Characterization of WWOX inactivation in murine mammary gland development

The WW domain‐containing oxidoreductase (WWOX) is commonly inactivated in multiple human cancers, including breast cancer. Wwox null mice die prematurely precluding adult tumor analysis. Nevertheless, aging Wwox‐heterozygous mice at C3H genetic background develop higher incidence of mammary tumors. We recently generated a Wwox conditional knockout mouse in which loxp sites flank exon 1 in the Wwox allele and showed that total ablation of WWOX in these mice resembles that of conventional targeting of Wwox. Here, we report the characterization of WWOX ablation in mouse mammary gland using MMTV‐Cre transgenic line. We demonstrated that WWOX ablation leads to impaired mammary ductal growth. Moreover, targeted deletion of WWOX is associated with increased levels of fibronectin, a component of the extracellular matrix. In addition, we showed that shRNA knockdown of WWOX in MCF10A breast epithelial cells dramatically increased fibronectin and is associated with enhanced cell survival and impaired growth in three‐dimensional culture Matrigel assay. Taken together our results are consistent with a critical role for WWOX in normal breast development and tumorigenesis. J. Cell. Physiol. 228: 1391–1396, 2013. © 2012 Wiley Periodicals, Inc.     

Generation and Characterization of Mice Carrying a Conditional Allele of the Wwox Tumor Suppressor Gene

WWOX, the gene that spans the second most common human chromosomal fragile site, FRA16D, is inactivated in multiple human cancers and behaves as a suppressor of tumor growth. Since we are interested in understanding WWOX function in both normal and cancer tissues we generated mice harboring a conditional Wwox allele by flanking Exon 1 of the Wwox gene with LoxP sites. Wwox knockout (KO) mice were developed by breeding with transgenic mice carrying the Cre-recombinase gene under the control of the adenovirus EIIA promoter. We found that Wwox KO mice suffered from severe metabolic defect(s) resulting in growth retardation and all mice died by 3 wk of age. All Wwox KO mice displayed significant hypocapnia suggesting a state of metabolic acidosis. This finding and the known high expression of Wwox in kidney tubules suggest a role for Wwox in acid/base balance. Importantly, Wwox KO mice displayed histopathological and hematological signs of impaired hematopoeisis, leukopenia, and splenic atrophy. Impaired hematopoeisis can also be a contributing factor to metabolic acidosis and death. Hypoglycemia and hypocalcemia was also observed affecting the KO mice. In addition, bone metabolic defects were evident in Wwox KO mice. Bones were smaller and thinner having reduced bone volume as a consequence of a defect in mineralization. No evidence of spontaneous neoplasia was observed in Wwox KO mice. We have generated a new mouse model to inactivate the Wwox tumor suppressor gene conditionally. This will greatly facilitate the functional analysis of Wwox in adult mice and will allow investigating neoplastic transformation in specific target tissues.     

Conditional inactivation of the mouse Wwox tumor suppressor gene recapitulates the null phenotype

WW domain‐containing oxidoreductase (WWOX) is highly conserved in both human and murine. WWOX spans the second most common human chromosomal fragile site, FRA16D, and is commonly inactivated in multiple human cancers. Modeling WWOX inactivation in mice revealed a complex phenotype including postnatal lethality, defects in bone metabolism and steroidogenesis and tumor suppressor function resulting in osteosarcomas. For better understanding of WWOX roles in different tissues at distinct stages of development and in pathological conditions, Wwox conditional knockout mice were generated in which loxp sites flank exon 1 in the Wwox allele. We demonstrated that Cre‐mediated recombination using EIIA‐Cre, a Cre line expressed in germline, results in postnatal lethality by age of 3 weeks and decreased bone mineralization resembling total ablation of WWOX as in conventional null mice. This animal model will be useful to study distinct roles of WWOX in multiple tissues at different ages. J. Cell. Physiol. 228: 1377–1382, 2013. © 2012 Wiley Periodicals, Inc.     

