Gene transfer to inflorescence and flower meristems using ballistic micro-targeting

Direct gene transfer to floral meristems could contribute to cell-fate mapping, to the study of flower-specific genes and promoters, and to the production of transgenic gametes via the transformation of sporogenic tissues. Despite the wide potential of its applications, direct gene transfer to floral meristems has not been achieved so far because of the lack of suitable technology. We show in this paper that ballistic micro-targeting is the technique of choice for this purpose, and in this way, we were able to transfer genes efficiently into excised wheat immature spikes. Particle size was adjusted for optimal penetration into the L1 and L2 cell layers of the spikes with limited cell damage. Spikes at different developmental stages were shot either with a plasmid containing two genes involved in anthocyanin biosynthesis or with a plasmid bearing the uidA (-glucuronidase) gene. The transient expression of these marker genes was observed in the different developmental stages tested and in cells of both the L1 and the L2 layers. The transient expression of the uidA gene was significantly increased when the sucrose concentration in the culture medium was increased from 0.06 to 0.52 M. At the highest concentration, 100% of the targeted spikes expressed the uidA gene, with an average of 69 blue cells per spike. Twelve days after microtargeting, multicellular sectors showing transgene expression and containing up to 17 cells were found in 85% of the shot immature inflorescences. This indicated that targeted cells survived particle bombardment. Sectors were found in primordia of both vegetative and reproductive organs.     

A pre-formed Pyrogenic Factor Released by Lipopolysaccharide Stimulated Macrophages

The aim of this study was to investigate the pyrogenic activity of factor(s) released by rat peritoneal macrophages following a brief stimulation with LPS. The effect of this factor on the number of circulating leukocytes and serum Fe, Cu and Zn levels, was also evaluated. The possibility that the content of interleukin (IL)-1beta, IL-6 and tumour necrosis factor (TNF) in the supernatant could explain the observations was investigated. Supernatant produced over a period of 1 h by peritoneal macrophages, following a 30 min incubation with LPS at 37 degrees C, was ultrafiltered through a 10 000 MW cut-off Amicon membrane, sterilized, and concentrated 2.5, 5, 10 and 20 times. The intravenous (i.v.) injection of this supernatant induced a concentration-dependent fever in rats with a maximal response at 2 h. The pyrogenic activity was produced by macrophages elicited with thioglycollate and by resident cells. The supernatants also induced neutrophilia and reduction in Fe and Zn 6 h after the injection. Absence of activity in boiled supernatants, or supernatants from macrophages incubated at 4 degrees C with LPS, indicates that LPS was not responsible for the activity. In vitro treatment with indomethacin (Indo), dexamethasone (Dex), or cycloheximide (Chx) did not modify the release of pyrogenic activity into the supernatant or its effects on the reduction in serum metal levels. Although Chx abolished the production of mediator(s) inducing neutrophilia, and Dex reduced the induction of IL-1beta, TNF and IL-6, injection of the highest concentration of these cytokines detected in the supernatants did not induce fever. In vivo treatment with Dex, but not Indo, abolished the fever induced by the supernatant. These results suggest that macrophages contain pre-formed pyrogenic mediator(s), not related to IL-1beta, IL-6 or TNF, that acts indirectly and independently of prostaglandtn. It also seems likely that the pyrogenic activity is related to the factor responsible for the reduction of serum Fe and Zn levels, but not the neutrophilia.     

Chaetomella acutiseta produces chaetomellic acids A and B which are reversible inhibitors of farnesyl-protien transferase

Chaetomellic acids A and B, isolated from Chaetomella acutiseta, are specific inhibitors of farnesyl-protein transferase that do not inhibit geranylgeranyl transferase type 1 or squalene synthase. Chaetomellic acids A and B are reversible inhibitors, resemble farnesyl diphosphate and probably inhibit FPTase by substituting for farnesyl diphosphate. Chaetomellic acid production appears to be widespread within the genus Chaetomella. Correspondence to: R. B. Lingham     

Tum—mutation P35B generates the MHC-binding site of a new antigenic peptide

Immunogenic tumor cell variant P35 was obtained by mutagen treatment of mouse mastocytoma P815. It express a potent new antigen recognized by syngeneic cytolytic T lymphocytes (CTL). This antigen is the result of a point mutation in a gene that is expressed by most healthy cells. A decapeptide encoded by the region spanning the mutation sensitized P815 cells to the relevant CTL, whereas the homologous decapeptide corresponding to the normal sequence did not. Only the mutant decapeptide was capable of enhancing the expression of the D^d-presenting molecule at the cell surface, indicating that the mutation generates a motif which enables the antigenic peptide to bind to D^d. Correspondence to: T. Boon.     

