Morphometric and ultrastructural characterization of Bos indicus preantral follicles

The aim of the present study was to characterize the ultrastructure of zebu cow preantral follicles (PAFs). Ovarian cortex samples were processed for light and transmission electron microscopy. Primordial follicles consisted of an oocyte surrounded by one layer of flattened or flattened-cuboidal granulosa cells. The oocyte contained a large and usually eccentric nucleus. Most organelles were located at the perinuclear ooplasm. Round shaped mitochondria, which contained electron-dense granules, smooth and rough endoplasma reticulum and a Golgi apparatus were also observed. Vesicles and coated pits were often observed in the cortical ooplasm. In primary follicles, the oocyte was surrounded by one layer of cuboidal granulosa cells. Short microvilli were observed on the oolema. Secondary follicles consisted of an oocyte surrounded by a variable number of layers of cuboidal granulosa cells. Small secondary follicles had an ultrastructure very similar to that observed in primary follicles. At this follicular stage, the zona pellucida was beginning to form around the oocyte. In large secondary follicles, the zona pellucida was totally developed around the oocyte. Several granulosa cell projections could be detected that were encroaching into the zona pellucida and protruding towards the oocyte, where gap junctions were observed between oocyte and granulosa cell membranes. Organelles within the oocyte were located at the periphery of the ooplasm, and clusters of cortical granules were observed. Round mitochondria were abundant in all developmental stages. In conclusion, this study described the ultrastructure of zebu cow PAFs, and some unique characteristics could be observed as compared with what has been reported for follicles of Bos taurus cattle.     

Heterologous Production of Functional Forms of Rhizopus oryzae Lipase in Escherichia coli

To date, expression of the lipase from Rhizopus oryzae (ROL) in Escherichia coli always led to the formation of inclusion bodies and inactive protein. However, the production of active ROL and its precursor ProROL in soluble form was achieved when E. coli Origami(DE3) and pET-11d were used as expression systems.     

Role of tyrosine 238 in the active site of Rhodotorula gracilis D-amino acid oxidase. A site-directed mutagenesis study.

Y238, one of the very few conserved residues in the active site of d-amino acid oxidases (DAAO), was mutated to phenylalanine and serine in the enzyme from the yeast Rhodotorula gracilis. The mutated proteins are catalytically competent thus eliminating Tyr238 as an active-site acid/base catalyst. Y238F and Y238S mutants exhibit a threefold slower turnover on d-alanine as substrate, which can be attributed to a slower rate of product release relative to the wild-type enzyme (a change of the rate constants for substrate binding was also evident). The Y238 DAAO mutants have spectral properties similar to those of the wild-type enzyme but the degree of stabilization of the flavin semiquinone and the redox properties in the free form of Y238S are different. The binding of the carboxylic acid competitive inhibitors and the substrate d-alanine are changed only slightly, suggesting that the overall substrate binding pocket remains intact. In agreement with data from the pH dependence of ligand binding and with the protein crystal structure, site-directed mutagenesis results emphasize the importance of residue Y238 in controlling access to the active site instead of a role in the substrate/ligand interaction.     

In Vivo Dynamics of Nuclear Pore Complexes in Yeast

While much is known about the role of nuclear pore complexes (NPCs) in nucleocytoplasmic transport, the mechanism of NPC assembly into pores formed through the double lipid bilayer of the nuclear envelope is not well defined. To investigate the dynamics of NPCs, we developed a live-cell assay in the yeast Saccharomyces cerevisiae. The nucleoporin Nup49p was fused to the green fluorescent protein (GFP) of Aequorea victoria and expressed in nup49 null haploid yeast cells. When the GFP–Nup49p donor cell was mated with a recipient cell harboring only unlabeled Nup49p, the nuclei fused as a consequence of the normal mating process. By monitoring the distribution of the GFP–Nup49p, we could assess whether NPCs were able to move from the donor section of the nuclear envelope to that of the recipient nucleus. We observed that fluorescent NPCs moved and encircled the entire nucleus within 25 min after fusion. When assays were done in mutant kar1-1 strains, where nuclear fusion does not occur, GFP–Nup49p appearance in the recipient nucleus occurred at a very slow rate, presumably due to new NPC biogenesis or to exchange of GFP– Nup49p into existing recipient NPCs. Interestingly, in a number of existing mutant strains, NPCs are clustered together at permissive growth temperatures. This has been explained with two different hypotheses: by movement of NPCs through the double nuclear membranes with subsequent clustering at a central location; or, alternatively, by assembly of all NPCs at a central location (such as the spindle pole body) with NPCs in mutant cells unable to move away from this point. Using the GFP–Nup49p system with a mutant in the NPCassociated factor Gle2p that exhibits formation of NPC clusters only at 37°C, it was possible to distinguish between these two models for NPC dynamics. GFP– Nup49p-labeled NPCs, assembled at 23°C, moved into clusters when the cells were shifted to growth at 37°C. These results indicate that NPCs can move through the double nuclear membranes and, moreover, can do so to form NPC clusters in mutant strains. Such clusters may result by releasing NPCs from a nuclear tether, or by disappearance of a protein that normally prevents pore aggregation. This system represents a novel approach for identifying regulators of NPC assembly and movement in the future.     

