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.     

Properties of endogenous β-glucosidase of a Pichia anomala strain isolated from Sicilian musts and wines

The AL 112 strain, isolated from 361 yeast strains in Sicilian musts and wines, has been identified by biochemical and molecular methods as belonging to Pichia anomala, and your endogenous β-glucosidase (βG, EC 3.2.1.21) subsequently characterised. This strain not only has extremely high specific productivity of βG, but above all shows arabinosidase (Ara, EC 3.2.1.55) activity, essential for aroma enhancement of wine. βG from Al 112 is activated by ethanol at the concentrations typically found in wine; it is not inhibited by fructose, whilst glucose, a non-competitive inhibitor, despite lowering activity, actually protects the enzyme from factors that could damage it. It has an optimum temperature of 20 °C, compatible with typical cellar conditions, and stability in model must-wine and wine solutions ≥40 days.     

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.     

Microsomal epoxide hydrolase polymorphisms and lung cancer risk: a quantitative review

To investigate the role of microsomal epoxide hydrolase (mEH) polymorphisms in the aetiology of lung cancer and to assess the interaction between mEH polymorphisms and smoking, we performed a meta-analysis of seven published studies, which included 2078 cases and 3081 controls, and a pooled analysis of eight studies (four published and four unpublished at that time) with a total of 986 cases and 1633 controls. The combined metaanalysis odds ratios (ORs) were 0.98 (95% confidence interval [CI] = 0.72-1.35) for polymorphism at amino acid 113 in exon 3 (His/His versus Tyr/Tyr genotype) and 1.00 (95% CI= 0.71-1.41) for polymorphism at amino acid 139 in exon 4 (Arg/Arg versus His/ His genotype). In the pooled analysis, we observed a significant decrease in lung cancer risk (OR = 0.70, 95% CI = 0.51-0.96) for exon 3 His/His genotype after adjustment for age, sex, smoking and centre. The protective effect of exon 3 polymorphism seems stronger for adenocarcinoma of the lung than for other histological types. The OR for high predicted mEH activity, compared with low activity, was 1.54 (95% CI = 0.77-3.07) in the meta analysis and 1.18 (95% CI = 0.92-1.52) in the pooled analysis. We did not find a consistent modification of the carcinogenic effect of smoking according to mEH polymorphism, although the risk of lung cancer decreased among never smokers with high mEH activity and among heavy smokers with the exon 3 His/His genotype. In conclusion, this study suggests a possible effect of mEH polymorphisms at exon 3 in modulating lung cancer. If present, this effect may vary among different populations, possibly because of interaction with genetic or environmental factors.     

Endosome to Golgi Transport of Ricin Is Independent of Clathrin and of the Rab9- and Rab11-GTPases

The plant toxin ricin is transported to the Golgi and the endoplasmic reticulum before translocation to the cytosol where it inhibits protein synthesis. The toxin can therefore be used to investigate pathways leading to the Golgi apparatus. Except for the Rab9-mediated transport of mannose 6-phosphate receptors from endosomes to the trans-Golgi network (TGN), transport routes between endosomes and the Golgi apparatus are still poorly characterized. To investigate endosome to Golgi transport, we have used here a modified ricin molecule containing a tyrosine sulfation site and quantified incorporation of radioactive sulfate, a TGN modification. A tetracycline-inducible mutant Rab9S21N HeLa cell line was constructed and characterized to study whether Rab9 was involved in transport of ricin to the TGN and, if not, to further investigate the route used by ricin. Induced expression of Rab9S21N inhibited Golgi transport of mannose 6-phosphate receptors but did not affect the sulfation of ricin, suggesting that ricin is transported to the TGN via a Rab9-independent pathway. Moreover, because Rab11 is present in the endosomal recycling compartment and the TGN, studies of transient transfections with mutant Rab11 were performed. The results indicated that routing of ricin from endosomes to the TGN occurs by a Rab11-independent pathway. Finally, because clathrin has been implicated in early endosome to TGN transport, ricin transport was investigated in cells with inducible expression of antisense to clathrin heavy chain. Importantly, endosome to TGN transport (sulfation of endocytosed ricin) was unchanged when clathrin function was abolished. In conclusion, ricin is transported from endosomes to the Golgi apparatus by a Rab9-, Rab11-, and clathrin-independent pathway.     

Transformation of white poplar (Populus alba L.) with a novel Arabidopsis thaliana cysteine proteinase inhibitor and analysis of insect pest resistance

Transgenic white poplar (Populus alba L.) plants expressing a novel Arabidopsis thaliana cysteine proteinase inhibitor (Atcys) gene have been produced using Agrobacterium tumefaciens-mediated gene transfer. Internodal stem segments of cv. Villafranca were co-cultivated with the EHA105 pBI-Atcys A. tumefaciens strain. Sixteen putative transgenic plant lines were regenerated from different calli with a transformation efficiency of 11%. The integration and expression of the cysteine proteinase inhibitor (Atcys) gene into the plant genome was confirmed by Southern and northern blot analyses. Papain inhibitory activity was detected in poplar transgenic tissues by means of a specific in vitro assay. Such activity was sufficient to inhibit most of the digestive proteinase activity of chrysomelid beetle (Chrysomela populi L.) and confer resistance to C. populi larvae on selected transgenic plants. A close correspondence between the inhibition of papain and resistance to poplar leaf beetle was observed in all tested transgenic lines. Our results indicate that Atcys could be succesfully employed in breeding programmes aimed at the selection of new poplar genotypes resistant to major insect pests.     

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     

Molecular properties and metabolic effect on blood cells produced by a new toxin of Enterobacter cloacae

A new toxin of Enterobacter cloacae able to lyse erythrocytes and leukocytes was found. Purification of the toxin was performed by salt precipitation, gel filtration, ion exchange and HPLC in C₈ column. SDS-PAGE electrophoresis showed more than one bank corresponding to the leukotoxin able to form polymers and aggregate like some pore-forming cytotoxins (RTX). In culture supernatant the toxin showed 1 HU/ml (hemolytic unit) and 1.5 LU/ml (leukotoxic unit); after purification it reached 15 HU/ml and 20 LU/ml. The ratio between HU and percentage red cells affected the lytic capacity. E. cloacae toxin stimulated the oxidative metabolism of neutrophils, but over 50 μg toxin/ml the stimulus ceased as it was shown by NBT assay due to cell death. Chemiluminescence evidenced an increase in superoxide anion generation, but an excess of toxin interfered with this stimulus, as was previously observed in HlyA Escherichia coli toxin. Cross-reaction was found by immunoblotting with this HlyA. E. cloacae toxin presented higher amounts of proline, valine, aspartic and glutamic acids than HlyA. E. cloacae toxin was similar to HlyA in the prescence of a glycine-rich DNA sequence and in the observed effect of calcium on toxin activity. E. cloacae toxin did not cross-react by immunoblotting with hemolysin HmpA of Proteus. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.