iNOS ablation does not improve specific force of the extensor digitorum longus muscle in dystrophin-deficient mdx4cv mice

Nitrosative stress compromises force generation in Duchenne muscular dystrophy (DMD). Both inducible nitric oxide synthase (iNOS) and delocalized neuronal NOS (nNOS) have been implicated. We recently demonstrated that genetic elimination of nNOS significantly enhanced specific muscle forces of the extensor digitorum longus (EDL) muscle of dystrophin-null mdx4cv mice (Li D et al J. Path. 223:88-98, 2011). To determine the contribution of iNOS, we generated iNOS deficient mdx4cv mice. Genetic elimination of iNOS did not alter muscle histopathology. Further, the EDL muscle of iNOS/dystrophin DKO mice yielded specific twitch and tetanic forces similar to those of mdx4cv mice. Additional studies suggest iNOS ablation did not augment nNOS expression neither did it result in appreciable change of nitrosative stress markers in muscle. Our results suggest that iNOS may play a minor role in mediating nitrosative stress-associated force reduction in DMD.     

Sibling competition does not exacerbate inbreeding depression in the burying beetle Nicrophorus vespilloides

Inbreeding results from matings between relatives and can cause a reduction in offspring fitness, known as inbreeding depression. Previous work has shown that a wide range of environmental stresses, such as extreme temperatures, starvation and parasitism, can exacerbate inbreeding depression. It has recently been argued that stresses due to intraspecific competition should have a stronger effect on the severity of inbreeding depression than stresses due to harsh physical conditions. Here, we tested whether an increase in the intensity of sibling competition can exacerbate inbreeding depression in the burying beetle Nicrophorus vespilloides. We used a 2 × 3 factorial design with offspring inbreeding status (outbred or inbred) and brood size (5, 20, or 40 larvae) as the two factors. We found a main effect of inbreeding status, as inbred larvae had lower survival than outbred larvae, and a main effect of brood size, as larvae in large broods had lower survival and mass than larvae in medium‐sized broods. However, there was no effect of the interaction between inbreeding status and brood size, suggesting that sibling competition did not influence the severity of inbreeding depression. Since we focused on sibling competition within homogeneous broods of either inbred or outbred larvae, we cannot rule out possible effects of sibling competition on inbreeding depression in mixed paternity broods comprising of both inbred and outbred offspring. More information on whether and when sibling competition might influence inbreeding depression can help advance our understanding of the causes underlying variation in the severity of inbreeding depression.     

Skeletal myogenesis in the mouse esophagus does not occur through transdifferentiation

To determine the developmental history of murine esophageal skeletal muscle, smooth muscle cells were fate mapped by lineage-specific recombination and phenotypically marked by eGFP. Examination of embryonic and postnatal tissues revealed that esophageal skeletal muscle does not arise from transdifferentiation of committed smooth muscle cells.     

Rescue of skeletal muscle α-actin–null mice by cardiac (fetal) α-actin

Skeletal muscle α-actin (ACTA1) is the major actin in postnatal skeletal muscle. Mutations of ACTA1 cause mostly fatal congenital myopathies. Cardiac α-actin (ACTC) is the major striated actin in adult heart and fetal skeletal muscle. It is unknown why ACTC and ACTA1 expression switch during development. We investigated whether ACTC can replace ACTA1 in postnatal skeletal muscle. Two ACTC transgenic mouse lines were crossed with Acta1 knockout mice (which all die by 9 d after birth). Offspring resulting from the cross with the high expressing line survive to old age, and their skeletal muscles show no gross pathological features. The mice are not impaired on grip strength, rotarod, or locomotor activity. These findings indicate that ACTC is sufficiently similar to ACTA1 to produce adequate function in postnatal skeletal muscle. This raises the prospect that ACTC reactivation might provide a therapy for ACTA1 diseases. In addition, the mouse model will allow analysis of the precise functional differences between ACTA1 and ACTC.     

