Inactivation of the murine Transferrin Receptor 2 gene using the Cre recombinase: loxP system

Transferrin Receptor 2 (TfR2) is a key molecule involved in the regulation of iron homeostasis. Mutations in TfR2 lead to type 3 hemochromatosis in humans. We have developed mice with a targeted deletion of TfR2. The Cre-recombinase:loxP system used to create the mice allows both full deletion and tissue-specific deletion of TfR2. The development of these mice will provide new models for type 3 hemochromatosis and assist in determining the role of TfR2 in iron metabolism.     

基因枪转化的bar基因表达盒在水稻中遗传行为复杂

基因枪介导基因表达盒(仅包括启动子、编码区和终止子)转化是基因枪转化植物的新趋势,它能消除质粒载体主干序列对转基因植物的不利影响。本文研究了基因枪转化的bar基因表达盒在转基因水稻T1～T3世代中的遗传行为。结果发现:作为筛选标记的bar基因表达盒在水稻基因组中多拷贝整合,遗传分离行为复杂,还出现了Basta抗感分离比在35:1～144:1之间的"假纯合体",但50%转基因株系中(5/10)bar基因可作为一个显性基因按孟德尔方式稳定遗传至自交T2代。虽然bar基因为多拷贝整合,30%的转基因株系(3/10)在自交低世代(T1)能获得纯合体。Southern杂交分析发现,多拷贝的bar基因表达盒倾向于连接成转基因串联子整合在水稻基因组内。我们发现在Basta抗性正常分离的株系后代中bar基因表达盒Southern杂交模式能稳定遗传,但异常分离的株系后代中bar基因表达盒的一些拷贝发生了丢失。我们推测,bar基因表达盒在水稻中遗传分离行为的复杂原因可能是bar基因表达盒多拷贝整合、基因丢失和基因表达互作。     

Functional Genomics Reveals Synthetic Lethality between Phosphogluconate Dehydrogenase and Oxidative Phosphorylation

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Overexpression of mutant HSP27 causes axonal neuropathy in mice

Background

Mutations in heat shock 27 kDa protein 1 (HSP27 or HSPB1) cause distal hereditary motor neuropathy (dHMN) or Charcot-Marie-Tooth disease type 2 F (CMT2F) according to unknown factors. Mutant HSP27 proteins affect axonal transport by reducing acetylated tubulin.

Results

We generated a transgenic mouse model overexpressing HSP27-S135F mutant protein driven by Cytomegalovirus (CMV) immediate early promoter. The mouse phenotype was similar to dHMN patients in that they exhibit motor neuropathy. To determine the phenotypic aberration of transgenic mice, behavior test, magnetic resonance imaging (MRI), electrophysiological study, and pathology were performed. Rotarod test showed that founder mice exhibited lowered motor performance. MRI also revealed marked fatty infiltration in the anterior and posterior compartments at calf level. Electrophysiologically, compound muscle action potential (CMAP) but not motor nerve conduction velocity (MNCV) was reduced in the transgenic mice. Toluidine staining with semi-thin section of sciatic nerve showed the ratio of large myelinated axon fiber was reduced, which might cause reduced locomotion in the transgenic mice. Electron microscopy also revealed abundant aberrant myelination. Immunohistochemically, neuronal dysfunctions included elevated level of phosphorylated neurofilament and reduced level of acetylated tubulin in the sural nerve of transgenic mice. There was no additional phenotype besides motor neuronal defects.

Conclusions

Overexpression of HSP27-S135F protein causes peripheral neuropathy. The mouse model can be applied to future development of therapeutic strategies for dHMN or CMT2F.

Electronic supplementary material

The online version of this article (doi:10.1186/s12929-015-0154-y) contains supplementary material, which is available to authorized users.     

Dose – response relationship of bevacizumab in hereditary hemorrhagic telangiectasia

Hereditary hemorrhagic telangiectasia (HHT), a genetic vascular disorder associated with epistaxis and hepatic shunts, is responsible for high-output cardiac failure in rare cases. Bevacizumab, which targets vascular endothelial growth factor, was shown to decrease both cardiac index (CI) and epistaxis duration in HHT patients with severe liver involvement. The relationship between its serum concentration and change in both CI and epistaxis duration was investigated to design the bevacizumab maintenance dosing regimen of future therapeutic studies. Twenty-five HHT patients with dyspnea and high CI were included in a prospective non-comparative study. They received bevacizumab at a dose of 5 mg/kg per infusion every 14 days for a total of 6 injections. The relationships between bevacizumab serum concentration and both CI and epistaxis duration were described using transit compartments and direct inhibition pharmacokinetic-pharmacodynamic models. The performances of different maintenance regimens were evaluated using simulation. Infusions every 3, 2 and one months were predicted to maintain 41%, 45% and 50% of patients with CI <4 L/min/m² at 24 months, respectively. The fraction of patients with <20 min epistaxis per month was predicted to be 34%, 43% and 60%, with infusion every 3, 2 or one months, respectively. Simulations of the effects of different maintenance dosing regimens predict that monthly 5 mg/kg infusions of bevacizumab should allow sustained control of both cardiac index and epistaxis.     

