Tissue-specific expression of the β-glucuronidase reporter gene in transgenic strawberry (Fragaria × ananassa) plants

The strawberry ( Fragaria spp) is regarded as a false fruit because it originates from the receptacle, which is a non-ovarian tissue. For this reason, fruit-specific promoters isolated from plant species in which the fruit is derived from the ovary wall might not be suited to control gene expression in a fruit-specific way in strawberry. In order to achieve (false) fruit-specific expression in strawberry, we tested the petunia FBP7 (floral binding protein7) promoter, which proved to be active in the receptacles of petunia flowers, in transgenic strawberry fruits. In strawberry plants containing the FBP7 promoter fused to the ß-glucuronidase (GUS) reporter gene ( gus), GUS activity was found in floral and fruit tissues of all developmental stages tested but not in leaf, petiole and root tissue . Surprisingly, Northern blot analysis showed the presence of gus-derived mRNAs in root (strong) and petiole (weak) tissue of fbp7- gus plants in addition to the floral and fruit tissues. Therefore, it is concluded that the histological GUS phenotype does not necessarily correspond with expression at the mRNA level. mRNA quantification using the TaqMan polymerase chain reaction technology confirmed the Northern results and showed that in red strawberry tissue the cauliflower mosaic virus 35S promoter is at least sixfold stronger than the FBP7 promoter.     

Sex-restricted non-Mendelian inheritance of mouse Chromosome 11 in the offspring of crosses between C57BL/6J and (C57BL/6J × DBA/2J)F1 mice

We report on the observation of sex-restricted, non-Mendelian inheritance over a region of mouse Chromosome (Chr) 11, occurring in the offspring of crosses between two commonly used Mus musculus-derived inbred strains, C57BL/6J and DBA/2J. In the surviving backcross progeny of reciprocal matings between (C57BL/6J × DBA/2J)F₁ hybrids and the C57BL/6J parental strain, we observed the preferential appearance of C57BL/6J alleles along a region of Chr 11. The deviation from Mendelian predictions was observed only in female offspring from both reciprocal backcrosses, and not in males from either cross. The sex-specificity of the observed non-Mendelian inheritance points to an explanation based on embryonic or neonatal lethality. Our data add to previously obtained evidence for a Chr 11 locus or loci with sex-specific and allele-specific effects on viability. Received: 19 December 1997 / Accepted: 10 June 1998     

Inherited non-autosomal effects on body fat in F2 mice derived from an AKR/J × SWR/J cross

In this study we describe the contribution of matrilineal and patrilineal effects on the adiposity, body weight, and on the weights of individual fat pads in F₂ male mice derived from an SWR/J × AKR/J cross. AKR/J mice become obese after 12 weeks on a high-fat diet, whereas SWR/J mice remain relatively lean. Here we report that mice with AKR maternal and AKR paternal grandmothers have significantly larger epidydimal and retroperitoneal fat pads than those with SWR maternal and paternal grandmothers. However, grandparental strain had no effect on the overall adiposity (AI) or the weights of the inguinal, subcutaneous or mesenteric fat pads. The strain of the paternal grandparents had a small but significant effect on body weight. These effects can be attributed to in utero effects, imprinting effects, cytoplasmic and/or Y chromosome transmission of factors controlling body fat. We also describe the presence of a quantitative trait locus (QTL) on Chromosome X, close to DXMit174, which is linked to adiposity, body weight, and to the weights of the individual fat depots. However, this QTL is not responsible for the grandparental strain effects described above. Received: 3 March 1997 / Accepted: 5 May 1997     

A Non segregation F2 Population Derived from the Cross of Triploid×Diploid in Rice

A non-segregating F2 population was obtained from the cross of tfiploid × diploid in rice ( Oryza sativa L. ). The maternal parent used in cross was SAR-3 which is an autotriploid occurring in the field, and the paternal was a restored line Sheng 47. The F2 population from the same F1 plant appears uniform, and so does F3. Results from coefficients of variation (CV) of F2, Ducan’s test of F3 and SSRs marker analysis showed that the F2 of SAR-3 × Sheng 47 was non-segregating. Markers for either parent could be present in F2 progenies. In some cases, both markers for each parent could be found on one chromosome. The authors presume that after crossing there might be recombination of chromosome followed by homozygotization. Stability of early generation could be utilized in breeding, as it is a new approach to shorten the breeding procedures.     

