Antiplasmodial activity of Artemisia annua plant cell cultures

Extracts of Artemisia annua cultures have been assessed for in vitro activity against the malarial parasite Plasmodium falciparum. Callus and suspension cells and medium were analysed and examined for their activity at different stages of growth and development. Time-course experiments were carried out to investigate the influence of various basal media, plant growth regulators and light on both growth and possible artemisinin production. Two active fractions were obtained but artemisinin was not detected.     

Expression of insulin-like growth factor-I and its receptor by SV40-transformed rat granulosa cells

Cellular proliferation is a dominant aspect of ovarian follicular development in the rat, and insulin-like growth factor I (IGF-I) has been proposed as a mediator of cellular growth and differentiation in the ovary. An SV40-transformed rat granulosa cell line (RGA-41S) has been established as a model for studies on dividing cells of granulosa origin. Granulosa cells from the ovaries of immature diethylstilbestrol-treated rats were infected with the tsA255 mutant of SV40, followed by cloning in serum-free medium to select transformed cell lines which were serum independent. At the permissive temperature (33 C), RGA-41S cells exhibited a transformed phenotype and rapidly formed high density multilayers of compact cells that readily overgrew nontransformed cells. At the nonpermissive temperature (40 C) cell replication declined and division ceased after 4 days. Furthermore, at 40 C the cells grew as a monolayer and assumed a tetrahedral shape with a high cytoplasm-to-nucleus ratio, and displayed reduced ability to overgrow nontransformed cells. The transformed ovarian cells did not express detectable gonadotropin receptors and steroidogenic activity but retained their epithelial phenotype as demonstrated by cytokeratin staining of the cytoskeleton, the presence of microvilli, and the formation of tight junctions between cells. In support of the proposed autocrine-paracrine actions of IGF-I in the ovary, assay of conditioned serum-free culture medium revealed secretion of IGF-I-immunoreactive material by RGA-41S cells. HPLC-purified IGF-I immunoreactivity from these cells eluted with the same retention time as recombinant human IGF-I. When hybridized with a 32P-labeled rat IGF-I cDNA probe, poly(A)+ mRNA prepared from RGA-41S cells grown at both temperatures showed the typical three size classes of IGF-I mRNA on Northern blots (7.5, 1.7, and 0.8-1.2 kilobase kb), although the levels were somewhat higher at 33 C. The presence of IGF-I receptors in transformed cells was demonstrated by specific 125I-IGF-I binding to intact cells. Scatchard analysis indicated a single class of high affinity receptors at a density of 10(5) binding sites per cell and a dissociation constant (Kd) = 0.52 x 10(-9) M. Furthermore, hybridization of a 32P-labeled IGF-I receptor probe to Northern blots of poly(A+) RNA prepared from cells grown at 33 C and 40 C revealed an 11-kilobase rat IGF-I receptor mRNA. Physiological concentrations of IGF-I increased [3H]aminoisobutyric acid uptake by RGA-41S cells grown at either temperature, attesting to the retention of responsiveness to IGF-I in these transformed granulosa cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Economic impact of a nationwide outbreak of salmonellosis: cost-benefit of early intervention.

The recognition and investigation of an outbreak of food poisoning in 1982 due to chocolate contaminated with Salmonella napoli enabled the food that carried the salmonella to be identified and four fifths of the implicated consignment of chocolate to be withdrawn. The economic benefits of prompt intervention in the outbreak have been assessed. The cost of the outbreak was over 0.5 pounds m. It is estimated that five deaths were prevented by the intervention and that 185 admissions to hospital and 29,000 cases of S napoli enteritis were avoided. This successful investigation yielded a 3.5-fold rate of return to the public sector and a 23.3-fold return to society on an investment in public health surveillance. A methodology is described that can be used to estimate the benefits of early intervention in outbreaks of foodborne illness and topics for further research are suggested. It is concluded that public health authorities and industry have much to gain by collaborating in the research into the design of cost effective programmes to prevent foodborne infections.     

Endocytosis of wheat germ agglutinin binding sites from the cell surface into a tubular endosomal network

