Stringent regulation of human growth hormone expression in cultured murine C2C12 myoblasts by theE. coli lac repressor

Summary Gene transfer techniques can be used to encode the production of a polypeptide product, such as human growth hormone (hGH), that is missing in an acquired or inherited disease state such as growth hormone deficiency. In one model system, engineered C2C12 myoblasts are injected intramuscularly into a mouse and subsequently secrete hGH into the circulation. In this regard, a gene-expression regulatory system that functions in myoblasts would be of interest. We demonstrate that theEscherichia coli lac operon system can be used to stringently regulate the expression of hGH in engineered C2C12 myoblasts in tissue culture. A DNA segment encoding hGH was linked to a DNA segment containing an SV40 enhancer and promoter. The latter components were positioned between two syntheticlac operators.Lac repressor expression was driven by a simian cytomegalovirus promoter. In transient co-transfection assays, hGH expression from cultured C2C12 myoblasts could be modulated up to 60-fold (P = 0.002) with the inducing agent, isopropyl-β-d-thiogalactoside (IPTG). In the absence of IPTG, hGH expression was almost fully repressed. These results show that the components of theE. coli lac operon provide a stringent regulatory system for use in myoblasts. The system might prove to be useful for the regulation of transferred genes in animals.     

Intra- and interobserver concordance in scoring Harris lines: A test on bone sections and radiographs

Little attention has been devoted to assessing the reproducibility of (paleo)pathological observations. Harris lines (HL) are among the markers most used to determine chronology of stresses suffered during growth. Nevertheless, their scoring entails remarkable methodological difficulty. Bone sections (S) and radiographs (R) of 29 adult tibiae of archeological provenance (medieval) were scored for HL by five observers. At regular intervals of time, each observer gave two independent counts on both series. Results show a) a substantial interobserver disagreement of HL estimates for both sectional and radiographic records, and b) a high level of intraobserver error. © 1994 Wiley-Liss, Inc.     

Physiologic target of the Air-1 trans-activator revealed by stable transfection assay

RJ 2.2.5 is a human B cell mutant, derived from Raji cells, which has lost expression of major histocompatibility complex (MHC) class II genes because of a defect in the AIR1 locus function. The MHC class II-positive phenotype can be restored by introducing an active AIR1 locus or its mouse equivalent, Air-1. An example of the latter is the H4 cell hybrid, derived by somatic cell fusion between RJ 2.2.5 and mouse class II-positive spleen cells. H4 contains a single mouse chromosome, autosome 16, in which the Air-1 locus maps, and an entire RJ 2.2.5-derived genome. In the present study we show that the physiologic target of the Air-1 locus product is contained within a limited HLA-DRA promoter sequence of 300 base pairs, encompassing the crucial Y, X, and W cis-acting elements. A plasmid construct, pDRA300neo, containing the HLA-DRA promoter sequence which drives the expression of the neomycin resistance gene, has been stably integrated in the genome of the H4 hybrid. Transfectants selected in the presence of G418 retain mouse chromosome 16 and express the DR genes. On the other hand, transfectants grown in a non-selective medium segregate mouse chromosome 16; this is accompanied by a loss of DRA gene expression and G418 resistance, although pDRA300neo is still integrated in the genome. These results offer scope for using this experimental model to clone the Air-1 gene in a straightforward way. Correspondence to: R. S. Accolla.     

Exogenous polyamines improve rooting of hazel microshoots

A strong positive effect of polyamines on rooting of microshoots of adult hazel (Corylus avellana L., cv. Gironell) is described. The effect of polyamines, both in the root induction solution and in the actual rooting medium, was assessed in order to study the effect on the successive rooting phases. Polyamines improved rooting of indole-3-butyric acid-treated microshoots in a synergistic fashion, perhaps by favouring a better induction of roots, with an acceleration of the response (only half the time required for rooting compared to the control). When applied without indole-3-butyric acid, polyamines had only a limited positive effect on rooting, although longer exposure times and/or higher concentrations could increase their effect. Possible rapid uptake and translocation of polyamines in the xylem in our system is discussed. The results offer a new approach to enhance rooting ability of species that are normally difficult to root.Abbreviations BM basal medium - IAA indole-3-acetic acid - IBA indole-3-butyric acid - NAA 1-naphthaleneacetic acid - Put putrescine - Spd spermidine - Spm spermine     

Speciation in the Artemia genus: Mitochondrial DNA analysis of bisexual and parthenogenetic brine shrimps

