Growth of a human carcinoma (HEp3) in nude mice: rapid and efficient metastasis

Our aim was to identify conditions which would permit the development of spontaneous metastasis of a human tumor in nude mice in a rapid and predictable manner and to explore ways to quantitate metastasis. Using a human squamous carcinoma--HEp3--we determined that invasiveness and metastasis were influenced by the host. HEp3 cells grew very rapidly and without a significant lag period in Balb/c and NIH(S)-II nude mice kept in aseptic conditions; a much longer lag period was observed in NIH-Swiss mice kept in conventional conditions. The HEp3 tumor displayed a highly invasive behavior in N-NIH(S)-II mice, in which it invaded the body wall, gaining access to the peritoneal cavity. Microinvasion was noted in all strains of mice inoculated with HEp3 cells. To prolong survival of the mice until metastases became evident, primary tumors were excised when they weighed 1-2 gm. N-NIH(S)-II and Balb/c nude mice, maintained in germ-free conditions, were most receptive to the development of metastases-lung metastases developed in 80% of these mice. Over 60% of all metastases were present within 4 weeks following the removal of the primary. Only 26% of tumor bearing NIH-Swiss developed lung metastases. Lung metastases were observed in some mice in the absence of local microinvasion, local tumor recurrence, and regardless of the presence of lymph node involvement. They were also noted in mice from which primary tumors were not excised. We compared three methods of lung metastasis detection: histology, detection of tumor cells in the cultures of lung minces, and the measurement of the levels of human urokinase-type plasminogen activator directly in the lysates of lungs. The detection of tumor cells in cultures of lung minces appeared to be the most sensitive of these methods and the determination of enzyme in lung lysates seemed to hold most promise for a quantitative approach. These data indicate that, the type of tumor, as well as the genetic background and the maintenance conditions of the host, have to be carefully selected to ensure the successful outcome of the particular tumor-host interaction being studied. Adherence to these guidelines allowed us, in the case of the HEp3 tumor, to develop a rapid, predictable, and efficient model in which to study factors affecting metastasis of this human tumor.     

Physical and Chemical Correlates of Microbial Activity and Biomass in Composting Municipal Sewage Sludge

Various physical and chemical parameters were monitored to evaluate their influence on the microbial communities present in composting municipal sewage sludge. Temperature, moisture content, depth, pH, protein content, total nitrogen, total carbon, lipid phosphate biomass, and the rates of microbial incorporation of substrates into lipids were measured at several times throughout the 17- to 19-day composting runs. Temperature was found to have the most consistent and dramatic effect on microbial activity and biomass. When temperatures exceeded 55 to 60°C, microbial activity fell dramatically, usually by more than 1 order of magnitude. Microbial activity was generally greatest in samples taken from the 35 to 50°C areas of the composting piles. Changes in the composition of the compost over time included increased pH, increased protein content, and decreased total organic content. The changes in these parameters appeared to reflect the microbial activity and biomass present. The results of this study indicate that the rate of composting may best be optimized by controlling the composting temperatures, provided that the other parameters fall within reasonable limits in the starting material.     

Improvements in and Environmental Applications of Double-Vial Radiorespirometry for the Study of Microbial Mineralization

Several variations in the scintillation mixture and the filter paper arrangements for double-vial radiorespirometry were compared. Improved efficiencies (44%) and shorter response times were found by adding wetting agents and methanolic NaOH to the scintillation mixture in the filter paper. The scintillation chemicals used did not contain dioxane and were found to be nontoxic to the test microbiota in this system. Covering the inner reaction vial with aluminum foil minimized the reduction in counting efficiency when testing colored or dense environmental samples. Mineralization rates were determined with ¹⁴C-labeled glucose, acetate, and glutamate and [¹⁴C]cellulose- and [¹⁴C]lignin-labeled lignocellulose for composting cow manure, forest soil, and arctic lake sediment microbiota. This improved method can be used in a variety of procedures involving the measurement of microbial mineralizations of organic compounds. Since no liquid scintillation cocktail is used for counting, the radioactive wastes are aqueous or can be incinerated, making disposal easy.     

Effect of chromium nicotinic acid supplementation on selected cardiovascular disease risk factors

The effects of daily supplemental chromium (200 μg) complexed with 1.8 mg nicotinic acid on plasma glucose and lipids, including total cholesterol, HDL cholesterol, LDL cholesterol, and triglycerides, were assessed in 14 healthy adults and 5 adults with noninsulin-dependent diabetes mellitus (NIDDM) using a double-blind crossover study with 8-wk experimental periods. Eight of the 14 healthy subjects and all 5 subjects with NIDDM also underwent an oral glucose tolerance test with assessment of 90 min postprandial plasma glucose and insulin concentrations. No statistically significant effects of chromium nicotinic acid supplementation were found on plasma insulin, glucose, or lipid concentrations, although chromium nicotinic acid supplementation slightly lowered fasting plasma total and LDL cholesterol, triglycerides, and glucose concentrations, and 90-min postprandial glucose concentrations in individuals with NIDDM.     