Three new HLA-G alleles and their linkage disequilibria with HLA-A

Three new allelic forms of the HLA-G DNA sequence (HLA-G*II, HLA-G*III, and HLA-G*IV) have been identified. With the HLA-G*I sequence (previously designated HLA 6.0) as a reference, HLA-G*II shows a silent (G A) mutation at the third base of codon 57, HLA-G*III bears a non-synonymous (A T), but conservative, (Thr Ser) substitution at the first base of codon 31, and HLA-G*IV shows two silent substitutions: (A T) at the third base of codon 107 and (G A) at the third base of codon 57. A rapid method of singling out each allele on genomic DNA has been developed by using polymerase chain reaction amplification followed by restriction endonuclease treatment. Also, more or less strong linkage disequilibria has been found between most HLA-A alleles and either HLA-G*I or *II, both being the most prevalent alleles in the population, with a genotypic frequency of 0.55 and 0.38, respectively; HLA-G*III is very rare and HLA-G*IV has a genotypic frequency of 0.07. An evolutive classification of HLA-A alleles results according to their association with either HLA-G*I or HLA-G*II, which does not correlate with the classical serological cross-reacting groups classification. The finding of a strong and selective A/G linkage disequilibria with most HLA-A alleles, together with the existence of less frequent random A/G associations, may suggest that there exist in different haplotypes true and varied A/G genetic distances (and not a recombinational hotspot). It may be inferred from preliminary data that in primates HLA-A/G haplotypes bearing G*II may have appeared later than those bearing G*I.The nucleotide sequence data reported in this paper have been submitted to the GenBank and EMBL nucleotide sequence databases and have been assigned the following accession numbers: EMBL-X60983 (HLA-G*II), GenBank-M99048 (HLA-G*III), and GenBank-L07784 (HLA-G*IV).The contribution to this paper by P. Morales and A. Corell is equal, and the order of authorship is arbitrary. Correspondence to: A. Arnaiz-Villena.     

Biomass structure and dry matter dynamics of subtropical alluvial and exotic Ligustrum forests at the Río de la Plata,Argentina

Biomass, litterfall, litter standing crop, and decomposition was studied in a native subtropical alluvial forest locally known as Selva Marginal (SM) and an exotic Ligustrum lucidum forest (LF) at the Reserva Integral de Punta Lara, Buenos Aires Province, 34°47S and 58°1W. The alluvial forest site was at the southern limit of distribution of subtropical forests in South America. The Ligustrum forest was invading disturbed areas. Total biomass was 147.7 Mg/ha (86% aboveground and 14% belowground) in the SM, and 71.4 Mg/ha (93% and 7%, respectively) in the LF. Litterfall was 10.3 Mg/ha·yr and 13.8 Mg/ha·yr respectively. Annual leaf decomposition rate was greater for Ligustrum (k=4.07) than for SM species (k=1.48). The mean residence time of aboveground biomass was 12 yr for the SM and 5 yr for the LF. The k₁ values (litterfall/standing crop) were 1.9 and 2.0 for SM and LF respectively. The influence of coastal road and wall in the hydroperiod, native forested wetland ecosystem survival and exotic forest invasion is discussed.     