Genetic regulation of the class conversion of dsDNA-specific antibodies in (NZB × NZW)F1 hybrid

To investigate the possible effects of NZW genes on the class conversion of dsDNA-specific antibodies in NZB X NZW (B/W)F1 hybrids, we measured IgM, IgG1, and IgG2 dsDNA-specific antibodies, using the Crithidia luciliae kinetoplast immunofluorescence test, in NZB, NZW, B/W F1 hybrid, B/W F1 X NZB backcross, and B/W F1 X NZW backcross mice at 4, 7, and 10 months of age. The highest serum levels of IgM dsDNA-specific antibodies were observed in NZB mice at the ages tested; however, the amounts of IgG1 and IgG2 antibodies were scanty. In contrast, a large amount of both IgG1 and IgG2 dsDNA-specific antibodies was produced in B/W F1 hybrids, in which the serum IgM antibodies were lower than those observed in NZB mice. NZW mice were virtually negative for these antibodies. Progeny testing suggested that a combined effect of two unlinked dominant genes of the NZB strain determines the production of dsDNA-specific antibodies and that these genes only act to produce IgM antibodies. These traits are to a great degree modified by the NZW loci in B/W F1 hybrids, and a combined effect of two unlinked dominant genes leads to conversion of the class of the antibodies from IgM to IgG, which, in turn, increases the serum levels of dsDNA-specific antibodies. The F1 hybrid of C57BL/6 and NZW strains produced no dsDNA-specific antibodies, indicating that the relevant NZB predisposing genes are required for the NZW gene action. Linkage studies showed that one of such NZW genes is to some extent linked to the H-2 complex on chromosome 17, but not to Mup-1 (chromosome 4) or a coat color locus (chromosome 2). The appearance of IgG dsDNA-specific antibodies correlated well with the incidence of renal disease in B/W F1 X NZB backcross mice.     

Collagenl α1 promoter drives the expression of Cre recombinase in osteoblasts of transgenic mice

Osteoblasts participate in bone formation,bone mineralization,osteoclast differentiation and many pathological processes.To study the function of genes in osteoblasts using Cre-LoxP system,we generated a mouse line expressing the Cre recombinase under the control of the rat Collagenlal (Coilal) promoter(Coilatl-Cre).Two founders were identified by genomic PCR from 16 offsprings.and the integration efficiency is 12.5%.In order tO determine the tissue distribution and the activity of Cre rccombinase in the transgenic mice,the Collal-Cre transgenic mice were bred with the ROSA26 reporter strain and a mouse strain that carries Smad4 conditional alleles (Smad4co/co).Multiple tissue PCR of Collal-Cre;Smad4co/ mice revealed the restricted Cre activity in bone tissues containing osteoblasts and tendon.LacZ staining in the Coilal-Cre;ROSA26 double transgenic mice revealed that the Cre recombinase began to express in the osteoblasts of calvaria at E14.5.Cre activity was observed in the osteoblasts and osteocytes of P10 double transgenic mice.All these data indicated that the Collal-Cre transgenic mice could Serve as a valuabletool for osteoblast lineage analysis and conditional gene knockout in osteoblasts.     

Low expression of Neu2 sialidase in the thymus of SM/J mice—existence of neuraminidase positive cells “Neu-medullocyte” in the murine thymus

We have already reported that the homogenate of the A/J mouse thymus shows a high sialidase activity at the neutral pH region and that in both soluble and membrane fractions optimal pH was 6.5–7 (Kijimoto-Ochiai et al., Glycoconj. J., 20:375–384, 2004). In the present study, we investigated the level of sialidase activities in the thymus of the SM/J mouse, a mouse strain that we know to have a Neu1^a allele that reveals a low level of sialidase activity in the liver. We found that while in the A/J thymus the soluble sialidase activity at pH 6.5 was high, the SM/J thymus lacked all such activity. A QTL analysis of SMXA recombinant inbred strains showed that soluble sialidase activity correlated well with the D1Mit8/9 marker on chromosome 1. The murine whole DNA-sequence data and the results of our FISH analysis (Kotani et al., Biochem. Biophys. Res. Comm., 286:250–258, 2001) showed that this location is consistent with the position of Neu2 gene. We confirmed that it is hard to detect the Neu2 enzyme of the SM/J mouse thymus by an anti-Neu2 antibody using a Western blot analysis. We also found that while the mRNA expression of Neu2 was quite normal in the SM/J mouse liver, it was very low in the SM/J mouse thymus. We therefore conclude that the lack of soluble sialidase activity in the SM/J mouse thymus is due to the thymus-specific low expression level of the Neu2 gene. We have previously shown that the sialidase positive cell which contains the Mac-1 and immunoglobulin, and which is located sparsely in the corticomedullar region or medullary region of the A/J mouse thymus (Kijimoto-Ochiai et al., Glycoconj. J., 20:375–384, 2004). We showed now in this paper that the detection of this cell in the SM/J mouse thymus at pH 7.0 was difficult. We propose, therefore, to name the cell “Neu-medullocyte”.     

