Sub-cellular Localization of Calcium in Azolla-Anabaena Symbiosis by Chlortetracycline,ESI and EELS

The subcellular localization of calcium in cells of symbiotic partners located within leaf cavities of Azolla was investigated by using chlorotetracycline, ESI and EELS analysis. Loosely membrane-bound calcium was evidenced by using CTC or EGTA and CTC, in cytoplasmic regions of Azolla hair cells and in cytoplasm of the cyanobiont. Tightly membrane-bound calcium revealed by CTC, and ESI and EELS analysis, was observed in cyanophycin granules and carboxysomes of the cyanobiont. A third calcium type, revealed by ESI and EELS analysis, was localized at the level of cell walls of simple and branched Azolla hairs, in the envelope of heterocysts, and in the cell walls of the cyanobiont.     

The oxygenase component of phenol hydroxylase from Acinetobacter radioresistens S13.

Phenol hydroxylase (PH) from Acinetobacter radioresistens S13 represents an example of multicomponent aromatic ring monooxygenase made up of three moieties: a reductase (PHR), an oxygenase (PHO) and a regulative component (PHI). The function of the oxygenase component (PHO), here characterized for the first time, is to bind molecular oxygen and catalyse the mono-hydroxylation of substrates (phenol, and with less efficiency, chloro- and methyl-phenol and naphthol). PHO was purified from extracts of A. radioresistens S13 cells and shown to be a dimer of 206 kDa. Each monomer is composed by three subunits: alpha (54 kDa), beta (38 kDa) and gamma (11 kDa). The gene encoding PHO alpha (named mopN) was cloned and sequenced and the corresponding amino acid sequence matched with that of functionally related oxygenases. By structural alignment with the catalytic subunits of methane monooxygenase (MMO) and alkene monooxygenase, we propose that PHO alpha contains the enzyme active site, harbouring a dinuclear iron centre Fe-O-Fe, as also suggested by spectral analysis. Conserved hydrophobic amino acids known to define the substrate recognition pocket, are also present in the alpha-subunit. The prevalence of alpha-helices (99.6%) as studied by CD confirmed the hypothized structural homologies between PHO and MMO. Three parameters (optimum ionic strength, temperature and pH) that affect kinetics of the overall phenol hydroxylase reaction were further analyzed with a fixed optimal PHR/PHI/PHO ratio of 2/1/1. The highest level of activity was evaluated between 0.075 and 0.1 m of ionic strength, the temperature dependence showed a maximum of activity at 24 degrees C and finally the pH for optimal activity was determined to be 7.5.     

Genetic Diversity of Isolates of Glomus mosseae from Different Geographic Areas Detected by Vegetative Compatibility Testing and Biochemical and Molecular Analysis

We detected, for the first time, the occurrence of vegetative incompatibility between different isolates of the arbuscular mycorrhizal fungal species Glomus mosseae. Vegetative compatibility tests performed on germlings belonging to the same isolate showed that six geographically different isolates were capable of self-anastomosing, and that the percentage of hyphal contacts leading to fusions ranged from 60 to 85%. Successful anastomoses were characterized by complete fusion of hyphal walls, protoplasm continuity and occurrence of nuclei in the middle of hyphal bridges. No anastomoses could be detected between hyphae belonging to different isolates, which intersected without any reaction in 49 to 68% of contacts. Microscopic examinations detected hyphal incompatibility responses in diverse pairings, consisting of protoplasm retraction from the tips and septum formation in the approaching hyphae, even before physical contact with neighboring hyphae. Interestingly, many hyphal tips showed precontact tropism, suggesting that specific recognition signals may be involved during this stage. The intraspecific genetic diversity of G. mosseae revealed by vegetative compatibility tests was confirmed by total protein profiles and internal transcribed spacer-restriction fragment length polymorphism profiles, which evidenced a higher level of molecular diversity between the two European isolates IMA1 and BEG25 than between IMA1 and the two American isolates. Since arbuscular mycorrhizal fungi lack a tractable genetic system, vegetative compatibility tests may represent an easy assay for the detection of genetically different mycelia and an additional powerful tool for investigating the population structure and genetics of these obligate symbionts.     

Cloning and comparative mapping of a gene from the commonly deleted region of DiGeorge and Velocardiofacial syndromes conserved in C. elegans

We have identified and cloned a gene, ES2, encoding a putative 476 amino acid protein with a predicted M _r of 52,568. The gene is localized within the DiGeorge/Velocardiofacial syndrome locus on 22q11.2 and is deleted in all the patients in which a deletion within 22q11 could be demonstrated, with the exception of one patient. ES2 is expressed in all the tissues studied. Sequence comparison showed identity with five ESTs and at the amino acid level the sequence was highly similar to, and collinear with, a hypothetical C. elegans protein of unknown function. Mutation analysis was performed in 16 patients without deletion, but no mutation has been found. The cDNA sequence is conserved in mouse and is localized on MMU16B1-B3, known to contain a syntenic group in common with HSA 22q11.2. Received: 25 March 1996 / Accepted: 15 May 1996     