The tumor suppressor WW domain-containing oxidoreductase modulates cell metabolism

The WW domain-containing oxidoreductase (WWOX) encodes a tumor suppressor that is frequently altered in cancer. WWOX binds several proteins and thus is postulated to be involved in a variety of cellular processes. Interestingly, Wwox-knockout mice develop normally in utero but succumb to hypoglycemia and other metabolic defects early in life resulting in their death by 3–4 weeks of age. Cumulative evidence has linked WWOX with cellular metabolism including steroid metabolism, high-density lipoprotein cholesterol (HDL-C) metabolism, bone metabolism and, more recently, glucose metabolism. In this review, we discuss these evolving functions for WWOX and how its deletion affects cellular metabolism and neoplastic progression.     

Caprine blood groups. 2. The C,G, H,I, J,K, L,N, and Q systems

Nine blood group systems of goats were identified using 12 caprine reagents produced by absorption of alloimmune antisera. The caprine C blood group system, possibly homologous to the ovine C blood group system, was characterized by two reagents and shown to be controlled by three alleles,C ¹²,C ²⁵, andC ⁻. A more complex blood group system of goats, designated G, was identified using three reagents and shown to be controlled by six codominant alleles (G ^10.19.20,G ^10.19,G ^10.20,G ¹⁰,G ¹⁹,G ²⁰) and a recessive allele (G ⁻). A further seven one-factor two-allelic systems were identified by seven reagents. The nine genetic systems provided exclusion probabilities of 0.479, 0.492, 0.548, and 0.572 in Australian Angora, Dairy, Cashmere, and Texan Angora goat breeds, respectively. This work was supported by a grant from the Australian Stud Book, Alison Road, Randwick, New South Wales 2031, Australia.     

HETEROCHROMATIN BANDING IN BOYKINIA,HEUCHERA, MITELLA,SULLIVANTIA, TIARELLA,AND TOLMIEA (SAXIFRAGACEAE)

Karyotypic differences were sought among species of Boykinia, Heuchera, Mitella, Sullivantia, Tiarella, and Tolmiea utilizing a modification of the Hy-banding technique. Prominent centromeric and some telomeric heterochromatin banding was observed. Boykinia aconitifolia and species of Sullivantia possess an identical banded karyotype, while four species of Heuchera, Mitella diphylla, Tiarella cordifolia, and Tolmiea menziesii (the latter at the tetraploid level) are characterized by a second, slightly different banded karyotype. In Sullivantia, Giemsa C-banding stains the same chromosomal regions revealed by Hy-banding. Larger amounts of heterochromatin are present in chromosomes of species of Heuchera, Mitella, Tiarella, and Tolmiea than in chromosomes of Sullivantia species and Boykinia aconitifolia. These karyological observations confirm generic relationships and demonstrate the systematic applicability of chromosome banding techniques to plants with very small chromosomes.     

Evolutionary chromosomal differentiation among four species of Conoderus Eschscholtz, 1829 (Coleoptera, Elateridae, Agrypninae, Conoderini) detected by standard staining, C-banding, silver nitrate impregnation, and CMA3/DA/DAPI staining

The speciose Brazilian Elateridae fauna is characterized by high karyotypic diversity, including one species (Chalcolepidius zonatus Eschscholtz, 1829) with the lowest diploid number within any Coleoptera order. Cytogenetic analysis of Conoderus dimidiatus Germar, 1839, C. scalaris (Germar, 1824,) C. ternarius Germar, 1839, and C. stigmosus Germar, 1839 by standard and differential staining was performed with the aim of establishing mechanisms of karyotypic differentiation in these species. Conoderus dimidiatus, C. scalaris, and C. ternarius have diploid numbers of 2n(♂) = 17 and 2n(♀) = 18, and a X0/XX sex determination system, similar to that encountered in the majority of Conoderini species. The karyotype of C. stigmosus was characterized by a diploid number of 2n=16 and a neoXY/neoXX sex determination system that was highly differentiated from other species of the genus. Some features of the mitotic and meiotic chromosomes suggest an autosome/ancestral X chromosome fusion as the cause of the neoXY system origin in C. stigmosus. C-banding and silver impregnation techniques showed that the four Conoderus species possess similar chromosomal characteristics to those registered in most Polyphaga species, including pericentromeric C band and autosomal NORs. Triple staining techniques including CMA₃/DA/DAPI also provided useful information for differentiating these Conoderus species. These techniques revealed unique GC-rich heterochromatin associated with NORs in C. scalaris and C. stigmosus and CMA₃-heteromorphism in C. scalaris and C. ternarius.     

