Identification of Selected Gamma-Ray Induced Deficiencies in Zebrafish Using Multiplex Polymerase Chain Reaction

The ease with which mutations can be generated in zebrafish makes this vertebrate an important resource for developmental genetics and genome studies. We have developed a PCR-based screening method that allows the efficient identification of gamma-ray induced deficiencies targeted to selected sequences. We describe three mutants characteristic of our findings and show that these mutations include deletions and translocations that can affect as much as 1% of the genome. These deficiencies provide a basis for analyzing the functions of cloned zebrafish genes using noncomplementation screens for point mutations induced by high-efficiency chemical mutagenesis.     

A SIGNAL GLYCOPROTEIN WITH α-d-MANNOSYL RESIDUES IS INVOLVED IN THE WOUND-HEALING RESPONSE OF ANTITHAMNION SPARSUM (CERAMIALES,RHODOPHYTA)1

A variety of fluorescein isothiocyanate-labeled lectins specific for different sugar moieties were examined as probes for the wound-healing response in the filamentous red alga Antithamnion sparsum Tokida. Among them, only concanavalin A (ConA) and Lens culrinaris agglutinin (LCA), which have specificity to α-D-mannosyl residues, bound specifically to repair cells during the wound-healing process. When ConA or LCA was added at various time intervals after wounding, it first bound (3 h post-wounding) as a thin layer at the tips of the adjacent cells. Later (4–5 h post-wounding) labeling also appeared at the tips of the repair cells. Intense labeling at these sites continued throughout the healing process until repair cell fusion, at which time the lectin labeling was reduced to a narrow ring around the area of fusion. When added to plants prior to wounding and continually monitored, these same lectins acted as inhibitors to the wound-healing response. Other control lectins showed no inhibitory effects. A crude extract solution obtained from decapitated filaments stimulated the wound-healing response, and a lectin-binding component bound strongly to a protein-binding transfer membrane. These results suggest that the labeled compound is a glycoprotein that has α-D-mannosyl residues and is similar to the repair hormone rhodomorphin found in Griffithsia pacifica Kylin.     

Anatomisch-physiologische Untersuchungen an Blütennectarien

Ohne Zusammenfassung     

Nutrient balance affects foraging behaviour of a trap-building predator

Predator foraging may be affected by previous prey capture, but it is unknown how nutrient balance affects foraging behaviour. Here, we use a trap-building predator to test whether nutrients from previous prey captures affect foraging behaviour. We fed orb-weaving spiders (Zygiella x-notata) prey flies of different nutrient composition and in different amounts during their first instar and measured the subsequent frequency of web building and aspects of web architecture. We found that both the likelihood of web building and the number of radii in the web were affected by prey nutrient composition while prey availability affected capture area and mesh height. Our results show that both the balance of nutrients in captured prey and the previous capture rate may affect future foraging behaviour of predators.     

Glycopeptide-preferring Polypeptide GalNAc Transferase 10 (ppGalNAc T10), Involved in Mucin-type O-Glycosylation, Has a Unique GalNAc-O-Ser/Thr-binding Site in Its Catalytic Domain Not Found in ppGalNAc T1 or T2

