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.     

Morphine and Enkephalins Potently Inhibit [3H]Noradrenaline Release from Rat Brain Cortex Synaptosomes: Further Evidence for a Presynaptic Localization of μ-Opioid Receptors

Synaptosomes prepared from rat cerebral cortex and labeled with [3H]noradrenaline (NA) were superfused with calcium-free Krebs-Ringer-bicarbonate medium and exposed to 10 mM K+ plus 0.1 mM Ca2+ so that [3H]NA release was induced. 6,7-Dihydroxy-N,N-dimethyl-2-aminotetralin (TL-99) strongly inhibited synaptosomal K+-induced [3H]NA release (EC50 = 5-10 nM) by activating alpha 2-adrenoceptors. Release was also inhibited (maximally by 40-50%) by morphine (EC50 = 5-10 nM), [Leu5]enkephalin (EC50 = approximately 300 nM), [D-Ala2,D-Leu5]enkephalin (DADLE), and Tyr-D-Ala-Gly-(NMe)Phe-Gly-ol (DAGO) (EC50 values = approximately 30 nM). In contrast to the mu-selective opioid receptor agonists morphine and DAGO, the highly delta-selective agonist [D-Pen2,D-Pen5]enkephalin (1 microM) did not affect [3H]-NA release. Furthermore, the inhibitory effect of DADLE, an agonist with affinity for both delta- and mu-opioid receptors, was antagonized by low concentrations of naloxone. The findings strongly support the view that, like alpha 2-adrenoceptors, mu-opioid receptors mediating inhibition of NA release in the rat cerebral cortex are localized on noradrenergic nerve terminals.     

Flavonoid synthesis in Petunia hybrida: partial characterization of dihydroflavonol-4-reductase genes

In this paper we describe the organization and expression of the genes encoding the flavonoid-biosynthetic enzyme dihydroflavonol-4-reductase (DFR) in Petunia hybrida. A nearly full-size DFR cDNA clone (1.5kb), isolated from a corolla-specific cDNA library was compared at the nucleotide level with the pallida gene from Antirrhinum majus and at the amino acid level with enzymes encoded by the pallida gene and the A1 gene from Zea mays.The P. hybrida and A. majus DFR genes transcribed in flowers contain 5 introns, at identical positions; the three introns of the A1 gene from Z. mays coincide with first three introns of the other two species. P. hybrida line V30 harbours three DFR genes (A, B, C) which were mapped by RFLP analysis on three different chromosomes (IV, II and VI respectively).Steady-state levels of DFR mRNA in the line V30 follow the same pattern during development as chalcone synthase (CHS) and chalcone flavanone isomerase (CHI) mRNA. Six mutants that accumulate dihydroflavonols in mature flowers were subjected to Northern blot analysis for the presence of DFR mRNA. Five of these mutants lack detectable levels of DFR mRNA. Four of these five also show drastically reduced levels of activity for the enzyme UDPG: flavonoid-3-O-glucosyltransferase (UFGT), which carries out the next step in flavonoid biosynthesis; these mutants might be considered as containing lesions in regulatory genes, controlling the expression of the structural genes in this part of the flavonoid biosynthetic pathway. Only the an6 mutant shows no detectable DFR mRNA but a wild-type level for UFGT activity. Since both an6 and DFR-A are located on chromosome IV and DFR-A is transcribed in floral tissues, it is postulated that the An6 locus contains the DFR structural gene. The an9 mutant shows a wild-type level of DFR mRNA and a wild-type UFGT activity.     

Intentional cranial vault deformation and induced changes of the cranial base and face

Three morphologically distinct populations of Peruvian crania (n = 130) were metrically analysed to quantify changes resulting from intentional artificial vault deformation. Two of these samples are artificially deformed (anteroposterior [AP] and circumferential [C] types). Measurements taken from lateral radiographs demonstrated that alternative forms of the cranial base angle (N-S-Ba, planum angle, planum sphenoidale to plane of the clivus and PANG angle, planum sphenoidale to basion-sella plane) and the orbital and OANG angles (orbital roof to plane of the clivus and basion-sella plane, respectively) of both deformed groups increased while the angle S-Ba-O decreased significantly with respect to the undeformed (N) sample. Changes in the AP group are largely due to anteroinferior displacement of the basion-sella plane. Similar changes in group C are amplified by this group's posterosuperior frontal migration. This migration results in a relatively shallow orbit at the orbital plate/frontal squama interface. Unlike the deformation experienced by the external vault plates, the basion-sella plane orientation remains stable with respect to the Frankfort Horizontal. Additionally, nasal region measurements such as maximum nasal aperture breadth and nasal height were largely stable between each deformed group and the undeformed group. However, facial (bimaxillary and bizygomatic), basicranial, cranial, and frontal breadths decreased significantly from group AP to group N to group C. Thus, gross morphological facial changes between each undeformed group and the control group are largely accounted for by dimensional changes in peripheral structures. These results stress the importance of the dynamic interrelationship between the cranial vault and base in the development of the craniofacial complex.     

