Acid Sphingomyelinase Gene Knockout Ameliorates Hyperhomocysteinemic Glomerular Injury in Mice Lacking Cystathionine-β-Synthase

Acid sphingomyelinase (ASM) has been implicated in the development of hyperhomocysteinemia (hHcys)-induced glomerular oxidative stress and injury. However, it remains unknown whether genetically engineering of ASM gene produces beneficial or detrimental action on hHcys-induced glomerular injury. The present study generated and characterized the mice lacking cystathionine β-synthase (Cbs) and Asm mouse gene by cross breeding Cbs^+/− and Asm^+/− mice. Given that the homozygotes of Cbs^−/−/Asm^−/− mice could not survive for 3 weeks. Cbs^+/−/Asm^+/+, Cbs^+/−/Asm^+/− and Cbs^+/−/Asm^−/− as well as their Cbs wild type littermates were used to study the role of Asm^−/− under a background of Cbs^+/− with hHcys. HPLC analysis revealed that plasma Hcys level was significantly elevated in Cbs heterozygous (Cbs^+/−) mice with different copies of Asm gene compared to Cbs^+/+ mice with different Asm gene copies. Cbs^+/−/Asm^+/+ mice had significantly increased renal Asm activity, ceramide production and O₂.⁻ level compared to Cbs^+/+/Asm^+/+, while Cbs^+/−/Asm^−/− mice showed significantly reduced renal Asm activity, ceramide production and O₂.⁻ level due to increased plasma Hcys levels. Confocal microscopy demonstrated that colocalization of podocin with ceramide was much lower in Cbs^+/−/Asm^−/− mice compared to Cbs^+/−/Asm^+/+ mice, which was accompanied by a reduced glomerular damage index, albuminuria and proteinuria in Cbs^+/−/Asm^−/− mice. Immunofluorescent analyses of the podocin, nephrin and desmin expression also illustrated less podocyte damages in the glomeruli from Cbs^+/−/Asm^−/− mice compared to Cbs^+/−/Asm^+/+ mice. In in vitro studies of podocytes, hHcys-enhanced O₂.⁻ production, desmin expression, and ceramide production as well as decreases in VEGF level and podocin expression in podocytes were substantially attenuated by prior treatment with amitriptyline, an Asm inhibitor. In conclusion, Asm gene knockout or corresponding enzyme inhibition protects the podocytes and glomeruli from hHcys-induced oxidative stress and injury.     

The C57BL/6J Niemann–Pick C1 mouse model with decreased gene dosage has impaired glucose tolerance independent of body weight

The human Niemann–Pick C1 (NPC1) gene has been found to be associated with extreme (early-onset and morbid-adult) obesity and type 2 diabetes independent of body weight. We previously performed growth studies using BALB/cJ Npc1 normal (Npc1^+/+) and Npc1 heterozygous (Npc1^+/−) mice and determined that decreased Npc1 gene dosage interacts with a high-fat diet to promote weight gain and adiposity. The present study was performed using both BALB/cJ and C57BL/6J Npc1^+/+ and Npc1^+/− mice to determine if decreased Npc1 gene dosage predisposes to metabolic features associated with type 2 diabetes. The results indicated that C57BL/6J Npc1^+/− mice, but not BALB/cJ Npc1^+/− mice, have impaired glucose tolerance when fed a low-fat diet and independent of body weight. The results also suggest that an accumulation of liver free fatty acids and hepatic lipotoxicity marked by an elevation in the amount of plasma alanine aminotransferase (ALT) may be responsible for hepatic insulin resistance and impaired glucose tolerance. Finally, the peroxisome-proliferator activated receptor α (PPARα) and sterol regulatory element-binding protein-1 (SREBP-1) pathways known to have a central role in regulating free fatty acid metabolism were downregulated in the livers, but not in the adipose or muscle, of C57BL/6J Npc1^+/− mice compared to C57BL/6J Npc1^+/+ mice. Therefore, decreased Npc1 gene dosage among two different mouse strains interacts with undefined modifying genes to manifest disparate yet often related metabolic diseases.     