New quantitative trait loci that regulate wound healing in an intercross progeny from DBA/1J and 129×1/SvJ inbred strains of mice

Wound healing/regeneration mouse models are few, and studies performed have mainly utilized crosses between MRL/MPJ (a good healer) and SJL/J (a poor healer) or MRL/lpr (a good healer) and C57BL/6J (a poor healer). Wound healing is a complex trait with many genes involved in the expression of the phenotype. Based on data from previous studies that common and additional quantitative trait loci (QTL) were identified using different crosses of inbred strains of mice for various complex traits, we hypothesized that a new cross would identify common and additional QTL, unique modes of inheritance, and interacting loci, which are responsible for variation in susceptibility to fast wound healing. In this study, we crossed DBA/1J (DBA, a good healer) and 129/SvJ (129, a poor healer) and performed a genome-wide scan using 492 (DBA×129) F2 mice and 98 markers to identify QTL that regulate wound healing/regeneration. Four QTL on chromosomes 1, 4, 12, and 18 were identified which contributed toward wound healing in F2 mice and accounted for 17.1% of the phenotypic variation in ear punch healing. Surprisingly, locus interactions contributed to 55.7% of the phenotype variation in ear punch healing. In conclusion, we have identified novel QTL and shown that minor interacting loci contribute significantly to wound healing in DBA×129 mice cross. The authors Masinde, Li, and Nguyen contributed equally to this article.     

Autoimmune-induced preferential depletion of myelin-associated glycoprotein (MAG) is genetically regulated in relapsing EAE (B6 × SJL) F1 mice

Background

The physiological function of the cellular prion protein (PrP^C) remains unknown. However, PrP^C has been reported to possess a cytoprotective activity that prevents death of neurons and other cells after a toxic stimulus. To explore this effect further, we attempted to reproduce several of the assays in which a protective activity of PrP had been previously demonstrated in mammalian cells.

Results

In the first set of experiments, we found that PrP over-expression had a minimal effect on the death of MCF-7 breast carcinoma cells treated with TNF-α and Prn-p ^0/0 immortalized hippocampal neurons (HpL3-4 cells) subjected to serum deprivation. In the second set of assays, we observed only a small difference in viability between cerebellar granule neurons cultured from PrP-null and control mice in response to activation of endogenous or exogenous Bax.

Conclusion

Taken together, our results suggest either that cytoprotection is not a physiologically relevant activity of PrP^C, or that PrP^C-dependent protective pathways operative in vivo are not adequately modeled by these cell culture systems. We suggest that cell systems capable of mimicking the neurotoxic effects produced in transgenic mice by N-terminally deleted forms of PrP or Doppel may represent more useful tools for analyzing the cytoprotective function of PrP^C     

β-Glucanase specific expression in the parotid gland of transgenic mice

The feasibility of using the pig parotid secretory protein promoter to drive the β-glucanase transgene expression in mouse parotid glands was examined in this study. The parotid gland-specific vector expressing β-glucanase gene (GLU, from Paenibacillus polymyxa CP7) was constructed. Transgenic mice were produced by the pronuclear microinjection. Both PCR and Southern blot analysis showed that the mice carried the β-glucanase gene and the β-glucanase gene could be stably inherited. Furthermore, RT-PCR and northern blot analysis indicated that it was specifically expressed in the parotid. The β-glucanase activity in the saliva was found to be 0.18 U/mL. After feeding a diet containing 2 % β-glucan, the average daily gain of transgenic was significantly higher than non-transgenic mice. The crude protein and crude fat concentration in faeces of transgenic mice were significantly reduced compared with that of the non-transgenic mice. These results suggest that the successful expression of foreign β-glucanase in the animal parotid would offer a promising biological approach to reduce the anti-nutritional effect of β-glucans in feed.     

