Expression and function of the ghrelin axis, including a novel preproghrelin isoform, in human breast cancer tissues and cell lines.

While oestrogen, progesterone and growth factors, including growth hormone (GH), are clearly implicated in the pathogenesis of breast cancer, there is now evidence that the newly described ghrelin axis is also involved. The aims of this study were to investigate the expression of the ghrelin axis in breast cancer tissues and cell lines and to examine the effect of ghrelin on breast cancer cell proliferation in vitro. Ghrelin and its functional receptor, the growth hormone secretagogue receptor (GHSR) type 1a, were expressed in normal breast tissue and breast cancer specimens and cell lines. In contrast, the truncated GHSR type 1b isoform was exclusively expressed in breast carcinoma, suggesting that it has potential as a diagnostic marker. Ghrelin treatment significantly increases the proliferation of the MDA-MB-435 and MDA-MB-231 breast cancer cell lines in vitro. In addition, we have described the expression of a human preproghrelin isoform, exon 3-deleted preproghrelin, which encodes mature ghrelin plus a novel C-terminal peptide. Quantitative RT-PCR was used to demonstrate that this mRNA isoform is highly expressed in the MDA-MB-435 metastatic breast cancer cell line relative to the benign MCF-10A breast epithelial cell line. The unique C-terminal peptide of exon 3-deleted preproghrelin is expressed in the glandular epithelium of breast cancer tissues, with high-grade carcinoma exhibiting the strongest immunoreactivity. The data presented here suggest that components of the ghrelin axis may represent novel markers for breast cancer and potential therapeutic targets.

Chloramphenicol inhibition of denitrifying enzyme activity in two agricultural soils.

Chloramphenicol, at concentrations greater than 0.1 g/liter (0.3 mM), inhibited the denitrifying enzyme activity (DEA) of slurries of humisol and sandy loam soils by disrupting the activity of existing nitrate reductase enzymes. When the concentration of chloramphenicol was increased from 0.1 to 2.0 g/liter (6.0 mM), the rate of nitrite production from nitrate decreased by 25 to 46%. The rate of NO production from nitrate decreased by 20 to 39%, and the rate of N(2)O production from nitrate, in the presence of acetylene (DEA), decreased by 21 to 61%. The predicted values of DEA at 0 g of chloramphenicol/liter computed from linear regressions of DEA versus chloramphenicol concentration were 18 to 43% lower than DEA measurements made in the absence of chloramphenicol and within a few per cent of DEA rates measured in the presence of 0.1 g of chloramphenicol/liter. We conclude that DEA assays should be carried out with a single (0.1-g/liter) chloramphenicol concentration. Chloramphenicol at concentrations greater than 0.1 g/liter inhibits the activity of existing denitrifying enzymes and should not be used in DEA assays.

Polymorphisms of the interleukin-6 gene are associated with bone mineral density.

Genetic factors play an important role in determining bone mass and several genes probably act as regulators of this process. Interleukin-6 (IL-6) is a candidate gene for regulation of bone density, since it has stimulatory effects on cells of the osteoclast lineage and has been implicated in the pathogenesis of bone loss associated with estrogen deficiency. Here we studied the relationship between bone mineral density (BMD) and a polymorphic AT rich minisatellite repeat in the 3' flank of the IL-6 gene in a cohort of 200 women. Six length variants were identified (designated A-F), but four of these were rare such that the last majority of individuals fell into one of two common genotypes: F/F (58.5%) and C/F (27.5%). There was a significant relationship between IL-6 genotype and bone mass at the lumbar spine as determined by analysis of variance (p = 0.04) and a similar trend for bone mass at the femoral neck (p = 0.11). When BMD values were compared in the two common genotypes, we found that spine BMD values were significantly higher in the C/F genotype (mean +/- SEM = 0.94 +/- 0.04 g/cm2) as compared with the F/F genotype (0.81 +/- g/cm2; p = 0.012). A similar trend was seen for hip BMD values, but here, the difference failed to reach statistical significance (p = 0.06). Further analysis showed that genotype-specific effects on bone mass were observed in both premenopausal and postmenopausal women and did not increase with age, suggesting that the association between IL-6 polymorphisms and bone density may be mediated by an effect on peak bone mass, rather than rate of bone loss. We conclude that bone mass is associated with two common polymorphisms of the IL-6 gene. Although the mechanisms that underlie this association will require further research, our data suggest that polymorphic variation at the IL-6 gene locus may contribute to the genetic regulation of bone mass.

