Regeneration of garlic callus as affected by clonal variation, plant growth regulators and culture conditions over time

 A long-term regeneration system for garlic (Allium sativum L.) clones of diverse origin was developed. Callus was initiated on a modified Gamborg's B-5 medium supplemented with 4.5 μM 2,4-D and maintained on the same basal medium with 4.7 μM picloram+0.49 μM 2iP. Regeneration potential of callus after 5, 12 and 16 months on maintenance medium was measured using several plant growth regulator treatments. The 1.4 μM picloram+13.3 μM BA treatment stimulated the highest rate of shoot production. Regeneration rate decreased as callus age increased, but healthy plantlets from callus cultures up to 16-months-old were produced for all clones. Regeneration of long-term garlic callus cultures could be useful for clonal propagation and transformation. Received: 24 September 1998 / Revision received: 27 January 1999 / Accepted: 26 February 1999     

Helix-helix transitions in DNA: fibre X-ray study of the particular cases poly(dG-dC) · poly(dG-dC) and poly(dA) · 2poly(dT)

The helix-helix transitions which occur in poly(dG-dC) · poly(dG-dC) and in poly (dG-m⁵dC) · poly(dG-m⁵dC) are commonly assumed to be changes between the right-handed A- or B-DNA double helices and the left-handed Z-DNA structure. The mechanisms for such transconformations are highly improbable, especially when they are supposed to be active in long polynucleotide chains organised in semicrystalline fibres. The present alternative possibility assumes that rather than the Z-DNA it is a right-handed double helix (S-DNA) which actually takes part in these form transitions. Two molecular models of this S form, in good agreement with X-ray measurements, are proposed. They present alternating C(2′)-endo and C(3′)-endo sugar puckering like the “alternating B-DNA” put forward some years ago. Dihedral angles, sets of atomic coordinates and stereo views of the two S-DNA structures are given, together with curves of calculated diffracted intensities. Furthermore, we question the possibility of obtaining semicrystalline fibres with triple helices of poly(dA) · 2poly(dT) in a way which renders X-ray diffraction efficient. It is suggested that, up to now, only double helices of poly(dA) · poly(dT) can actually be observed by fibre X-ray diffraction measurements. Received: 30 March 1999 / Revised version: 30 June 1999 / Accepted: 30 June 1999     

On the stall force for growing microtubules

The assembly of microtubules generates forces that play a role in cellular motility processes such as the motion of chromosomes during mitosis. Recently, Mogilner and Oster proposed a model for the growth of microtubules that agrees quantitatively with the force-velocity relation measured for individual microtubules. In addition, the authors predicted that the stall force for any polymer consisting of N independently growing protofilaments should increase as the square root of N. We simulated this model and found that the stall force increases linearly with N, and is in fact consistent with the maximum force predicted by thermodynamic arguments. We show that this discrepancy can be explained by a more careful treatment of the “off-term” in the Mogilner-Oster model. Received: 27 September 1999 / Revised version: 12 December 1999 / Accepted: 20 December 1999     

Isolation of two genes that were induced upon the initiation of somatic embryogenesis on carrot hypocotyls by high concentrations of 2,4-D

 This study describes the formation of somatic embryos directly on the surface of hypocotyl sections of Daucus carota L. after exposure to 450 μM of 2,4-D for 2 h, followed by culturing without 2,4-D for 2 weeks. A search for differentially expressed genes immediately after the 2,4-D treatment resulted in the identification of two genes, Dchsp-1 and Dcarg-1. Dchsp-1 has homology to low-molecular-weight heat-shock proteins, while Dcarg-1 has homology to auxin-regulated genes. Dchsp-1 was expressed upon the exposure to 2,4-D treatment but also responded to general stress responses. Dcarg-1 responded primarily to auxin; however, a parallel relationship between the expression of Dcarg-1 and the formation of somatic embryos on the hypocotyl sections was observed. It seems the exposure to auxin shock could serve as a trigger in inducing cell division in the epidermal cells and promoting their further differentiation to somatic embryos. These results are discussed in relationship to our current understanding of the molecular basis of somatic embryogenesis. Received: 3 May 1999 / Accepted: 9 August 1999     

