PTX3 plays a key role in the organization of the cumulus oophorus extracellular matrix and in in vivo fertilization

PTX3 is a prototypic long pentraxin that plays a non-redundant role in innate immunity against selected pathogens and in female fertility. Here, we report that the infertility of Ptx3(-/-) mice is associated with severe abnormalities of the cumulus oophorus and failure of in vivo, but not in vitro, oocyte fertilization. PTX3 is produced by mouse cumulus cells during cumulus expansion and localizes in the matrix. PTX3 is expressed in the human cumulus oophorus as well. Cumuli from Ptx3(-/-) mice synthesize normal amounts of hyaluronan (HA), but are unable to organize it in a stable matrix. Exogenous PTX3 restores a normal cumulus phenotype. Incorporation in the matrix of inter-alpha-trypsin inhibitor is normal in Ptx3(-/-) cumuli. PTX3 does not interact directly with HA, but it binds the cumulus matrix hyaladherin tumor necrosis factor alpha-induced protein 6 (TNFAIP6, also known as TSG6) and thereby may form multimolecular complexes that can cross-link HA chains. Thus, PTX3 is a structural constituent of the cumulus oophorus extracellular matrix essential for female fertility.     

Characterization of the Participation of Sodium Channels on the Rise in Na+ Induced by 4-Aminopyridine (4-AP) in Synaptosomes

The participation of voltage-sensitive Na+ channels (VSSC) on the changes on internal (i) Na+, K+, Ca2+, and on DA, Glu, and GABA release caused by different concentrations of 4-AP was investigated in striatum synaptosomes. TTX, which abolished the increase in Na(i) (as determined with SBFI), induced by 0.1 mM 4-AP only inhibited by 30% the rise in Na(i) induced by 1 mM 4-AP. One millimolar 4-AP markedly decreased the fluorescence of the K+ indicator dye PBFI but 0.1 mM 4-AP did not. Like 1 mM 4-AP, ouabain decreased PBFI fluorescence and increased a considerable fraction of Na(i) in a TTX-insensitive manner. In contrast with the different TTX sensitivity of the rise in Na(i) induced by 0.1 and 1 mM 4-AP, the rise in Ca(i) (as determined with fura-2) induced by the two concentrations of 4-AP was markedly inhibited by TTX, as well as by omega-agatoxin in combination with omega-conotoxin GVIA, indicating that only the TTX-sensitive fraction of the rise in Na(i) induced by 4-AP is linked with the activation of presynaptic Ca2+ channels. It is concluded that the TTX-sensitive fraction of neurotransmitter release evoked by 4-AP is released by exocytosis, and the TTX insensitive fraction involves reversal of the neurotransmitters transporters. This contrasts with the exocytosis evoked by high K+ that is unchanged by TTX and with the neurotransmitter-transporter-mediated release evoked by veratridine, which is highly TTX sensitive and does not require activation of Ca2+ channels.     

Survey of black howler (<Emphasis Type="Italic">Alouatta pigra</Emphasis>) and spider (<Emphasis Type="Italic">Ateles geoffroyi</Emphasis>) monkeys in the Mayan sites of Calakmul and Yaxchilán,Mexico and Tikal,Guatemala

Surveys of populations of spider and howler monkeys were conducted at the Mayan sites of Calakmul and Yaxchilán, Mexico and Tikal, Guatemala. The forests in which these sites are found are part of the largest landmass of tropical rain forests present in Mesoamerica, encompassing about 4 million ha. Triangulation of monkey vocalization combined with ground surveys was used to determine the presence of howler and spider monkey groups. Howler monkey mean troop size at these sites varied from 6.6±2.1 individuals in Yaxchilán to 7.5±1.9 in Calakmul to 8.7±2.2 in Tikal. Density estimates varied from 12.8 individuals/km² in Yaxchilán to 15.2 individuals/km² in Calakmul to 17.8 individuals/km² in Tikal. Mean spider monkey subgroup size varied from 4.7±2.6 individuals in Tikal to 5.6±3.0 individuals in Yaxchilán to 7.7±3.8 individuals in Calakmul. Spider monkey density varied from 17.0 individuals/km² in Yaxchilán to 17.2 individuals/km² in Calakmul to 56.4 individuals/km² in Tikal. All sightings of both howler and spider monkeys at the three sites were in undisturbed rain forest vegetation and spider monkeys in general were more frequently sighted at higher tree heights than howlers. We discuss the value of further acquiring data on howler and spider monkey populations existing in extensive forest tracts and on the conservation value for both primate species of the forests surrounding the Mayan ruins found in this area of Mesoamerica.     