Analysis of Sharksucker, Echeneis naucrates, Induced Behavior Patterns in the Blacktip Shark, Carcharhinus limbatus

I analyzed and quantified the behavior of blacktip sharks, Carcharhinus limbatus, irritated by sharksuckers, Echeneis naucrates, attached to their bodies. The sharksucker induced behavior patterns were divided into simple rotational patterns, expressed by body turns along their main axes (roll, yaw, pitch); and complex patterns (wiggle, shake, wind, chafe), consisting of a combination or a repetition of simple behavior patterns. Three nonrotational behavior patterns were also observed (freeze, yawn, flicker). Of the 78 blacktip sharks examined, 70 performed either a simple or a complex pattern, or a combination of the two types. Of the total 195 rotational behavior patterns observed, only three wind patterns, performed as chaffing, led to the dislocation of a sharksucker, implying that the shark's primary intention may not be to shake off the teleost, but to initiate a position change to a less sensitive part of its body. Three sharks showed behavior patterns resembling typical agonistic display patterns, but they were induced by sharksuckers, not triggered by the presence of humans.     

Purification, characterization, and sequencing of antimicrobial peptides, Cy-AMP1, Cy-AMP2, and Cy-AMP3, from the Cycad (Cycas revoluta) seeds

Novel antimicrobial peptides (AMP), designated Cy-AMP1, Cy-AMP2, and Cy-AMP3, were purified from seeds of the cycad (Cycas revoluta) by a CM cellulofine column, ion-exchange HPLC on SP COSMOGEL, and reverse-phase HPLC. They had molecular masses of 4583.2 Da, 4568.9 Da and 9275.8 Da, respectively, by MALDI–TOF MS analysis. Half of the amino acid residues of Cy-AMP1 and Cy-AMP2 were cysteine, glycine and proline, and their sequences were similar. The sequence of Cy-AMP3 showed high homology to various lipid transfer proteins. For Cy-AMP1 and Cy-AMP2, the concentrations of peptides required for 50% inhibition (IC₅₀) of the growth of plant pathogenic fungi, Gram-positive and Gram-negative bacteria were 7.0–8.9 μg/ml. The Cy-AMP3 had weak antimicrobial activity. The structural and antimicrobial characteristics of Cy-AMP1 and Cy-AMP2 indicated that they are a novel type of antimicrobial peptide belonging to a plant defensin family.     

Phylogenetic hypotheses: Cladoniaceae,Stereocaulaceae, Baeomycetaceae,and Icmadophilaceae revisited

Parsimony analyses of SSU rDNA sequences were conducted to examine phylogenetic relationships of selected genera within the families Cladoniaceae, Stereocaulaceae, Icmadophilaceae and Baeomycetaceae (lichen-forming ascomycetes). The analyses included 93 taxa (84 species) representing various groups of ascomycetous fungi. Analyses of the matrix with pre-aligned sequences were performed using heuristic and parsimony ratchet searches, and support values for the same matrix were calculated using parsimony jackknifing. The results support the recognition of the four families. Cladoniaceae are recircumscribed to accommodate Cladia, Cladonia, Heterodea, Metus, Pilophorus, Pycnothelia, Ramalea, Thysanothecium and the newly erected genus Carassea. Myelorrhiza is excluded from the family, while the status of other potential members, Calathaspis, Gymnoderma s.str. and Squamella, remains unresolved. Baeomycetaceae include Baeomyces and Phyllobaeis. Stereocaulaceae include Stereocaulon only, although the status of Muhria is still unclear. Finally, Icmadophilaceae include Dibaeis, Endocena, Knightiella, Icmadophila, Siphula and Thamnolia, while the status of Pseudobaeomyces and Siphulella requires further elucidation. The genus Cladonia appeared to be a polyphyletic assemblage, and accordingly, a new genus Carassea S. Stenroos, gen. nov., represented by C. connexa (Vain.) S. Stenroos, comb. nov., is described. Carassea is most closely related to Pycnothelia and Metus in the Cladoniaceae. Siphula, represented in the present analysis by six species, is not monophyletic, and is in need of reclassification.     