Engineering Pichia pastoris for stereoselective nitrile hydrolysis by co-producing three heterologous proteins

A Pichia pastoris strain with stereoselective nitrile hydratase activity has been constructed by engineering the co-expression of three genes derived from Pseudomonas putida. Using a technique that could be widely applicable, the genes encoding nitrile hydratase α and β structural subunits and P14K accessory protein were first assembled as individual expression cassettes and then incorporated onto one plasmid, which was integrated into the P. pastoris chromosome. The resulting strain can be used as a catalyst for bioconversions requiring stereospecific nitrile hydrolysis. Received: 3 November 1998 / Received revision: 25 February1999 / Accepted: 14 March 1999     

Local cytokine therapy of cancer: interleukin-2, interferons and related cytokines

 Local therapy with interleukin-2 (IL-2) and other cytokines may be a very effective way to treat cancer. This was the theme of the First Symposium on Local Cytokine Therapy of Cancer: Interleukin-2, Interferons and Related Cytokines, in Hamburg, 29 April–1 May 1999. The abstracts are published in Anticancer Research 19: 1995–2016 (1999) [1]. Here we present a report. Received: 28 October 1999 / Accepted: 2 December 1999     

Effect of yeast elicitor on the secondary metabolism of Ti-transformed Salvia miltiorrhiza cell suspension cultures

 Salvia miltiorrhiza contains two groups of biologically active secondary metabolites termed phenolic compounds (e.g. rosmarinic acid) and tanshinones (e.g. cryptotanshinone). Their roles in plant defense responses were examined using a simplified system consisting of a yeast elicitor and a Ti C58 transformed S. miltiorrhiza cell line. Both dosage and time course studies were carried out on the effects of yeast elicitor on the formation of rosmarinic acid and cryptotanshinone. It was found that the yeast elicitor reduced the constituent level of rosmarinic acid (from ca. 5% to ca. 3.0% of dry cell weight) whereas the level of cryptotanshinone was enhanced greatly (from a negligible amount to ca. 20 mg/l). These results suggest that in S. miltiorrhiza, rosmarinic acid and cryptotanshinone may take part in plant passive and active defense responses, respectively, against pathogen attack. Cryptotanshinone was identified as a phytoalexin in S. miltiorrhiza for the first time. Results of the treatment of cell cultures with 2-aminoindan-2-phosphonic acid, a highly specific and potent inhibitor of phenylalanine ammonia-lyase (EC 4.3.1.5), indicated that this compound did not inhibit yeast elicitor induced tanshinone formation, but did inhibit rosmarinic acid biosynthesis. Received: 22 May 1999 / Revision received: 27 August / Accepted: 1 September 1999     

Cloning and functional expression of the d-β-hydroxybutyrate dehydrogenase gene of Rhodobacter sp. DSMZ 12077

Nucleotide sequence and biochemical analysis of d-β-hydroxybutyrate dehydrogenase (EC 1.1.1.30), isolated from Rhodobacter sp., indicate functional oligomers composed of subunits of 257 amino acids with a calculated M _r of 26,800 and a pI of 5.90. Compared to mammalian short-chain alcohol dehydrogenases, the bacterial enzyme lacks a C-terminal lipid anchor domain and was found to be highly active upon expression in Escherichia coli even without lipid supplement. The recombinant enzyme could be highly enriched using a single chromatography step and was shown to be stable over a broad range of pH and temperature. Received: 1 April 1999 / Received last revision: 11 June 1999 / Accepted: 11 June 1999     

Genomic organization and chromosomal mapping of mouse nuclear factor kappa B 2 (NFKB2)

