The complete primary structure of the boar spermadhesin AQN-1, a carbohydrate-binding protein involved in fertilization.

Gamete recognition and adhesion are essential steps in the complex process of fertilization. In mammals and in other species, increasing evidence indicates that carbohydrate-binding proteins on the sperm surface play a pivotal role as counter-receptors for certain oligosaccharide moieties attached to the oocyte zona pellucida glycoproteins. Although different sperm-associated zona-pellucida-binding proteins have been identified in a number of species, few of them have been isolated and structurally characterized. In this paper we report the primary structural characterization of AQN-1, a 12-kDa boar-sperm-associated carbohydrate-binding and zona-pellucida-binding protein. The molecular mass of AQN-1 was determined by time-of-flight plasma-desorption mass spectrometry. Determination of its amino acid sequence and location of disulphide bridges were accomplished by a combination of proteochemical and mass spectrometric methods. The primary structure of AQN-1 failed to show any significant similarity to the protein structures deposited with the Martinsried Institute for Protein Sequences data bank, indicating that it may belong to a novel protein family involved in fertilization. AQN-1 shares extensive structural, as well as functional, similarity with two other boar sperm zona-pellucida-binding proteins, AQN-3 and AWN, which we have recently characterized. To name this protein family, we have coined the term spermadhesin. Our data may be relevant for identification of spermadhesins in other species, and thus may contribute to a better understanding of the species-specific sperm-egg recognition mechanism.     

Contribution to the study of cytokinin production by phytopathogenic fungi

The excretion of cytokinins into the cultivating medium, which are produced by the phytopathogenic fungiMonilia sp. andCytospora sp. has been investigated. All the isolates of the fungi used in the experiments (Monilia fructicola, Monilia fructigena, isolate 2 and 4,Monilia laxa isolate 3 andCytospora sp. isolate CPL and C₁) have been found to produce cytokinins. The production is increased during the formation of the fructification organs. Among the isolates investigatedMonilia fructigena isolate 4 andCytospora sp. isolate CPL showed the highest production of cytokinins. After chromatographic separation, cytokinin activity was found at R_F 0.7–0.8 values by the biological test as well as by identification according to UV spectra. Application of purified cytokinins produced by the fungi evoked the formation of “green islands” on isolated barley leaves underin vitro conditions.     

RANBP2 and USP9x regulate nuclear import of adenovirus minor coat protein IIIa

As intracellular parasites, viruses exploit cellular proteins at every stage of infection. Adenovirus outbreaks are associated with severe acute respiratory illnesses and conjunctivitis, with no specific antiviral therapy available. An adenoviral vaccine based on human adenovirus species D (HAdV-D) is currently in use for COVID-19. Herein, we investigate host interactions of HAdV-D type 37 (HAdV-D37) protein IIIa (pIIIa), identified by affinity purification and mass spectrometry (AP-MS) screens. We demonstrate that viral pIIIa interacts with ubiquitin-specific protease 9x (USP9x) and Ran-binding protein 2 (RANBP2). USP9x binding did not invoke its signature deubiquitination function but rather deregulated pIIIa-RANBP2 interactions. In USP9x-knockout cells, viral genome replication and viral protein expression increased compared to wild type cells, supporting a host-favored mechanism for USP9x. Conversely, RANBP2-knock down reduced pIIIa transport to the nucleus, viral genome replication, and viral protein expression. Also, RANBP2-siRNA pretreated cells appeared to contain fewer mature viral particles. Transmission electron microscopy of USP9x-siRNA pretreated, virus-infected cells revealed larger than typical paracrystalline viral arrays. RANBP2-siRNA pretreatment led to the accumulation of defective assembly products at an early maturation stage. CRM1 nuclear export blockade by leptomycin B led to the retention of pIIIa within cell nuclei and hindered pIIIa-RANBP2 interactions. In-vitro binding analyses indicated that USP9x and RANBP2 bind to C-terminus of pIIIa amino acids 386–563 and 386–510, respectively. Surface plasmon resonance testing showed direct pIIIa interaction with recombinant USP9x and RANBP2 proteins, without competition. Using an alternative and genetically disparate adenovirus type (HAdV-C5), we show that the demonstrated pIIIa interaction is also important for a severe respiratory pathogen. Together, our results suggest that pIIIa hijacks RANBP2 for nuclear import and subsequent virion assembly. USP9x counteracts this interaction and negatively regulates virion synthesis. This analysis extends the scope of known adenovirus-host interactions and has potential implications in designing new antiviral therapeutics.     

Species specificity and intraspecific variation in the chemical profiles of Heliconius butterflies across a large geographic range

In many animals, mate choice is important for the maintenance of reproductive isolation between species. Traits important for mate choice and behavioral isolation are predicted to be under strong stabilizing selection within species; however, such traits can also exhibit variation at the population level driven by neutral and adaptive evolutionary processes. Here, we describe patterns of divergence among androconial and genital chemical profiles at inter‐ and intraspecific levels in mimetic Heliconius butterflies. Most variation in chemical bouquets was found between species, but there were also quantitative differences at the population level. We found a strong correlation between interspecific chemical and genetic divergence, but this correlation varied in intraspecific comparisons. We identified “indicator” compounds characteristic of particular species that included compounds already known to elicit a behavioral response, suggesting an approach for identification of candidate compounds for future behavioral studies in novel systems. Overall, the strong signal of species identity suggests a role for these compounds in species recognition, but with additional potentially neutral variation at the population level.     

Cultivating <Emphasis Type="Italic">in vitro</Emphasis> coconut palms (<Emphasis Type="Italic">Cocos nucifera</Emphasis>) under glasshouse conditions with natural light,improves <Emphasis Type="Italic">in vitro</Emphasis> photosynthesis nursery survival and growth

Plantlets of coconut were cultured in vitro under three different ambient conditions including a standard culture room, a culture room inside a glasshouse with natural light but controlled temperature, and a standard glasshouse with natural light and natural fluctuations of temperature. Plantlets from the 3 treatments were compared in terms of growth, plant survival as well as net photosynthesis and efficiency of PSII (Fv/Fm ratio) both at the end of the in vitro stage and at 3 stages of ex vitro acclimatization. At the end of the in vitro stage, plantlets cultured in vitro under glasshouse conditions showed the best performance showing the highest photosynthesis rate, dry weight and number of leaves. Plantlets from the standard culture room showed the lowest photosynthesis and growth rate. After 6 months of ex vitro acclimatization, plantlets originally grown in vitro under glasshouse conditions maintained better field survival and growth rates in terms of fresh weight, dry weight and leaf number than plantlets originally grown in vitro in the standard culture room. Although more studies are required to define the reason for this effect, it is clear that the conditions of standard culture rooms are not the best for in vitro cultivation of coconut and perhaps other tropical species.     

Isolation and biochemical characterization of a zona pellucida-binding glycoprotein of boar spermatozoa.

Lectin-like molecules on the sperm surface are implicated in the process of gamete recognition and adhesion. We have isolated and biochemically characterized a 15 kDa glycoprotein from ejaculated boar sperm which possess zona pellucida-binding- and haemagglutinating-activity. The zona/15 kDa protein interaction is inhibited by fucoidan, suggesting that the glycoprotein is one of the sperm components which participate in the initial gamete interaction. N-Terminal sequence analysis of the isolated 15 kDa glycoprotein showed that it may belong to the same sperm/egg recognition-mediating protein family as the sea urchin sperm protein binding.