Occurrence of airborne bacteria and pathogen indicators during land application of sewage sludge.

Glass impingers (AGI-30) were used at a commercial sludge application site to determine the levels of airborne bacteria and pathogen indicators. Even though heterotrophic bacteria averaged 10(5) CFU/m3, none of the sites showed the presence of Salmonella spp. or indicators such as fecal coliforms or coliphages. Indicators such as H2S producers and pathogenic clostridia were present in locations having significant physical agitation of the sludge material. PCR-based ribotyping using the 16S-23S interspacer region is a promising method to identify the genetic relatedness and origins of airborne clostridia.     

Preadipocyte Recruitment in Stromal Vascular Cultures After Depletion of Committed Preadipocytes by Immunocytotoxicity

Glucocorticoids or the glucocorticoid analog dexamethasone (DEX) enhances the differentiation of preadipocytes in the presence of insulin and influences preadipocyte proliferation. The purpose of the present study was to determine if DEX can induce the recruitment of preadipocytes. Using monoclonal antibodies for complement-mediated cytotoxicity, preadipocytes were removed from porcine stromal vascular (S-V) cell cultures. Our experiments demonstrated for the first time that after removal of preadipocytes by cytotoxicity, preadipocytes or fat cells could be induced by DEX or DEX plus insulin but not by insulin alone. However, many more fat cells were induced (258 ± 15/unit area) when DEX was added with fetal bovine serum (FBS) followed with insulin treatment, compared to DEX with insulin (21.3 ± 5.1/ unit area) after removal of preadipocytes. Immunocyto-chemistry with AD-3, a preadipocyte marker, showed that DEX with FBS for 3 days after seeding (i.e., the proliferation phase) produced many more preadipocytes (AD-3 positive, 223 ± 45/unit area) than FBS alone (10.5 ± 1.4/unit area). Bromodeoxyuridine (BrdU) incorporation assays demonstrated that the efficiency of DEX with FBS (i.e., during proliferation) was mitosis dependent. Accordingly, we conclude that: porcine S-V cultures contain preadipocytes at different stages of differentiation and that DEX induced early preadipocyte differentiation depends on mitosis.     

Human papilloma virus E6/E7 genes can expand the lifespan of human corneal fibroblasts

Summary Human corneal fibroblasts were infected with a retroviral delivery vector containing the E6 and E7 genes from human Papilloma virus type 16 in order to produce cell lines that have an expanded lifespan in culture. Morphologically, some of the trasfected corneal fibroblast lines appeared to have the normal spindle-shape morphology of diploid fibroblasts, whereas other lines appeared to have a more elongated morphology. All the cell lines were anchorage-dependent. Cells that had a normal morphology grew at a rate similar to normal diploid human corneal fibroblasts and had a population doubling time of 48 h. All E6/E7 expressing cell lines, regardless of morphology, produce types I, III, and V collagen, at levels similar to those observed in the parent corneal diploid fibroblast. These corneal fibroblast lines will be a usefulin vitro system to study collagen expression and fibril formation, as well as normal stroma development. These results also demonstrate that the use of E6/E7 genes to expand a cell’s lifespan can be a powerful tool because it does not appear to alter either the growth rate of the cell or collagen expression.     

Rapid Stimulation of EAAC1-Mediated Na+-Dependent l-Glutamate Transport Activity in C6 Glioma Cells by Phorbol Ester

Abstract: C6 glioma cells were used as a model system to study the regulation of EAAC1-mediated Na⁺-dependent l -[³H]glutamate transport. Although a 30-min preincubation with forskolin had no effect on transport activity, preincubation with phorbol 12-myristate 13-acetate (PMA) increased transport activity two- to threefold. PMA caused a time-dependent and concentration-dependent increase in EAAC1-mediated l -[³H]glutamate transport activity. A 2-min preincubation with PMA was sufficient to cause more than a twofold increase in transport activity and the protein synthesis inhibitor cycloheximide had no effect on the increase. These data suggest that this increase is independent of protein synthesis. The EC₅₀ value of PMA for stimulation of transport activity was 80 nM. Kinetic analyses demonstrated that the increase in transport activity was due to a 2.5-fold increase in V_max with no change in K_m. PMA also increased the transport of the nonmetabolizable analogue, d -[³H]aspartate to the same extent. In parallel assays, PMA did not, however, increase Na⁺-dependent glycine transport activity in C6 glioma. The inactive phorbol ester 4α-phorbol 12,13-didecanoate, did not stimulate l -[³H]glutamate transport activity, and the protein kinase C inhibitor chelerythrine blocked the stimulation caused by PMA. Okadaic acid and cyclosporin A, which are phosphatase inhibitors, had no effect on the stimulation of transport activity caused by PMA. The Ca²⁺ ionophore A23187 did not act synergistically to increase PMA stimulation. In previous studies, PMA caused a rapid increase in amiloride-sensitive Na⁺/H⁺ transport activity in C6 glioma. In the present study, pre- and coincubation with amiloride had no effect on the stimulation of transport activity caused by PMA. These studies suggest that activation of protein kinase C causes a rapid increase in EAAC1-mediated transport activity. This rapid increase in Na⁺-dependent l -[³H]-glutamate transport activity may provide a novel mechanism for protection against acute insults to the CNS.     

