δ-Aminolevulinate dehydratase from Raphanus sativus cotyledons

Phytochrome induces δ-aminolevulinate dehydratase (ALAD) activity in radish seedling cotyledons under continuous far red light. Analysis of the enzymatic activity in etioplasts vs total activity shows a constant ALAD level in these organelles (10 %) in etiolated seedlings. In far red irradiated seedlings, the percentage of enzyme detected into etioplasts increases up to 45 % of the total. Comparative kinetic studies of ALAD activity detected in the cytoplasm and the etioplasts indicate an increase in both compartments with a maximum value reached respectively at 96 and 120 hr from sowing. Treatment with cycloheximide shows a very fast abolition of cytoplasmic ALAD activity which is always correlated to an etioplast decrease with a time shift of ca 24 hr. Erythromycin acts only on the cytoplasmic level of ALAD, and only for far red irradiated seedlings, with an increase of activity twice the level detected in untreated ones. This unexpected effect is discussed.     

Diversity at two genetic loci associated with the major histocompatibility complex in the golden snub-nosed monkey (Rhinopithecus roxellana)

Habitat loss via human activity has fragmented populations of the golden snub-nosed monkey (Rhinopithecus roxellana), and thus affected patterns of gene flow. We investigated in-depth a single troop in the Qinling Mountains, central China, two major histocompatibility complex (MHC) class II loci, DQA1 and DQB1, and compared the resulting data with data from troops from the wider Qinling Mountains region. We found that a novel DQB1 allele was only present in the study troop and relatively few divergent alleles at the DQA1 and DQB1 loci were present compared with the wider population. The inbreeding coefficient (F_is) at the MHC region was lower than previous measurements, which may have reflected different sampling strategies. However, R. roxellana has relatively high diversity in MHC genes, even though it has probably experienced serious past population bottlenecks and reduced gene flow between populations. We also found that some alleles present in the wider population had been lost in the study troop, and suggest that conservation management strategies be implemented to increase gene flow between troops in order to increase genetic variation.     

Deletion of sterol O-acyltransferase 2 (SOAT2) function in mice deficient in lysosomal acid lipase (LAL) dramatically reduces esterified cholesterol sequestration in the small intestine and liver

Sterol O-acyltransferase 2 (SOAT2), also known as ACAT2, is the major cholesterol esterifying enzyme in the liver and small intestine (SI). Esterified cholesterol (EC) carried in certain classes of plasma lipoproteins is hydrolyzed by lysosomal acid lipase (LAL) when they are cleared from the circulation. Loss-of-function mutations in LIPA, the gene that encodes LAL, result in Wolman disease (WD) or cholesteryl ester storage disease (CESD). Hepatomegaly and a massive increase in tissue EC levels are hallmark features of both disorders. While these conditions can be corrected with enzyme replacement therapy, the question arose as to what effect the loss of SOAT2 function might have on tissue EC sequestration in LAL-deficient mice. When weaned at 21 days, Lal^−/−:Soat2^+/+ mice had a whole liver cholesterol content (mg/organ) of 24.7 mg vs 1.9 mg in Lal^+/+:Soat2^+/+ littermates, with almost all the excess sterol being esterified. Over the next 31 days, liver cholesterol content in the Lal^−/−:Soat2^+/+ mice increased to 145 ± 2 mg but to only 29 ± 2 mg in their Lal^−/−:Soat2^−/− littermates. The level of EC accumulation in the SI of the Lal^−/−:Soat2^−/− mice was also much less than in their Lal^−/−:Soat2^+/+ littermates. In addition, there was a >70% reduction in plasma transaminase activities in the Lal^−/−:Soat2^−/− mice. These studies illustrate how the severity of disease in a mouse model for CESD can be substantially ameliorated by elimination of SOAT2 function.     

Inhibitors of sphingolipid metabolism enzymes

Sphingolipids are a family of lipids that play essential roles both as structural cell membrane components and in cell signalling. The cellular contents of the various sphingolipid species are controlled by enzymes involved in their metabolic pathways. In this context, the discovery of small chemical entities able to modify these enzyme activities in a potent and selective way should offer new pharmacological tools and therapeutic agents.     

Efficient temporally-controlled targeted mutagenesis in smooth muscle cells of the adult mouse

To generate temporally-controlled targeted somatic mutations selectively and efficiently in smooth muscles, we have established a transgenic SMA-Cre-ER(T2) mouse line in which the expression of the Tamoxifen-dependent Cre-ER(T2) recombinase is under the control of a large genomic DNA segment of the mouse smooth muscle alpha actin (SMA) gene, contained in a Bacterial artificial chromosome (Bac). In this transgenic mouse line, Cre-ER(T2)-mediated recombination of LoxP-flanked target DNA is strictly Tamoxifen-dependent, and efficient in both vascular and visceral smooth muscle cells. Moreover, with the exception of few cardiomyocytes, LoxP-flanked DNA excision is restricted to smooth muscle cells. Thus, SMA-Cre-ER(T2) mice should be of great value to analyze gene function in smooth muscles, and to establish new animal models of human smooth muscle disorders.     