Mucin-type O-gly co sy la tion is initiated by a large family of UDP-GalNAc:polypeptide α-N-acetylgalactosaminyltransferases (ppGalNAc Ts) that transfer GalNAc from UDP-GalNAc to the Ser and Thr residues of polypeptide acceptors. Some members of the family prefer previously gly co sylated peptides (ppGalNAc T7 and T10), whereas others are inhibited by neighboring gly co sy la tion (ppGalNAc T1 and T2). Characterizing their peptide and glycopeptide substrate specificity is critical for understanding the biological role and significance of each isoform. Utilizing a series of random peptide and glycopeptide substrates, we have obtained the peptide and glycopeptide specificities of ppGalNAc T10 for comparison with ppGalNAc T1 and T2. For the glycopeptide substrates, ppGalNAc T10 exhibited a single large preference for Ser/Thr-O-GalNAc at the +1 (C-terminal) position relative to the Ser or Thr acceptor site. ppGalNAc T1 and T2 revealed no significant enhancements suggesting Ser/Thr-O-GalNAc was inhibitory at most positions for these isoforms. Against random peptide substrates, ppGalNAc T10 revealed no significant hydrophobic or hydrophilic residue enhancements, in contrast to what has been reported previously for ppGalNAc T1 and T2. Our results reveal that these transferases have unique peptide and glycopeptide preferences demonstrating their substrate diversity and their likely roles ranging from initiating transferases to filling-in transferases.Mucin-type O-glycosylation is a common post-translational modification of secreted and membrane-associated proteins. O-Glycan biosynthesis is initiated by the transfer of GalNAc from UDP-GalNAc to the hydroxyl groups of serine or threonine residues in a polypeptide, catalyzed by a family of polypeptide N-α-acetylgalactosaminyltransferases (ppGalNAc Ts).⁵ To date, 16 mammalian members have been reported in the literature (1–16) with a total of at least 20 members currently present in the human genome data base. Multiple members of the ppGalNAc T family have also been identified in Drosophila (9, 10, 14), Caenorhabditis elegans (3, 8), and single and multicellular organisms (17–20). Several members show close sequence orthologues across species suggesting that the ppGalNAc Ts are responsible for biologically significant functions that have been conserved during evolution. For example, in Drosophila four isoforms have close sequence orthologues to the mammalian transferases. Of the two that have been recently compared, nearly identical peptide substrate specificities have been observed between the fly and mammals, suggesting common but presently unknown functions preserved across these diverse species (21).Recently, several ppGalNAc T isoforms have been shown to be important for normal development or cellular processes. For example, inactive mutations in the fly PGANT35A (the T11 orthologue in mammals) are lethal because of the disruption of the tracheal tube structures (9, 10, 22), whereas mutations in PGANT3 alter epithelial cell adhesion in the Drosophila wing blade resulting in wing blistering (23). In humans, mutations in ppGalNAc T3 are associated with familial tumoral calcinosis, the result of the abnormal processing and secretion of the phosphaturic factor FGF23 (24, 25). Human ppGalNAc T14 has been suggested to modulate apoptotic signaling in tumor cells by its glycosylation of the proapoptotic receptors DLR4 and DLR5 (26), and very recently the specific O-glycosylation of the TGFB-II receptor (ActR-II) by the GalNTL1 has been shown to modulate its signaling in development (16).Historically, the major targets of the ppGalNAc Ts have been thought to be heavily O-glycosylated mucin domains of membrane and secreted glycoproteins. Such domains typically contain 15–30% Ser or Thr, which are highly (>50%) substituted by GalNAc. One question in the field is as follows. How is this high degree of peptide core glycosylation achieved and is it related to the large number of ppGalNAc isoforms, some of which may even have specific mucin domain preferences? Interestingly, some members of the ppGalNAc T family are known to prefer substrates that have been previously modified with O-linked GalNAc on nearby Ser/Thr residues, hence having so-called glycopeptide or filling-in activities, i.e. ppGalNAc T7 and T10 (8, 27–29). Others simply possess altered preferences against glycopeptide substrates, i.e. ppGalNAc T2 and T4 (30–33), or may be inhibited by neighboring glycosylation, i.e. ppGalNAc T1 and T2 (29, 34, 35). These latter transferases have been called early or initiating transferases, preferring nonglycosylated over-glycosylated substrates. Presently, little is known about which factors dictate the different peptide/glycopeptide specificities among the ppGalNAc Ts.The ppGalNAc Ts consist of an N-terminal catalytic domain tethered by a short linker to a C-terminal ricin-like lectin domain containing three recognizable carbohydrate-binding sites (36). Because ppGalNAc T7 and T10 prefer to transfer GalNAc to glycopeptide acceptors, it has been widely assumed that their C-terminal lectin domains would play significant roles in this activity, as has been demonstrated for other family members (27, 28, 32). Recently, Kubota et al. (37) solved the crystal structure of ppGalNAc T10 in complex with Ser-GalNAc specifically bound to its lectin domain. In this work (37), the authors further demonstrated that a T10 lectin domain mutant indeed had altered specificity against GalNAc-containing glycopeptide substrates when the acceptor Ser/Thr site was distal from the pre-existing glycopeptide GalNAc site. However, it was also observed that the lectin mutant still possessed relatively unaltered glycopeptide activity when the acceptor Ser/Thr site was directly N-terminal of a pre-existing glycopeptide GalNAc site. Kubota et al. (37) therefore concluded that for ppGalNAc T10, both its lectin and indeed its catalytic domain must contain distinct peptide GalNAc recognition sites. In support of this, Raman et al. (33) have shown that the complete removal of the ppGalNAc T10 lectin domain only slightly alters its specificity against distal glycopeptide substrates while showing no difference in its ability to glycosylate residues directly N-terminal of an existing site of glycosylation. Thus, it seems that the catalytic domain of ppGalNAc T10 may have specific requirements for a peptide O-linked GalNAc in at least the +1 position (toward the C terminus) of residues being glycosylated. As no systematic determination of the glycopeptide binding properties of the ppGalNAc Ts catalytic domain has been performed, it is unknown whether additional GalNAc peptide-binding sites exist in T10 or, for that matter, any of the other ppGalNAc Ts.We have recently reported the use of oriented random peptide substrates, GAGA(X)_nT(X)_nAGAGK (where X indicates randomized amino acid positions and n = 3 and 5) for determining the peptide substrate specificities of mammalian ppGalNAc T1, T2, and their fly orthologues (21, 38). In the present work, we extend this approach to the determination of the catalytic domain glycopeptide (Ser/Thr-O-GalNAc) substrate preferences for ppGalNAc T1, T2, and T10 employing two n = 4 oriented random glycopeptide libraries (21). Interestingly, ppGalNAc T10 displays few significant enhancements and specifically lacks the Pro residue enhancements observed for ppGalNAc T1 and T2. These findings further demonstrate the vast substrate diversity of the catalytic domains of the ppGalNAc T family of transferases.