Multidrug-resistance (MDR) phenotype of human osteosarcoma cells evaluated by quantitative morphological and electron microscopy analyses

Summary— Multidrug-resistant (MDR) variants of a human osteosarcoma cell line (U-2 OS) have been recently obtained by continuous exposure to doxorubicin (DX). The growth and phenotypic characteristics of these cell lines have been demonstrated to be related to the level of expression of P-glycoprotein. In this work, the morphological changes associated with MDR have been evaluated by quantitative image analysis and transmission electron microscopy. Resistant cells present morphological changes with respect to sensitive cells at both cytoplasmic and nuclear level. Some of these changes appear to be related to the degree of resistance but not to the direct presence of DX, since deprived cells maintain some modified characters, while others are partly lost. These findings suggest that DX exposure affects cell metabolism causing progressive changes of the cell morphotype.     

Leaf characteristics and optical properties of different woody species

 Light partition has been examined and evaluated on five woody species (Olea europaea, Ficus carica, Pittosporum tobira, Hedera helix maculata, Persica vulgaris) in relation to their leaf morpho-histological characteristics, water and chlorophyll contents. Leaf parameters and optical properties (reflectance, transmittance, absorbance) in PAR, FR and NIR wavebands (400–1100 nm) were preliminarily submitted to a canonical correlation analysis where lamina thickness and water content showed a leading role in determining all the optical properties, while chlorophyll, influential in the PAR region, was remarkably effective only in an extreme pigment situation when green and albino patches of ivy leaves were compared. Transmittance appeared inversely related to lamina thickness in accordance with the Lambert Beer law. Significant correlations were found also between mesophyll water content and both transmittance (positive) and reflectance (negative). Olive leaves showed peculiar optical patterns because of the dense and continuous trichome layer on their abaxial surface. Received: 3 January 1997 / Accepted: 5 May 1997     

Comparative mapping of the DiGeorge syndrome region in mouse shows inconsistent gene order and differential degree of gene conservation

We have constructed a comparative map in mouse of the critical region of human 22q11 deleted in DiGeorge (DGS) and Velocardiofacial (VCFS) syndromes. The map includes 11 genes potentially haploinsufficient in these deletion syndromes. We have localized all the conserved genes to mouse Chromosome (Chr) 16, bands B1-B3. The determination of gene order shows the presence of two regions (distal and proximal), containing two groups of conserved genes. The gene order in the two regions is not completely conserved; only in the proximal group is the gene order identical to human. In the distal group the gene order is inverted. These two regions are separated by a DNA segment containing at least one gene which, in the human DGS region, is the most proximal of the known deleted genes. In addition, the gene order within the distal group of genes is inverted relative to the human gene order. Furthermore, a clathrin heavy chain-like gene was not found in the mouse genome by DNA hybridization, indicating that there is an inconsistent level of gene conservation in the region. These and other independent data obtained in our laboratory clearly show a complex evolutionary history of the DGS-VCFS region. Our data provide a framework for the development of a mouse model for the 22q11 deletion with chromosome engineering technologies. Received: 8 July 1997 / Accepted 11 August 1997     

Characterization of a Small Family (CAIII) of Microsatellite-Containing Sequences with X-Y Homology

Four X-linked loci showing homology with a previously described Y-linked polymorphic locus (DYS413) were identified and characterized. By fluorescent in situ hybridization (FISH), somatic cell hybrids, and YAC screening, the X-linked members of this small family of sequences (CAIII) all map in Xp22, while the Y members map in Yq11. These loci contribute to the overall similarity of the two genomic regions. All of the CAIII loci contain an internal microsatellite of the (CA)_n type. The microsatellites display extensive length polymorphism in two of the X-linked members as well as in the Y members. In addition, common sequence variants are found in the portions flanking the microsatellites in two of the X-linked members. Our results indicate that, during the evolution of this family, length variation on the Y chromosome was accumulated at a rate not slower than that on the X chromosome. Finally, these sequences represent a model system with which to analyze human populations for similar X- and Y-linked polymorphisms. Received: 29 July 1996 / Accepted: 15 January 1997     

Haploinsufficiency of cytosolic serine hydroxymethyltransferase in the Smith-Magenis syndrome.

Folate-dependent one-carbon metabolism is critical for the synthesis of numerous cellular constituents required for cell growth, and serine hydroxymethyltransferase (SHMT) is central to this process. Our studies reveal that the gene for cytosolic SHMT (cSHMT) maps to the critical interval for Smith-Magenis syndrome (SMS) on chromosome 17p11.2. The basic organization of the cSHMT locus on chromosome 17 was determined and was found to be deleted in all 26 SMS patients examined by PCR, FISH, and/or Southern analysis. Furthermore, with respect to haploinsufficiency, cSHMT enzyme activity in patient lymphoblasts was determined to be approximately 50% that of unaffected parent lymphoblasts. Serine, glycine, and folate levels were also assessed in three SMS patients and were found to be within normal ranges. The possible effects of cSHMT hemizygosity on the SMS phenotype are discussed.