Effects of strain and age on hepatic gene expression profiles in murine models of HFE-associated hereditary hemochromatosis

Hereditary hemochromatosis is an iron overload disorder most commonly caused by a defect in the HFE gene. While the genetic defect is highly prevalent, the majority of individuals do not develop clinically significant iron overload, suggesting the importance of genetic modifiers. Murine hfe knockout models have demonstrated that strain background has a strong effect on the severity of iron loading. We noted that hepatic iron loading in hfe−/− mice occurs primarily over the first postnatal weeks (loading phase) followed by a timeframe of relatively static iron concentrations (plateau phase). We thus evaluated the effects of background strain and of age on hepatic gene expression in Hfe knockout mice (hfe−/−). Hepatic gene expression profiles were examined using cDNA microarrays in 4- and 8-week-old hfe−/− and wild-type mice on two different genetic backgrounds, C57BL/6J (C57) and AKR/J (AKR). Genes differentially regulated in all hfe−/− mice groups, compared with wild-type mice, including those involved in cell survival, stress and damage responses and lipid metabolism. AKR strain-specific changes in lipid metabolism genes and C57 strain-specific changes in cell adhesion and extracellular matrix protein genes were detected in hfe−/− mice. Mouse strain and age are each significantly associated with hepatic gene expression profiles in hfe−/− mice. These affects may underlie or reflect differences in iron loading in these mice.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-014-0443-1) contains supplementary material, which is available to authorized users.     

Effect of glycine substitutions on alpha5(IV) chain structure and structure-phenotype correlations in Alport syndrome

The phenotype variety caused by glycine substitutions in alpha5(IV) chain in X-linked Alport syndrome (XLAS) prompted the complexity of structure changes of alpha5(IV) chain that was little to know now. In this study, we expressed a domain of alpha5(IV) chain containing different glycine substitutions (G1015V and G1030S, respectively) which were revealed in two XLAS pedigrees with different phenotype severities and the corresponding domain of a control in Escherichia coli. The recombinant proteins were characterized by immunoblot and mass spectrometry and analyzed the secondary structure by using circular dichroism (CD) spectroscopy. CD analysis showed that the recombinant protein containing G1015V mutation identified in the pedigree of juvenile-onset XLAS exhibited 12.9% alpha-helix that was not found in the control recombinant protein. The spectrum of the recombinant protein containing G1030S mutation identified in the pedigree of adult-onset XLAS was slightly different from that of the control, that is, mostly with the random coil and the beta-sheet, while without alpha-helix. These results demonstrated that two kinds of glycine substitutions, although in the same domain of alpha5(IV) chain, displayed the distinctly different secondary structures. The changes of the secondary structure could explain the phenotypic diversities of XLAS, which would be hardly understood solely by analyzing genomic DNA or mRNA of alpha5(IV) chain.     

Transferrin [corrected] receptor association and endosomal localization of soluble HFE are not sufficient for regulation of cellular iron homeostasis

Iron uptake and storage are tightly regulated to guarantee sufficient iron for essential cellular processes and to prevent the production of damaging free radicals. A non-classical class I MHC molecule, the hemochromatosis factor HFE, has been shown to regulate iron metabolism, potentially via its direct interaction with the transferrin receptor (TfR). In this study, we demonstrate that a soluble beta2microglobulin-HFE monochain (sHFE) folds with beta2microglobulin (beta2m) and associates with the TfR, indicating that the transmembrane and cytoplasmic domains are not necessary for assembly and trafficking through the ER-Golgi network. We also demonstrate human TfR-specific uptake and accumulation of extracellular sHFE by treated cells. The sHFE localized to the endosomal compartment albeit we observed variation in the time taken for endosomal trafficking between different cell types. The sHFE monochain was effective in reducing Tf uptake into cells, however this did not correlate to any changes in TfR or ferritin synthesis, in contrast to the HFE-induced increase and decrease of TfR and ferritin, respectively. These findings of incongruent sHFE activity suggest that either variation in affinity binding of sHFE to TfR prevents efficient modulation of iron-regulated proteins or that HFE has multiple functions some of which may be independent of TfR but dependent on interactions within the endosomal compartment for effective modulation of iron metabolism.     

Increases of T-type Ca2+ current in heart cells of the cardiomyopathic hamster

In the present study, the whole-cell voltage clamp technique was used in order to record the T- and L-type Ca²⁺ currents in single heart cells of newborn and young normal and hereditary cardiomyopathic hamsters. Our results showed that the I/V relationship curve as well as the kinetics of the L-type Ca²⁺ currents (I_Ca(L)) in both normal and cardiomyopathic heart cells were the same. However, the proportion of myocytes from normal heart hamster that showed L-type I_Ca was less than that of heart cells from cardiomyopathic hamster. The I/V relationship curve of the T-type I_Ca (I_Ca(T)) was the same in myocytes of both normal and cardiomyopathic hamsters. The main differences between I_Ca(T) of cardiomyopathic and normal hamster are a larger window current and the proportion of ventricular myocytes that showed this type of current in cardiomyopathic hamster. The high density of I_Ca(T) as well as the large window current and proportion of myocytes showing I_Ca(T) may explain in part Ca²⁺ overload observed in cardiomyopathic heart cells of the hamster.     

Coming to terms with heritability

The complex mechanisms of heredity are little appreciated by non-specialists, in some measure, because of misunderstandings that are perpetuated when words used for technical terms have other, more widely understood, folk meanings. When a word has both technical and folk meanings, it is the responsibility of the specialist to avoid promoting confusion by either using extremely cautious and precise language when using the term or, in cases when confusion is inevitable, abandoning the term in favor of one without a widely understood folk meaning. The study of heredity is beset by such confusion, and the term heritability appears to be at the heart of some of the confusion. In this article, I discuss both the technical and folk meanings of heritability and examine the bridge between them. By continuing to use the term heritability, we risk promulgating serious misunderstanding about the workings of heredity, therefore I suggest selectability as an alternative term to avoid such pitfalls. This revised version was published online in August 2006 with corrections to the Cover Date.