Packing forces in ribonuclease crystals

Packing in Ribonuclease A and Ribonuclease S crystals have been compared in order to determine the possible role of the precipitant on lattice contacts. Both proteins have similar tertiary structures, but they crystallize in different space groups depending on the precipitating agent. It is found that packing differs either by the number of nearest neighbours or by the size of surface areas buried in individual contacts. Ammonium sulfate seems to promote hydrophobic interactions with interfaces similar to those found in oligomeric proteins. Organic precipitants favour electrostatic interactions.     

Tyrosine protein kinase activity of the EGF receptor is required to induce activation of receptor-operated calcium channels

We have investigated the effects of epidermal growth factor (EGF) on calcium ion channels in A431 epidermoid carcinoma cells. We have found that: -1- EGF stimulates Ca2+ channels. -2- EGF stimulated Ca2+ channels are voltage independent, exhibit a low conductance (8 pS) and a bursting multichannels activity (BMC). -3- Activation of the tyrosine-kinase function of the EGF receptor is required to generate Ca2+ current. -4- Inositol (1,4,5) triphosphate (Ins (1,4,5) P3) and EGF have similar effect on the channel activation. These results suggest that: stimulation of tyrosine-kinase activity of the EGF receptor, production of Ins (1,4,5)P3 and calcium entry via voltage independent channels are important connected steps in mediating the mitogenic effect of this growth factor.     

Evidence for a novel growth factor in Xenopus oocytes

Xenopus oocytes contain a novel growth factor, as determined by its effect on the anchorage-dependent and -independent growth of various rat cells and on a Xenopus cell line; it is destroyed by trypsin, acidification (pH 2.0), heating (100 degrees C, 3 min), 8 M urea but not by dithiothreitol. Gel filtration of this activity in nondissociating conditions suggests the existence of aggregates and the presence of a very high (approximately 2000 Kd) and a much lower (approximately 30 Kd) form.     

Nucleotide sequence of Acinetobacter baumannii aphA-6 gene: evolutionary and functional implications of sequence homologies with nucleotide-binding proteins, kinases and other aminoglycoside-modifying enzymes

A new kanamycin-resistance gene, detected in Acinetobacter baumannii and designated aphA-6, was sequenced. It specifies a 30319 Dalton 3'-aminoglycoside phosphotransferase (APH(3'] that mediates resistance to kanamycin and structurally related aminoglycosides, including amikacin. Pairwise comparisons of the six types of APH(3') so far detected in human pathogens (types I, II, III and VI) and in amino-glycoside-producing microorganisms (types IV and V), confirm that APH(3') enzymes have diverged from a common ancestor. Three highly retained motifs (1: V--HGD----N; 2: G--D-GR/K-G and 3: D--K/R--Y/F---LDE) located in the C-terminal part of the enzymes were defined. Screening of protein sequence data bases fore each of these motifs revealed that motifs 1 and 2 are both found in nucleotide-binding phosphotransferases associated with a variety of biological processes, namely adenylate kinase, viral oncogenic protein kinases, elongation factors, Na+/K+-transporting ATPase, myosin and antibiotic-modifying enzymes. Motif 2 probably corresponds to the MgATP binding site, while motifs 3 and 1 could be involved in the splitting of the phosphodiester bond and in the phosphate transfer, respectively. Moreover, an additional motif, almost invariably centrally located, was found in all aminoglycoside-modifying enzymes. The occurrence of this motif, possibly a recombination site which would have allowed the association of units of separate functions, is compatible with a modular concept for the structure of aminoglycoside-modifying enzymes.     

Direct regeneration in vitro and transient GUS expression in Mentha×piperita

Genetic transformation of peppermint is known to be very difficult essentially because of low efficiency regeneration. A regeneration protocol allowing 51% shooting frequency is proposed. Transient -glucuronidase expression and adjustment of selection pressure with kanamycin are also reported. The final retained method to attempt peppermint transformation is:Agrobacterium inoculation or biolistic treatment of the first apical leaves ofin vitro clones, regeneration in the dark with kanamycin (1 mg l^–1) and 6-benzylaminopurine (2 mg l^–1), followed by selection of regenerated shoots with 200 mg 1^–1 kanamycin.Abbreviations BA 6-benzylaminopurine - GUS -glucuronidase - MS Murashige and Skoog (1962) - NAA -naphthalenacetic acid - PIG particle inflow gun - SEM scanning electron microscope     

Evidence for in vitro induced mutation which improves somatic embryogenesis in Asparagus officinalis L.

