The axolotl thymus: cell types of the microenvironment

Summary The three-dimensional structure of the axolotl (urodele amphibian) thymus was studied by combined scanning and transmission electron microscopy. The epithelial cell is the major component of the microenvironment forming the meshwork where thymocytes differentiate. Three different types of epithelial cells could be defined by their intracytoplasmic organelles and their localization in the subcapsular or deeper part of the organ. These epithelial cells participate in various types of lymphostromal interactions. Other stromal elements, such as interdigitating reticular cells, macrophages, eosinophil granulocytes and epithelial cysts were also defined. The absence of a true cortico-medullary differentiation in the axolotl thymus, the presence of different stromal elements and the physiological significance of these various microenvironments are discussed.     

Melanin as a natural germ cell marker for intraspecific transplantation experiments in Ambystoma mexicanum (Urodela,Amphibia)

Summary In urodele amphibians, the lack of a reliable germ cell marker restricts the experimental study of the germ lineage. In the present work, we conducted genetic and histological analyses in order to demonstrate that melanin from oocytes constitutes a germ cell marker available for intraspecific experiments in Ambystoma mexicanum. Then, using this marker, we implanted germ cells from undifferentiated gonads (stage 48) into the blastocoel of host embryos and investigated their fate and determined state. Our results show that, from this stage on, the donor cells do not differentiate into other cell types; therefore, they are restricted in developmental capacity and irreversibly determined as germ cells. On the other hand, exogenous germ cells were found in an isotopic position until the young tail-bud stage, and then were found in an ectopic position; these results suggest that, from the middle tail-bud stage on, an active process contributes to migration of primordial germ cells to the gonadal territory.     

The cytotoxicity of chrysotile asbestos fibers to pulmonary alveolar macrophages I. Effects of inhibitors of ADP-ribosyl transferase

Pulmonary alveoler macrophages exposedto very short chrysotile asbestos fibers present a typical cytotoxic response: extracellular releases of lactate dehydrogenase and -galactosidase, and a decrease in cellular ATP content. The objective of this study was to determine if nicotinamide and 3-aminobenzamide, two inhibitors of the ADP-ribosyl transferase, could modify the in vitro toxicity of chrysotilee fibers. After 30 min of pre-exposure with each of the two inihibitors, pulmonary alveolar macrophage monolayers were concominantly exposed for 18 hours to 50g of fibers. It was observed that, in a dose-effect relationship (5 to 30 mM), nicotinamide was very effective in reducing the extracellular liberation of the marker enzymes. At 30 mM, the enzyme releases in the medium had returned to control values; the restoration of cell viability was confirmed by ATP levels. Up to 5 mM 3-aminobenzamide did not provide any protection against chrysotile cytotoxicity. Nicotinic acid, a structural analogue of nicotinamide, but not an inhibitor of the ADP-ribosyl transferase, also showed no protective effect. Nicotinamide and 3-aminobenzamide increased the intracellular NAD⁺ pools, respectively by 350% and 250%. However, with or without additives, the chrysotile fibers caused a constant and significant decrease in NAD⁺ levels (40–55 pmoles). These results suggest that the inhibition of the nuclear ADP-ribosyl transferase is not the major mechanism by which nicotinamide protects pulmonary alveolar macrophages against the chrysotile asbestos fibers.Abbreviations 3-AB 3-aminobenzamide - ADPRT ADP-ribosyl transferase - -GAL -galactosidase - DTT dithiothreitol - FBS fetal bovine serum - FMN flavin mononucleotide - HEPES N-2-hydroxyethyl piperazine-N-2-ethanesulfonic acid - LDH lactate dehydrogenase - NAD⁺ nicotinamide adenine dinucleotide (oxidized form) - NADH nicotimide adenine dinucleotide (reduced forms) - NADPH nicotimide adenine dinucleotide phosphate (reduced form) - NAM nicotinamide - NIC nicotinic acid - ORS oxygen radical species - PAM pulmonary alveolar macrophages - S.E. standard error of the mean - TAPS tris (hydroxymethyl) methylamino-propane sulfonic acid - TRIS tris (hydroxymethyl) aminomethane - VSF very short chrysotile fibers     

Human recombinant insulin-like growth factor I. I. Development of a serum-free medium for clonal density assay of growth factors using balb/c 3T3 mouse embryo fibroblasts

Summary A serum-free clonal density growth assay was developed for the quantification of the biological activity of human recombinant insulin-like growth factor I (IGF-I). The assay measures IGF-I stimulated growth of Balb/c 3T3 cells cultured over 4 d on poly-d-lysine-coated plastic surfaces in a serum-free medium formulation composed of a 1∶1 (vol/vol) mixture of Ham's F12 and Dulbecco's modified Eagle's media, supplemented with 3.0 ng/ml bovine basic fibroblast growth factor (bFGF), 10 μg/ml human transferrin, 100 μg/ml ovalbumin, and 1.0 μM dexamethanose. Low-temperature trypsinization of serum-supplemented stock cultures combined with the use of poly-d-lysine-coated plates made it unnecessary to use serum or fibronectin to promote cell attachment and survival. Serum-free growth conditions were optimized with respect to the concentrations of the supplements. Addition of IGF-I resulted in 3.5-fold more cells than control cultures without IGF-I after 4 d. Deletion of bFGF resulted in no IGF-I stimulation of growth. The concentrations of various preparations of IGF-I required to achieve one-half maximal stimulation of cell number (ED₅₀), ranged between 1.25 and 4.7 ng/ml. In parallel assays, IGF-I was 6.6 times more potent than human recombinant insulin-like growth factor II and 32 times more potent than insulin. When cells were seeded into medium containing IGF-I, transferrin, ovalbumin, and dexamethasone but no bFGF, growth was minimal. Dose-response addition of bFGF showed an ED₅₀, of 0.9 ng/ml. The methods reported are useful to monitor the biological potency of recombinant and natural-source growth factors as well as providing a new means of studying the multiple growth factor requirements of Balb/c 3T3 cells in cultures. This work was supported by a contract from IMCERA Bioproducts, Inc.     