By using fluorescence and electron microscopy, the endocytic pathway encountered by cell surface components after they had bound wheat germ agglutinin (WGA) was visualized. The majority of these components are thought to consist of sialylated glycoproteins (HMWAG) that represent a subpopulation of the total cell surface proteins but most of the externally disposed plasma membrane proteins of the cell. Examination of semi-thin sections by medium- and high-voltage electron microscopy revealed the three-dimensional organization of vesicular and tubular endosomes. Binding of either fluorescein isothiocyanate-, horseradish peroxidase-, or ferritin-conjugated WGA to cells at 4 degrees C showed that the HMWAG were distributed uniformly over the cell surface. Warming of surface-labeled cells to 37 degrees C resulted in the endocytosis of WGA into peripheral endosomes via invagination of regions of both coated and uncoated membrane. The peripheral endosome appeared as isolated complexes comprising a vesicular element (300-400 nm diam.) surrounded by and continuous with tubular cisternae (45-60 nm diam.), which did not interconnect the endosomes. After 30 min or more label also became localized in a network of anastomosing tubules (45-60 nm diam.) that were located in the centrosomal region of the cell. Endocytosed WGA-HMWAG complexes did not become associated with cisternae of the Golgi apparatus, although tubular and vesicular endosomes were noted in the vicinity of the trans-Golgi region. The accumulation of WGA-HMWAG in the endosomes within the centrosomal region was inhibited when cells were incubated at 18 degrees C. None of these compartments contained acid phosphatase activity, a result that is consistent with other data that the HMWAG do not pass through lysosomes initially. The kinetics of labeling were consistent with the interpretation that recycling of most of the WGA binding surface glycoproteins occurred rapidly from early peripheral endosomes followed by the late trans-Golgi compartment. In conclusion, a portion of cell surface glycoproteins are routed to a complex arrangement of tubular and vesicular compartments following endocytosis that includes a putative post-endosomal, tubular reticulum that appears to be separate from the trans-most Golgi saccule.     

Structural basis for variations in the sensitivity of human decay accelerating factor to phosphatidylinositol-specific phospholipase C cleavage.

Human decay-accelerating factor (DAF) proteins expressed on E and nucleated cells differ in their susceptibility to phosphatidylinositol (PI)-specific phospholipase C (PLC) cleavage/release. To investigate the mechanism of this difference, the glycoinositol-phospholipid anchoring moieties of E DAF, and of HeLa cell, polymorphonuclear cell, and lymphocyte DAF were structurally compared. Labeling of PI-PLC-resistant E DAF with 3-(trifluoromethyl)-3-(m-[125I]-iodophenyl)-diazirine ([125I]TID) and TLC analysis of nitrous acid deamination anchor fragments showed a predominant phospholipid species with less polar migration than the 125I-TID-labeled PI. Gas chromatographic analyses of methanolyzed E protein revealed 2.20 +/- 0.16 mol of fatty acids [16:0, 18:0, 18:1, 20:4, 22:4, and 22:5 (0.76, 0.36, 0.25, 0.15, 0.40, 0.28 mol, respectively)] and 0.86 +/- 0.05 mol of inositol per mol of N-terminal Asp. Gas chromatography-mass spectroscopy demonstrated principally myo-inositol but also variable amounts of the chiro-isomer. Nondenaturing polyacrylamide gel electrophoresis of 14C-radiomethylated E protein revealed that pretreatment with hydroxylamine, a reagent which removes ester-linked lipids, rendered it PI-PLC susceptible. In contrast, parallel analyses of 35S-cys-labeled PI-PLC-sensitive HeLa DAF protein revealed only minor amounts of the hydroxylamine-sensitive phospholipid species. Similar results were obtained with 125I-surface-labeled DAF from polymorphonuclear cells, as well as from unstimulated peripheral blood and anti-CD3-activated lymphocytes. These findings demonstrate that, rather than PI, the E DAF anchor contains an inositol alkylacylglycerol-phospholipid which is heterogeneous with respect to acyl groups and inositol isomers, that an ester-linked substitution in this inositolphospholipid underlies the resistance of E DAF protein to PI-PLC cleavage/release, and that this structural modification is cell-specific.     

Two-dimensional polyacrylamide gel electrophoresis of proteins synthesized and released by conceptuses and endometria from pony mares

Conceptuses were obtained from pony mares on each day of pregnancy between Days 12 and 28, and on Days 39, 45, 65 and 100. Endometrium was obtained from mares at Days 12, 14, 16, 18, 39, 45, 65 and 100 of pregnancy, and from non-pregnant mares during anoestrus, during transition into the breeding season, at oestrus, or during dioestrus. Tissues were incubated in vitro for 24 h with L-[3H]leucine. Proteins synthesized and released into the culture medium were analysed by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and fluorography. Conceptuses obtained before Day 14 after ovulation released a characteristic pattern of labelled proteins. These included two groups of apparent isoelectric variants of relative molecular weights (Mr) 30,000-40,000 (pI values 4.5-5.5 and 6-7), one group of Mr approximately 22,000 (pI 6.5-7), and large protein(s) that did not enter the 10% polyacrylamide gel. After Day 14 the array of labelled proteins had changed and resembled that produced by isolated yolk sac at the later stages of pregnancy studied. Included amongst these were several acidic polypeptides with Mr 20,000 (pI 5-6). The endometrial samples released an array of non-dialysable polypeptides into the culture medium. Fluorograms could be assigned to one of three general groups, with endometrium from mares within each group producing similar patterns of labelled proteins. The first group consisted of anoestrous, transitional and ovariectomized mares, and mares at oestrus or Day 1 or Day 18 after ovulation. The second group was comprised of mares at Days 12-16 of dioestrus or Days 12-18 of pregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nucleotide sequences of Caenorhabditis elegans core histone genes. Genes for different histone classes share common flanking sequence elements