Prolonged submaximal exercise and L-carnitine in humans

Changes in the main physiological parameters and circulating indicators of carbohydrate, protein, lipid (and ketone body) metabolism were measured in ten exercising subjects before L-carnitine (L-carn) loading, after 4 weeks of daily loading with 2 g L-carn, and 6-8 weeks after terminating L-carn administration. Measurements were made on venous blood samples collected during each experiment at fixed time intervals over an initial rest of 45 min, 60 min bicycle exercise performed near 50% VO2max and 120 min recovery. Free and total plasma carnitine levels reached a plateau corresponding to an average rise of 25% for both fractions, 9-10 days after the beginning of the L-carn diet. These levels returned to their initial values 6-8 weeks after cessation of the supply. Generally L-carn supplementation did not significantly modify the physiological parameters and circulating metabolites. No distinct increase of the relative participation of endogenous lipids in the fuel supply of prolonged submaximal exercise was observed. In normal human subjects the increased demand for fatty acid oxidation resulting from exercise seems to be adequately supported by endogenous levels of carnitine.     

Transient states of geosmin,pigments, carbohydrates and proteins in continuous cultures of Oscillatoria brevis induced by changes in nitrogen supply

Transitions in the growth limiting factor from light (I) to nitrogen (N) and vice versa caused changes in geosmin production, protein and carbohydrate content, and the synthesis of pigments such as chlorophyll a (Chl a), phycobiliproteins (PBPs), and -carotene of the cyanobacterium Oscillatoria brevis. Following IN transition the first 150h, the decrease in protein content was compensated for by an increase of carbohydrates, and thereby, a constant biomass level was maintained in this period. Thereafter, biimass dropped to 15% of its initial level. A decrease in geosmin and pigment content was observed during transition from IN-limited growth. However, geosmin increased relative to phytol (Chl a) and -carotene which may indicate that a lowered demand for phytol and -carotene during N-limited growth allows isoprenoid precursors to be directed to geosmin rather than to pigment synthesis. Synthesis of Chl a and -carotene at the expense of geosmin was suggested for the observed start of increase in geosmin production only at the time that Chl a and -carotene had reached their I-limited steady state. Transition from nitrogen to light limited growth caused an acceleration of metabolism shown by a rapid decrease in carbohydrate content accompanied by an increase in protein content. The growth rate of the organisms temporarily exceeded the dilution rate of the culture and the biomass level increased 6-fold. Due to the only modest changes in geosmin production (2-fold) compared to changes in biomass level (6-fold) during I-or N-limited growth, environmental factors seem to have limited effect on geosmin production.Abbreviations Chl a chlorophyll a - dry wt dry weight; - I-limited light-limited - N-limited nitrogen-limited - PBP phycobiliprotein This research was performed at the Department of Microbiology, University of Amsterdam, with finacial support provided by the Royal Norwegian Ministry of Foreign Affairs and the Royal Norwegian Council for Scientific and Industrial Research     

The chalcone synthase multigene family of Petunia hybrida (V30): sequence homology,chromosomal localization and evolutionary aspects

Chalcone synthase (CHS) genes in Petunia hybrida comprise a multigene family containing at least 7 complete members in the strain Violet 30 (V30). Based on a high sequence homology in both coding and non-coding sequence, a number of CHS genes can be placed into two subfamilies. By restriction fragment length polymorphism (RFLP) analysis it was shown that both chromosomes II and V carry one of these subfamilies, in addition to the other CHS genes identified so far. Members of a subfamily were found to be closely linked genetically. Analysis of the Petunia species that contributed to the hybrid nature of P. hybrida (P. axillaris, P. parodii, P. inflata and P. violacea) shows that none of the CHS gene clusters is specific for either one of the parents and therefore did not arise as a consequence of the hybridization. The number of CHS genes within a subfamily varies considerably among these Petunia species. From this we infer that the CHS subfamilies arose from very recent gene duplications.     

