The occurrence of reactive oxygen species in the semen of males from infertile couples

The aim of this pilot study was to establish a reactive oxygen species (ROS) evaluation method as a step in the routine diagnosis of men from infertile couples, which attend the Centre of Assisted Reproduction at the Teaching Hospital in Olomouc. Standard semen analyses were performed manually according to WHO guidelines. The number of peroxidase-positive leukocytes in the semen was determined using the Endtz test. The levels of ROS were estimated by chemiluminescence assay using luminol (5-amino-2,3 dihydro-1,4 phthalazinedione) as a probe. The semen samples were collected from 68 patients. Normospermia was found in 15 patients (22.1 %). The semen samples of 3 normospermic patients were classified as ROS-positive. Elevated ROS production was recorded in all subgroups of patients irrespective of any pathology found. We confirmed that spermatozoa might be the source of ROS as well as the seminal leukocytes. Apart from the leukocytes, sperm cells with residual cytoplasm and immature spermatozoa are considered to be a major source of ROS. Thus it is suggested that sperm morphology abnormalities should be evaluated more carefully.     

Changes of electrical impedance in vaginal vestibule in cyclic sows

The changes of the electrical impedance in the vaginal vestibule during the oestrous cycle and the influence of sow parity on the vestibular impedance in oestrus were examined. Primiparous and multiparous sows of the Large White breed were used. Oestrus was tested via exposure of sows to a sexually mature boar. The criterion for conformation of ovulation was the increase in plasma progesterone levels above 12.5nmoll(-1) on day 8 and 12 after oestrus onset. A two-terminal method was used to measure the impedance. The vestibular impedance rose slightly in the first day after weaning. The impedance increased markedly during oestrus (P<0.01) and decreased during early dioestrus (P<0.01). No significant changes were observed thereafter. The individual sows reached the peak of vestibular impedance between 1 and 3 days after oestrus onset. The parity of sows did not significantly influence the impedance values during oestrus. The study showed that the impedance changes in the vaginal vestibule during peri-oestrus are considerably different from those described earlier in the vagina and that sow parity does not affect the vestibular impedance in oestrus.     

Fusion with HDEL protects cell wall invertase from early degradation when N-glycosylation is inhibited

Previous data obtained in different suspension-cultured plant cells have clearly illustrated that N-glycans are absolutely required for transport of glycoproteins to the extracellular compartment, regardless of their oligosaccharide structure [see Lerouge et al. (1998) Plant Mol. Biol. 38: 31 for review]. In the present study the role of N-glycosylation in the transport of glycoproteins to the cell surface was studied in BY2 tobacco cells using both endogenous and recombinant cell wall invertases as markers. When synthesized without their N-glycans, both invertases were very rapidly degraded. This degradation did not occur in an acidic compartment and was brefeldin A-insensitive. Therefore, it most probably represents a pre-Golgi event. However, the low efficiency of specific inhibitors did not favor a strong contribution of proteasomes in this proteolysis. In contrast, addition of a C-terminal His-Asp-Glu-Leu (HDEL) extension prevented arrival of these non-glycosylated glycoproteins in the compartment where they are degraded. These results argue for the presence of an endoplasmic reticulum (ER) domain specialized in protein degradation. Consistent with our results and the well-known stabilization of recombinant proteins retained in the ER, the addition of an ER retention signal to a protein would prevent its targeting to an ER domain devoted to degradation.     

Translokin is an intracellular mediator of FGF-2 trafficking

Basic fibroblast growth factor (bFGF or FGF-2) exerts its pleiotropic activities both as an exogenous and an intracellular factor. FGF-1 and FGF-2 are prototypes for this dual signalling, but the mechanisms of their intracellular actions remain unknown. Here we show that Translokin, a cytoplasmic protein of relative molecular mass 55,000 (M(r) 55K), interacts specifically with the 18K form of FGF-2. Translokin is ubiquitously expressed and colocalizes with the microtubular network. As Translokin does not interact with FGF-1, we used a strategy based on FGF-1-FGF-2 chimaeras to map the interacting regions in FGF-2 and to generate Nb1a2, a non-interacting variant of FGF-2. Although most of the FGF-2 properties are preserved in Nb1a2, this variant is defective in intracellular translocation and in stimulating proliferation. The fusion of a nuclear localization signal to Nb1a2 restores its mitogenic activity and its nuclear association. Inhibiting Translokin expression by RNA interference reduces the translocation of FGF-2 without affecting the intracellular trafficking of FGF-1. Our data show that the nuclear association of internalized FGF-2 is essential for its mitogenic activity and that Translokin is important in this translocation pathway.     

Ecological Life Table of Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae)

An ecological life table was constructed, aiming to determine the critical stages and key mortality factors of Tuta absoluta (Lepidoptera: Gelechiidae). The total population mortality of this tomato leafminer was 92.3%. During the egg stage the mortality was 58.7%, mainly due to egg inviability. A total of 8.6% egg parasitism by Trichogramma pretiosum (Riley) (Hymenoptera: Trichogrammatidae) and 5.0% egg predation by Xylocoris sp. (Heteroptera: Anthocoridae), Cycloneda sanguinea (L.) (Coleoptera: Coccinellidae) and members of the family Phlaeothripidae (Thysanoptera) was observed. The mortality of the larval stage was 33.0%. This was considered to be the critical stage as it showed the highest apparent mortality (79.8%). Larval parasitism was low (0.1%), and was only found with Goniozus nigrifemur Ashmead (Hymenoptera: Bethylidae). Predators were responsible for 79.4% of larval mortality. Therefore, their attraction to and maintenance in the target area are important management tactics to be considered for T. absoluta control. The first and second instars were considered to be the most critical, and predation by the above mentioned species was the key mortality factor. The mortality at the pupal stage was low (0.6%) and was due to malformation.     