Summary Somatic embryogenesis from different genotypes of Asparagus officinalis L. could be obtained by in vitro culture of shoot apices. Apices were first cultured on an auxin-rich inducing medium and then transferred onto a hormone-free development medium. All genotypes tested in this way produced a few somatic embryos. In some experiments, during the development phase, a new kind of friable highly embryogenic tissue appeared in a random manner. These tissues could be continuously subcultured on a hormone-free medium and were named embryogenic lines. Five of these embryogenic lines regenerated plants from somatic embryos. These regenerated plants exhibited an increased embryogenic response compared to the parent plants; e.g. apex culture produced somatic embryos without any auxin treatments. For one of the embryogenic lines, a genetic analysis showed that the improved embryogenic response of regenerated plants was controlled by a mendelian dominant monogenic mutation.Abbreviations LSEA low somatic embryogenesis ability - HSEA high somatic embryogenesis ability - NAA 1-naphthaleneacetic acid     

Agrobacterium-mediated transformation of Asparagus officinalis L. long-term embryogenic callus and regeneration of transgenic plants

Summary Twenty-three independent kanamycin resistant lines were obtained after cocultivation of longterm embryogenic cultures of three Asparagus officinalis L. genotypes with an Agrobacterium tumefaciens strain harboring ß-glucuronidase and neomycin phosphotransferase II genes. All the lines showed ß-glucuronidase activity by histological staining. DNA analysis by Southern blots of the kanamycin resistant embryogenic lines and of a plant regenerated from one of them confirmed the integration of the T-DNA.Abbreviations GUS ß-glucuronidase - X-Gluc 5-bromo-4-chloro-3indolyl ß-D-glucuronic acid - NPT II neomycin phosphotransferase II     

HIV-1 Nef protein: purification, crystallizations, and preliminary X-ray diffraction studies.

Human immunodeficiency virus Nef protein accelerates virulent progression of AIDS by its interaction with specific cellular proteins involved in cellular activation and signal transduction. Here we report the purification and crystallization of the conserved core of HIV-1LAI Nef protein in the unliganded form and in complex with the wild-type SH3 domain of the P59fyn protein-tyrosine kinase. One-dimensional NMR experiments show that full-length protein and truncated fragment corresponding to the product of HIV-1 protease cleavage have a well-folded compact tertiary structure. The ligand-free HIV-1 Nefcore protein forms cubic crystals belonging to space group P23 with unit cell dimensions of a = b = c = 86.4 A. The Nef-Fyn SH3 cocrystals belong to the space group P6(1)22 or its enantiomorph, P6(5)22, with unit cell dimensions of a = b = 108.2 A and c = 223.7 A. Both crystal forms diffract to a resolution limit of 3.0 A resolution using synchrotron radiation, and are thus suitable for X-ray structure determination.     

Long-term gene delivery into the livers of immunocompetent mice with E1/E4-defective adenoviruses.

We have compared the in vitro and in vivo behaviors of a set of isogenic E1- and E1/E4-defective adenoviruses expressing the lacZ gene of Escherichia coli from the Rous sarcoma virus long terminal repeat. Infection of tumor-derived established cell lines of human origin with the doubly defective adenoviruses resulted in (i) a lower replication of the viral backbone that correlated with reduced levels of E2A-specific RNA and protein, (ii) a significant shutoff of late gene and protein expression, and (iii) no apparent virus-induced cytotoxicity. Independently of the extent of the deletion, the additional inactivation of E4 from the viral backbone therefore drastically disabled the virus in vitro, with no apparent effect on transgene expression. A lacZ-transgenic model was used to compare the different recombinant adenoviruses in the livers of C57BL/6 mice. The immune response to the virally encoded beta-galactosidase was minimal in this model, as infusion of the E1-defective adenovirus resulted in a time course of transgene expression that mimicked that in immunodeficient (nu/nu) mice, with very little inflammation and necrosis in the liver. Administration of a doubly defective adenovirus to the transgenic animals led to long-term extrachromosomal persistence of viral DNA in the liver, with no detectable methylation of CpG dinucleotides. However, transient transgene expression was observed independently of the extent of the E4 deletion, suggesting that the choice of the promoter may be critical to maintain transgene expression from these attenuated adenovirus vectors.     

Transforming growth factor type beta 1 (TGF-beta 1) down-regulates interleukin-2 production and up-regulates interleukin-2 receptor expression in a thymoma cell line

Transforming growth factor type beta 1 (TGB-beta 1) belongs to a family of polypeptides with regulatory effects on growth and differentiation of a variety of cell types. TGB-beta 1 plays an important role in regulation of immune response by acting as a negative control signal for T cell proliferation through still unknown mechanisms. In this study we have analysed the effects of TGB-beta 1 on EL 4-6.1, a variant of the murine EL 4 thymoma, which can be induced by phorbol 12-myristate 13-acetate (PMA) and/or interleukin 1 (IL-1) to secrete interleukin 2 (IL-2) and express IL-2 receptors (IL-2R). Using this defined model system, we show that TGB-beta 1 simultaneously down-regulates IL-2 expression and up-regulates the number of both high and low affinity IL-2R. These changes correlate with changes at the mRNA level, suggesting an effect at the pre-translational level. The specificity of both TGF-beta 1 effects was demonstrated using a neutralizing antiserum to TGF-beta 1. Our data also suggest that TGF-beta 1 does not interfere with early activation signals of PMA and/or IL-1. This model might be useful for elucidating the complex role of TGF-beta 1 in the regulation of T cell responses.