Fine structure of cells specialized for secretion of aggregation pheromone in a nitidulid beetle Carpophilus freemani (coleoptera: Nitidulidae)

The cells that secrete the aggregation pheromone of the male nitidulid beetle Carpophilus freemani are exceptionally large and lie within the body cavity. These secretory cells share many ultrastructural features with cells of other pheromone and defense glands, but they also have several unique features. A deep invagination of the surface of each of these cells acts as the secretory surface for the pheromone. The invaginated surface is highly convoluted and surrounds a narrow cuticular ductule that is connected to the tracheal system. This surface is not covered with microvilli as the comparable surfaces are in other insect secretory cells. Each secretory cell is filled with an abundance of lipid spheres that presumably contain precursors for the pheromone. Examining cells from beetles producing different levels of pheromone showed that sizes of secretory cells are positively correlated with rates of pheromone production. Whereas secretory and ductule cells of other insect glands are usually epidermal cells, these cells of nitidulid beetles represent the first pheromone glands in which oenocytes are believed to have been recruited for pheromone production and tracheal cells have been recruited as ductules for these cells.     

Use of inexpensive pressure transducers for measuring water levels in wells

Frequent measurement of below ground water levels at multiple locations is an important component of many wetland ecosystem studies. These measurements, however, are usually time consuming, labor intensive, and expensive. This paper describes a water-level sensor that is inexpensive and easy to construct. The sensor is placed below the expected low water level in a shallow well and, when connected to a datalogger, uses a pressure transducer to detect groundwater or surface water elevations. Details of pressure transducer theory, sensor construction, calibration, and examples of field installations are presented. Although the transducers must be individually calibrated, the sensors have a linear response to changing water levels (r ² .999). Measurement errors resulting from temperature fluctuations are shown to be about 4 cm over a 35°C temperature range, but are minimal when the sensors are installed in groundwater wells where temperatures are less variable. Greater accuracy may be obtained by incorporating water temperature data into the initial calibration (0.14 cm error over a 35C temperature range). Examples of the utility of these sensors in studies of groundwater/surface water interactions and the effects of water level fluctuations on tree growth are provided.     

Fasciola hepatica: A secreted cathepsin L-like proteinase cleaves host immunoglobulin

Adult Fasciola hepatica secrete a cysteine proteinase capable of cleaving host IgG close to the papain cleaving site. The proteinase was separated by size permeation chromatography. Gelatinsubstrate polyacrylamide gel electrophoresis analysis revealed that the proteinase migrates as 6 proteolytic bands in the apparent molecular size range 60–90 kDa. Based on pH profiles of activity, inhibition studies using diethylpyrocarbonate and the diazomethylketone Z-phe-ala-CHN₂, and characterising the substrate specificity of the enzymes using fluorogenic peptide substrates we have shown that the 60–90-kDa proteinases are cathepsin L-Iike proteinases.     

Multinuclear NMR studies of intracellular cations in perfused hypertensive rat kidney.

We have investigated hypertension-associated alterations in intracellular cations in the kidney by measuring intracellular pH, free Mg2+, free Ca2+, and Na+ concentrations in perfused normotensive and hypertensive rat (8-14 weeks old) kidneys using 31P, 19F, and double quantum-filtered (DQ) 23Na NMR. The effects of both anoxia and ischemia on the 23Na DQ signal confirmed its ability to detect changes in intracellular Na+. However, there was a sizable contribution of the extracellular Na+ to the 23Na DQ signal of the kidney. The intracellular free Ca2+ concentration, measured using 19F NMR and 5,5'difluoro-1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid, also increased dramatically during ischemia; the increase could be partly reversed by reperfusion. No significant differences were found between normotensive and hypertensive kidneys in the ATP level, intracellular pH, intracellular free Mg2+, and the 23Na DQ signal or in the extent of the extracellular contribution to the 23Na DQ signal. Oxygen consumption rates were also similar for the normotensive (5.02 +/- 0.46 mumol of O2/min/g) and hypertensive (5.47 +/- 0.42 mumol O2/min/g) rat kidneys. The absence of a significant difference in intracellular pH, Na+ concentration, and oxygen consumption between normotensive and hypertensive rat kidneys suggests that an alteration in the luminal Na+/H+ antiport activity in hypertension is unlikely. However, a highly significant increase (64%, p less than 0.01) in free Ca2+ concentration was found in perfused kidneys from hypertensive rats (557 +/- 48 nM, blood pressure = 199 +/- 5 mmHg, n = 6) compared with normotensive rats (339 +/- 21 nM, blood pressure = 134 +/- 6, n = 4) indicating altered renal calcium homeostasis in essential hypertension. An increase in intracellular free Ca2+ concentration without an accompanying change in the intracellular Na+ suggests, among many possibilities, that the Ca2+/Mg(2+)-ATPase may be inhibited in the hypertensive renal tissue.     

Effects of dietary fat on cholesterol movement between tissues in CBA/J and C57BR/cdJ mice.

Differences in dietary fats cause differences in cholesterol metabolism in mice. CBA/J mice are resistant to diet-induced hypercholesterolemia and atherosclerosis; they adjust hepatic hydroxymethyl-glutaryl-CoA reductase activity (HMGR) to maintain homeostasis; C57BR/cdJ mice are susceptible, but young animals are thought to maintain homeostasis by changing fecal excretion of sterols. Compartmental modelling of movement of [4-14C]cholesterol was used to analyze movement of cholesterol between serum and liver, heart, and carcass in mice fed 40 en% fat, polyunsaturated to saturated fatty acid ratio (P/S) = 0.24 (US74) or 30 en% fat, P/S = 1 (MOD). Dietary effects were quite pronounced, while strain effects were more subdued. The C57/cdJ animals appear to regulate the overall cholesterol balance by reducing synthesis, as do the CBA/J animals, even though synthesis is not reduced to the same degree as in the CBA/J animals. Both diet and strain influence the whole-animal turnover rate, with slower turnover occurring for C57BR/cdJ animals and animals fed the US74 diet.