Spatial heterogeneity in the distribution of denitrifying bacteria associated with denitrification activity zones.

Denitrifying enzyme activity (DEA) in artificially established denitrification activity zones was, on average, 3.5-fold greater than in bulk soil. Denitrifier biomass was between 1 and 4 orders of magnitude greater in activity zones. Denitrifier specific activity was lower in activity zones and more similar to specific activity values reported for pure cultures of denitrifying bacteria added to soil. Measurements of DEA and biomass carried out on a homogeneous subsample derived from a soil core containing a denitrification activity zone will overestimate the DEA and biomass of the bulk soil and underestimate the DEA and biomass of the activity zone.

Competition between Two Isolates of Denitrifying Bacteria Added to Soil.

We examined the competitive relationship between two isolates of denitrifying bacteria, both of which grow well under aerobic conditions but differ in their ability to grow under denitrifying conditions. The growth and persistence of the two isolates, added to sterile soil or added to soil previously colonized by the other isolate, were monitored under aerobic and denitrifying (anaerobic) conditions. When isolates were added together to sterile soil, the isolate added at the higher density reduced the growth of the isolate added at the lower density. The magnitude of the growth reduction varied depending on the competitive abilities of the individual isolates and the aeration state of the soil. Prior colonization of soil with one of the isolates conferred a competitive advantage on the colonized isolate but did not lead to the disappearance of the challenging isolate. Fluctuations in aeration state caused large changes in the population density of one isolate and altered the competitive relationship between the two isolates. The competitive effectiveness of each isolate varied with cell density, the degree of prior colonization of the soil by the other isolate, and the aeration state of the soil.

Impact of changing attitudes in carotid surgery on community hospital practice.

In 1985, institutional guidelines for the evaluation and performance of carotid surgery were established in our community hospital. During the 5-year period from 1985 through 1989, 159 carotid reconstructions were done. There were four major strokes (3%), one eventually resulting in death, with the second death in this series from a myocardial infarction (mortality 1%). The combined mortality/major stroke morbidity incidence was 3%. Three transient ischemic attacks (2%) postoperatively cleared promptly without residua. During the latter 1980s, an increasing number of vascular surgeons were doing less carotid surgery. Monitoring institutional quality assurance and individual surgeon performance within the community hospital is becoming a reality. Our experience with institutional guidelines for the evaluation and conduct of carotid surgery, together with an assessment of results and ongoing individual surgeon performance, is presented. Maintaining acceptable morbidity and mortality statistics can be enhanced by having a plan for assessment, management, and concurrent review.

Aerobic and anaerobic growth of rifampin-resistant denitrifying bacteria in soil.

The growth and survival of several rifampin-resistant isolates of denitrifying bacteria were examined under anaerobic (denitrifying) and aerobic conditions. Two isolates added to nonsterile Bruno soil at densities of between 10(4) and 10(6) CFU g dry soil-1 exhibited an initial period of growth followed by a gradual decline in numbers. After 28 days, both isolates maintained viable populations of between 10(4) and 10(5) CFU g dry soil-1 under both denitrifying and aerobic conditions. One of the isolates consistently grew better under denitrifying conditions, and the other isolate consistently grew better under aerobic conditions. The relative pattern of denitrifying versus aerobic growth for each organism was not affected by the addition of glucose. The growth yields of the two isolates varied with soil type, but the relative pattern of denitrifying versus aerobic growth was consistent in three soils with greatly different properties. Five of nine isolates introduced into Bruno soil at low population densities (approximately 10(5) CFU g dry soil-1) exhibited better growth after 2 days under denitrifying conditions. It was not possible to predict the prevalence of the denitrifying or aerobic mode of growth in nonsterile soil from the growth characteristics of the isolates in pure cultures or sterile soil.

Kinetics of nitrate utilization by mixed populations of denitrifying bacteria.