Sialylation and Muscle Performance: Sialic Acid Is a Marker of Muscle Ageing

Sialic acids (Sia) are widely expressed as terminal monosaccharides on eukaryotic glycoconjugates. They are involved in many cellular functions, such as cell–cell interaction and signal recognition. The key enzyme of sialic acid biosynthesis is the bifunctional UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (GNE), which catalyses the first two steps of Sia biosynthesis in the cytosol. In this study we analysed sialylation of muscles in wild type (C57Bl/6 GNE ^+/+) and heterozygous GNE-deficient (C57Bl/6 GNE ^+/−) mice. We measured a significantly lower performance in the initial weeks of a treadmill exercise in C57Bl/6 GNE ^+/− mice compared to wild type C57Bl/6 GNE ^+/+animals. Membrane bound Sia of C57Bl/6 GNE ^+/− mice were reduced by 33–53% at week 24 and by 12–15% at week 80 in comparison to C57Bl/6 GNE ^+/+mice. Interestingly, membrane bound Sia concentration increased with age of the mice by 16–46% in C57Bl/6 GNE ^+/+, but by 87–207% in C57Bl/6 GNE ^+/−. Furthermore we could identify specific morphological changes in aged muscles. Here we propose that increased Sia concentrations in muscles are a characteristic feature of ageing and could be used as a marker for age-related changes in muscle.     

Role of mitochondrial superoxide dismutase in contraction-induced generation of reactive oxygen species in skeletal muscle extracellular space

Contractions of skeletal muscles produce increases in concentrations of superoxide anions and activity of hydroxyl radicals in the extracellular space. The sources of these reactive oxygen species are not clear. We tested the hypothesis that, after a demanding isometric contraction protocol, the major source of superoxide and hydroxyl radical activity in the extracellular space of muscles is mitochondrial generation of superoxide anions and that, with a reduction in MnSOD activity, concentration of superoxide anions in the extracellular space is unchanged but concentration of hydroxyl radicals is decreased. For gastrocnemius muscles from adult (6–8 mo old) wild-type (Sod2^+/+) mice and knockout mice heterozygous for the MnSOD gene (Sod2^+/-), concentrations of superoxide anions and hydroxyl radical activity were measured in the extracellular space by microdialysis. A 15-min protocol of 180 isometric contractions induced a rapid, equivalent increase in reduction of cytochrome c as an index of superoxide anion concentrations in the extracellular space of Sod2^+/+ and Sod2^+/- mice, whereas hydroxyl radical activity measured by formation of 2,3-dihydroxybenzoate from salicylate increased only in the extracellular space of muscles of Sod2^+/+ mice. The lack of a difference in increase in superoxide anion concentration in the extracellular space of Sod2^+/+ and Sod2^+/- mice after the contraction protocol supported the hypothesis that superoxide anions were not directly derived from mitochondria. In contrast, the data obtained suggest that the increase in hydroxyl radical concentration in the extracellular space of muscles from wild-type mice after the contraction protocol most likely results from degradation of hydrogen peroxide generated by MnSOD activity. hydroxyl radicals; microdialysis     

The white tigers of Rewa and gene homology in the felidae

The white tiger is a chinchillated variety, with the yellow pigment completely eliminated and the black pigment altered to a dark speia-brown. The causative mutant gene is inherited as an autosomal recessive and is seemingly a homologue of chinchilla mutants in other mammalian species. It is proposed that the gene be re-symbolized asc ^ch .     

Heme Oxygenase-1 Regulates the Progression of K/BxN Serum Transfer Arthritis

Background

Heme oxygenase-1 (HO-1) is induced in many cell types as a defense mechanism against stress. We have investigated the possible role of endogenous HO-1 in the effector phase of arthritis using the K/BxN serum transfer model of arthritis in HO-1 heterozygous and homozygous knock-out mice.

