receptor activity as the cells mature. To characterize the differences in the carbohydrate moieties of IMTGP-1 and IMTGP-2, we developed a procedure for purifying the receptors, using phenol extraction followed by serial lectin affinity chromatography. Carbohydrate compositional analysis of the purified receptors indicated that the carbohydrate moieties of IMTGP-1 and IMTGP-2 consist of both N- and O-glycans containing galactose, glucose, sialic acid, mannose, N-acetylgalactosamine, N-acetylglucosamine, and fucose. The major difference between the two receptors is that IMTGP-2 contains a higher percentage of monosaccharides (mannose and glucose) commonly found in N-glycans.

Adhesins, Escherichia coli↗

PubMed1998

Distinct localization of renin and GLUT-4 in juxtaglomerular cells of mouse kidney.

The insulin-responsive glucose transporter, GLUT-4, is found primarily in adipocytes and skeletal muscle cells, where it is sequestered in a specialized recycling compartment, from which it can be recruited to the cell surface following insulin stimulation. Lower levels of GLUT-4 are also expressed in other tissues, including the kidney, where it is present particularly in cells of the afferent arteriole and juxtaglomerular apparatus (JGA). The exact nature of GLUT-4-containing compartments and their relationship to other regulated trafficking pathways in different cells are not yet well defined. The trafficking of GLUT-4 has been studied in different cells with regulated secretory pathways, and a recent study shows that, in cardiomyocytes, GLUT-4 is sorted and packaged into multiple regulated pathways (J. W. Slot, G. Garruti, S. Martin, V. Oorschot, G. Pshuma, E. W. Kraegen, R. Laybutt, G. Thibault, and D. E. James. J. Cell Biol. 137: 1243-1254, 1997). In the kidney, cells of the JGA synthesize and secrete their major product, renin, via a well-established, regulated, secretory pathway. These cells also express GLUT-4 and thus offer the potential to directly compare the localization and trafficking of GLUT-4 and renin in a unique cell type. The present study was undertaken to investigate the intracellular distribution of GLUT-4 in mouse kidney cortex and to determine whether GLUT-4 and renin are trafficked in the same or in separate regulated pathways. Ultrathin cryosections of mouse kidney were labeled by the immunogold technique and viewed by electron microscopy, demonstrating the distribution of GLUT-4 in cells of the JGA, afferent arteriole, and distal tubule. In granular cells of the JGA, renin was localized in secretory granules of the regulated secretory pathway, whereas GLUT-4 labeling in the same cells was found in a distinct tubulovesicular compartment located adjacent to the trans-Golgi network. We show that granular cells have separate, morphologically distinct compartments for the sequestration of renin and GLUT-4, providing evidence that there may be distinct pathways for the sorting and trafficking of these two proteins.

Animals↗

PubMed1998

Normal breast: myths, realities, and prospects.

Modern concepts of the breast acknowledge the extent of physiologic changes and attempt to relate them to pathogenesis. The natural life cycle of the lobule is incompletely understood, but the influences of the menstrual cycles, pregnancy, and involution are more clearly defined from morphologic studies. The extensive variety of modulating influences is a confounding factor, but an important principle is acceptance that breast and uterus respond differently to the mitogenic stimuli of steroid hormones. This has implications for the use of oral contraceptives and hormone replacement therapy. Greater understanding of the cytokines and growth factors that regulate cell cycle activity has potential relevance to the field of breast cancer chemoprevention. These and other pathways are now being explored more intensively in connection with the breast cancer genes BRCA1 and BRCA2. As the awareness increases of the complexities involved in cell signal transduction and transcription, it is useful to reflect on what evolution might tell us. A theory is developed herein to link the erect posture of humans with greater local breast estrogen synthesis and an inevitable association with cancer risk. The complex and variable structure of the breast requires cautious interpretations of conclusions reached with limited numbers or undue simplifications.

Adult↗

PubMed1997

Modification of Schwann cell phenotype with Plp transgenes: evidence that the PLP and DM20 isoproteins are targeted to different cellular domains.

The X-linked proteolipid protein (Plp) gene encodes PLP, the major protein of central nervous system myelin, and its alternative RNA splice product, termed DM20. Schwann cells also express the Plp gene but, in contrast to oligodendrocytes, neither protein is incorporated into peripheral myelin. In the present study, we use different transgenes encoding PLP and DM20 to modify the expression of these proteins in myelin-forming Schwann cells of wild-type and jimpy mice. Increasing the level of PLP, either singly or in combination with DM20, leads to the incorporation of PLP into the compacted myelin sheath; however, DM20 always remains restricted to cytoplasmic regions of the Schwann cell. The insertion of PLP into the membrane does not appear to depend on a cooperativity of the two isoproteins. The presence of PLP does not visibly alter the ultrastructure and periodicity of peripheral nervous system (PNS) myelin. The results indicate that the absence of PLP in the peripheral myelin of normal animals most probably reflects the very low amounts of this isoprotein synthesised by Schwann cells. The preferential incorporation of PLP, as opposed to DM20, in peripheral myelin may indicate that a myelin targeting signal is present in the PLP-specific region of the molecule.

