Identification of EcoRV fragments spanning the N alpha-tubulin gene of Physarum

The N alpha-tubulin gene of Physarum polycephalum has an EcoRV site at codons 252/253. EcoRV digestion of physarum DNA generated two EcoRV fragments per gene copy comprising both coding and flanking sequences. Hybridisation probes which included coding sequences upstream from the central EcoRV site cross-hybridised with another alpha-tubulin gene. Probes derived from either 5'- or 3'-flanking regions were gene-specific. These probes identified two EcoRV fragments in the haploid strain CLdAXE viz 5.4 kb (5'-fragment) and 6.2 kb (3'-fragment). The same two fragments were identified in EcoRV digests of DNA of the diploid strain M3CVIII, and a second form of the gene was also identified comprising two fragments viz 5.0 kb (5'-end) and 5.5 kb (3'-end). Both forms gave rise to an identical 4.65 kb HindIII fragment as judged by restriction mapping.     

Lack of correlation between loss of anchorage-independent growth and levels of transformation-specific p53 protein in retinoic acid-treated F9 embryonal carcinoma cells

It has been shown that differentiated derivatives of retinoic acid (RA)-treated F9 embryonal carcinoma cells become non-malignant. In the present study it is asked whether this loss of malignancy is due to cellular differentiation. Because the ability of cells to grow in suspension correlates with in vivo tumorigenicity, we determined the time course of the loss of this property, after RA treatment, with relation to the differentiation to parietal endoderm and the acquisition of normalcy in several common transformation-specific properties of F9 cells. Our results show that pretreatment with RA for 24 h caused 80% inhibition of anchorage-independent growth in F9 cells, and this inhibition reached its highest level (98%) after pretreatment with RA for 48 h and longer. However, all other observed transformation-related properties, and the levels of plasminogen activator (marker for parietal endoderm) remained unaltered at this early post-treatment stage. These observations suggest that the loss of malignancy is a relatively early event in the biochemical pathways involved in the RA-induced differentiation of F9 cells. Furthermore, our data show that the presence of elevated levels of p53 alone may not be sufficient to maintain the anchorage-independent growth and the rapid proliferation of F9 cells.     

Liver connective tissue cells isolated from human schistosomal fibrosis or alcoholic cirrhosis represent a modified phenotype of smooth muscle cells

Hepatic connective tissue cells associated with schistosomal fibrosis and alcoholic cirrhosis were studied in vitro. Primary cell lines were isolated from all biopsies: they were identified as specific homogeneous cell populations, named liver connective tissue cells (LCTC). They were recognized as analogous to smooth muscle cells, different from true fibroblasts by morphological and physiological criteria. The proliferative capacity of LCTC is directly proportional to the degree of fibrosis in hepatic tissues. LCTC are able to secrete type I, III and IV collagen, fibronectin, laminin and amyloid P component. Their relationship with specific pathology of intrahepatic vascular tree in schistosomiasis is hypothesized.     

Sex expression in floral buds of Acorn squash

Summary Staminate flowers of the Acorn squash (Cucurbita pepo, cv. Table Queen) are initiated monosexually, pistillate flowers are initiated bisexually. Pistillate flower primordia are physiologically bisexual from a stage below 0.3 mm until they are 0.8 mm in width.Male and female floral buds were cultivated on White's medium, modified according to Galun et al. (1963). In excised female floral buds the relative development of stamens is better than in in-situ floral buds; the opposite relationship holds for ovary development. The smaller the bud at excision, the greater the stamen growth and the lesser the ovary growth, both in absolute and relative terms. Although smaller pistillate floral buds cultured in vitro showed a greater increase in maleness than larger ones, there was no difference in the percentage of female buds between explants of the two sizes when originating from the mixed stem region (that is, the stem region carrying male and female flowers). Addition of 3-indoleacetic acid and the anti-auxin p-chlorophenoxyisobutyric acid to standard medium in equal amounts resulted in somewhat better development of excised floral buds than addition of each of these compounds alone.The percentage of female buds among explants originating from identical nodes was not significantly influenced by 3-indoleacetic acid or p-chlorophenoxyisobutyric acid.Weizmann Memorial Fellow. The studies reported in this publication were mainly undertaken at the Weizmann Institute of Science     