Genetic dissection of herbicide tolerance in maize by molecular markers

In order to detect the genetic architecture of maize tolerance to Alachlor, a widely used chloroacetanilide, linkage analysis between the expression of the trait and allelic composition of molecular markers was performed. The experiment was carried out on a population of 142 recombinant inbred lines, developed starting from the F₁ between two lines with different reactivity to the herbicide, and self-fertilized for 10 generations; the lines were typed by 48 RFLP markers and 66 microsatellites (SSR). Besides seedling tolerance, evaluated as proportion of normal (non-injured) plants after herbicide treatment, other minor components of tolerance were studied: seed germination ability, pollen germination and tube growth in the presence of the herbicide. The analysis, performed by three statistical methods, revealed the presence of factors controlling seedling tolerance on seven chromosomal regions. Five QTLs appeared to be involved in seed germination ability in the presence of Alachlor, four QTLs in pollen tolerance in terms of germination and four in tube growth under stress were detected. Three loci, on chromosomes 1, 7 and 10, explained most of the variation of seedling tolerance, thus being interesting candidate for marker-assisted selection.     

A 9-bp deletion (2320del9) on the background of the arylsulfatase A pseudodeficiency allele in a metachromatic leukodystrophy patient and in a patient with nonprogressive neurological symptoms

A 9-bp deletion (2320del9) was detected in the arylsulfatase A genes of a patient with late infantile metachromatic leukodystrophy and of a patient with nonprogressive neurological symptoms and very low arylsulfatase A activity. Both patients are heterozygous for the deletion, which involves codons 406–408 and causes loss of a Ser-Asp-Thr tract in the predicted protein. In both patients the 9-bp deletion lies in a pseudodeficiency allele. The patient with metachromatic leukodystrophy carries the common 459 + 1G > A mutation in the other allele. The other patient is homozygous for the pseudodeficiency allele, and consequently is a compound heterozygote for a metachromatic leukodystrophy allele and a pseudodeficiency allele. We hypothesize that the compound heterozygosity predisposes to the development of nonprogressive neurological symptoms in the presence of additional, still unknown, genetic or nongenetic factors. Received: 18 April 1997 / Accepted: 16 August 1997     

Ragweed pollen: The aeroallergen is spreading in Italy

Presently in Europe, ragweed pollen as an aeroallergen is not as important as Poaceae,Parietaria or Betulaceae, even if in some countries the plant is beginning to influence the local composition of the airborne pollen spectra. In northern Italy, the presence of ragweed airborne pollen has only been reported since the beginning of the 1980’s and it is increasingly spreading from year to year. Given this situation, the allergologists have begun to regard the potential risk of sensitisation to ragweed pollen with much attention. Up to now, such pollen has not been included in the routine allergological tests. In 1995 in some sites of northern Italy (Turin, Milan, Trieste), the concentration values of ragweed pollen were remarkable (∼ 20–30 p/m³) and on the increase with respect to the previous years. This investigation aims at focusing the atmospheric concentration trend on this new aeroallergen (Ambrosia sp.) in Italy from 1991 throughout 1995.     

Life history of two abundant populations of Dipetalogaster maximus (Uhler, 1894) (Hemiptera: Reduviidae: Triatominae) in northwestern Mexico

We focused on the analysis of biological parameters of two different abundant populations of Dipetalogaster maximus (Uhler) (Hemiptera: Reduviidae: Triatominae) from northwestern Mexico. The biological parameters were related to hatching of eggs, life cycle, and meal needed for molting by each instar of two populations from environmentally similar areas (El Fandango and San Dionisio) with a similar number of available hosts as blood meal sources. The D. maximus populations from the two locations were evaluated and compared. No significant differences (P>0.05) were recorded for the average hatching time of the two cohorts. The median egg‐to‐adult development time and the number of blood meals at each nymphal group were significantly shorter (P<0.05) for the El Fandango cohort. The number of obtained females at the end of the cycles, number of eggs laid, and rate of egg hatching were significantly higher (P<0.05) for the El Fandango cohort. These results contribute to the estimation of abundances of the studied populations of D. maximus in areas where many tourists go for camping and have an increased risk of being bitten and infected by this species.