Animals↗

PubMed1997

Molecular staging of cancer.

Explore the source record for details and available documents.

Axilla↗

PubMed1997

Relation of pacing-induced coronary resistance vessel dilation to total serum cholesterol and heart rate-blood pressure product.

Coronary risk factors adversely affect coronary resistance vessel dilation to acetylcholine, but little is known about the effect of risk factors on coronary blood flow (CBF) responses to physiologic stimuli. CBF was derived from Doppler flow velocity (0.018-inch Doppler wire) and coronary diameter (quantitative angiography) in response to rapid atrial pacing in 50 patients (mean age 52 +/- 12 years). Patients were prospectively divided into 3 groups based on their angiograms: group 1 (n = 17), normal coronary arteries; group 2 (n = 18), 1-vessel coronary artery disease (CAD) with a smooth study artery; group 3 (n = 15), 1-vessel CAD and an irregular study artery (<20% stenosis). Pacing produced a significant increase in CBF compared with baseline in groups 1 and 2 (34 +/- 40%, 42 +/- 35%, p < 0.0001), respectively, but not in group 3 (21 +/- 33%), but there was no difference in the pacing response among the 3 groups. The increase in CBF to pacing was inversely related to serum cholesterol (p = 0.01) and triglycerides (p = 0.06) and directly related to the increase in heart rate-blood pressure product (p = 0.007). By multivariate analysis, total cholesterol and the increase in double product were the only factors related to the increase in CBF. Increases in CBF to atrial pacing are inversely related to serum total cholesterol and are not related to the angiographic presence of atherosclerosis in patients with mild CAD.

Adenosine↗

PubMed1997

Expression and phylogenetic relationships of a novel lacZ homologue from Actinobacillus pleuropneumoniae.

To learn more about the genetics and physiology of the important swine pathogen, Actinobacillus pleuropneumoniae, we cloned the lacZ gene by complementation of an Escherichia coli delta lac mutant. The A. pleuropneumoniae lacZ gene has an open reading frame of 3015 bp which could encode a protein with a predicted molecular mass of 117022. The deduced protein shares 26.8-34.8% identity with beta-galactosidases from both Gram-positive and Gram-negative bacteria. Sequences with homology to seven regions commonly found in beta-galactosidases are present and amino acids corresponding to active site residues Tyr-503 and Glu-537 in E. coli LacZ are also conserved; however, there is a leucine in the place of Gly-794, a residue which has been implicated in substrate recognition. The sequences flanking the A. pleuropneumoniae lacZ gene do not share homology with known transport or regulatory genes nor do they share homology with cAMP receptor protein (CRP) or LacI binding sites. Low levels of beta-galactosidase activity could be detected when the protein was expressed from a multicopy plasmid in E. coli delta lac and when it was measured in A. pleuropneumoniae. The level of activity was not markedly reduced in the presence of glucose. Although the A. pleuropneumoniae LacZ shares some features with other beta-galactosidases, its constitutive expression and an unusual active site residue suggest that it may have a unique function.

Actinobacillus pleuropneumoniae↗

PubMed1997

The impact of core-biopsy on pre-operative diagnosis rate of screen-detected breast cancers.

Explore the source record for details and available documents.

Biopsy, Needle↗

PubMed1997

Ductal carcinoma in situ of the breast: reproducibility of histological subtype analysis.

Historically, two major strata of ductal carcinoma in situ (DCIS) have been linked to outcome, the presence or absence of comedo type and size. Our initial approach in classification was dichotomous, often favoring the comedo type with most worrisome implications to foster agreement in diagnosis. We have now tested guidelines that foster agreement in the modified Lagios three-tiered system. Sixteen cases of DCIS were selected, reflecting a spectrum of histological subtypes, with specific inclusion of cases in which consensus in classification using a dichotomous (comedo/noncomedo) scheme would be difficult. Six independent observers reviewed a minimum of five color 35-mm slides from each case at two separate occasions. The aim was to subclassify each case based on architectural pattern, nuclear grade, and presence or absence of tumor necrosis (Modified Lagios Classification, Lagios et al, Cancer 1989). After initial review, emphasizing placement of each case into a high- or low-grade category, there was disagreement in seven cases (44%), confirming our aim to choose cases with uncertain cues for classification. Agreement was achieved in 94% of cases by allowing re-review with emphasis on inclusion of an intermediate-grade category. Our study also suggests that pure micropapillary DCIS and apocrine DCIS warrant independent classification as "special type" DCIS. Our small pilot study suggests that, with adherence to specific criteria, most DCIS cases can be easily and consistently classified into the following five categories: (1) high grade, (2) intermediate grade, (3) low grade, (4) pure or predominantly micropapillary, and (5) pure apocrine. Our six observers independently reached a final concordance of 94% despite selection of cases in which consensus in a dichotomous classification was difficult. This was achieved predominantly by accepting an intermediate category of DCIS with intermediate nuclear features and limited necrosis. Confirmation of the applicability of the Modified Lagios Classification awaits completion of a much larger multi-institutional study in which statistical significance and interobserver variation can be better defined.

