The freshwater planarian Dugesia (G.) tigrina contains a great diversity of homeobox genes

To identify potential pattern control and cell determination and/or differentiation genes in the freshwater planarian Dugesial (G.) tigrina, we searched for homeobox genes of different types in the genome of this primitive metazoan. We applied two basic approaches: 1) Screening the cDNA library with degenerate oligonucleotides corresponding to the most conserved amino acid sequence from helix-3 of the homeodomain of each family; and 2) PCR amplification of genomic DNA or cDNA, using two sets of degenerated oligonucleotides corresponding to helices 1 and 3 of the homeodomain or two specific domains of the POU family. Using the first strategy we have identified and characterized two tissue-specific cell determination and/or differentiation NK-type homeobox genes. Using the second strategy we have identified several homeobox genes that belong to the HOM/Hox, paired (prd) or POU families.     

Trehalose-6-phosphate synthase as an intrinsic selection marker for plant transformation

Insertion of foreign DNA into plant genomes occurs randomly and with low frequency. Hence, a selectable marker is generally required to identify transgenic plants. Until now, all selection systems have been based on the use of non-plant genes, derived from microorganisms and usually conferring antibiotic or herbicide resistance. The use of microorganism-derived genes however has raised biosafety concerns. We have developed a novel selection system based on enhancing the expression of a plant-intrinsic gene and the use of a harmless selection agent. Selection takes advantage of the reduced glucose sensitivity of seedlings with enhanced expression of AtTPS1, a gene encoding trehalose-6-P synthase. As a result, transformants can be identified as developing green seedlings amongst the background of small, pale non-transformed plantlets on high glucose medium. In addition, vegetative regeneration of tobacco leaf explants is very sensitive to high external glucose. Overexpression of AtTPS1 in tobacco allows selecting glucose insensitive transgenic shoots.     

Evaluation of matrix-assisted laser desorption ionization-time of flight whole cell profiles for assessing the cultivable diversity of aerobic and moderately halophilic prokaryotes thriving in solar saltern sediments

The moderately halophilic, cultivable fraction of prokaryotes thriving in hypersaline sediments of a solar saltern in Mallorca, Spain, has been studied by means of different cultivation media. A set of 374 isolates retrieved with six different culture conditions was screened, using whole-cell MALDI-TOF MS analysis to classify them into 25 phenotypic clusters at 52% similarity. The phylogenetic inference, made from comparative sequence analyses of the 16S rRNA genes of selected strains, indicated that each phenotypic cluster was comprised of a genealogically homogeneous set of strains. DNA-DNA hybridization (DDH) results among selected strains confirmed that each MALDI-TOF cluster encompassed members of the same species. On the other hand, the intra-cluster diversity, measured by several RAPD (Random Amplified Polymorphic DNA) amplifications, indicated that the clusters corresponded to several populations of the same phylogenetic unit coexisting in the same environment. The results encourage the use of MALDI-TOF MS for further exhaustive studies of the cultivable diversity of hypersaline environments.     

Entomopathogenic Nematodes for Control of Insect Pests Above and Below Ground with Comments on Commercial Production

Entomopathogenic nematodes (EPNs) have been utilized in classical, conservation, and augmentative biological control programs. The vast majority of applied research has focused on their potential as inundatively applied augmentative biological control agents. Extensive research over the past three decades has demonstrated both their successes and failures for control of insect pests of crops, ornamental plants, trees and lawn and turf. In this paper we present highlights of their development for control of insect pests above and below ground. The target insects include those from foliar, soil surface, cryptic and subterranean habitats. Advances in mass-production and formulation technology of EPNs, the discovery of numerous efficacious isolates/strains, and the desirability of reducing pesticide usage have resulted in a surge of commercial use and development of EPNs. Commercially produced EPNs are currently in use for control of scarab larvae in lawns and turf, fungus gnats in mushroom production, invasive mole crickets in lawn and turf, black vine weevil in nursery plants, and Diaprepes root weevil in citrus in addition to other pest insects. However, demonstrated successful control of several other insects, often has not lead to capture of a significant share of the pesticide market for these pests.     