TABLE 1

ppGalNAc transferase random substrates utilized in this workPVI, PVII, GP-I, and GP-II random (glyco)peptide substrates.

Repair of hydrolytic DNA deamination damage in thermophilic bacteria: cloning and characterization of a Vsr endonuclease homolog from Bacillus stearothermophilus

Hydrolytic deamination of 5-methyl cytosine in double stranded DNA results in formation of a T/G mismatch that—if left unrepaired—leads to a C→T transition mutation in half of the progeny. In addition to several mismatch-specific glycosylases that have been found in both pro- and eukaryotes to channel this lesion into base excision repair by removing the T from the mismatch, Vsr endonuclease from Escherichia coli has been described which initiates repair by an endonucleolytic strand incision 5′ to the mismatched T. We have isolated a gene coding for a homolog of E.coli Vsr endonuclease from the thermophilic bacterium Bacillus stearothermophilus H3 (Vsr.Bst) using a method that allows PCR amplification with degenerated primers of gene segments which code for only one highly conserved amino acid region. Vsr.Bst was produced heterologously in E.coli and purified to apparent homogeneity. Vsr.Bst specifically incises heteroduplex DNA with a preference for T/G mismatches. The selectivity of Vsr.Bst for the sequence context of the T/G mismatch appears less pronounced than for Vsr.Eco.     

Natural use of heterothermy by a small, tree-roosting bat during summer

Little is known about the use of heterothermy by wild bats during summer, especially for tree-roosting species. Because thermal conditions within tree roosts can fluctuate widely with ambient temperature, which affects thermoregulatory energy expenditure during diurnal roosting, we measured skin temperatures of free-ranging male Nyctophilus geoffroyi (8 g) to quantify the relation between summer torpor use and roost thermal conditions. Bats roosted under bark on the northern (sunny) side of trees and entered torpor every day, usually near sunrise. Bats exhibited two bouts of torpor on most days: the first occurred in the morning, was terminated by partially passive rewarming, and was followed by a period of normothermy during the warmest part of the day; a second torpor bout occurred in the late afternoon, with arousal near sunset. On the warmest days, bats had only a single, short morning bout. On the coolest days, bats remained torpid throughout the day, and one 2-d bout was observed. Thus, presumably owing to their poorly insulated roosts and the high energetic cost of normothermy at temperatures below 30 degrees C, the extent and timing of heterothermy was closely related to the cycle of diurnal temperatures. Our study indicates that torpor use is important for energy maintenance during summer diurnal roosting of N. geoffroyi and likely of other small, tree-roosting bats.     

Effect of nitrogen fertilizer level on infestations of lepidopterous stemborers and yields of maize in Zanzibar

The effect of nitrogen levels of 0, 60, 120, and 250 kg/ha and insecticide treatment (Furadan) on population densities and parasitism of lepidopteran stemborers, and maize yields were studied in Zanzibar during 2004/05. Chilo partellus (Swinhoe) (Lepidoptera: Crambidae) dominated by 3-fold over Sesamia calamistis Hampson (Lepidoptera: Noctuidae) and 42 fold over Chilo orichalcociliellus Strand (Lepidoptera: Crambidae). Stemborer density per plant and parasitism by Cotesia flavipes (Cameron) (Hymenoptera: Braconidae) increased with nitrogen application level. Percentage of bored internodes per plant caused by stemborer decreased with N levels during the short rainy season. Pesticide application reduced densities of all stemborer species during the short rainy season, when infestations were high. Maize yield increased 2 to 8 times with N level, compared to the zero treatment, but the effect was less pronounced in the protected plots.     

Cryptosporidium and Giardia in marine-foraging river otters (Lontra canadensis) from the Puget Sound Georgia Basin ecosystem

Species of Cryptosporidium and Giardia can infect humans and wildlife and have the potential to be transmitted between these 2 groups; yet, very little is known about these protozoans in marine wildlife. Feces of river otters (Lontra canadensis), a common marine wildlife species in the Puget Sound Georgia Basin, were examined for species of Cryptosporidium and Giardia to determine their role in the epidemiology of these pathogens. Using ZnSO4 flotation and immunomagnetic separation, followed by direct immunofluorescent antibody detection (IMS/DFA), we identified Cryptosporidium sp. oocysts in 9 fecal samples from 6 locations and Giardia sp. cysts in 11 fecal samples from 7 locations. The putative risk factors of proximate human population and degree of anthropogenic shoreline modification were not associated with the detection of Cryptosporidium or Giardia spp. in river otter feces. Amplification of DNA from the IMS/DFA slide scrapings was successful for 1 sample containing > 500 Cryptosporidium sp. oocysts. Sequences from the Cryptosporidium 18S rRNA and the COWP loci were most similar to the ferret Cryptosporidium sp. genotype. River otters could serve as reservoirs for Cryptosporidium and Giardia species in marine ecosystems. More work is needed to better understand the zoonotic potential of the genotypes they carry as well as their implications for river otter health.