From the cloned mitochondrial DNAs (mtDNAs) isolated from two bisexual species, one Mediterranean, Artemia salina, and one American, Artemia franciscana, and two parthenogenetic (diploid and tetraploid) strains of Artemia parthenogenetica collected in Spain, physical maps have been constructed and compared. They are extremely different among themselves, much more than the differences between Drosophila melanogaster and D. yakuba and in the same range of different mammalian species such as mouse/rat or man/cow. The nucleotide sequences of two regions of mtDNA encoding parts of the cytochrome c oxidase subunit I (COI) and cytochrome b (Cytb) genes have been determined in the two bisexual species and the two parthenogenetic strains. Comparisons of these sequences have revealed a high degree of divergence at the nucleotide level, averaging more than 15%, in agreement with the differences found in the physical maps. The majority of the nucleotide changes are silent and there is a strong bias toward transitions, with the CT substitutions being highly predominant. The evolutionary distance between the two Artemia parthenogenetica is high and there is no clear relationship with any of the bisexual species, including the one present nowadays in Spain. Using a combination of molecular (mtDNA) and morphological markers it is possible to conclude that all of these Artemia isolates should be actually considered as belonging to different species, even the two Artemia parthenogenetica diploidica and tetraploidica.On sabbatical leave from Departamento de Bioquímica, Facultad de Veterinaria, Universidad Complutense de Madridearly Italian artemiologists to designate the Medi-Beatriz Batuecas died in an accident during the Christmas holy days of 1988 after she had initiated this workCorrespondence to: R. Garesse     

Evaluation of rice straw hemicellulose hydrolysate in the production of xylitol byCandida guilliermondii

Summary Rice straw was used as a lignocellulosic source to provide rich pentose media. By using a well characterized yeast strain,Candida guilliermondii FTI 20037, the hydrolysate obtained was converted to xylitol with an efficiency of 75% and production of 27 g of xylitol per liter in 48 hours. The satisfactory results reported here can be attributed to the low concentrations of toxic components generated throughout the chemical depolymerization of this raw material.     

Effects of recombinant human gamma interferon on intracellular survival of Bordetella pertussis in human phagocytic cells

Abstract Several studies have demonstrated that Bordetella pertussis has the ability to enter and survive intracellularly within human polymorphonuclear leukocytes (PMNL) and human monocytes/macrophages. The effects of human recombinant gamma interferon (IFN-γ) on the survival of B. pertussis in PMNL and human monocytes, and on the oxidative burst activity of PMNL and human monocytes in response to B. pertussis were assessed in this study. IFN-γ partially increased intracellular killing of phagocytosed B. pertussis in human monocytes, as determined by an orange acridine-crystal violet assay. In contrast, IFN-γ did not enhance intracellular killing of B. pertussis in PMNL. No significant increase of superoxide production was noted in human monocytes in response to B. pertussis when stimulated with various concentrations of IFN-γ. The partial increase of B. pertussis killing by IFN-γ within monocytes, together with poor production of superoxide may explain how B. pertussis can survive within human phagocytic cells, and thus cause a more prolonged course of the disease.     

DNA supercoiling and thermal regulation of unsaturated fatty acid synthesis in Bacillus subtilis

Bacillus subtilis growing at 37° C synthesizes, almost exclusively, saturated fatty acids. However, when a culture growing at 37°C is transferred to 20°C, the synthesis of unsaturated fatty acids is induced. The addition of the DNA gyrase inhibitor novobiocin specifically prevented the induction of unsaturated fatty acid synthesis at 20° C. Furthermore, it was determined that plasmid DNA isolated from cells growing at 20°C was significantly more negatively supercoiled than the equivalent DNA isolated from cells growing at 37°C. The overall results agree with the hypothesis that an increase in DNA supercoiling associated with a temperature downshift could regulate the unsaturated fatty acids synthesis in B. subtilis.     

Binding of BiP to an assembly-defective protein in plant cells

The binding protein (BiP) has been implicated as a mediator of protein folding and assembly in the endoplasmic reticulum of mammalian cells and has often been found in stable association with structurally defective proteins. To acquire information on the activity of BiP in plant cells, we have expressed in tobacco protoplasts the wild type form and an assembly-defective form of bean phaseolin. Phaseolin (PHSL) is a soluble, trimeric, storage glycoprotein co-translationally inserted into the lumen of the endoplasmic reticulum and then transported along the secretory pathway to the protein storage vacuoles. We have previously shown that a PHSL mutant in which the last 59 amino acids have been deleted (Δ363PHSL) is unable to form trimers and is retained in a pre-Golgi compartment when synthesized in Xenopus oocytes. When transiently expressed in tobacco leaf protoplasts, wild-type PHSL is correctly glycosylated and assembles efficiently and rapidly into trimers. Δ363PHSL is also correctly glycosylated but does not trimerize. Tobacco BiP and Δ363PHSL are co-immunoselected using either anti-PHSL or anti-BiP antibodies. Under the same conditions, co-immunoselection of BiP with wild-type PHSL is not detectable. The BiP bound to Δ363PHSL can be released by treatment of the complex with ATP, indicating that the binding is related to the proposed function of BiP in protein folding and assembly in the endoplasmic reticulum. These data indicate that BiP stably binds structurally defective proteins in plant cells.     

Pigment production from in vitro cultures of Alkanna tinctoria Tausch

Summary An in vitro culture of Alkanna tinctoria Tausch cells was set up in order to investigate the possibility of producing alkannin, a red naphthoquinone naturally present in the root bark of this plant. Furthermore, an in vitro culture of callusderived roots was established and the production of alkannin evaluated. In the different experimental conditions investigated, differences in the production of alkannin derivatives as well as in the type of pigments produced, were observed. The potential use of this technology is discussed.