 NFKB2 is a member of the NFKB/Rel gene family, which is known to be a pivotal regulator of the acute phase and immune responses. NF-κB2 is initially synthesized as a ∼100 000 M _r protein which needs to be processed in order to bind DNA, either as homodimer or as heterodimer with other members of the NF-κB/Rel family. The unprocessed form of NF-κB2 acts as an IκB-like protein. Therefore, NF-κB2 has a dual function. In this report we describe the genomic structure, expression pattern, and chromosomal localization of mouse NFKB2. Genomic clones were isolated, which span the entire gene of approximately 8.5 kilobases (kb) including 1.5 kb of the promoter region. Comparison to its human and avian homologues revealed a strong evolutionary conservation of the gene structure including the exon/intron borders, sequence, and position of the nuclear localization signal, the glycine-hinge region, and the ankyrin repeats. By fluorescence in situ hybridization, mouse NFKB2 was mapped to Chromosome (Chr) MMU 19C3-D2, which is homologous to human Chr 10q24, at which position the human NFKB2 was previously located. NFKB2 is ubiquitously expressed, highest in lymph nodes and thymus, underlining its role in the immune function. Received: 14 January 1999 / Revised: 29 March 1999     

Dominant negative mutations in the alpha-factor receptor, a G protein-coupled receptor encoded by the STE2 gene of the yeast Saccharomyces cerevisiae.

The α-mating pheromone receptor encoded by the STE2 gene of the yeast Saccharomyces cerevisiae is a G protein-coupled receptor (GPCR) that is homologous to the large family of GPCRs that mediate multiple types of signal transduction in mammals. We have screened libraries of mutant receptors to identify dominant negative alleles that are capable of interfering with the function of a co-expressed normal receptor. Two dominant negative alleles have been recovered in this manner. In addition, we find that previously isolated loss-of-function mutations in the α-factor receptor exhibit dominant negative effects. Detection of the dominant effects requires high-level expression of the mutant receptors but does not require a high ratio of mutant to normal receptors. Cellular levels of the normal receptors are not affected by co-expression of the dominant negative alleles. Expression of the mutant receptors does not interfere with constitutive signaling in a strain that lacks the G protein α subunit encoded by GPA1, indicating that interference with signaling occurs at the level of the receptor or the interacting G protein. Expression of increased levels of G protein subunits partially reverses the dominant negative effects. The dominant negative behavior of the mutant receptors is diminished by deletion of the SST2 gene, which encodes an RGS (Regulator of G protein Signaling) protein involved in desensitization of pheromone signaling. The most likely explanation for the dominant negative effects of the mutations appears to be the existence of an interaction between unactivated receptors and the trimeric G protein that titrates the G protein away from the normal receptors or renders the G protein insensitive to receptor activation. This interaction appears to be mediated by the SST2 gene product. Received: 15 January 1999 / Accepted: 25 March 1999     

Fixation of spent Saccharomyces cerevisiae biomass for lead sorption

Spent Saccharomyces cerevisiae cells from a beer fermentation process were evaluated for lead cation sorption. The crude biomass was washed with water and acetone prior to any other treatment. Although the washed biomass showed substantial lead ion sorption it was susceptible to microbial spoilage. Different aldehydes were tested as chemical fixation agents; however, most of them caused drastic lowering of the metal uptake capacity. However, benzaldehyde was not only an excellent fixation agent, but the biomass treated with it also retained its original lead sorption capacity. A mechanism for the fixation process is suggested. Received: 11 January 1999 / Received revision: 26 April 1999 / Accepted: 1 May 1999     

Screening of mannanases in actinomycetes and their potential application in the biobleaching of pine kraft pulps

Fifty actinomycete strains were screened for the production of mannanase activity during growth in both liquid and solid media. Streptomyces scabies CECT 3340 and Streptomyces ipomoea CECT 3341 were selected for their ability to produce high levels of mannanase (294.3 U/l and 242.9 U/l, respectively) during growth in liquid culture. β-Mannosidase (15.3 U/l) and α-galactosidase (7.7 U/l) activities were also detected in culture filtrate from S. scabies CECT 3340. Highest levels of mannanase activity for S. scabies CECT 3340 were achieved in media containing locust bean gum and asparagine (4.8 U mg⁻¹ protein) whilst in S. ipomoea CECT 3341 greatest activity was detected in media containing locust bean gum and yeast extract (13.2 U mg⁻¹ protein). No carboxymethylcellulase activity was detected. In biobleaching experiments, enzyme treatment, carried out with mannanase activity produced by S. ipomoea CECT 3341, followed by alkaline extraction of pine kraft pulp resulted in the release of colour (A ₄₆₅, 0.69) and chromophoric material from the pulp (A ₂₃₇, 12.9; A ₂₅₄, 6.9 and A ₂₈₀, 6.7). The ability of this enzyme complex to improve the bleaching of pine kraft pulps was also shown by a pulp brightness increase (2.4 units ISO) and a reduction in kappa number (from 21.4 units to 20.1 units) with the absence of variations on the viscosity values. Received: 23 February 1999 / Received revision: 1 April 1999 / Accepted: 6 April 1999     