Dominant-Negative Mutations in the G-Protein-Coupled α-Factor Receptor Map to the Extracellular Ends of the Transmembrane Segments

G-protein-coupled receptors (GPCRs) transduce the signals for a wide range of hormonal and sensory stimuli by activating a heterotrimeric guanine nucleotide-binding protein (G protein). The analysis of loss-of-function and constitutively active receptor mutants has helped to reveal the functional properties of GPCRs and their role in human diseases. Here we describe the identification of a new class of mutants, dominant-negative mutants, for the yeast G-protein-coupled α-factor receptor (Ste2p). Sixteen dominant-negative receptor mutants were isolated based on their ability to inhibit the response to mating pheromone in cells that also express wild-type receptors. Detailed analysis of two of the strongest mutant receptors showed that, unlike other GPCR interfering mutants, they were properly localized at the plasma membrane and did not alter the stability or localization of wild-type receptors. Furthermore, their dominant-negative effect was inversely proportional to the relative amount of wild-type receptors and was reversed by overexpressing the G-protein subunits, suggesting that these mutants compete with the wild-type receptors for the G protein. Interestingly, the dominant-negative mutations are all located at the extracellular ends of the transmembrane segments, defining a novel region of the receptor that is important for receptor signaling. Altogether, our results identify residues of the α-factor receptor specifically involved in ligand binding and receptor activation and define a new mechanism by which GPCRs can be inactivated that has important implications for the evaluation of receptor mutations in other G-protein-coupled receptors.G-protein-coupled receptors (GPCRs) comprise a large family of receptors that are found in a wide range of eukaryotic organisms from yeasts to humans (4, 10). These receptors respond to diverse stimuli including hormones, neurotransmitters, and other chemical messengers (48). GPCRs transduce their signal by stimulating the α subunit of a heterotrimeric guanine nucleotide binding protein (G protein) to bind GTP (4, 16). This releases the α subunit from the βγ subunits, and then either the α subunit or the βγ subunits go on to promote signaling depending on the specific pathway (28).GPCRs are structurally similar in that they contain seven transmembrane domains (TMDs) connected by intracellular and extracellular loops. Although many techniques have been applied to study receptor function, much of our knowledge on the mechanisms of GPCR activation comes from the characterization of mutant receptors. Loss-of-function and supersensitive mutants have helped to identify receptor regions needed for ligand binding, G-protein activation, and down-regulation of signaling (4, 49). Furthermore, the study of constitutively active receptor mutations has played a key role in the development of current models for receptor activation (26). Naturally occurring GPCR mutations have also been implicated in a number of human diseases (8, 25, 42). Interestingly, the analysis of different mutant receptors indicates that GPCRs utilize common structural domains for similar functions. In particular, the third intracellular loop has an essential role in G-protein activation in a wide range of GPCRs.The genetic approaches possible in the yeast Saccharomyces cerevisiae have been used to examine the relationship between structure and function of the G-protein-coupled mating pheromone receptors. The α-factor and a-factor pheromones induce conjugation in yeast by binding to receptors with seven TMDs that activate a G-protein signal pathway that is highly conserved with mammalian signaling pathways (24). In fact, some human GPCRs can activate the pheromone signal pathway when they are expressed in yeast (19, 29). The analysis of loss-of-function, supersensitive, and constitutively active α-factor receptor mutants has begun to reveal the mechanisms for activation and regulation of this receptor. For example, the analysis of constitutively active mutants indicates that movement in the transmembrane segments plays a key role in α-factor receptor activation (22). Constitutive mutations and loss-of-function mutations implicate the third intracellular loop in G-protein activation (7, 34, 44). Mutagenesis studies also indicate that the cytoplasmic C terminus is not needed for G-protein activation but is involved in down-regulation of receptors by endocytosis (17) and desensitization of receptors by phosphorylation (6). In addition, studies with chimeric receptors suggest that the specificity for α-factor binding is determined by discontinuous segments of the α-factor receptor that include the transmembrane and extracellular regions (36, 37). Although some of the important domains of the α-factor receptor have been identified in these studies, the molecular mechanism of receptor signaling remains to be determined.Dominant-negative (DN) mutants represent an important class of mutation in which a mutant receptor interferes with the function of the wild-type (WT) version of the receptor. Since the inhibitory phenotype in DN mutants implies loss of some but not all functions of the protein, these mutants have been used to great advantage in other receptor systems. For example, in the case of receptor tyrosine kinases, DN mutants have been used to assign particular functions to specific structural features or to study the effects of blocking receptor signaling (18). In view of the large number of mutations reported for GPCRs, it is intriguing that there are few examples of dominant GPCR mutations (42, 43). Furthermore, in cases where it has been examined, dominant mutations in GPCRs seem to affect primarily the targeting of receptors to the plasma membrane and not directly the function of the WT receptors. Therefore, we sought to determine if the analysis of DN mutants could be applied to GPCRs by taking advantage of the genetic accessibility of the yeast S. cerevisiae. In this report, we describe the identification of DN mutations in the α-factor pheromone receptor. Interestingly, our results indicate that these DN mutants interfere with the activity of the WT receptors by competing for the G protein. In addition, these mutations identify a new domain on the extracellular side of the TMDs that is important for receptor function.