Breast Neoplasms↗

PubMed1997

Host specificity, evolutionary relationships and macrogeographic differentiation among Ascaris populations from humans and pigs.

We describe a variety of restriction site polymorphisms in the introns of Ascaris nuclear genes and in the ribosomal DNA spacers. We use these markers, in addition to previously described mitochondrial variation, to clarify our understanding of the epidemiology of Ascaris in Guatemalan villages where humans and pigs occur in sympatry and to describe the genetic structure of host-associated Ascaris populations from world-wide locations. Intron sequences were amplified from individual worms and alleles defined by endonuclease digestion. Two loci were monomorphic, while 4 length variants and 22 point mutations were detected in the other 7 loci. Within sympatric Guatemalan populations no single locus from either the nuclear or mitochondrial genome was fixed for alternative alleles, although allele frequencies were significantly different at many loci. Phenograms constructed from multilocus nuclear genotypes of individual worms failed to reveal a single case of cross-infection, and demonstrate that divergent mtDNA genotypes are segregating within host-associated populations. On a world-wide scale, the data suggest that extant worm populations result from a single host shift, although characterization of genetic variation in additional loci will be necessary to confirm this. The direction and the geographical origin of the host shift were unresolved. Overall 65% of nuclear genetic variation was found within populations, host (human or pig) explained 18%, while geographical variation within host-associated populations explained 17%. The results (a) demonstrate the utility of introns for studying the epidemiology of parasites showing limited allozyme variation (b) suggest that programmes aiming to control Ascaris infection in the human population can safely ignore zoonotic infection from pigs and (c) illustrate the problems inherent in using single genetic markers to make inferences about the epidemiology of closely related parasite taxa.

Animals↗

PubMed1997

Tamoxifen induced apoptosis in ZR-75 breast cancer xenografts antedates tumour regression.

The ZR-75-1 ER positive breast cancer cell line, xenografted in female nude mice, has been used to determine the effect of tamoxifen on cell proliferation (as measured by mitosis) and cell death (as evidenced by apoptosis and necrosis). After 2 days treatment, there was a significant rise in apoptosis (p < 0.05), whereas a fall in mitosis was not apparent until 7 days (p < 0.05). Furthermore there was an increase in the apoptotic:mitotic ratio on day 7 (p < 0.05). These changes antedated tumour regression, which did not reach not significance until day 14. Tamoxifen did not increase necrosis (which significantly decreased in treated tumours once they had regressed (p < 0.01). In contrast tamoxifen treatment of xenografted MDA-MB-231 ER-negative breast cancer cells produced no significant effects on growth, apoptosis, or mitosis. This study presents clear evidence for tamoxifen inducing apoptosis in ZR-75-1 xenografts (but not MDA-MB-231 tumours). Since changes in apoptosis and mitosis antedate tumour regression, their assessment may provide the potential by which to predict tumour response to tamoxifen therapy.

Animals↗

PubMed1997

Primary systemic therapy for operable breast cancer--10-year survival data after chemotherapy and hormone therapy.

Between 1984 and 1990, 94 women presenting to the Edinburgh Breast Unit with operable breast cancer of 4 cm or greater in diameter (T2, T3, N0, N1, M0) were given preoperative systemic therapy. Initially, all women received hormone therapy, with CHOP (cyclophosphamide 1 g m(-2), doxorubicin 50 mg m(-2), vincristine 1.4 mg m(-2) to a maximum of 2 mg and prednisolone 40 mg per day orally for 5 days) chemotherapy being administered to those who failed to respond by 3 months. After April 1987, first-line hormone therapy was only offered to women with oestrogen receptor (ER)-moderate/-rich (> 20 fmol mg(-1) protein) tumours, and CHOP was reserved for those women whose tumours failed to respond to hormone therapy and for those with ER-negative/-poor tumours. Response data have been published previously (Anderson et al, 1991). After a median follow-up of 7.5 years, there is no difference in survival between those women given initial hormone therapy and those given chemotherapy, with neither group having yet reached its median survival. The two key factors that predicted for a poor survival were the number of involved axillary nodes after preoperative systemic therapy (P < 0.00001) and a lack of response to preoperative therapy (P < 0.05). These data suggest that many women with ER-moderate/-rich tumours will have a good prognosis after preoperative hormone therapy alone. However, it is possible to identify, by their post-systemic therapy axillary node status, a group of women who still have an appalling prognosis after preoperative chemotherapy or hormone therapy.

Adult↗