Expression of rolB in apomictic Hieracium piloselloides Vill. causes ectopic meristems in planta and changes in ovule formation, where apomixis initiates at higher frequency

The effects of the Agrobacterium rhizogenes rolB oncogene on apomixis were examined in the facultative apomictic plant Hieracium piloselloides because the oncogene has been shown to alter plant growth, morphogenesis and cellular sensitivity to auxin. Introduction of rolB under the control of either its own promoter or the CaMV35S promoter induced ectopic meristem formation from the inflorescence, confirming in planta a meristem-inducing role for this oncogene previously observed only in tissue culture. These ectopic meristems formed vegetative rosettes and floral plant organs. Upon immersion in water these meristems generated roots, suggesting that meristem commitment towards the generation of a specific organ type is a separate and later event that is dependent upon the developmental context. Ovule identity and form was altered in ectopically induced florets in plants expressing the CaMV35S::rolB construct. In contrast to the ovules of untransformed apomictic plants, the sexual process ceased earlier, prior to meiosis, yet surprisingly, apomixis initiated from a greater number of cells, and embryos and endosperm continued to develop in the structurally altered ovules. The alternative possibilities that the effects on reproduction might result from rolB influencing cellular response to auxin, or from alterations in cell signaling caused by changes in ovule morphology that are induced because of the expression of the oncogene are discussed.     

AtCYS1, a cystatin from Arabidopsis thaliana, suppresses hypersensitive cell death.

In plants, cysteine protease inhibitors are involved in the regulation of protein turnover and play an important role in resistance against insects and pathogens. AtCYS1 from Arabidopsis thaliana encodes a protein of 102 amino acids that contains the conserved motif of cysteine protease inhibitors belonging to the cystatin superfamily (Gln-Val-Val-Ala-Gly). Recombinant A. thaliana cystatin-1 (AtCYS1) was expressed in Escherichia coli and purified. AtCYS1 inhibits the catalytic activity of papain (Kd = 4.0 x 10-2 micro m, at pH 7.0 and 25 degrees C), generally taken as a molecular model of cysteine proteases. The molecular bases for papain inhibition by AtCYS1 have been analysed taking into account the three-dimensional structure of the papain-stefin B complex. AtCYS1 is constitutively expressed in roots and in developing siliques of A. thaliana. In leaves, AtCYS1 is strongly induced by wounding, by challenge with avirulent pathogens and by nitric oxide (NO). The overexpression of AtCYS1 blocks cell death activated by either avirulent pathogens or by oxidative and nitrosative stress in both A. thaliana suspension cultured cells and in transgenic tobacco plants. The suppression of the NO-mediated cell death in plants overexpressing AtCYS1 provides the evidence that NO is not cytotoxic for the plant, indicating that NO functions as cell death trigger through the stimulation of an active process, in which cysteine proteases and theirs proteinaceous inhibitors appear to play a crucial role.     

Home range dynamics of male roe deerCapreolus capreolus in a mountainous habitat

Male spacing behaviour of roe deer Capreolus capreolus (Linnaeus, 1758) was studied in a wooded mountainous habitat in the Casentinesi Forest National Park, Italy. Data were collected using radio-tracking techniques from March 1997 to February 1998. Annual, seasonal, and bimonthly home ranges were analysed. Different factors may influence male spacing behaviour throughout the year. Winter home range sizes may be dependent on environmental conditions, while social factors could determine a high level of individual variability during the territorial and reproductive period. Prime age males showed great spatial stability, in contrast to the ranging movements of yearlings.     

Forecasting airborne pollen concentrations: Development of local models

People's sensitivity to allergies may representone of the most important health factors of thenext century to which attention must be paid inorder to reduce the incidence of social costsand improve the quality of life.Taking into consideration the earnest requestsof the medical-scientific communityEmilia-Romagna ARPA (Regional Agency for thePrevention of the Environment) moved theattention from the monitoring to a short andmedium term prediction of the concentration ofallergenic pollens in the air in order toachieve a more effective therapeutic action.Our main objectives are to improve seasonalforecasts and to interpret anomalous years.A neural network model for grass pollenforecasting has been implemented. Inputvariables were meteorological situations, i.e.,daily temperature (max., min. and average) andrainfall, in addition to combinations ofindividual variables and their thresholds. Theoutput was daily pollen concentration.The model was able to understand and predictanomalous years. We demonstrate that therelationships between pollen concentrations andmeteorological situations are independent fromsite. This means that such models canunderstand the differences in differentareas.     

Ganglion cells and topographically related nerves in the vallate papilla/von Ebner gland complex.

