Transgenic mice with reduced numbers of functional sperm receptors on their eggs reproduce normally.

To initiate fertilization in mice, free-swimming sperm bind to mZP3, an approximately 83-kDa glycoprotein present in the ovulated egg zona pellucida (ZP). mZP3 is located periodically along the filaments that constitute the ZP. Sperm recognize and bind to specific oligosaccharides linked to one or more of five Ser residues clustered in the carboxy-terminal one-third of the mZP3 polypeptide. When all five Ser residues are converted to nonhydroxy amino acids by site-directed mutagenesis of the mZP3 gene, an inactive form of mZP3, called mZP3[ser], is secreted by embryonal carcinoma cells stably transfected with the mutated gene. Here, seven independent transgenic mouse lines were established that harbor the mutated mZP3 gene. In all lines, the mutant gene is expressed by growing oocytes and mZP3[ser] is synthesized, secreted, and incorporated into the ZP. Purified mZP3[ser] prepared from ovaries of transgenic mice, like mZP3[ser] from transfected embryonal carcinoma cells, is inactive in sperm binding assays in vitro. On the other hand, the presence of mZP3[ser] in the ZP does not significantly affect either the binding of sperm to ovulated eggs in vitro or the reproduction of the mice, i.e., the transgenic mice are fertile, breed at normal intervals, and produce litters of normal sizes. These results indicate that the number of functional sperm receptors in the ZP can be reduced by more than 50% without adversely affecting fertilization of eggs in vivo.     

Glycoprotein constituents of the vitelline coat of Phallusia mammillata (Ascidiacea) with fertilization inhibiting activity.

Vitelline coats (VCs) of Phallusia mammillata were isolated and purified following homogenization of live eggs in order to investigate the molecular basis of sperm-egg recognition. Clean VCs were partly solubilized by sonication in H2O and the soluble fraction (SFVC), derived from the outer surface of VCs, was used for further characterization. Electrophoretic analyses of radioiodinated VCs revealed that SFVC consists of two major glycoprotein components with apparent average Mr's of 450,000 and 180,000, respectively. The 450,000 Mr component is composed of several charge isomers, whereas the 180,000 Mr component is supposed to consist of two oligomers, both with acidic pI, held together by a disulfide linkage(s). Each of the two components possesses WGA-binding sites as shown by transblotting followed by WGA-peroxidase treatment. The amino acid composition of SFVC was determined after acid hydrolysis and its carbohydrate composition was analyzed and quantified by GLC. GlcNAc and GalNAc were found to predominate with 86% by weight of total sugar content and fucose, mannose, and glucose accounted for the remaining 14%. The susceptibility of SFVC to enzymatic (N-glycosidase F) and chemical (TFMS) deglycosylation as well as to protease (trypsin and chymotrypsin) digestion was investigated. Furthermore, sperm receptor activity of SFVC was tested in a fertilization assay. The fertilization rate decreased in a concentration-dependent manner when sperm were preincubated with SFVC. Additionally, sperm treated with SFVC showed binding for FITC-WGA or WGA-gold at the apical portion of the sperm head. Therefore, we strongly assume that one or both of the identified glycoprotein macromolecules of SFVC are involved in sperm-egg recognition.     

The ZP domain is a conserved module for polymerization of extracellular proteins

Many eukaryotic extracellular proteins share a sequence of unknown function, called the zona pellucida (ZP) domain. Among these proteins are the mammalian sperm receptors ZP2 and ZP3, non-mammalian egg coat proteins, Tamm-Horsfall protein (THP), glycoprotein-2 (GP-2), alpha- and beta-tectorins, transforming growth factor (TGF)-beta receptor III and endoglin, DMBT-1 (deleted in malignant brain tumour-1), NompA (no-mechanoreceptor-potential-A), Dumpy and cuticlin-1 (refs 1,2). Here, we report that the ZP domain of ZP2, ZP3 and THP is responsible for polymerization of these proteins into filaments of similar supramolecular structure. Most ZP domain proteins are synthesized as precursors with carboxy-terminal transmembrane domains or glycosyl phosphatidylinositol (GPI) anchors. Our results demonstrate that the C-terminal transmembrane domain and short cytoplasmic tail of ZP2 and ZP3 are not required for secretion, but are essential for assembly. Finally, we suggest a molecular basis for dominant human hearing disorders caused by point mutations within the ZP domain of alpha-tectorin.     

Variants in the interleukin-1 alpha and beta genes,and the risk for periodontal disease in dogs

Elevated levels of interleukin-1 (IL-1) have been shown to amplify the inflammatory response against periodontopathogenic bacteria. In humans, polymorphisms in the IL1A and IL1B genes are the most well-studied genetic polymorphisms associated with periodontal disease (PD). In contrast to human, there is a lack of knowledge on the genetic basis of canine PD. A case–control study was conducted in which a molecular analysis of dog IL1A and IL1B genes was performed. Of the eight genetic variants identified, seven in IL1A gene and one in IL1B gene, IL1A/1_g.388A >C and IL1A/1_g.521T >A showed statistically significant differences between groups (adjusted OR (95% CI): 0.15 (0.03–0.76), P= 0.022; 5.76 (1.03–32.1), P= 0.046, respectively). It suggests that in the studied population the IL1A/1_g.388C allele is associated with a decreased PD risk, whereas the IL1A/1_g.521A allele can confer an increased risk. Additionally, the IL1A/2_g.515G >T variation resulted in a change of amino acid, i.e. glycine to valine. In silico analysis suggests that this change can alter protein structure and function, predicting it to be deleterious or damaging. This work suggests that IL1 genetic variants may be important in PD susceptibility in canines.     