SPORE MORPHOLOGY IN THE CYATHEACEAE. II. THE GENERA LOPHOSORIA,METAXYA, SPHAEROPTERIS,ALSOPHILA, AND NEPHELEA

Scanning electron microscopy and transmitted light microscopy are used in a palynological study of Lophosoria, Metaxya, Sphaeropteris, Alsophila, and Nephelea of the tree fern family Cyatheaceae. The monotypic American genera Lophosoria and Metaxya each have a unique spore morphology which reinforces the taxonomic distinctness of these genera as indicated by their other characters. All investigated paleotropical species of Sphaeropteris develop a single type of perine characterized by coarse, pointed projections. In the neotropics, the Sphaeropteris horrida group shares this perine type, whereas all other neotropical Sphaeropteris species appear to have a different kind of perine with fine hair-like processes. The exine in paleotropical Sphaeropteris appears uniformly unsculptured, whereas in the neotropics several exine morphologies are found. In Alsophila all investigated neotropical species and the vast majority of the paleotropical species are characterized by a basically ridged perine morphology and an unsculptured exine. In several paleotropical Alsophila species, however, a perine with hair-like processes similar to those in neotropical Sphaeropteris is found, and the exine in several species is variously pitted. In at least one paleotropical Alsophila species, the porate exine morphology is indistinguishable from that in the neotropical genus Cnemidaria. The spores of the American genus Nephelea are similar to those of the majority of Alsophila species in ridged perine morphology and unsculptured exine. Several new instances of atypical spore numbers per sporangium are reported in Sphaeropteris and Alsophila. These and the palynological data are discussed in a taxonomic framework. The spore morphology in these genera is consistent with Tryon's recent generic revision of the family.     

Localization of growth arrest-specific genes on mouse Chromosomes 1, 7, 8, 11, 13, and 16

Growth arrest in NIH3T3 cells is associated with increased expression of a variety of mRNAs, several of which have been isolated as cDNA clones. Six of these growth arrest-specific (Gas) genes were mapped by following the inheritance of DNA restriction fragment length variants (RFLVs) associated with them in panels of recombinant inbred (RI) strains of mice and in the progeny of backcrosses both between laboratory mouse strains and between a laboratory strain and Mus spretus. The six genes are unlinked. Gas-1 maps to Chromosome (Chr) 13, Gas-2 to Chr 7, Gas-3 to Chr 11, Gas-4 to Chr 16, Gas-6 to Chr 8, and Gas-10 to Chr 1.     

Taxa, individuals, clusters and a few other things

The recognition of species proceeds by two fairly distinct phases: (1) the sorting of individuals into groups or basic taxa (‘discovery’) (2) the checking of those taxa as candidates for species-hood (‘justification’). The target here is a rational reconstruction of phase 1, beginning with a discussion of key terms. The transmission of ‘meaning’ is regarded as bimodal: definition states the intension of the term, and diagnosis provides a disjunction of criteria for recognition of its extension. The two are connected by a spectrum, with purely theoretical definition at one pole and purely ‘operational’ diagnosis at the other, with the more operational elements explained by the more theoretical. The current plethora of species concepts provides a good example. Accepting the Ghiselin–Hull thesis, that a species is an individual, a basic taxon is therefore also an individual with organisms as its parts. In a generalised synchronic individual its parts are conceptually integrated by an integrating principle (IP), which consists of a relation applying within a plan or rule. Fully developed, such an IP ensures the maximisation of the information content of the individual. A diachronic individual is then the set of its component synchronic parts, and its IP is provided by near-identity in an appropriate space-time (not necessarily physical). The integration of parts of an individual is illuminated by Gasking's concept of a proper group (in this case a chain-group), whose members are related by the relation serially fitting together with, the IP completed by an appropriate plan or rule. Gasking also applied the term cluster to a proper group persisting over a substantial period of time, individuated by any member acting as focus. A basic taxon is therefore a cluster of individuals, integrated by the relation significantly taxonomically similar and the rule in character-space-time. The nature of those concepts is discussed and defended. A species will inherit certain of the attributes of the preceding basic taxon (taxa). In at least the synchronic version its parts (individuals) will resemble each other significantly, providing the intuitive applicability of ‘members of’ and ‘instances of’ a species. Also, the notion that a species’ name is given by pure ostension, via a name-bearer (holotype) is empirically incoherent: the name cannot be applied in practice without an appropriate set of diagnostic traits.     