Loss of callose in the stigma papillae does not affect the Brassica self-incompatibility phenotype

As part of the Brassicaceae self-incompatibility response, callose is deposited in the stigma papillar cells. To determine if callose plays an important role in the rejection of incompatible pollen by the stigma, transgenic Brassica napus. L. plants were produced which express the tobacco β-1,3-glucanase cDNA (the enzyme which degrades callose) in the stigma papillae. Using aniline blue fluorescence, little or no callose was detected in the papillar cells of transgenic stigmas. However, the self-incompatibility system appeared to be unaffected based on the lack of pollen tube growth and the subsequent lack of seed set. The transgene had no effect on compatible pollinations. Thus, while callose deposition is associated with the B. napus self-incompatibility response, it is not required for the rejection of incompatible pollen. Received: 14 March 1997 / Accepted: 15 April 1997     

Cyclosporin A inhibits the production of gamma interferon (IFN gamma), but does not inhibit production of virus-induced IFN alpha/beta

The effect of cyclosporin A (CsA) on the production of gamma interferon (IFN gamma) versus IFN alpha/beta was studied using mouse and human lymphocytes and fibroblasts. Spleen cells from C57Bl/6 mice produced low but significant levels (40-60 U/ml) of IFN gamma after 2 to 3 days of culture with irradiated DBA spleen cells. The addition of CsA at concentrations as low as 0.1 microgram/ml completely inhibited (less than 10 U/ml) IFN gamma production in these cultures. High levels of IFN gamma (170-1200 U/ml) were produced when either C57Bl/6 spleen cells or Ficoll-Hypaque-purified human peripheral blood lymphocytes (PBL) were cultured with the T-cell mitogen staphylococcal enterotoxin A (SEA). The addition of CsA (0.1 microgram/ml) to these cultures also completely inhibited (less than 10 U/ml) IFN gamma production. This inhibition was shown not to be due to a change in the kinetics of IFN gamma production or to a change in the amount of SEA required for stimulation. IFN gamma production in SEA-stimulated mouse spleen cells was inhibited at 3 days of culture even when CsA was added at 24 or 48 hr postculture initiation. Thus, CsA inhibits IFN gamma production even when early events associated with lymphocyte activation have been allowed to take place. In contrast to IFN gamma production, IFN alpha/beta production by Newcastle disease virus (NDV)-infected mouse and human lymphocytes or fibroblasts was not inhibited by the addition of CsA (1 microgram/ml). CsA also did not block the action of IFN gamma or IFN alpha/beta since addition of CsA (1 microgram/ml) to reference IFN standards had no effect on their antiviral activity. Thus, CsA inhibits the production of IFN gamma by T cells but appears to have no effect on the production of IFN alpha/beta by virus-infected cells or on the antiviral action of already produced IFN gamma and IFN alpha/beta.     

Netropsin does not bind with the oligonucleotide d(GCGATCGC)

By the method of circular dichroism we have studied binding of netropsin in a solution with self-complementary oligonucleotide d(GCGATCGC). It is shown that the presence of two successively located A and T nucleotides is not enough for binding netropsin with B-DNA.     

Deficiency of the cystine-transporter gene, xCT, does not exacerbate the deleterious phenotypic consequences of SOD1 knockout in mice

Both α-lipoic acid (LA) and ascorbic acid (vitamin C) have been shown to improve endothelial dysfunction, a precursor of atherosclerosis. Since oxidant stress can cause endothelial dysfunction, we tested the interaction and efficacy of these antioxidants in preventing oxidant damage to lipids due to both intra- and extracellular oxidant stresses in EA.hy926 endothelial cells. LA spared intracellular ascorbate in culture and in response to an intracellular oxidant stress induced by the redox cycling agent menadione. Extracellular oxidant stress generated by incubating cells for 2 h in with 0.2 mg/ml LDL and 5 μM Cu²⁺ caused a time-dependent increase of the lipid peroxidation product malondialdehyde in both cells and LDL, preceded by rapid disappearance of` α-tocopherol in LDL. α-Lipoic acid at concentrations of 40–80 μM blunted these effects. Similarly, intracellular ascorbate concentrations of 1–2 mM also prevented Cu²⁺-induced lipid peroxidation in LDL and cells. Cu²⁺-dependent oxidation of LDL in the presence of ascorbate-loaded cells decreased intracellular ascorbate by 20%, but this decrease was not reversed by LA. Both LA and ascorbate protect endothelial cells and LDL from either intra- or extracellular oxidant stress, but that LA does not spare ascorbate in oxidatively stressed cells.     