Ganglion cells and topographically related nerves in the vallate papilla/von Ebner gland complex were investigated in rat tongue by cytochemical, immunocytochemical, and ultrastructural methods to evaluate the possible presence of different neuronal subpopulations. Immunostaining for neurofilaments and protein gene product 9.5 revealed ganglionic cell bodies and nerve fibers. A large part of the neurons were positive at immunostaining for neuronal nitric oxide synthase (NOS), vesicular acetylcholine transporter (VAChT), or vasoactive intestinal peptide (VIP). A small subset of nerve fibers revealed immunoreactivity for cholecystokinin. Axons traveling under the lingual epithelium were evidenced by their content of calcitonin gene-related peptide (CGRP) or substance P (SP). Cell bodies positive for SP or CGRP were not detected. Using methods of co-localization, three different neuronal classes were detected. The main population was composed of AChE/NADPH-diaphorase (NADPHd)-positive cells. Small groups of acetylcholine esterase (AChE)-positive/NADPHd-negative cells were visible. Isolated neurons were AChE-negative/NADPHd-positive. The results of co-localization experiments for VAChT/NOS were consistent with those obtained by cytochemical co-localization of AChE and NADPHd. Experiments of co-localization for peptidergic and nitrergic structures revealed CGRP- and SP-immunoreactive fibers in the vallate papilla/von Ebner gland ganglion. In conclusion, the results demonstrated in the VP/VEG complex peptidergic, cholinergic, and nitrergic neurons. The presence of different neuronal subclasses suggests that a certain degree of functional specialization may exist.     

Linkage disequilibrium between GABRB3 gene and nonsyndromic familial cleft lip with or without cleft palate

The malformation of nonsyndromic cleft lip with or without cleft palate (CL/P) is a common congenital disease that affects approximately 1/1000 newborns in Caucasian populations. Genetic studies indicate that CL/P has the characteristics of a complex genetic trait. Linkage analysis and mouse-model knockout studies have suggested several candidate genes mapping in different chromosome regions for CL/P malformation. On these grounds, we have investigated, by linkage disequilibrium (LD) and parametric and nonparametric linkage analyses, five different candidate genes, including those for the beta3 subunit of the gamma-aminobutyric acid receptor (GABRB3), glutamic acid decarboxylase 1 (GAD1), retinoic acid receptor alpha (RARA), transforming growth factor beta3 (TGFB3), and msh ( Drosophila) homeobox homolog 1 (MSX1). Interestingly, a significant LD between GABRB3 and CL/P was obtained ( P-value=0.008 in the allele-wise analysis for multiallelic markers), suggesting that the GABRB3 gene is involved in this congenital disease. This new finding in humans is in agreement with previously reported data obtained with the murine model. Indeed, mouse studies indicate a role for gamma-aminobutyric acid (GABA) and its receptor in normal palate development. Exclusion of the GAD1 gene, which encodes the GABA-producing enzyme, in CL/P pathogenesis was obtained in our study. Moreover, we were unable to confirm the involvement of the MSX1 gene in nonsyndromic CL/P. Modest evidence of LD between marker alleles and CL/P was found at the RARA and TGFB3 loci suggesting a minor role for these genes in our family set of nonsyndromic CL/P.     

Aberrant spermatogenesis and sex determination in Bourletiellidae (Hexapoda, Collembola), and their evolutionary significance

Light and electron microscopic evidence is provided to describe a new example of a postzygotic sex-determination system in two collembolan species, Bourletiella arvalis and B. hortensis. In B. arvalis, where chromosome number could be assessed, both sexes are homogametic (n=6) and all zygotes have an identical chromosome composition (2n=12). However, male embryos develop after the loss of two sex chromosomes, making the male genotype 2n=10 (4AAX₁0X₂0). On the other hand, female embryos develop if the zygote retains all chromosomes and the female genetic system is, therefore, 4AAX₁X₁X₂X₂ (2n=12). As an apparent consequence of the lack of two chromosomes in the male germ cells, spermatogenesis is aberrant. At the first meiotic division, in fact, the two resulting secondary spermatocytes receive a different number of chromosomes: six and four. The cells which receive six chromosomes (one haploid set of four autosomes and two sex chromosomes) proceed through the meiotic process and the two spermatids generated produce two spermatozoa by a normal spermiogenesis. The cells receiving only four chromosomes do not undergo the second meiotic division and soon degenerate. The degenerating cells can be considered a morphological marker for this process, as they are easily recognizable at the electron microscope from the functional secondary spermatocytes by the appearance of the nucleus (totally condensed), the reduction of the cytoplasm (limited to a thin layer surrounding the nucleus), and the lack of most cytoplasmic organelles (with the exception of a couple of centrioles). Electron microscopic evidence has been collected for both species, allowing to extend the same process to B. hortensis, even if chromosomes could not be counted in this species. Therefore, as a result of the spermatocyte elimination, the efficiency of spermatogenesis is reduced to 50%. This process is identical to that observed in other collembolan species of the suborder Symphypleona, and it is suggested that it represents a synapomorphic feature uniting the families Dicyrtomidae, Sminthuridae and Bourletiellidae (Sminthuriformia). It is also suggested that the process is related with the finding of a distorted sex ratio in natural populations and, possibly, with the evolution of parthenogenesis. This hypothesis is supported by the fact that chromosome pairing and genetic recombination occurs only during female meiosis, while chromosomes do not pair during male meiosis. Accepted: 27 December 2000     