Effect of n-3 and n-6 fatty acids on hepatic microsomal lipid metabolism: a time course study

The present study examines the time dependent effects of n-6 and n-3 polyunsaturated fatty acids on liver microsomal lipid metabolism in FVB mice fed a diet supplemented with a mixture of free fatty acids (mainly 18:3n-6 and 20:5n-3) at 25 mg/g diet. Significant changes in the fatty acid composition of total liver and microsomal lipids were observed after 7 days on the diets. Thereafter, some animals remained on the same diet while others were fed a diet supplemented with hydrogenated coconut oil (HCO). With the exception of 20:5n-3 which showed a slower recovery, establishment of the HCO pattern was rapid indicating that the diet-induced changes could be easily reversed. The unsaturation index, the cholesterol/phospholipid ratio and the microviscosity of the microsomal membranes were not affected by these dietary manipulations. Unsaturated fatty acid supplementation reduced the activity of ⁹ desaturase by 50%. Feeding the HCO diet to mice previously fed the EPA/GLA diet led to a progressive increase in ⁹ desaturase activity, reaching 80% of the day zero values after 14 days. The monoene content of hepatic total lipids reflected, in most cases, the changes in enzyme activity. This study shows that a low dose of a n-3 and n-6 free fatty acid mixture increases the quantities of members of the n-3 family, without loss of n-6 fatty acids in microsomal membranes and modifies the activity of ⁹ desaturase without altering the microsome physicochemical parameters.     

Application of a directed conformational search for generating 3-D coordinates for protein structures from alpha-carbon coordinates.

A directed conformational search algorithm using the program CONGEN (ref. 3), which samples backbone conformers, is described. The search technique uses information from the partially built structures to direct the search process and is tested on the problem of generating a full set of backbone Cartesian coordinates given only alpha-carbon coordinates. The method has been tested on six proteins of known structure, varying in size and classification, and was able to generate the original backbone coordinates with RMSs ranging from 0.30-0.87A for the alpha-carbons and 0.5-0.99A RMSs for the backbone atoms. Cis peptide linkages were also correctly identified. The procedure was also applied to two proteins available with only alpha-carbon coordinates in the Brookhaven Protein Data Bank; thioredoxin (SRX) and triacylglycerol acylhydrolase (TGL). All-atom models are proposed for the backbone of both these proteins. In addition, the technique was applied to randomized coordinates of flavodoxin to assess the effects of irregularities in the data on the final RMS. This study represents the first time a deterministic conformational search was used on such a large scale.     

A free-radical hypothesis for the instability and evolution of genotype and phenotypein vitro

It has been known for several decades that cultured murine cells undergo a defined series of changes, i.e., anin vitro evolution, which includes crisis, spontaneous transformation (immortalization), aneuploidy, and spontaneous neoplastic transformation. These changes have been shown to be caused by thein vitro environment rather than an inherent instability of the murine phenotype or genotype. Serum amine oxidases were recently identified as a predominant cause of crisis. These enzymes generate hydrogen peroxide from polyamine substrates that enter the extracellular milieu. This finding implicates free-radical toxicity as the underlying cause ofin vitro evolution. We propose an oxyradical hypothesis to explain each of the stages ofin vitro evolution and discuss its significance for cytotechnology and long-term cultivation of mammalian cell types.ORR, CDER, FDA Mod-1, Room 2023, 8301 Muirkirk Road, Laurel MD 20708, USA     

Macrophylloside, a flavone glucoside from Primula macrophylla

A new flavone glucoside macrophylloside has been isolated from the whole plant of Primula macrophylla and its structure was determined by spectroscopic methods as 2′-hydroxy-7-O-β- -glucopyranosyloxyflavone. Sitosterol glucoside was also isolated for the first time from this plant.     

In vitro OKT3-induced mitogenesis in selenium-deficient patients on a diet for phenylketonuria

Patients with phenylketonuria (PKU) are frequently deficient in the essential trace element selenium (Se), because of their very low protein diet. Using two approaches to investigate T-cell response to proliferative signaling, viz, mitogenesis caused by the monoclonal antibody OKT3 and the plant lectin phytohaemagglutinin (PHA), we demonstrated significantly reduced responses to optimal concentrations of OKT3 in a group of PKU patients with reduced serum Se compared with a normal group (p = 0.0005) and with a group of PKU patients whose serum Se was normal (p = 0.0023). The response of the Se-deficient group to optimal levels of PHA did not differ from that of the normal controls or from that of Se-normal PKU patients. A dose-dependent relationship between serum Se levels and mitogenic response was evident for OKT3 (r = 0.34, p = 0.0154), but not for PHA (r = -0.02, p = 0.9086). We suggest that the reduced response to OKT3 mitogenesis in Se-deficient PKU patients is possibly the consequence of impaired Se-dependent metabolic activity, which affects mitogenic signaling via the T cell antigen receptor (TCR/CD3) complex.