SLC30A family expression in the pancreatic islets of humans and mice: cellular localization in the β-cells

Zinc is a vital co-factor for insulin metabolism in the pancreatic β-cell, involved in synthesis, maturation, and crystallization. Two families of zinc transporters, namely SLC30A (ZNT) and SLC39A (ZIP) are involved in maintaining cellular zinc homeostasis in mammalian cells. Single nuclear polymorphisms or mutations in zinc transporters have been associated with insulin resistance and risk of type 2 diabetes (T2D) in both humans and mice. Thus, mice can be useful for studying the underlying mechanisms of zinc-associated risk of T2D development. To determine potential differences in zinc transporter expression and cellular localization in the pancreatic β-cells between humans and mice, we examined all members (ZNT1-10) of the ZNT family in pancreatic islets and in β-cell lines derived from both species using immunohistochemistry and immunofluorescence microscopic analysis. We found that there were no substantial differences in the expression of nine ZNT proteins in the human and mouse islets and β-cells with exception of ZNT3, which was only detected in human β-cells, but not in mouse β-cells. Moreover, we found that ZNT2 was localized on the cell surface of both human and mouse β-cells, suggesting a role of ZNT2 in direct export of zinc out of the β-cell. Together, our study suggests functional conservations of the ZNT proteins between humans and mice. We believe that our results are of interest for future studies in the association of zinc metabolism with risk of T2D in humans using mouse models.     

A message emerging from development: the repression of mitochondrial β-F1-ATPase expression in cancer

Mitochondrial research has experienced a considerable boost during the last decade because organelle malfunctioning is in the genesis and/or progression of a vast array of human pathologies including cancer. The renaissance of mitochondria in the cancer field has been promoted by two main facts: (1) the molecular and functional integration of mitochondrial bioenergetics with the execution of cell death and (2) the implementation of ¹⁸FDG-PET for imaging and staging of tumors in clinical practice. The latter, represents the bed-side translational development of the metabolic hallmark that describes the bioenergetic phenotype of most cancer cells as originally predicted at the beginning of previous century by Otto Warburg. In this minireview we will briefly summarize how the study of energy metabolism during liver development forced our encounter with Warburg’s postulates and prompted us to study the mechanisms that regulate the biogenesis of mitochondria in the cancer cell.     

Investigate of AQP gene expression in the liver of mice after ischemia–reperfusion

Ischemia–reperfusion (IR) injury usually occurs during liver transplantation. Aquaporins (AQPs) are transmembrane channels that facilitate water permeability through cell membranes and are essential for the regulation of water homeostasis. Changes in the AQPs expression have been correlated with several inflammatory diseases. Less is known about AQPs expression in hepatic ischemia reperfusion injury. To clarify the roles of AQPs in IR injury, in this current study we examined the gene expression patterns of AQP1, 8 and 9 in the liver after IR injury. Male balb/c mice were exposed to partial (70%) hepatic ischemia for 65 min and then randomized into five groups of reperfusion [0 h (A), 8 h (B), 1 day (C), 3 days (D), and 7 days (E)]. A surgical group was also selected as the sham group. Serum and liver tissue samples were collected for evaluation of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and liver histopathology. Real time PCR was performed to evaluate the AQPs expression. I/R injury resulted in a significant increase in ALT and AST (p?<?0.05) compared to sham mice in each group. The gene expression of AQPs was significantly increased in the IR group compared with the sham group (p?<?0.05). AQP8 and AQP1 after 8 h (group B) showed the highest gene expression in comparison with other groups, but the highest level of AQP9 gene expression was observed after 1 day (group C). Pathologic changes in the liver after reperfusion were confirmed the IR. In the IR group cytoplasmic vacuolization, inflammatory cell infiltration and focal necrosis were detected. In conclusion, our findings indicated that the damage caused by ischemia–reperfusion in the liver can change the expression of AQP genes, which can interfere with hepatocellular homeostasis and their function. Upregulation of AQP1, 8 and 9 could contribute to the development of hepatocellular swelling after hepatic IR injury.     

Isozyme Analysis of F 5 and BC 1F 4 from Cultivated Barley (Hordeum vulgare)× Roegneria ciliaris

栽培大麦,纤毛鹅观草,属间杂种,酯酶,过氧化物酶 ISOZYME ANALYSIS OF F5 AND BCiF4 FROM CULTIVATED BARLEY ( HORDEUM VULGARE ) ~ ROEGNERIA CILIARIS LI Wan-Ji LI Yi-Ping L1U Fang Abstract Esterase and peroxidase isozymes were analysed in the variants including 4 types, 16 lines of Fs, BC1F4 and the parents derived from cultivated barley ( Hordeum vulgare cv. Arupo) x Roegneria ciliaris (Trin.) Nevski in young roots, shoots, spikes and seeds. The zymogram patterns of esterase and peroxidase demonstrated that the 16 lines of F5 and BC1F4 had all or most bands of the cultivated barley parent cv. "Arupo", 1 to 3 bands from the male R. ciliaris, and new hybrid isozyme bands in various amount. Some bands of parent "Arupo" were lost. It suggested that the genetic substances come from R. ciliaris were stably inherited to the progenies of selfing and backcrossing, and there were some variations among the lines. There was certain relationship between isozyme variance and plant characters. Thus, in identifying the translocation lines by isozyme analysis, it would be preferable to study the various organ-specific isozymes or to trace one type of isozyme pattern in consequence.     