Thidiazuron-induced plant regeneration from hypocotyl cultures of St. John's wort (Hypericum perforatum. cv 'Anthos')

 St. John's wort (Hypericum perforatum. cv 'Anthos') is a medicinal plant with evidence of efficacy as an anti-depressant. The present report describes the development of an in vitro regeneration system that utilizes thidiazuron [N-phenyl-N′-(1,2,3-thidiazol-yl)urea] for the induction of de novo shoots on etiolated hypocotyl segments of St. John's wort seedlings. The optimum level of thidiazuron supplementation to the culture medium was 5 μmol·l^–1 for a 9-day induction period followed by subculture of induced hypocotyl explants on basal medium. Other plant growth regulators including benzyladenine and indoleacetic acid were not effective in inducing regeneration on St. John's wort hypocotyls. Histological examination of the cultures revealed that the regenerated plants were derived from de novo developed shoots. Transfer of the regenerated shoots into a liquid medium with no plant growth regulators resulted in the rapid and prolific growth of viable plantlets. The rapid and efficient micropropagation system for St. John's wort may be useful for both the genetic improvement of this crop and the production of high-quality phytopharmaceutical preparations for the treatment of neurological disorders. Received: 19 March 1999 / Revision received: 5 July 1999 · Accepted: 17 August 1999     

Characterization of Lactobacillus collinoides response to heat, acid and ethanol treatments

Tolerance to stress and cross-protection in Lactobacillus collinoides were examined after exposure to ethanol, acid or heat shock. Ethanol and heat-adapted cells demonstrate induced homologous␣tolerance and cross-resistance to acid stress. No cross-protection of acid-adapted cells against ethanol and heat stresses was observed. Heat was the only pretreatment leading to cross-protection against the two other stresses. Whole-cell protein extract analysis revealed that each treatment induced a battery of stress proteins; the synthesis of some of these polypeptides being induced by more than one condition. The greatest overlap was observed between ethanol and heat treatments. Ten proteins were found to be common to these stresses. Received: 7 November 1998 / Received revision: 10 February 1999 / Accepted: 12 February 1999     

HER-2/neu peptide specificity in the recognition of HLA-A2 by natural killer cells

Although natural killer (NK) cells have been described as non-MHC-restricted, new evidence suggests that NK activity can be either up- or down-regulated after interaction with the peptide–MHC-class-I complex expressed on target cells. However, the epitope(s) recognized by NK cells have remained ill-defined. We investigated NK cell recognition of synthetic peptides representing a portion of a self-protein encoded by the HER-2/neu (HER-2) proto-oncogene and presented by HLA-A2. HER-2 nonapeptides C85, E89, and E75 were found partially to protect T2 targets from lysis by freshly isolated and interleukin-2(IL-2)-activated NK cells (either HLA-A2⁺ or A2⁻). This inhibition was not solely due to changes in the level of HLA-A2 expression or conformation of serological HLA-A2 epitopes. Using single-amino-acid variants at position 1 (P1) of two HER-2 peptides, we observed that protection of targets was dependent on the sequence and the side-chain. These results suggest similarities in the mechanism of target recognition by NK and T cells. This information may be important for understanding the mechanisms of tumor escape from immunosurveillance and could help explain the aggressiveness of HER-2-overexpressing tumor cells. Received: 16 March 1999 / Accepted: 3 June 1999     