Methodology/Principal Findings

Arthritis was induced in C57/Black-6 xFVB (HO-1^+/+, HO-1^+/− and HO-1^−/−) mice by intraperitoneal injection of 150 µl serum from arthritic K/BxN mice at days 0 and 2. Blood was collected and animals were sacrificed at day 10. Histological analysis was performed in ankle sections. The levels of inflammatory mediators were measured in serum and paw homogenates by enzyme-linked immunosorbent assay or Multiplex technology. The incidence of arthritis was higher in HO-1^+/− and HO-1^−/− groups compared with HO-1^+/+. The inflammatory response was aggravated in HO-1^+/− mice as shown by arthritic score and the migration of inflammatory cells that could be related to the enhancement of CXCL-1 production. In addition, the HO-1^+/− group showed proteoglycan depletion significantly higher than HO-1^+/+ mice. Serum levels of matrix metalloproteinase-3, monocyte chemotactic protein-1, plasminogen activator inhibitor-1, E-selectin and intercellular adhesion molecule-1 were increased in arthritic HO-1^−/− mice, whereas vascular endothelial growth factor and some cytokines such as interferon-γ showed a reduction compared to HO-1^+/+ or HO-1^+/− mice. In addition, down-regulated gene expression of ferritin, glutathione S-reductase A1 and superoxide dismutase-2 was observed in the livers of arthritic HO-1^+/− animals.

Conclusion/Significance

Endogenous HO-1 regulates the production of systemic and local inflammatory mediators and plays a protective role in K/BxN serum transfer arthritis.     

Glycosaminoglycans in two mollusks, Aplysia californica and Helix aspersa, and in the leech, Nephelopsis obscura

The presence of glycosaminoglycans was examined in two mollusks (Pulmonates): the terrestrial garden snail, Helix aspersa, and the opishtobranchian sea slug, Aplysia californica and also in the leech (Hirudinea, Erpobdellidae, Nephelopsis obscura). Organs in the garden snail contained predominately chondroitin sulfate and heparan sulfate as a lesser component. The ctenidium of the sea slug contained mainly chondroitin sulfate and a compound which migrated on electrophoresis as heparin but additional data indicated that it could also represent a highly sulfated form of heparan sulfate. The foregut contained only the heparin-like polymer. No standard glycosaminoglycan could be identified in the leech although a polydispersed polysaccharide containing uronic acid, hexosamine and sulfate was shown to be present. A detailed analysis of the heparan sulfate isolated from the garden snail is also given.     

Determination of the mouse homologous region for the rat dmy locus

The autosomal recessive mutation dmy causes neurological changes which are characterized by a severe demyelination in the spinal cord, associated with paralysis of the hind limbs. Kuramoto et al. have already mapped the mutation between Hh1ltts and Agtr1a loci on rat chromosome 17 (Kuramoto et al. 1996). Toward the identification of the dmy mutation, we determined here the corresponding genomic region on mouse chromosome 13 and constructed its fine genetic and physical maps. We are currently producing further backcross progeny to narrow down the dmy interval on the rat genome. Determination of the homologous region on the mouse genome should help identifying the causative gene.     

Acridine orange stabilization of glycosaminoglycans in beginning endochondral ossification

Summary This study indicated that acridine orange, when combined with the initial fixative stabilized soluble matrix glycosaminoglycan in situ in areas where considerable glycosaminoglycan extraction is known to occur. Acridine orange was able to diffuse through bone into areas of undecalcified mineralizing cartilage and to bind with the glycosaminoglycans in these areas equally well as in growth plate cartilage matrix. Matrix staining was visible by light microscopy without further staining and was seen to vary territorially in intensity; although cellular definition was poor. This deficiency was overcome by the additional application of p phenylenediamine, which stained the cells intensely. At the ultrastructure level, glycosaminoglycan was present as electron dense structures in the cartilage matrix. Preliminary X-ray microanalysis studies confirmed that the acridine orange stained structures contain sulphur; this finding extends the use of acridine orange further to quantitative analysis of glycosaminoglycan.     

Biosynthesis of an acidic galactan and sulfated glycosaminoglycans during embryonic development of the mollusc Pomacea sp.

The synthesis of acidic polysaccharides by eggs of Pomacea sp. at different stages of development was studied. By day 3 after oviposition an acidic galactan became apparent. This compound reaches its maximum concentration by day 6 and then slowly decreases in concentration, no longer being detectable by day 12. Among the sulfated glycosaminoglycans, chondroitin sulfate is the first to appear (day 10), followed by heparan sulfate and other sulfate glycosaminoglycans, which were synthesized in large amounts until hatching (around day 15). Each one of the compounds was purified and characterized by chemical analysis and enzymatic degradation. The synthesis and characterization of the sulfated glycosaminoglycans was confirmed by the use of radioactive sulfate applied to intact eggs at different stages of development. These studies suggest that the main difference between vertebrate and mollusc development regarding the acidic polysaccharides is that hyaluronic acid seems to be absent during early mollusc development. It is proposed that the acidic galactan may be synthesized from the neutral galactan, already present in the eggs in high amounts, and may replace this glycosaminoglycan in the mollusc embryogenesis.     