The promoter of filamentation (POF1) protein from <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> is an ATPase involved in the protein quality control process

Background

The gene YCL047C, which has been renamed promoter of filamentation gene (POF1), has recently been described as a cell component involved in yeast filamentous growth. The objective of this work is to understand the molecular and biological function of this gene.

Results

Here, we report that the protein encoded by the POF1 gene, Pof1p, is an ATPase that may be part of the Saccharomyces cerevisiae protein quality control pathway. According to the results, Δpof1 cells showed increased sensitivity to hydrogen peroxide, tert-butyl hydroperoxide, heat shock and protein unfolding agents, such as dithiothreitol and tunicamycin. Besides, the overexpression of POF1 suppressed the sensitivity of Δpct1, a strain that lacks a gene that encodes a phosphocholine cytidylyltransferase, to heat shock. In vitro analysis showed, however, that the purified Pof1p enzyme had no cytidylyltransferase activity but does have ATPase activity, with catalytic efficiency comparable to other ATPases involved in endoplasmic reticulum-associated degradation of proteins (ERAD). Supporting these findings, co-immunoprecipitation experiments showed a physical interaction between Pof1p and Ubc7p (an ubiquitin conjugating enzyme) in vivo.

Conclusions

Taken together, the results strongly suggest that the biological function of Pof1p is related to the regulation of protein degradation.

     

Cerebral near infrared spectroscopy and acupuncture--results of a pilot study]

For the first time, we have studied the effects of manual acupuncture and of laser puncture on cerebral oxygenation in three healthy male volunteers using transcranial near-infrared spectroscopy (NIRO 300, Hamamatsu Photonics, Japan). The results indicate that acupuncture of specific acupuncture points leads to an increase in oxygenated haemoglobin and in the tissue oxygenation index. However, needling and laser puncture at placebo points does not produce the same effect on cerebral oxygenation. This fact provides further proof of specific quantifiable effects of acupuncture on the brain.     

Testing the quick meal hypothesis: The effect of pulp on hoarding and seed predation of Hymenaea courbaril by red‐rumped agoutis (Dasyprocta leporina)

Abstract: Red‐rumped agoutis (Dasyprocta leporina) are important seed dispersers/predators of Neotropical large‐seeded plants. Several species of seeds cached by agoutis have an edible reward, in contrast to temperate rodent‐dispersed diaspores. The quick meal hypothesis states that the presence of a reward such as edible pulp will enhance the efficiency of rodents as seed disperses by satiating the animal and, consequently, reducing seed predation and enhancing hoarding. In this study, this hypothesis was tested using as the reference system the pulp and seeds of Hymenaea courbaril. Seeds with and without pulp were offered to agoutis and the behaviour of each individual was recorded. Since the probability of predation and hoarding were complementary, we used the probability of predation. The proportion of agoutis that preyed on at least one seed was similar for seeds with (42.8% of individuals) and without (40.0% of individuals) pulp. In agoutis that preyed upon at least one seed, the probability that they killed a seed did not differ between seeds with (0.17 ± 0.03) and without (0.20 ± 0.08) pulp. Hence, these results do not support the ‘quick meal hypothesis’.     

Extrafloral nectaries as a deterrent mechanism against seed predators in the chemically protected weed Crotalaria pallida (Leguminosae)

Abstract In several plants, extrafloral nectaries (EFN) are located close to the reproductive structures, suggesting that ants may act as a defence against specialized seed predators that overcome chemical defences. Alternatively, ants may also deter herbivores in a generalized manner, thereby protecting the whole plant. In this work, we examined the relationship between the chemically protected weed Crotalaria pallida Ait. (Leguminosae) that bears EFN, its specialized seed predator, the larvae of the arctiid moth Utetheisa ornatrix L. (Arctiidae) and ants. We tested two hypotheses related to the type of deterrence caused by ants. The Seed Predator Deterrence Hypothesis predicts that ant deterrence is directed primarily towards herbivores that destroy seeds and other reproductive structures, without attacking herbivores on vegetative structures. The General Deterrence Hypothesis states that ants are general in their effects, equally deterring herbivores in vegetative and reproductive structures. Our results supported the predictions of the Seed Predator Deterrence Hypothesis, namely, that (i) ant activity on EFN was related to the vulnerability of reproductive structures to attack by U. ornatrix; (ii) ant patrolling was restricted almost entirely to racemes; (iii) ants removed termites used as baits more frequently on racemes than on leaves; and (iv) U. ornatrix larvae were often expulsed from the racemes. These results indicate that EFN can act as another deterrent mechanism in chemically protected plants by promoting the expulsion of specialist seed predators.