Kinetics of nitrate utilization by mixed bacterial populations from two agricultural soils and a pond sediment in Kentucky were measured by using progress curves of nitrous oxide production. Nitrous oxide production from anaerobic soil and sediment slurries containing added nitrate and acetylene exhibited first-order kinetics. Nitrate affinity (K(m)) for mixed populations of denitrifying bacteria in unfertilized agricultural soils and pond sediments ranged from 1.8 to 13.7 muM. The affinity of bacterial populations for nitrate did not vary with habitat, and the ability to use low concentrations of nitrate was retained by bacterial populations living in environments which received large inputs of nitrate.

Dynamics of Soil Denitrifier Populations: Relationships between Enzyme Activity, Most-Probable-Number Counts, and Actual N Gas Loss.

To better understand temporal variability in soil denitrification, denitrifying enzyme activity (DEA) and denitrifier populations (as determined by most-probable-number [MPN] counts) were measured in field and laboratory experiments. Measurements of DEA and MPN provided highly contradictory indications of denitrifier dynamics. In laboratory incubations, under conditions favoring active denitrification, the synthesis of new denitrifying enzymes and the actual amount of denitrification were closely related. In other experiments, however, both DEA and MPN counts were poor indicators of actual denitrification. In some cases, we found significant increases in DEA but no significant production of N gas. Except with unnaturally high substrate amendments, changes in DEA were small relative both to the persistently high DEA background and to changes in MPN. As estimated by MPN counts, denitrifier populations increased significantly during denitrification events. It was apparent that only a small fraction of the denitrifiers were included in the MPN counts, but it appeared that this isolatable fraction increased during periods of active denitrifier growth. Use of DEA as an index of biomass of cells which have synthesized denitrifying enzymes suggested that denitrifier populations were persistent, stable, and much larger than indicated by MPN procedures.

Influence of physical disruption on growth of attached bacteria.

Attached bacterial populations cultured without an exogenous carbon source or grown in conjunction with attached diatoms incorporated [H]thymidine at a rate between four and five times lower than that of replicate bacterial populations which were dispersed before being assayed.

Stimulation of bacterial DNA synthesis by algal exudates in attached algal-bacterial consortia.

Algal-bacterial consortia attached to polystyrene surfaces were prepared in the laboratory by using the marine diatom Amphora coffeaeformis and the marine bacterium Vibrio proteolytica (the approved name of this bacterium is Vibrio proteolyticus [W. E. C. Moore, E. P. Cato, and L. V. H. Moore, Int. J. Syst. Bacteriol. 35:382-407, 1985]). The organisms were attached to the surfaces at cell densities of approximately 5 x 10 cells cm (diatoms) and 5 x 10 cells cm (bacteria). The algal-bacterial consortia consistently exhibited higher rates of [H]thymidine incorporation than did biofilms composed solely of bacteria. The rates of [H]thymidine incorporation by the algal-bacterial consortia were fourfold greater than the rates of incorporation by monobacterial biofilms 16 h after biofilm formation and were 16-fold greater 70 h after biofilm formation. Extracellular material released from the attached Amphora cells supported rates of bacterial activity (0.8 x 10 to 17.9 x 10 mol of [H]thymidine incorporated cell h) and growth (doubling time, 29.5 to 1.4 days) comparable to values reported for a wide variety of marine and freshwater ecosystems. In the presence of sessile diatom populations, DNA synthesis by attached V. proteolytica cells was light dependent and increased with increasing algal abundance. The metabolic activity of diatoms thus appears to be the rate-limiting process in biofilm development on illuminated surfaces under conditions of low bulk-water dissolved organic carbon.

Influence of macrophyte decomposition on growth rate and community structure of okefenokee swamp bacterioplankton.

Dissolved substances released during decomposition of the white water lily (Nymphaea odorata) can alter the growth rate of Okefenokee Swamp bacterioplankton. In microcosm experiments dissolved compounds released from senescent Nymphaea leaves caused a transient reduction in the abundance and activity of water column bacterioplankton, followed by a period of intense bacterial growth. Rates of [H]thymidine incorporation and turnover of dissolved d-glucose were depressed by over 85%, 3 h after the addition of Nymphaea leachates to microcosms containing Okefenokee Swamp water. Bacterial activity subsequently recovered; after 20 h [H]thymidine incorporation in leachate-treated microcosms was 10-fold greater than that in control microcosms. The recovery of activity was due to a shift in the composition of the bacterial population toward resistance to the inhibitory compounds present in Nymphaea leachates. Inhibitory compounds released during the decomposition of aquatic macrophytes thus act as selective agents which alter the community structure of the bacterial population with respect to leachate resistance. Soluble compounds derived from macrophyte decomposition influence the rate of bacterial secondary production and the availability of microbial biomass to microconsumers.