We have determined the nucleotide sequence of core histone genes and flanking regions from two of approximately 11 different genomic histone clusters of the nematode Caenorhabditis elegans. Four histone genes from one cluster (H3, H4, H2B, H2A) and two histone genes from another (H4 and H2A) were analyzed. The predicted amino acid sequences of the two H4 and H2A proteins from the two clusters are identical, whereas the nucleotide sequences of the genes have diverged 9% (H2A) and 12% (H4). Flanking sequences, which are mostly not similar, were compared to identify putative regulatory elements. A conserved sequence of 34 base-pairs is present 19 to 42 nucleotides 3' of the termination codon of all the genes. Within the conserved sequence is a 16-base dyad sequence homologous to the one typically found at the 3' end of histone genes from higher eukaryotes. The C. elegans core histone genes are organized as divergently transcribed pairs of H3-H4 and H2A-H2B and contain 5' conserved sequence elements in the shared spacer regions. One of the sequence elements, 5' CTCCNCCTNCCCACCNCANA 3', is located immediately upstream from the canonical TATA homology of each gene. Another sequence element, 5' CTGCGGGGACACATNT 3', is present in the spacer of each heterotypic pair. These two 5' conserved sequences are not present in the promoter region of histone genes from other organisms, where 5' conserved sequences are usually different for each histone class. They are also not found in non-histone genes of C. elegans. These putative regulatory sequences of C. elegans core histone genes are similar to the regulatory elements of both higher and lower eukaryotes. The coding regions of the genes and the 3' regulatory sequences are similar to those of higher eukaryotes, whereas the presence of common 5' sequence elements upstream from genes of different histone classes is similar to histone promoter elements in yeast.     

Immunodetection and quantitation of the two forms of transforming growth factor-beta (TGF-beta 1 and TGF-beta 2) secreted by cells in culture

Transforming growth factor beta (TGF-beta), a potent modulator of cell growth, differentiation, and the expression of extracellular matrix components in a variety of cell types, exists as two distinct homodimers (TGF-beta 1 and TGF-beta 2), sharing 71% sequence homology. Radioreceptor and previously described radioimmunological assays using rabbit antibodies have not been able to distinguish between these two forms. We have developed antisera in turkeys against native TGF-beta 1 and TGF-beta 2, each of which specifically blocks both the receptor binding and biological activity of each of these peptides. With these immunological reagents we describe sensitive and specific immunological assays for TGF-beta 1 and TGF-beta 2 in complex biological fluids. Using these assays we show that both TGF-beta 1 and TGF-beta 2 are secreted by a variety of cultured cells, but that some cells secrete predominantly either TGF-beta 1 or TGF-beta 2 while others secrete both peptides in nearly equal amounts. Our results demonstrate that the expression of each of the two forms of TGF-beta is independently regulated.     

Acid giemsa technique for rapid identification of mitotic cells

We developed a rapid technique for differential staining of compacted chromatin as a tool for screening of large tissue culture cell populations for mitotic cells. With a combination of acid Giemsa staining and counterstaining, differential staining of mitotic cells and classification according to stage of mitosis can be accomplished at magnifications as low as x 50-100 (objectives of x 5-10). The mapped and classified cells can then be de-stained and re-studied for DNA content by Feulgen staining and/or for uptake of radioactive DNA precursors by autoradiography. The staining and de-staining procedures outlined do not affect the reproducibility and accuracy of DNA content measurements or measurements of radioactive uptake. Therefore, this technique can be used for cell kinetic analysis by the percentage labeled mitoses method and for cytophotometric studies of mitotic segregation.     

Analysis of dose-incidence relationships for marrow failure in different species, in terms of radiosensitivity of tissue-rescuing units

The analysis of 68 published sets of dose-incidence data for marrow failure in different species, using a double-log mortality function, indicates: (a) There is more heterogeneity, i.e. greater sums-of-squares per degree of freedom, within the data sets for mouse than for larger species (monkey, dog, sheep, goat, pig). (b) For mice the curves for acute doses are characterized by a D0 of about 100 cGy for tissue-rescuing units (or target cells), which are depleted at most to about 3 x 10(-4) at LD50. (c) Larger species are much less tolerant to target-cell depletion, the corresponding level being consistently in the range of 10(-2)-10(-3) at LD50. Also, the D0 is often lower (approximately 55 cGy), which is compatible in the dog with such a value for hemopoietic progenitor cells. (d) With larger species there is an unexpected reduction in heterogeneity when the dose rate is lower, which gives a D0 lower than expected and a higher extrapolate. It is concluded that the position and slope of the dose-incidence curves are compatible with interpretations based primarily on target-cell number and survival characteristics, modified by additional heterogeneity factors.