Incrimination of the mosquito,Aedes taeniorhynchus,as the primary vector of heartworm,Dirofilaria immitis,in coastal Yucatan,Mexico

Mosquito collections were carried out on microfilaraemic dogs, positive for Dirofilaria sp., for 18 consecutive nights in the coastal town of Celestún, Yucatan, southeast Mexico, during the rainy season (August) of 2007. A total of 292 female mosquitoes representing 12 species of dipteran Culicidae were collected: Anopheles albimanus (Wiedemann); Anopheles crucians (Wiedemann); Anopheles pseudopunctipennis (Theobald); Culex coronator (Dyar & Knab); Culex interrogator (Dyar & Knab); Culex nigripalpus (Theobald); Culex quinquefasciatus (Say); Culex salinarius (Coquillett); Aedes aegypti (Linnaeus); Aedes scapularis (Rondani); Aedes sollicitans (Walker), and Aedes taeniorhynchus (Wiedemann). Aedes taeniorhynchus and Cx. quinquefasciatus were the species found most commonly feeding on the dogs. Filarial nematodes were observed by microscopy in nine of the mosquito species collected; however, third‐instar larvae were only observed in Ae. taeniorhynchus and An. crucians. Of 76 Ae. taeniorhynchus specimens found positive for Dirofilaria sp. by dissection, 14 were confirmed to be positive for Dirofilaria immitis (Leidy) by polymerase chain reaction (PCR). The resulting infection rate for D. immitis confirmed by PCR (6.2%) is higher than any infection rate for Ae. taeniorhynchus previously reported from the Americas.     

Synthesis,oxygen radical absorbance capacity,and tyrosinase inhibitory activity of glycosides of resveratrol,pterostilbene, and pinostilbene

The stilbene compound resveratrol was glycosylated to give its 4′-O-β-D-glucoside as the major product in addition to its 3-O-β-D-glucoside by a plant glucosyltransferase from Phytolacca americana expressed in recombinant Escherichia coli. This enzyme transformed pterostilbene to its 4′-O-β-D-glucoside, and converted pinostilbene to its 4′-O-β-D-glucoside as a major product and its 3-O-β-D-glucoside as a minor product. An analysis of antioxidant capacity showed that the above stilbene glycosides had lower oxygen radical absorbance capacity (ORAC) values than those of the corresponding stilbene aglycones. The 3-O-β-D-glucoside of resveratrol showed the highest ORAC value among the stilbene glycosides tested, and pinostilbene had the highest value among the stilbene compounds. The tyrosinase inhibitory activities of the stilbene aglycones were improved by glycosylation; the stilbene glycosides had higher activities than the stilbene aglycones. Resveratrol 3-O-β-D-glucoside had the highest tyrosinase inhibitory activity among the stilbene compounds tested.     

Habitat specificity of littoral Chydoridae (Crustacea,Branchiopoda, Anomopoda) in Plastic Lake,Ontario, Canada

This study identified two scales of pattern in the assemblages of Chydoridae (Crustacea, Branchiopoda, Anomopoda) in the shallow (<2 m) littoral zone of Plastic Lake, Ontario, Canada in the autumn of 1987. Twenty chydorid species were collected in 15 over-night sets of funnel traps in each of four habitat types. Analysis of variance multivariate analysis of variance, and discriminant function analysis revealed that assemblages differed among the habitats. Alona intermedia, Alona quadrangularis and Chydorus bicornutus were particularly abundant in the most structurally diverse habitat type – muddy, rock-strewn areas with approximately 40% bottom cover by the pipewort, Eriocaulon septangulare. In contrast, Anchistropus cf. minor was caught most often on bare shelves of rock. A second set of analyses demonstrated that chydorid assemblages also differed at a smaller scale, i.e. with local patchiness in bottom cover by the dominant macrophyte (E. septangulare). The abundance of Alona affinis was positively correlated with cover by E. septangulare, whereas Anchistropus cf. minor was caught mainly in microhabitats without vegetation. Alona intermedia and A. quadrangularis were most abundant in microhabitats with intermediate amounts of vegetation, suggesting their abundance is influenced by habitat factors other than vegetation.     