Annual cycle of bacterial secondary production in five aquatic habitats of the okefenokee swamp ecosystem.

Rates of bacterial secondary production by free-living bacterioplankton in the Okefenokee Swamp are high and comparable to reported values for a wide variety of marine and freshwater ecosystems. Bacterial production in the water column of five aquatic habitats of the Okefenokee Swamp was substantial despite the acidic (pH 3.7), low-nutrient, peat-accumulating character of the environment. Incorporation of [H]thymidine into cold-trichloroacetic acid-insoluble material ranged from 0.03 to 2.93 nmol liter day) and corresponded to rates of bacterial secondary production of 3.4 to 342.2 mug of carbon liter day (mean, 87.8 mug of carbon liter day). Bacterial production was strongly seasonal and appeared to be coupled to annual changes in temperature and primary production. Bacterial doubling times ranged from 5 h to 15 days and were fastest during the warm months of the year, when the biomass of aquatic macrophytes was high, and slowest during the winter, when the plant biomass was reduced. The high rates of bacterial turnover in Okefenokee waters suggest that bacterial growth is an important mechanism in the transformation of dissolved organic carbon into the nutrient-rich bacterial biomass which is utilized by microconsumers.

Microbial biomass and utilization of dissolved organic matter in the okefenokee swamp ecosystem.

The Okefenokee Swamp exhibited levels of microbial biomass and aerobic glucose uptake comparable to those of other organically rich, detritus-based aquatic ecosystems. In contrast to other peat-accumulating systems, this acidic (pH 3.7), low-nutrient environment does not show diminished water column or surface sediment microbial biomass or heterotrophic activity. The total particular ATP varied between 0.03 and 6.6 mug liter (mean, 1.6 mug liter) in water and between 1 and 28 mug g (dry weight) (mean, 10.0 mug g [dry weight] in sediments. The turnover times for dissolved d-glucose were 1.26 to 701.25 h (mean, 110.25 h) in aerobic waters and 2.4 to 72 min (mean, 10.2 min) in aerobic surface sediments. Water column bacterial secondary production, measured as the incorporation of [H]thymidine into cold-trichloroacetic acid-insoluble material, ranged from 0.06 to 1.67 nmol liter day (mean, 0.45 nmol liter day). The kinetics of d-glucose uptake by water column microflora are multiphasic and suggest the presence of a diverse microbial population capable of using labile substrates at nanomolar concentrations and at substantial rates. The presence of a large and active aerobic microbial community in the Okefenokee Swamp is indicative of an important role for microbes in swamp geochemistry and strongly suggests the existence of a detritus-based food web.

Guidelines for performance of transluminal angioplasty in the hospital.

The expanding use of balloon catheter dilatation of arterial lesions has demonstrated its effectiveness as a relatively simple therapeutic method to improve the initial patency of focal, proximally located atherosclerotic stenoses. It is recommended that the vascular surgeon assume a leadership role in establishing guidelines within each institution for the assessment and conduct of transluminal angioplasty. The careful selection of patients with an appropriate indication for the procedure is a preferred alternative to its improper and indiscriminate use.

Transluminal angioplasty: adjunct or gimmick?

An experience with transluminal dilatation using the Gruntzig catheter in 94 patients during the past 2 years with follow-up studies of 1 month to 2 years has provided certain guidelines for applying this technique to the management of peripheral vascular atherosclerosis. While the immediate success rate for the use of transluminal dilatation as a method of improving blood flow is encouraging, further studies are necessary to determine the mechanism of healing and the duration of patency and compare them with present vascular reconstructive procedures. A cooperative effort between surgeons and their non-surgical colleagues is necessary for optimal assessment and use of this technique.