Interactions between age,sex, and hormones in experimental ischemic stroke

Age, sex, and gonadal hormones have profound effects on ischemic stroke outcomes, although how these factors impact basic stroke pathophysiology remains unclear. There is a plethora of inconsistent data reported throughout the literature, primarily due to differences in the species examined, the timing and methods used to evaluate injury, the models used, and confusion regarding differences in stroke incidence as seen in clinical populations vs. effects on acute neuroprotection or neurorepair in experimental stroke models. Sex and gonadal hormone exposure have considerable independent impact on stroke outcome, but these factors also interact with each other, and the contribution of each differs throughout the lifespan. The contribution of sex and hormones to experimental stroke will be the focus of this review. Recent advances and our current understanding of age, sex, and hormone interactions in ischemic stroke with a focus on inflammation will be discussed.     

Temporal regulation of Cre recombinase activity in neural stem cells

Neural stem cells are known to give rise to distinct subtypes of neurons and glial cells over time by changing their competency. However, precise characterization of neural stem cells at various developmental stages remains to be performed. For such analysis, a tool to manipulate neural stem cells at different time points is necessary. Here, we generated transgenic mice that express Cre-ER(T2) in the ventricular zone of the developing nervous system under the control of the nestin promoter and enhancer (Nes-CreER(T2)). In mice expressing Cre-ER(T2) at appropriate levels, Cre recombinase activity was mostly inactive but efficiently activated by tamoxifen within 1 day. When such mice were crossed with the ROSA-26 or Z/EG reporter mice, neural stem cells were permanently labeled after administration of tamoxifen. Thus, Nes-CreER(T2) mice offer a powerful tool to manipulate neural stem cells genetically at desired time points.     

Identification of a novel missense mutation in Brazilian patient with a severe form of mucopolysaccharidosis type IVA

Mucopolysaccharidosis type IVA (MPS IVA) or Morquio syndrome type A is an autosomal recessive disease caused by deficiency of the lysosomal enzyme N-acetylgalactosamine-6-sulfatase (GALNS). We report molecular characterization of a patient who presents the new missense mutation p.C165Y in homozygosis. Bioinformatics analysis predicted this mutation as being probably pathogenic. To evaluate the possibility that this alteration was a polymorphism we tested 100 alleles and all the results were negative. These findings together with the observation that this alteration is not present in controls, suggest that it is a disease-causing mutation, which was correlated with the severe phenotype observed in our patient. We conclude that molecular analysis of the GALNS gene, in addition to enzyme assays, is important for diagnosis and contributes to the better understanding of the relationship between genotype and phenotype, which is important as enzyme replacement therapy (ERT) will soon become available and treatment decisions will have to be take in such cases.     

Novel GAA sequence variant c.1211 A > G reduces enzyme activity but not protein expression in infantile and adult onset Pompe disease

Pompe disease is a clinically and genetically heterogeneous autosomal recessive disorder caused by lysosomal acid α-glucosidase (GAA) deficiency. We report on two affected members of a non-consanguineous Caucasian family, including a classical infantile-onset patient with severe cardiomyopathy (IO) and his paternal grandmother with the adult-onset (AO) form. Two compound heterozygous sequence variants of the GAA gene were identified in each patient by mutation analyses (IO = c.1211A > G and c.1798C > T; AO = c.1211A > G and c.692 + 5G > T). For this study, the biochemical phenotype resulting from the missense mutation c.1211A > G in exon 8, which converts a highly conserved aspartate to glycine (p.Asp404Gly), was of specific interest because it had not been reported previously. Western blotting revealed a robust expression of all GAA isoforms in quadriceps muscle of both patients (fully CRIM positive), while enzymatic activity was 3.6% (IO) and 6.6% (AO) of normal controls. To further validate these findings, the c.1211A > G sequence variant was introduced in wild type GAA cDNA and over-expressed in HEK293T cells. Site-directed mutagenesis analyses confirmed that the mutation does not affect processing or expression of GAA protein, but rather impairs enzyme function. Similar results were reported for c.1798C > T (p.Arg600Cys), which further supports the biochemical phenotype observed in IO. The third mutation (c.692 + 5G > T, in intron 3) was predicted to affect normal splicing of the GAA mRNA, and qPCR indeed verified a 4-fold lower mRNA expression in AO. It is concluded that the novel sequence variant c.1211A > G results in full CRIM but significantly lower GAA activity, which in combination with c.1798C > T leads to infantile-onset Pompe disease. We surmise that the difference in disease severity between the two family members in this study is due to a milder effect of the intronic mutation c.692 + 5G > T (vs. c.1798C > T) on phenotype, partially preserving GAA activity and delaying onset in the proband (paternal grandmother).