Prey stage preference and functional response of Euseius hibisci to Tetranychus urticae (Acari: Phytoseiidae, Tetranychidae)

The aims of this study were: (a) determine the prey stage preference of female Euseius hibisci (Chant) (Phytoseiidae) at constant densities of different stages of Tetranychus urticae Koch (Tetranychidae), (b) assess the functional response of the predator females to the varying densities of eggs, larvae, or protonymphs of T. urticae, and (c) estimate the functional response of E. hibisci when pollen of Ligustrum ovalifolium was present as well. We conducted experiments on excised pieces of strawberry leaf arenas (Fragaria ananassa) under laboratory conditions of 25 ± 2 °C, 60 ± 5% RH and 12 h photophase. Our results indicated that the predator consumed significantly more prey eggs than other prey stages. Consumption of prey deutonymphs and adults was so low that they were excluded from the non-choice functional response experiments. The functional response on all food items was of type II. The two parameters of the functional response were estimated for each prey type by means of the adjusted non-linear regression model. The highest estimated value a (instantaneous rate of discovery) and the lowest value of T_h (handling time, including digestion) were found for the predator feeding on prey eggs, and a was lowest and T_h highest when fed protonymphs. Using the jack-knife method, the values for the functional response parameters were estimated. The values of a and T_h produced by the model were similar among all prey types except for the eggs, which were different. Using pollen simultaneously with prey larvae decreased the consumption of the latter over the full range of prey densities The suitability of this predator for biological control of T. urticae on strawberry is discussed.     

Defective dendritic cell migration and activation of adaptive immunity in PI3Kgamma-deficient mice

Gene-targeted mice were used to evaluate the role of the gamma isoform of phosphoinositide 3-kinase (PI3Kgamma) in dendritic cell (DC) migration and induction of specific T-cell-mediated immune responses. DC obtained from PI3Kgamma-/- mice showed a reduced ability to respond to chemokines in vitro and ex vivo and to travel to draining lymph nodes under inflammatory conditions. PI3Kgamma-/- mice had a selective defect in the number of skin Langerhans cells and in lymph node CD8alpha- DC. Furthermore, PI3Kgamma-/- mice showed a defective capacity to mount contact hypersensitivity and delayed-type hypersensitivity reactions. This defect was directly related to the reduced ability of antigen-loaded DC to migrate from the periphery to draining lymph nodes. Thus, PI3Kgamma plays a nonredundant role in DC trafficking and in the activation of specific immunity. Therefore, PI3Kgamma may be considered a new target to control exaggerated immune reactions.     

Marked inhibition of retinal neovascularization in rats following soluble-flt-1 gene transfer

BACKGROUND: In mouse models of retinopathy of prematurity (ROP) inhibitors of vascular endothelial growth factor (VEGF) functions administered systemically completely block retinal neovascularization. In contrast, selective ocular VEGF depletion has achieved an approx. 50% inhibition of retinal neovascular growth. It is unclear whether a more complete inhibition of new blood vessel development can be obtained with an anti-VEGF therapy localized to the eye. Therefore, the objective of the present study was to determine the effect of local anti-VEGF therapy in a different animal model which closely mimics human ROP. METHODS: Rats were exposed to alternating cycles of high and low levels of oxygen for 14 days immediately after birth; thereafter, they were intravitreally injected with an adenoviral vector expressing a secreted form of the VEGF receptor flt-1 (Ad.sflt), which acts by sequestering VEGF. Contralateral eyes were injected with the control vector carrying the reporter gene expressing beta-galactosidase (Ad.betaGal). RESULTS: At the peak of retinal neovascular growth, i.e. post-natal day 21 (P21), we observed up to 97.5% decrease in retinal neovascularization in animals injected with Ad.sflt. At the end of observation (P28), no significant difference in retinal vessel number was detected in both oxygen-injured and normoxic Ad.sflt-treated retinas compared with untreated or Ad.betaGal-treated retinas. CONCLUSION: Adenoviral-mediated sflt-1 gene transfer induces a near-complete inhibition of ischemia-induced retinal neovascularization in rats without affecting pre-existing retinal vessels.     