A danaid butterfly, Ideopsis similis, overcomes parasitization by a tachinid fly, Sturmia bella

The danaid butterfly Ideopsis similis, which occurs in the Ryukyu Islands, Japan, is a sedentary and monophagous species, whose larval food plant, Tylophora tanakae (Asclepiadaceae), is shared with the migratory and polyphagous danaid Parantica sita. The tachinid Sturmia bella is known to be a principal parasitoid of larval P. sita, whereas there have been no records of parasitization of I. similis by this tachinid. We conducted laboratory experiments and field surveys at seven sites in the Ryukyu Islands to determine how I. similis evades parasitization by this tachinid. We found eggs of S. bella at four sites and eggs and/or larvae of I. similis on the leaves of T. tanakae at all seven sites in the field survey. Out of 40 I. similis allowed to ingest S. bella eggs, 28 (70%) emerged as normal host adults and nine (22.5%) emerged as host adults with crippled wings. Only three (7.5%) died of parasitization in the pupal stage, whereas 27 (87%) of 31 P. sita given S. bella eggs yielded parasitoid flies and eventually died. Melanized dead larvae of S. bella were found in the abdomens of the I. similes adults that had been parasitized but did not yield parasitoid flies. When I. similis females parasitized by S. bella were released in a greenhouse with males, one of them laid fertile eggs. Out of 125 field‐collected I. similis adults, nine had melanized dipteran larvae within their abdomens, which were considered to be S. bella. These results demonstrate that I. similis has the ability to overcome parasitization by S. bella and develop into a fertile adult. It is possible that I. similis remains sedentary and monophagous because it has a strong defense mechanism against S. bella, whereas P. sita escapes from parasitization using its migratory and polyphagous capability.     

Differential expression of eight defensin genes of N. benthamiana following biotic stress, wounding, ethylene, and benzothiadiazole treatments

Eight Nicotiana benthamiana defensin genes were identified that could be divided into two classes with class II defensins being longer than class I defensins due to an additional acidic C-terminal domain. Class I defensins were NbDef1.1, NbDef1.2, NbDef1.3, NbDef1.4, NbDef1.5, and NbDef1.6, and class II were Nbdef2.1 and NbDef2.2. Relative RT-PCR showed that NbDef1.1, NbDef1.2, and NbDef1.4 had relatively similar expression levels in healthy leaves, stems, roots, flowers, and seeds. However, Nbdef1.3, NbDef1.5, and NbDef1.6 had varying degrees of tissue specific expression, and Nbdef2.1 and NbDef2.2 had strictly flower-specific expression. None of the defensins were significantly induced by infection by Colletotrichum destructivum or C. orbiculare. However, infection by Pseudomonas syringae pv. tabaci resulted in increased expression of Nbdef1.2 and Nbdef2.2, and decreased expression of NbDef1.1, NbDef1.4, and NbDef1.6. In the hypersensitive response of N. benthamiana containing Pto with P. syringae pv. tabaci containing AvrPto, only NbDef2.2 was significantly up-regulated. Expression of the genes was also affected by abiotic treatments. Both wounding and ethylene treatments resulted in a strong induction of NbDef2.2 and a moderate to weak induction of NbDef1.1, NbDef1.2, and NbDef1.4. Only weak or no induction was observed with treatment with benzothiadiazole. The expression of these eight defensin genes demonstrates that only a small fraction of the members of a defensin gene family will respond to a particular hemibiotrophic pathogen as well as to abiotic stress or signaling molecules. An erratum to this article can be found at