Effects of Acid on Plant Litter Decomposition in an Arctic Lake

The effects of acid on the microbial decomposition of the dominant aquatic macrophyte (Carex sp.) in Toolik Lake, Alaska were studied in microcosms during the ice-free season of 1980. Toolik Lake is slightly buffered, deep, and very oligotrophic. Microbial activities, as determined by ¹⁴C-acetate incorporation into extractable lipids, associated with Carex litter were significantly (P < 0.01) reduced within 2 days at pHs of 3.0 and 4.0, but not 5.0, 5.5, or 6.0, as compared with ambient controls (pH 7.4). ATP levels were significantly reduced at pH 3.0, but not at the other pHs tested. After 18 days, microbial activity significantly correlated with weight loss (P < 0.05), nitrogen content (P < 0.01), and C/N ratios (P < 0.01) of the litter, but did not correlate with ATP levels. Scanning electron microscopy of the litter surface revealed that the fungi present at ambient pH did not become dominant at pHs below 5.5, diatoms were absent below pH 4.0, and bacterial numbers and extracellular slime were greatly reduced at pH 4.0 and below. Mineralization of Carex¹⁴C-lignin-labeled or ¹⁴C-cellulose-labeled lignocellulose was reduced at pH 2.0, but not at pH 4.0, 5.0, or 6.0, compared with controls (pH 7). We concluded that if the pH of the water from this slightly buffered lake was sufficiently reduced, rates of litter decomposition would be significantly reduced.     

In vivo paracrine interaction between urokinase and its receptor: effect on tumor cell invasion

Numerous studies have linked the production of increased levels of urokinase type plasminogen activator (uPA) with the malignant phenotype. It has also been shown that a specific cell surface receptor can bind uPA through a domain distinct and distant from the proteolytic domain. In an in vivo model of invasion, consisting of experimentally modified chorioallantoic membrane (CAM) of a chick embryo, only cells that concurrently expressed both uPA and a receptor for uPA, and in which the receptor was saturated with uPA, were efficient in invasion. To test whether uPA produced by one cell can, in a paracrine fashion, affect the invasive capacity of a receptor-expressing cell, we transfected LB6 mouse cells with human uPA (LB6[uPA]), or human uPA-receptor cDNA (LB6[uPAR]). LB6(uPA) cells released into the medium 1-2 Ploug units of human uPA per 10(6) cells in 24 h. The LB6(uPAR) cells expressed on their surface approximately 12,000 high affinity (Kd 1.7 x 10(-10) M uPA binding sites per cell. Unlabeled LB6(uPA) and 125-IUdR-labeled LB6(uPAR) cells were coinoculated onto experimentally wounded and resealed CAMs and their invasion was compared to that of homologous mixtures of labeled and unlabeled LB6(uPAR) or LB6(uPA) cells. Concurrent presence of both cell types in the CAMs resulted in a 1.8-fold increase of invasion of the uPA-receptor expressing cells. A four-fold stimulation of invasion was observed when cells were cocultured in vitro, prior to in vivo inoculation. Enhancement of invasion was prevented in both sets of experiments by treatment with specific antihuman uPA antibodies, indicating that uPA was the main mediator of the invasion-enhancing, paracrine effect on the receptor-expressing cells.     

Studies on the Assembly Characteristics of Large Subunit Ribosomal Proteins in S. cerevisae

During the assembly process of ribosomal subunits, their structural components, the ribosomal RNAs (rRNAs) and the ribosomal proteins (r-proteins) have to join together in a highly dynamic and defined manner to enable the efficient formation of functional ribosomes. In this work, the assembly of large ribosomal subunit (LSU) r-proteins from the eukaryote S. cerevisiae was systematically investigated. Groups of LSU r-proteins with specific assembly characteristics were detected by comparing the protein composition of affinity purified early, middle, late or mature LSU (precursor) particles by semi-quantitative mass spectrometry. The impact of yeast LSU r-proteins rpL25, rpL2, rpL43, and rpL21 on the composition of intermediate to late nuclear LSU precursors was analyzed in more detail. Effects of these proteins on the assembly states of other r-proteins and on the transient LSU precursor association of several ribosome biogenesis factors, including Nog2, Rsa4 and Nop53, are discussed.