The mouse polycystic kidney disease mutation (cpk) is located on proximal chromosome 12

The mouse congenital polycystic kidney (cpk) mutation produces a condition that resembles human autosomal recessive polycystic kidney disease (ARPKD) in its pattern of inheritance, clinical progression, and histopathology. Inheritance of this mouse mutation in crosses segregating the Rb(12.14)8Rma translocation chromosome and various DNA markers of Chromosome 12 have localized cpk to a site near D12Nyu2, approximately 7 cM from the centromere of Chromosome 12. This result suggests that the homologous PKD2 gene should be localized to either human chromosome 2p23-p25 or chromosome 7q22-q31.     

Digenic Inheritance in Cystinuria Mouse Model

Cystinuria is an aminoaciduria caused by mutations in the genes that encode the two subunits of the amino acid transport system b^0,+, responsible for the renal reabsorption of cystine and dibasic amino acids. The clinical symptoms of cystinuria relate to nephrolithiasis, due to the precipitation of cystine in urine. Mutations in SLC3A1, which codes for the heavy subunit rBAT, cause cystinuria type A, whereas mutations in SLC7A9, which encodes the light subunit b^0,+AT, cause cystinuria type B. By crossing Slc3a1 ^-/- with Slc7a9 ^-/- mice we generated a type AB cystinuria mouse model to test digenic inheritance of cystinuria. The 9 genotypes obtained have been analyzed at early (2- and 5-months) and late stage (8-months) of the disease. Monitoring the lithiasic phenotype by X-ray, urine amino acid content analysis and protein expression studies have shown that double heterozygous mice (Slc7a9 ^+/- Slc3a1 ^+/-) present lower expression of system b^0,+ and higher hyperexcretion of cystine than single heterozygotes (Slc7a9 ^+/- Slc3a1 ^+/+ and Slc7a9 ^+/+ Slc3a1 ^+/-) and give rise to lithiasis in 4% of the mice, demonstrating that cystinuria has a digenic inheritance in this mouse model. Moreover in this study it has been demonstrated a genotype/phenotype correlation in type AB cystinuria mouse model providing new insights for further molecular and genetic studies of cystinuria patients.     

Conidial germination and germ tube elongation of Phomopsis amaranthicola and Microsphaeropsis amaranthi on leaf surfaces of seven Amaranthus species: Implications for biological control

Microsphaeropsis amaranthi and Phomopsis amaranthicola are potential biological control agents for several Amaranthus species. In an effort to understand the initial infection processes with these pathogens, a study was conducted of the conidial germination and germ tube length (μm) on the weed leaf surfaces at 21 °C and 28 °C. Weeds included Amaranthus rudis, A. palmeri, A. powellii, A. retroflexus, A. spinosus, A. hybridus, and A. albus. For P. amaranthicola, conidial germination and germ tube length varied among the seven weed species at both temperatures, while for M. amaranthi the differences in germ tube lengths were significant among weed species only at 21 °C. While the conidia of M. amaranthi and P. amaranthicola germinated on the leaf surfaces of all seven weed species, temperature appeared to impact the number and length of germ tubes on the leaf surfaces. The percentage of germinated conidia and the length of germ tubes at both temperatures were often greater for M. amaranthi than for P. amaranthicola. In order for the fungal pathogen to successfully infect and kill a weedy host, conidia must germinate and form a germ tube, two processes that vary with host species and temperature for M. amaranthi and P. amaranthicola. The extent to which successive infection processes, e.g., penetration, invasion and colonization, contribute to host specificity warrants study.     