Osmotic water permeability of isolated vacuoles

We measured the osmotic water permeability (P _os) of vacuoles isolated from onion (Allium cepa L.), rape (Brassica napus L.), petunia (Petunia hybrida Hook.) and red beet (Beta vulgaris L.). For all the vacuolar types investigated, P _os values were in the range 200–1000 μm s⁻¹. The change in membrane surface area induced by an osmotic gradient was smaller than 2–6%. The vacuolar P _os values for red beet and onion were reduced by 1 mM HgCl₂, to 14% and 30% of the control values, respectively, but were partially restored to 51% and 76% by 5 mM β-mercaptoethanol. These results suggest that aquaporins were present in all the vacuoles tested. In HgCl₂-treated onion vacuoles, the reduced P _os (56 μm s⁻¹) had a low activation energy (approx. 6 kJ mol⁻¹), indicating that water permeation was still occurring mainly via aquaporins, and that the water permeability of the lipid part of the vacuolar membrane is probably very low. Received: 18 February 1999 / Accepted: 21 June 1999     

In vitro plantlet regeneration from mature zygotic embryos of Pinus wallichiana A.B. Jacks

 Plantlet regeneration was achieved in blue pine (Pinus wallichiana A.B. Jacks) by organogenesis of mature zygotic embryos. The effect of various basal media and five cytokinins on adventitious bud induction, development and elongation was investigated. Half-strength Douglas fir cotyledon revised medium (DCR) supplemented with 2.5 μm N⁶-benzyladinine (BA) and 0.025 μM thidiazuron was found to be most effective in inducing adventitious buds. The effect of a BA pulse treatment was also tested, and the bud-forming capacity of each treatment was quantified. The elongation of adventitious buds was achieved on hormone-free half-strength DCR medium containing 2% sucrose and 0.05% activated charcoal. Rooting was induced in the elongated shoots with a 6-h treatment of indoleacetic acid and indolebutyric acid solutions (1 mM each). Rooted shoots were transplanted in the greenhouse for hardening and their survival percentage was 64.4 after 5 weeks and 45.7 after 6 months. Received: 11 September 1998 / Revision received: 10 February 1999 / Accepted: 26 February 1999     

Involvement of Tcf/Lef in establishing cell types along the animal-vegetal axis of sea urchins

Members of the Tcf/Lef family interact with β-catenin to activate programs of gene expression during development. Recently β-catenin was shown to be essential for establishing cell fate along the animal-vegetal axis of the sea urchin embryo. To examine the role of Tcf/Lef in sea urchins we cloned a Strongylocentrotus purpuratus Tcf/Lef homolog. Expression of SpTcf/Lef was maximal when β-catenin became localized to nuclei of vegetal blastomeres, consistent with its acting in combination with β-catenin to specify vegetal cell fates. Expression of a dominant-negative SpTcf/Lef inhibited primary and secondary mesenchyma, endoderm, and aboral ectoderm formation in a manner similar to that observed when nuclear accumulation of β-catenin was prevented. Our results suggest that SpTcf/Lef functions by interacting with β-catenin to specify cell fates along the sea urchin animal-vegetal axis. Received: 6 July 1999 / Accepted: 27 August 1999     

Drug treatments to reduce excitotoxicity in vivo: a potential for α 2-adrenoceptor antagonists?

Summary. It is hypothesized that the locus coeruleus-noradrenergic system controls compensatory and repair mechanisms in the CNS, and that its dysfunction is a critical factor in the progression of central neurodegenerative diseases. Pharmacological activation of locus coeruleus neurons can be achieved with α ₂-adrenoceptor antagonists, and such compounds are protective in vivo in some models of brain injury where excitotoxicity is thought to be a causative factor. To further explore this neuroprotective potential, the effects of a 7-day treatment with the α ₂-antagonists, (+)-efaroxan and (±)-idazoxan, were evaluated in rats undergoing a unilateral lesioning of the striatum with the excitotoxin, quinolinic acid. The α ₂-antagonist treatments reduced both the ipsiversive circling response to apomorphine and the deficit of choline acetyltransferase in the lesioned animals. To elucidate the mechanisms underlying this neuroprotective effect, a modulation of the extracellular levels of amino acids within the striatum was investigated using in vivo microdialysis. Intrastriatal injection of quinolinic acid increased taurine and tyrosine levels by 2–2.5 fold, while most other amino acids were not significantly altered; the effect of (+)-efaroxan on these changes is being investigated. Further research is required to identify which of several possible mechanisms is involved in the neuroprotective action of α ₂-antagonists in vivo. Received August 31, 1999 Accepted September 20, 1999