Binding of β-lactam antibiotics to the exocellular dd-carboxypeptidase–transpeptidase of Streptomyces R39

Benzylpenicillin and cephaloridine reacted with the exocellular dd-carboxypeptidase–transpeptidase from Streptomyces R39 to form equimolar and inactive antibiotic–enzyme complexes. At saturation, the molar ratio of chromogenic cephalosporin 87-312 to enzyme was 1.3:1, but this discrepancy might be due to a lack of accuracy in the measurement of the antibiotic. Spectrophotometric studies showed that binding of cephaloridine and cephalosporin 87-312 to the enzyme caused opening of their β-lactam rings. Benzylpenicillin and cephalosporin 87-312 competed for the same site on the free enzyme, suggesting that binding of benzylpenicillin also resulted in the opening of its β-lactam ring. In Tris–NaCl–MgCl₂ buffer at pH7.7 and 37°C, the rate constants for the dissociation of the antibiotic–enzyme complexes were 2.8×10⁻⁶, 1.5×10⁻⁶ and 0.63×10⁻⁶s⁻¹ (half-lives 70, 130 and 300h) for benzylpenicillin, cephalosporin 87-312 and cephaloridine respectively. During the process, the protein underwent reactivation. The enzyme that was regenerated from its complex with benzylpenicillin was as sensitive to fresh benzylpenicillin as the native enzyme. With [¹⁴C]benzylpenicillin, the released radioactive compound was neither benzylpenicillin nor benzylpenicilloic acid. The Streptomyces R39 enzyme thus behaved as a β-lactam-antibiotic-destroying enzyme but did not function as a β-lactamase. Incubation at 37°C in 0.01m-phosphate buffer, pH7.0, and in the same buffer supplemented with sodium dodecyl sulphate caused a more rapid reversion of the [¹⁴C]benzylpenicillin–enzyme complex. The rate constants were 1.6×10⁻⁵s⁻¹ and 0.8×10⁻⁴s⁻¹ respectively. Under these conditions, however, there was no concomitant reactivation of the enzyme and the released radioactive compound(s) appeared not to be the same as before. The Streptomyces R39 enzyme and the exocellular dd-carboxypeptidase–transpeptidase from Streptomyces R61 appeared to differ from each other with regard to the topography of their penicillin-binding site.     

A Monoclonal Antibody that Specifically Recognizes m'A Nucleoside

Abstract

A hybridoma against the nucleoside m⁶A has been obtained from mouse spleen. This hybridoma was named H65 and it secretes monoclonal antibodies anti-m⁶A. The competition assays showed that the monoclonal antibody was highly specific for m⁶A nucleoside.     

Note: A Convenient Method for the Preparation of N2, N2-Dimethylguanosine

Abstract

The preparation of N², N²-dimethylguanosine is described. The use of the 2-(p-nitrophenyl)ethyl group instead of the benzyl protecting group for the O⁶ position of the guanine ring resulted in better yields and shorter protocols.     

Fragile X‐associated tremor/ataxia syndrome: Regional decrease of mitochondrial DNA copy number relates to clinical manifestations

Fragile X‐associated tremor/ataxia syndrome (FXTAS) is a late‐onset neurodegenerative disorder that appears in at least one‐third of adult carriers of a premutation (55‐200 CGG repeats) in the fragile X mental retardation 1 (FMR1) gene. Several studies have shown that mitochondrial dysfunction may play a central role in aging and also in neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, Huntington's disease as well as in FXTAS. It has been recently proposed that mtDNA copy number, measured by the number of mitochondrial genomes per nuclear genome (diploid), could be a useful biomarker of mitochondrial dysfunction. In order to elucidate the role of mtDNA variation in the pathogenesis of FXTAS, mtDNA copy number was quantified by digital droplet Polymerase chain reaction. In human brain samples, mtDNA levels were measured in the cerebellar vermis, dentate nucleus, parietal and temporal cortex, thalamus, caudate nucleus and hippocampus from a female FXTAS patient, a FMR1 premutation male carrier without FXTAS and from three male controls. The mtDNA copy number was further analyzed using this technology in dermal fibroblasts primary cultures derived from three FXTAS patients and three controls as well as in cortex and cerebellum of a CGG knock in FXTAS mice model. Finally, qPCR was carried out in human blood samples. Results indicate reduced mtDNA copy number in the specific brain region associated with disease progression in FXTAS patients, providing new insights into the role of mitochondrial dysfunction in the pathogenesis of FXTAS.     

Prevalence and microdiversity of Alteromonas macleodii-like microorganisms in different oceanic regions

The presence, prevalence and variability of microorganisms related to the species Alteromonas macleodii, a well known culturable gamma-Proteobacterium, has been studied in different seawater samples from diverse geographical locations, in both the Northern and Southern hemispheres, and tested with two molecular techniques (rRNA hybridization and gene cloning and sequencing). Results show that A. macleodii-like microorganisms are present in high proportions in North Atlantic and, especially, Mediterranean waters, being higher at deep samples and particle-associated fractions, in agreement with previous findings. In contrast, Southern samples (all from very cold areas near Antarctica) presented no significant hybridization signals. The analysis of the ribosomal ITS (16S-23S internal transcribed spacers) revealed that A. macleodii-like microorganisms from Mediterranean, North Atlantic, Caribbean and Red Sea waters differed in both size and sequence, mostly depending on their geographical origin, with Mediterranean and North Atlantic clones clustering into two main groups whereas Caribbean and Red Sea clones appeared separated.