Pre- and post-weaning cold exposure does not lead to an obese phenotype in adult Brandt's voles (Lasiopodomys brandtii)

Evidence has shown that postnatal undernutrition, overnutrition and cold stress are associated with imbalanced metabolic regulation as rodents achieve adulthood. In this study, we used a breeding colony of Brandt's voles (Lasiopodomys brandtii), a wild rodent species from the Inner Mongolia grasslands in China, to examine the effects of pre- and post-weaning cold exposure on the adult body (fat) mass, serum hormones and hypothalamic neuropeptides. Unlike laboratory rodents, vole offspring exposed to pre-weaning cold did not exhibit overweight or obese phenotypes in adulthood compared with unexposed controls. Moreover, adult male voles that remained in colder conditions had less body mass and lower serum leptin levels despite having higher food intake compared to other groups. To understand the mechanism of this unexpected regulation, hypothalamic gene expression was assessed for pre- and post-weaning cold exposure. Voles exposed to cold before weaning increased hypothalamic, orexigenic agouti-related protein (AgRP) and decreased anorexigenic proopiomelanocortin (POMC) mRNA expression at weaning. These expression changes were associated with hyperphagia and catch-up growth after weaning. Interestingly, these changes in hypothalamic neuropeptides were short lasting because in adult voles these differences were no longer apparent, which might explain why the pre-weaning, cold-exposed voles did not become obese in adulthood. These data suggest that some species do not develop an obese phenotype in response to early life cold stress.     

中国不产曲籽芋属植物

曲籽芋属在中国的分布源自对采自海南的一号标本———W.T.Tsang（曾怀德）553=L.U.16052（1927年8月27日采）的错误鉴定。这号标本被E.D.Merrill定名为曲籽芋Cyrtosperma lasioides Griffith,并分藏于国内外的标本馆。因此,《海南植物志》第四卷、《中国植物志》第十三卷第二分册、《中国种子植物科属词典》以及吴征镒的《中国种子植物属的分布区类型》一文等都记录了曲籽芋属。收藏在中国科学院华南植物研究所、英国邱皇家植物园和爱丁堡皇家植物园的W.T.Tsang（     

Culture on basement membrane does not reverse the phenotype of lens derived mesenchyme-like cells

Definitive epithelia suspended within type I collagen gel give rise to individual, freely migrating cells that express the mesenchymal phenotype. They become elongate in shape, invade collagenous matrices and develop abundant RER. We investigated whether mesenchyme-like cells that derive from lens epithelia retain the mesenchymal phenotype or revert to epithelial phenotype when cultured on basement membrane (BM). Mesenchyme-like cells placed on top of BM gel or lens capsule BM retain the elongate, bipolar morphology of mesenchymal cells. They migrate individually along and into the BM matrix. Mesenchyme-like cells on or in BM ultrastructurally resemble true mesenchymal cells. They extend pseudopodia and filopodia, exhibit a circumferential actin cortex, and contain well developed RER. Mesenchymal products, such as type I collagen, continue to be expressed. We conclude that the phenotype of mesenchyme-like cells derived from definitive epithelia is stable even in or on matrix known to promote the epithelial genetic program. Their behavior, thus, is similar to that of true (secondary) mesenchymal cells in the embryo.     

Comprehensive assessment of the photomutagenicity, photogenotoxicity and photo(cyto)toxicity of azulene

The polycyclic aromatic hydrocarbon azulene and its naturally occurring derivative guaiazulene (1,4-dimethyl-7-isopropylazulene) are known to absorb light in the UV-vis region of the spectrum. Both compounds were reported to be mutagenic in the Salmonella typhimurium bacterial mutagenicity assay (Ames test) in strain TA102, and to cause DNA damage in the comet assay in vitro upon exposure to UVA light. In contrast, another study reported a photoprotective effect in vitro of guaiazulene. We present here a comprehensive assessment of the photo(cyto)toxicity (3T3 fibroblast Neutral Red uptake test), the photomutagenicity (Ames test) and photogenotoxicity (comet assay and micronucleus test in L5178Y cells in vitro) of azulene. In the Ames test, the mutagenicity of azulene was assessed in the presence and absence of UV light by use of the Salmonella strains TA102, TA104, TA2638 and E. coli WP2. Azulene was irradiated before being plated with bacteria (pre-irradiation), or concomitantly with the bacteria either after plating or while in suspension. Guaiazulene was included in some of the experiments. Neither in the photo-Ames test nor in the other photogenotoxicity tests, azulene or guaiazulene showed any photomutagenic or photogenotoxic activity. Weak photo(cyto)toxicity (estimate of PIF≥1.67) was observed with azulene in the 3T3 NRU test, the Alamar Blue test and the relative cell count, which may be due to the generation of reactive oxygen species, as reported recently.     