The karyology of the cyprinid genera <Emphasis Type="Italic">Scardinius</Emphasis> and <Emphasis Type="Italic">Rutilus</Emphasis> in southern Europe

The chromosomes of eight species of Rutilus and Scardinius, mostly endemic to the Italian and the Balkan peninsula, were analyzed by conventional and other banding techniques. Parallel analyses were conducted also on two leuciscine species, Alburnus albidus, for which we provide the first karyological analysis, and Leuciscus cephalus. All species examined displayed the same karyotype (2n=50 chromosomes, 8 metacentric+13 submetacentric+4 subtelo/acrocentric pairs) with nucleolus organizer regions (NORs) on the ends of the shorter arms of a medium-sized submetacentric pair. In contrast, interspecific variation was observed in the distribution of constitutive heterochromatin. The variation observed in this genomic material proved to be systematically and phylogenetically informative. Indeed, a peritelomeric C-band on the first telocentric pair characterizes species of Rutilus and Scardinius. In both genera heterochromatin differentiation appears to be directed to a centromere–telomere direction, particularly evident along the metacentric elements of their karyotypes.An erratum to this article can be found at     

Determination of protein markers in human serum: Analysis of protein expression in toxic oil syndrome studies

Toxic oil syndrome (TOS) is a disease that appeared in Spain in 1981. It affected more than 20 000 people and produced over 300 deaths in the first 2 years. In this paper, a prospective study on the differences in gene expression in sera between a control versus a TOS-affected population, both originally exposed to the toxic oil, is presented. Differential protein expression was analyzed by two-dimensional electrophoresis (2-DE). Several problems related with serum analysis by 2-DE were addressed in order to improve protein detection in the gel images. Three new commercial systems for albumin depletion were tested to optimize the detection of minor proteins that can be obscured by the presence of a few families of high abundance proteins (albumin, immunoglobulins). Other factors, such as the use of nonionic reductants or the presence of thiourea in the gels, were also tested. From these optimized images, a group of 329 major gel spots was located, matched and compared in serum samples. Thirty-five of these protein spots were found to be under- or overexpressed in TOS patients (> three-fold increase or decrease). Proteins in the differential spots were identified by matrix-assisted laser desorption/ionization-time of flight peptide map fingerprinting and database search. Several haptoglobin isoforms were found to be differentially expressed, showing expression phenotypes that could be related with TOS affection. Haptoglobin phenotypes have been previously reported to have important biological and clinical consequences and have been described as risk factors for several diseases.     

Floxed allele for conditional inactivation of the GABAB(1) gene

GABA(B) receptors are the G-protein-coupled receptors for the neurotransmitter GABA. GABA(B) receptors are broadly expressed in the nervous system. Their complete absence in mice causes premature lethality or--when mice are viable--epilepsy, impaired memory, hyperalgesia, hypothermia, and hyperactivity. A spatially and temporally restricted loss of GABA(B) function would allow addressing how the absence of GABA(B) receptors leads to these diverse phenotypes. To permit a conditional gene inactivation, we flanked critical exons of the GABA(B(1)) gene with lox511 sites. GABA(B(1)) (lox511/lox511) mice exhibit normal levels of GABA(B(1)) protein, are fertile, and do not display any behavioral phenotype. We crossed GABA(B(1)) (lox511/lox511) with Cre-deleter mice to produce mice with an unrestricted GABA(B) receptor elimination. These GABA(B(1)) (-/-) mice no longer synthesize GABA(B(1)) protein and exhibit the expected behavioral abnormalities. The conditional GABA(B(1)) allele described here is therefore suitable for generating mice with a site- and time-specific loss of GABA(B) function.