Improvement of somatic embryogenesis inFeijoa sellowiana berg (Myrtaceae) by manipulation of culture media composition

Summary Somatic embryos could be induced from the cotyledons of zygotic embryos from immature fruits ofFeijoa sellowiana Berg (Feijoa) in the presence of a wide range of concentrations of fructose, glucose, maltose, and sucrose. Mannitol or sorbitol alone were ineffective. The highest frequencies of induction (99%) and the greatest number of somatic embryos per explant (134) were obtained with 0.4M fructose and 0.3M sucrose, respectively. This sucrose concentration also showed greater induction capacity than equimolar combinations of its monosaccharide constituents combined. Somatic embryo development was arrested at the globular stage at concentrations higher than 0.5M of all the sugars tested. When transferred to solid germination medium containing 2.0 mg/liter (5.77μM) gibberellic acid, 0.5 mg/liter (2.32μM) kinetin, and 0.029M sucrose, somatic embryos formed under 0.3 or 0.4M sucrose had better germination capacity than those induced under lower (0.1 and 0.2M) concentrations, as assessed by the frequency of explants presenting germinated embryos and by the number of plants obtained from those explants. On liquid media of similar composition somatic embryos did not germinate. Our data suggest that high (0.3 to 0.4M) carbohydrate levels improve somatic embryogenesis by acting both as carbon source and as osmotic regulator.     

A τ Fragment Containing a Repetitive Sequence Induces Bundling of Actin Filaments

Abstract: Much indirect evidence suggests that the interconnections of actin microfilaments with the microtubule system are mediated by microtubule-associated proteins (MAPs). In this study we provide new data to support the interaction of a specific tubulin-binding domain on τ with actin in vitro. In actin polymerization assays, the synthetic peptide VRSKIGSTENLKHQPGGG, corresponding to the first repetitive sequence of τ protein, increased turbidity at 320 nm in a dose-dependent fashion. A salient feature of the τ peptide-induced assembly process is the formation of a large amount of actin filament bundles, as revealed by electron microscopic analysis. An increase in the τ peptide concentration resulted in a proportional increase in the bundling of actin filaments. It is interesting that a gradual decrease of pH within the range 7.6–4.7 resulted in a higher effect of τ peptide in promoting bundles of actin filaments. A similar pH-dependent effect was observed for τ protein-induced bundling. An analysis of the mechanisms that operate in the peptide induction of actin filament bundles suggests the involvement of electrostatic forces, because the neutralization of ɛ-aminolysyl residues by selective carbamoylation resulted in a complete loss of the peptide induction of actin bundles. The data suggest that a τ repetitive sequence (also found in MAP-2 and MAP-4) containing a common tubulin binding motif may constitute a functional domain on τ for the dynamics of the interconnections between actin filaments and microtu-bules.     

Regulation of ammonium ion assimilation enzymes inNeurospora crassa nit-2 andms-5 mutant strains

InNeurospora crassa thenit-2 andnmr-1 (ms-5) loci represent the major control genes encoding regulatory proteins that allow the coordinated expression of various systems involved with the utilization of a secondary nitrogen source. In this paper we examine the effect of thenit-2 andms-5 (nmr-1 locus) mutations on the regulation of the ammonium assimilation enzymes, glutamine synthetase and glutamate dehydrogenase, which are regulated by the products of these genes; however, glutamate synthase is not so regulated. Glutamine synthetase and glutamate dehydrogenase levels are also regulated by the amino nitrogen content. We present evidence that thems-5 andgln ^r strains, which behave very similarly in their resistance to glutamine repression, are different and map in different loci.     

Phospholipase A2 inhibitors from marine algae

Twelve out of twenty-nine compounds isolated from benthic marine algae from the phyla Chlorophyta, Phaeophyta and Rhodophyta have been found to be potent inhibitors of bee venom derived phospholipase A₂ (PLA₂) (> 50%) in the M range. The compounds investigated were from: Bryopsis pennata, Rhipocephalus phoenix, Caulerpa prolifera, C. racemosa, C. bikinensis, Cymopolia barbata, Laurencia cf. palisada, Laurencia sp., Ochtodes crockeri, Liagora farinosa, Sphaerococcus coronipifolius, Phacelocarpus labillardieri, Dictyota sp., B furcaria galapagensis, Stypopodium zonale, Dictyopteris undulata, Stoechospermum marginatum, Dictyopteris divaricata, Dilophus fasciola and Dilophus sp. This is the first report of bee venom PLA₂ inhibition in vitro by pure compounds isolated from marine algae.