Deletion of a lectin gene does not affect the phenotype of the nematode-trapping fungus Arthrobotrys oligospora

A number of filamentous fungi are known to produce high levels of saline-soluble and low-molecular-mass lectins. The function of these proteins are not clear but it has been proposed that they are involved in storage of nutrients, development, recognition of other organisms, and defense reactions. A gene encoding such a lectin (AOL) was deleted in the nematode-trapping fungus Arthrobotrys oligospora by homologous recombination. The deletion mutants did not express any hemagglutinating activity or protein cross-reacting with AOL antibodies. There were no significant differences between the DeltaAOL and wild-type strains in spore (conidia) germination, saprophytic growth, and pathogenicity. Furthermore, there was no significant difference in the growth and reproduction of collembolan feeding on the various strains of A. oligospora. Thus either the previous proposed functions of AOL are not correct, or the fungus can compensate for the absence of the lectin by expressing other proteins with similar function(s) as AOL.     

Toxicity phenotype does not correlate with phylogeny of Cylindrospermopsis raciborskii strains

Cylindrospermopsis raciborskii is a species of freshwater, bloom-forming cyanobacterium. C. raciborskii produces toxins, including cylindrospermopsin (hepatotoxin) and saxitoxin (neurotoxin), although non toxin-producing strains are also observed. In spite of differences in toxicity, C. raciborskii strains comprise a monophyletic group, based upon 16S rRNA gene sequence identities (greater than 99%). We performed phylogenetic analyses; 16S rRNA gene and 16S-23S rRNA gene internally transcribed spacer (ITS-1) sequence comparisons, and genomic DNA restriction fragment length polymorphism (RFLP), resolved by pulsed-field gel electrophoresis (PFGE), of strains of C. raciborskii, obtained mainly from the Australian phylogeographic cluster. Our results showed no correlation between toxic phenotype and phylogenetic association in the Australian strains. Analyses of the 16S rRNA gene and the respective ITS-1 sequences (long L, and short S) showed an independent evolution of each ribosomal operon. The genes putatively involved in the cylindrospermopsin biosynthetic pathway were present in one locus and only in the hepatotoxic strains, demonstrating a common genomic organization for these genes and the absence of mutated or inactivated biosynthetic genes in the non toxic strains. In summary, our results support the hypothesis that the genes involved in toxicity may have been transferred as an island by processes of gene lateral transfer, rather than convergent evolution.     

Loss of the peroxisome proliferation-activated receptor gamma (PPARgamma ) does not affect mammary development and propensity for tumor formation but leads to reduced fertility

The peroxisome proliferation-activated receptor gamma (PPARgamma) is expressed in many cell types including mammary epithelium, ovary, macrophages, and B- and T-cells. PPARgamma has an anti-proliferative effect in pre-adipocytes and mammary epithelial cells, and treatment with its ligands reduced the progression of carcinogen-induced mammary tumors in mice. Because PPARgamma-null mice die in utero it has not been possible to study its role in development and tumorigenesis in vivo. To investigate whether PPARgamma is required for the establishment and physiology of different cell types, a cell-specific deletion of the gene was carried out in mice using the Cre-loxP recombination system. We deleted the PPARgamma gene in mammary epithelium using WAP-Cre transgenic mice and in epithelial cells, B- and T-cells, and ovary cells using MMTV-Cre mice. The presence of PPARgamma was not required for functional development of the mammary gland during pregnancy and for the establishment of B- and T-cells. In addition, no increase in mammary tumors was observed. However, loss of the PPARgamma gene in oocytes and granulosa cells resulted in impaired fertility. These mice have normal populations of follicles, they ovulate and develop corpora lutea. Although progesterone levels are decreased and implantation rates are reduced, the exact cause of the impaired fertility remains to be determined.