Tetracycline production byStreptomyces aureofaciens: the time lag of production

Summary The exact time course of phosphate consumption in a tetracycline production byStreptomyces aureofaciens has been determined. The data have been compared with model simulations according to a model proposed by Votruba et al. (1984). This led to a revision of his equation for the rate of phosphate consumption and to the proposal that phosphate is consumed proportionally to the growth rate. In contradiction to the model simulations it was found that the length of the time lag of the production is independent of the initial phosphate concentration. While the model explains the time lag through inhibition of the production by phosphate, the measured data show that there must be another or an additional reason for the lag. Simultaneously with the start of the production the organism changes from an organic substrate to ammonia as nitrogen source.All experiments have been carried out in a bubble column of 651 working volume as fed batch fermentation. An autoanalyzer and a HPLC was coupled to the reactor for automatic measurement of phosphate, ammonia, sucrose and products in short intervals. Composition of the outlet gas, pH, pO₂, temperature and weight of the substrate flasks were monitored on-line.     

Isolation and characterization of a cDNA that encodes the peptide core of the secretory granule proteoglycan of human promyelocytic leukemia HL-60 cells

A cDNA that encodes the peptide core of the secretory granule proteoglycan of the human promyelocytic leukemic cell line, HL-60, has been isolated and analyzed. When human genomic DNA was digested and probed under conditions of low stringency with a rat cDNA that encodes a Mr = 18,600 serine/glycine-rich proteoglycan peptide core in L2 yolk sac tumor cells (Bourdon, M. A., Oldberg, A., Pierschbacher, M., and Ruoslahti, E. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 1321-1325) and basophilic leukemia-1 cells (Avraham, S., Stevens, R. L., Gartner, M. C., Austen, K. F., Lalley, P. A., and Weis, J. H. (1988) J. Biol. Chem. 263, 7292-7296), a number of DNA fragments were identified. A HL-60 cell-derived cDNA library was therefore screened under conditions of low stringency with the rat probe to identify and isolate a human homologue of this rat proteoglycan peptide core. Analysis of the resulting human cDNA clones indicated that the proteoglycan peptide core that is expressed in HL-60 cells is Mr = 17,600 and contains an 18-amino acid glycosaminoglycan attachment region that consists primarily of alternating serin and glycine. Northern blot analysis of total RNA probed with the human cDNA revealed that the major message for this proteoglycan peptide core in HL-60 cells is approximately 1.3 kilobase pairs in size. When a Southern blot of digested human genomic DNA was probed with the human cDNA, three bands of approximately 6, 9, and 12 kilobase pairs were detected. However, when the Southern blot was probed with the XmnI----3' fragment of this human cDNA, one prominent band was detected, indicating that a single gene encodes this protein in the human. Analysis of the DNA from human/mouse and human/hamster somatic cell hybrids probed with the human cDNA demonstrated that the gene that encodes this molecule resides on human chromosome 10. Because the proteoglycans that are present in the secretory granules of different types of rat and mouse mast cells possess small peptide cores that are rich in serine and glycine, we propose that this HL-60 cell-3 derived cDNA encodes the peptide core of the proteoglycan that is expressed in the secretory granules of this human promyelocytic cell.     

An Ichthyophonus-typefungus in the deep-sea pelagic fish Scopelogadus beanii (Günther) (Pisces: Melamphaidae)pathology, geographic distribution and ecological implications

Parasitological examination of 400 specimens of the lower mesopelagic-bathypelagic melamphaid fish Scopelogadus beanii (Günther) collected from throughout the species'geographic range in the Atlantic Ocean revealed cysts ranging in size from 1 to > 20 mm diameter in the livers of 50 individuals (12.5%). Histological preparations showed that the encysted material was an Ichthyophonus-type fungus, which destroyed the liver tissue in infected individuals by progressive necrosis, the extent of which varied with cyst number and size. Infections were found primarily in individuals > 80 mm s.l . (size of sexual maturity in S. beanii ), with a significantly higher rate of infection among females. Liver weight was significantly reduced in infected fishes. Body wet weights tended to be lower in infected females. Fish from the western Atlantic showed a significantly greater prevalence of infection (17.0%) than those from either the eastern (1.1%) or South Atlantic (7.1 %). Within the western Atlantic, individuals collected from the slope waters of the Mid-Atlantic Bight showed a significantly higher percentage of infected individuals than those collected from more northerly areas (27.8% v. 9.9%, respectively); regional variations may be accounted for by differences in habitat and diet of S. beanii among these regions. The high percentage of infected individuals in the western North Atlantic, coupled with the pathogenic nature of the fungus, suggests the possible loss of reproductively competent individuals from the population of this region.