Aberrant expression of IFN-γ in Th2 cells from Th2 LCR-deficient mice

The Th2 locus control region (LCR) has been shown to be a crucial cis-acting element for Th2 cytokine expression and Th2 cell differentiation. To study the role of Th2 LCR in ifng locus regulation, we examined the expression of IFN-γ in Th2 cells from Th2 LCR-deficient mice. We found IFN-γ to be aberrantly up-regulated. In addition, histone 3(H3)-acetylation and histone 3 lysine 4 (H3-K4)-methylation greatly increased at the ifng locus of the Th2 cells. GATA-3 and STAT6 bound to the ifng promoter in Th2 cells from the wild type but not from the Th2 LCR-deficient mice, and they directly repressed ifng expression in transient reporter assay. Moreover, ectopic expression of GATA-3 and STAT6-VT repressed the aberrant expression of the ifng gene and restored repressive chromatin state at the ifng locus in Th2 cells from Th2 LCR-deficient mice. These results suggest that expression of the ifng gene and chromatin remodeling of the ifng locus are under the control of a Th2 LCR-mediated Th2 differentiation program.     

Neuregulin 1-β regulates cell adhesion molecule L1 expression in the cortex and hippocampus of mice

Neuregulin 1 (Nrg1) functions in neuronal migration, survival and differentiation as well as synaptogenesis during ontogenetic development and maintenance of synaptic functions in the adult mammalian brain. The neural adhesion molecule L1 (L1CAM) functions in similar overlapping, but also non-overlapping roles in the nervous system. In the present study, we therefore investigated some aspects of the functional relationship between Nrg1 and L1 in mammalian neural cells. Nrg1 regulates the expression of L1 in cultures of both human neuroblastoma SK-N-SH cells and mouse cortical and hippocampal neurons. To analyze the role of Nrg1 on L1 expression in vivo, young adult male mice received intraperitoneal injections of Nrg1 or PBS (vehicle control). The correlation between Nrg1 and L1 expression was tested by qPCR, Western blot analysis, and immunocytology. Our data indicate that neuregulin 1-β (Nrg1β) increases L1 expression in neurons of the cerebral cortex, and decreases expression in neurons of the hippocampus in vitro and in vivo. In addition, Nrg1 induces phosphorylation of its receptors, ErbB2 and ErbB4, the predominant ErbB receptors in the nervous system. These results show that Nrg1β affects expression of L1 in the central nervous system and in parallel activates the ErbB receptors for Nrg1, suggesting a crosstalk between molecules that are of prime importance for nervous system functions.     

Quantitative trait loci for growth traits in C57BL/6J × DBA/2J mice

Quantitative trait loci (QTLs) for body weight and tail length are mapped in an F₂ population of 927 C57BL/6J × DBA/2J mice. We test the concordance between the locations of the mapped QTLs with those detected by changes of marker frequency under artificial selection in a previous experiment with the same base population. The directions of effects of the QTLs are generally in agreement, and in many cases significant QTLs are found in similar map positions, but there are also discrepancies between the two experiments. There are indications of age-specific QTL effects on growth. For body weight traits, the genetic variation in the F₂ appears to result from many loci with relatively small effects. For tail length at 10 weeks, however, a single QTL on Chromosome (Chr) 1 with a peak LOD score of ∼33 contributes most of the genetic variation detected, changes the trait value by about 6%, and explains about 20% of the phenotypic variance of the trait. Received: 4 August 1998 / Accepted: 17 November 1998     

Oxytrofalcatins A–F,N-benzoylindole analogues from the roots of Oxytropis falcata (Leguminosae)

Indole alkaloids, oxytrofalcatins A–F (1–6), together with five other known alkaloids (7–11), were isolated from the roots of Oxytropis falcata. Their structures were elucidated by comprehensive spectroscopic analyses, including using 1D and 2D NMR spectroscopy and mass spectrometry. This is the first report of N-benzoylindoles from a natural source. Compounds 1–6 lacked significant cytotoxicity against SGC-7901 and HL-60 tumor cell lines.