Die genetische Grundlage der Monogenie bei der Schmei?fliege Chrysomya rufifacies (Calliphoridae, Diptera)

Summary Recently in our wild stock of the monogenic blowfly Chrysomya rufifacies a recessive mutation white (w) causing white instead of red-brown eyes spontaneously appeared (Fig. 1). This marker gene enabled us to clarify the genetic basis of monogeny in this species. F₁ offspring produced by reciprocal crossings between normal (+/+) and white-eyed (w/w) flies were phenotypically wildtype (Table 1). In F₂ offspring of female-producing (thelygenic) and male-producing (arrhenogenic) F₁ females wildtype and white-eyed flies appeared in the expected 3:1 ratio; in several crossings a slight deviation of this ratio indicated a reduced viability of the w/w individuals (Table 2). Examination of F₂ progeny of thelygenic F₁+/w females, which had received the w allele from their father, showed that most of the F₂+/+ females were thelygenic, whereas most of the F₂ w/w females were arrhenogenic; among F₂+/w females thelygenic and arrhenogenic individuals occurred in almost equal numbers (Table 3). On the other hand, when F₂ offspring of thelygenic F₁+/w females which had inherited the w allele from their mother were tested, most of the F₂+/+ females turned out to be arrhenogenic and most of the F₂ w/w females thelygenic; among F₂+/w females thelygenic and arrhenogenic flies also were found in almost equal frequencies (Table 4). the sex-linked inheritance of the factor w following from these results was also confirmed by an analysis of the progeny of thelygenic F₁+/w females backcrossed with w/w males. Among the R₁ offspring of F₁+/w females, which had received the w allele from their father, the +/w females were predominantly thelygenic compared to their predominantly arrhenogenic w/w sisters (Table 5). Analysis of R₁ progeny of F₁+/w females, which had inherited the w allele from their mother, yielded reciprocal results (Table 6).This mode of incomplete sex-linkage of the mutation white observed in C. rufifacies (Figs. 2–5) supports the hypothesis that thelygenic females are heterozygous for a dominant female sex realizer (F') with predetermined sex-determining properties, and that arrhenogenic females as well as the males are homozygous for the recessive allele f (Fig. 6). The recombination frequency between F'/f and the w-Locus was calculated to be 12.72±0.72 per cent.

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Mit Unterstützung durch die Deutsche forschungsgemeinschaft.     

The chronic proliferative dermatitis mouse mutation (cpdm): mapping of the mutant gene locus

The chronic proliferative dermatitis (cpdm) mouse mutation was mapped to mouse Chromosome 15 using an intraspecific cross between C57BL/KaLawRij cpdm/cpdm and BALB/cJ mice. A second autosomal recessive mutation that resulted in a phenotype similar to that of cpdm arose spontaneously in a colony of OcB-3/Dem recombinant congenic mice. This new mutation was found to be allelic with cpdm. Therefore, the gene symbol for the allelic mutation is cpdm^Dem. The phenotype of these mutant mice consists of eosinophil-driven severe and progressive inflammatory changes in multiple organs including the skin and lungs.     

In vitro studies of the effect of Environmental Conditions on the anthacnose pathogen of bananas, Colletotrichum musae

In vitro studies were undertaken to determine the effect of pH, temperature, water availability and carbon dioxide (CO₂) concentration on germination and growth of Colletotrichum musae, the causal pathogen of anthracnose of bananas. The optimum pH for germination and growth varied between 4·0 and 5·0 depending on temperature. At low pH (< 3·0) and 15°C, both germination and growth were significantly reduced, with a marked increase in the lag time, in days, prior to growth. C. musae germinated and grew over a wide range of water activities (a_w; 0·995−0·94 and 0·995−0·92, respectively) at 20, 25 and 30°C. In all cases where germination occurred appresoria were subsequently produced. Optimum growth occurred at 30°C and 0·995 a_w, although this changed to 0·98 a_w at 35°C. Increasing CO₂ concentration to 15% or reducing oxygen concentration to 1% resulted in a significant (P < 0·05) reduction in growth, but did not inhibit growth completely.     