NOR polymorphism in the Iberian species Chondrostoma lusitanicum (Pisces: Cyprinidae)

Chromosomal polymorphism regarding number of NOR sites in the cyprinid fish Chondrostoma lusitanicum was examined using C-banding, silver-staining (Ag), and fluorescent staining with chromomycin A₃ (CMA₃). The analysis of heterochromatic regions allowed a more precise identification of the centromeric regions and the proposal of a revised haploid chromosome formula (7M: 15S: 3A). We describe variability in the number of NOR regions per genome, number of active NOR sites per cell, and relative size of individual NORs. Individuals expressed two or four NOR-bearing chromosomes. Polymorphism was detected in all the populations studied and sex-related differences were not found. The observed chromosomal NOR phenotypes suggest the occurrence of structural rearrangements during the evolutionary process of this diploid leuciscine cyprinid.     

Catabolism of caffeine and related purine alkaloids in leaves ofCoffea arabica L.

In a study of purine alkaloid catabolism pathways in coffee,¹⁴C-labelled theobromine, caffeine, theophylline and xanthine were incubated with leaves ofCoffea arabica. Incorporation of label into¹⁴CO₂ was determined and methanol-soluble metabolites were analysed by high-performance liquid chromatography-radiocounting. The data obtained demonstrate catabolism of caffeine theophylline 3-methylxanthine xanthine. Xanthine is degraded further by the conventional purine catabolism pathway to CO₂ and NH₃ via uric acid, allantoin and allantoic acid. The conversion of caffeine to theophylline is the rate-limiting step in purine alkaloid catabolism and provides a ready explanation for the high concentration of endogenous caffeine found inC. arabica leaves. Although theobromine is converted primarily to caffeine, a small portion of the theobromine pool appears to be degraded to xanthine by a caffeine-independent pathway. In addition to being broken down to CO₂, via the purine catabolism pathway, xanthine is metabolised to 7-methylxanthine. Metabolism of [2-¹⁴C]xanthine byC. arabica leaves in the presence of 5 mM allopurinol results in very large increases in incorporation of radioactivity into 7-methylxanthine as degradation of the substrate via the purine catabolism pathway is blocked. The identity of 7-methylxanthine in these studies was confirmed by gas chromatography-mass spectrometry analysis.Abbreviations HPLC-RC high-performance liquid chromatography-radiocounting This work was supported by the British Council which provided H.A. with Japan-UK travel grants. F.M.G. was supported by a Biotechnology and Biological Sciences Research Council grant to A.C.     

Segregation analysis indicates a major gene in the control of interleukine-5 production in humans infected with Schistosoma mansoni.

The interleukine-5 (IL-5) is a hormone of the immune system that is the main regulator of eosinopoiesis, eosinophil maturation and activation, and immunoglobulin A production. Thus, IL-5 contributes in several ways to human immune defenses against various pathogens, including helminths and infectious agents of the digestive and respiratory tracts. On the other hand, the increase in eosinophil number and the activation of these cells, which both have been related to elevated IL-5 production, are the cause of severe pathological disorders, as in asthma or hypereosinophilic syndromes. Although the immunological pathways leading to IL-5 synthesis have been identified, the reasons for the large variability observed in IL-5 production among subjects exposed to comparable antigenic stimulation are unknown. To investigate the role of genetic factors in this variability, we conducted a segregation analysis in a Brazilian population infected by the helminth parasite Schistosoma mansoni. The analysis was performed on IL-5 levels produced by blood mononuclear cells of these subjects after in vitro restimulation with either parasite extracts (IL-5/schistosomula sonicates [SS] phenotype) or a T-lymphocyte mitogen (IL-5/phytohemagglutin [PHA]). The results provide clear evidence for the segregation of a codominant major gene controlling IL-5/SS and IL-5/PHA production and accounting for 70% and 73% of the phenotypic variance, respectively; the frequency of the allele predisposing to low IL-5 production was approximately .22 for both phenotypes. No significant relationship was found between these genes and the gene controlling infection intensities by S. mansoni detected in a previous study. Linkage studies are ongoing to locate those genes that would help to characterize the genetic factors involved in pathological conditions such as severe helminth infections and allergic diseases.