Relaxometric characterization of human hemalbumin

Hemalbumin [i.e., Fe(III)-protoporphyrin IX-human serum albumin; Fe(III)heme-HSA] is an important intermediate in the recovery of heme iron following hemolysis. Relaxometric data are consistent with the occurrence of a hexacoordinated high-spin Fe(III) center with no water in the inner coordination sphere. The relatively high relaxation enhancement observed for an aqueous solution of Fe(III)heme-HSA (r1p=4.8 mM(-1)s(-1) at 20 MHz, pH 7, and 25 C) is ascribed to the occurrence of a strong contribution from water molecules in the second coordination sphere. Structural analysis of the putative binding region has been performed by a Monte Carlo simulated annealing procedure, which allowed us to identify His105 and Tyr148 as axial ligands. The role of a tyrosinate as the sixth Fe(III)heme ligand is supported by the pH-dependent analysis. Interestingly, when Fe(III) is replaced by Mn(III), the occurrence of a fast exchanging water molecule at pH values close to neutrality is detected. As the pH is increased, the Mn(III) containing system behaves analogously to Fe(III)heme-HSA. At higher pH, the phenolate ligand is eventually displaced by OH- from both Fe(III) and Mn(III) centers. Support for the proposed bonding scheme has been gained also from competitive binding assays for the sixth coordination site by fluoride, azide, and imidazole ligands.     

A novel regulatory mechanism for amino acid absorption in lepidopteran larval midgut

A number of methyl and ethyl esters of naturally occurring amino acids exert a potent stimulatory effect on the cotransport system responsible for the absorption of most essential amino acids along the midgut of the silkworm Bombyx mori. L-Leucine methyl ester (Leu-OMe), one of the most effective activators, induces a large increase of the initial rate of leucine uptake in midgut brush border membrane vesicles (BBMV) from the anterior-middle (AM) region, and a small effect in BBMV from the posterior (P) region. Nonetheless, the methyl ester causes in both regions a relevant K(+)-, Deltapsi- and pH-independent increase of the intravesicular accumulation of the amino acid. The activation by Leu-OMe proves that amino acid absorption can be modulated all along the B. mori larval midgut and that the AM region, where the ability to transport and concentrate the substrate is very low, is more susceptible than the P region. Leucine uptake in AM-BMMV can be activated by amino acid methyl esters with definite structural requisites, with the following order of potency: L-leucine>L-phenylglycine>L-methionine>L-phenylalanine>L-norleucinez.Gt;L-isoleucine. The activation is stereospecific and occurs also with some ethyl esters (e.g. leucine and phenylalanine). No activation was observed with esters of amino acids with short hydrophobic or polar side-chains. The activation mechanism here described plays a fundamental role in larval growth since silkworms reared on artificial diets supplemented with leucine or methionine methyl esters reach maximum body weight 12-18 h before control larvae and spin cocoons with a larger shell weight. This novel regulatory mechanism of an amino acid transport protein appears to be widespread among lepidopteran larvae.     

Proteolytic activity of bovine lactoferrin

Bovine lactoferrin catalyzes the hydrolysis of synthetic substrates (i.e., Z-aminoacyl-7-amido-4-methylcoumarin). Values of Km and kcat for the bovine lactoferrin catalyzed hydrolysis of Z-Phe-Arg-7-amido-4-methylcoumarin are 50 microM and 0.03 s(-1), respectively, the optimum pH value is 7.5 at 25 degrees C. The bovine lactoferrin substrate specificity is similar to that of trypsin, while the hydrolysis rate is several orders of magnitude lower than that of trypsin. The bovine lactoferrin catalytic activity is irreversibly inhibited by the serine-protease inhibitors PMSF and Pefabloc. Moreover, both iron-saturation of the protein and LPS addition strongly inhibit the bovine lactoferrin activity. Interestingly, bovine lactoferrin undergoes partial auto-proteolytic cleavage at positions Arg415-Lys416 and Lys440-Lys441. pKa shift calculations indicate that several Ser residues of bovine lactoferrin display the high nucleophilicity required to potentially catalyze substrate cleavage. However, a definitive identification of the active site awaits further studies.