The Mouse Severe Combined Immune Deficiency (scid) Mutation Is Closely Linked to the B-Cell-Specific Developmental Genes VpreB and λ5

The mouse severe combined immune deficiency (scid) phenotype is due to a recessive, autosomal mutation which results in failed development of lymphocytes. An important step during normal lymphocyte development is the germline rearrangement of DNA segments to assemble functional immunoglobulin or T cell receptor genes. scid lymphocytes fail to rearrange these genes properly, resulting in the absence of mature B and T lymphocytes. This mutation was originally mapped to chromosome 16 by linkage to the immunoglobulin λ light chain genes (Igl-1) and the coat color mutation mahoganoid . We have typed 288 progeny from backcrosses between MOLF/Ei or CAST/Ei and C.B-17-scid for the scid phenotype and nine other loci mapped to the centromeric region of MMU16. We have established a refined map of this region which places the scid gene between Prm-2 and Igl-1 . In addition, no recombinations were found between scid and three other loci, VpreB, λ5, and D16Mit31, providing markers useful for isolating the scid gene by positional cloning.     

Heterozygosity for Fibrinogen Results in Efficient Resolution of Kidney Ischemia Reperfusion Injury

Fibrinogen (Fg) has been recognized to play a central role in coagulation, inflammation and tissue regeneration. Several studies have used Fg deficient mice (Fg^−/−) in comparison with heterozygous mice (Fg^+/−) to point the proinflammatory role of Fg in diverse pathological conditions and disease states. Although Fg^+/− mice are considered ‘normal’, plasma Fg is reduced to ∼75% of the normal circulating levels present in wild type mice (Fg^+/+). We report that this reduction in Fg protein production in the Fg^+/− mice is enough to protect them from kidney ischemia reperfusion injury (IRI) as assessed by tubular injury, kidney dysfunction, necrosis, apoptosis and inflammatory immune cell infiltration. Mechanistically, we observed binding of Fg to ICAM-1 in kidney tissues of Fg^+/+ mice at 24 h following IRI as compared to a complete absence of binding observed in the Fg^+/− and Fg^−/− mice. Raf-1 and ERK were highly activated as evident by significantly higher phosphorylation in the Fg^+/+ kidneys at 24 h following IRI as compared to Fg^+/− and Fg^−/− mice kidneys. On the other hand Cyclin D1 and pRb, indicating higher cell proliferation, were significantly increased in the Fg^+/− and Fg^−/− as compared to Fg^+/+ kidneys. These data suggest that Fg heterozygosity allows maintenance of a critical balance of Fg that enables regression of initial injury and promotes faster resolution of kidney damage.     

Role of nucleotide excision repair deficiency in intestinal tumorigenesis in multiple intestinal neoplasia (Min) mice

Mice deficient in the Xeroderma pigmentosum group A (Xpa) gene are defective in nucleotide excision repair (NER) and highly susceptible to skin carcinogenesis after dermal exposure to UV light or chemicals. Min (multiple intestinal neoplasia) mice, heterozygous for a germline nonsense mutation in the tumor suppressor gene adenomatous polyposis coli (Apc), develop intestinal tumors spontaneously and show additional intestinal tumors after exposure to the food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). In this study, we investigated the impact of loss of XPA function on PhIP-induced intestinal tumorigenesis in F1 offspring of Min/+ (Apc^+/−) mice crossed with Xpa gene-deficient mice. Apc^+/− mice lacking both alleles of Xpa had higher susceptibility towards toxicity of PhIP, higher levels of PhIP–DNA adducts in the middle and distal small intestines, as well as in liver, and a higher number of small intestinal tumors at 11 weeks, compared with Apc^+/− mice with one or two intact Xpa alleles. Localization of tumors was not affected, being highest in middle and distal small intestines in all genotypes. At 11 weeks of age, the number of spontaneous intestinal tumors was not significantly increased by homozygous loss of Xpa, but untreated Apc^+/−/Xpa^−/− mice had significantly shorter life-spans than their XPA-proficient littermates. Heterozygous loss of Xpa did not affect any of the measured end points. In conclusion, the Xpa gene and the NER pathway are involved in repair of bulky PhIP–DNA adducts in the intestines and the liver, and most probably of DNA lesions leading to spontaneous intestinal tumors. These results confirm a role of the NER pathway also in protection against cancer in internal organs, additional to its well-known importance in protection against skin cancer. An effect of Apc^+/− on adduct levels, additional to that of Xpa^−/−, indicates that the truncated APC protein may affect a repair pathway other than NER.