Toc159- and Toc75-independent import of a transit sequence-less precursor into the inner envelope of chloroplasts

Chloroplast envelope quinone oxidoreductase (ceQORH) is an inner plastid envelope protein that is synthesized without cleavable chloroplast transit sequence for import. In the present work, we studied the in vitro-import characteristics of Arabidopsis ceQORH. We demonstrate that ceQORH import requires ATP and is dependent on proteinaceous receptor components exposed at the outer plastid surface. Competition experiments using small subunit precursor of ribulose-bisphosphate carboxylase/oxygenase and precursor of ferredoxin, as well as antibody blocking experiments, revealed that ceQORH import does not involve the main receptor and translocation channel proteins Toc159 and Toc75, respectively, which operate in import of proteins into the chloroplast. Molecular dissection of the ceQORH amino acid sequence by site-directed mutagenesis and subsequent import experiments in planta and in vitro highlighted that ceQORH consists of different domains that act concertedly in regulating import. Collectively, our results provide unprecedented evidence for the existence of a specific import pathway for transit sequence-less inner plastid envelope membrane proteins into chloroplasts.     

Functional expression and characterisation of human cytochrome P45017alpha in Pichia pastoris

Human cytochrome P45017alpha (CYP17), present in mammalian adrenal and gonadal tissues, catalyses both steroid 17-hydroxylation and C17,20 lyase reactions, producing intermediates for the glucocorticoid and androgenic pathways, respectively. The characterisation of this complex enzyme was initially hampered due to low level in vivo expression of CYP17. Heterologous expression systems have contributed greatly to our current knowledge of CYP17's dual catalytic activity. However, due to the hydrophobic nature of this membrane-bound protein, primarily truncated and modified forms of CYP17 are currently being expressed heterologously. Although the N-terminally modified enzyme has been well characterised, protein structure and function studies still necessitate the expression of unmodified, wild-type CYP17. We report here the expression of a catalytically active, unmodified human CYP17 in the industrial methylotrophic yeast, Pichia pastoris. A typical P450 carbon monoxide difference spectrum, with an absorption maximum at 448nm and a substrate-induced type I spectrum were recorded using a detergent-solubilised cellular fraction containing CYP17. The expressed enzyme catalysed the conversion of progesterone to 17-hydroxyprogesterone as well as 16-hydroxyprogesterone, a product unique to human and chimpanzee CYP17. This is the first report showing the heterologous expression of a fully functional human steroidogenic cytochrome P450 enzyme in P. pastoris.     

Proteomic analysis of the U1 snRNP of Schizosaccharomyces pombe reveals three essential organism-specific proteins

Characterization of spliceosomal complexes in the fission yeast Schizosaccharomyces pombe revealed particles sedimenting in the range of 30–60S, exclusively containing U1 snRNA. Here, we report the tandem affinity purification (TAP) of U1-specific protein complexes. The components of the complexes were identified using (LC-MS/MS) mass spectrometry. The fission yeast U1 snRNP contains 16 proteins, including the 7 Sm snRNP core proteins. In both fission and budding yeast, the U1 snRNP contains 9 and 10 U1 specific proteins, respectively, whereas the U1 particle found in mammalian cells contains only 3. Among the U1-specific proteins in S. pombe, three are homolog to the mammalian and six to the budding yeast Saccharomyces cerevisiae U1-specific proteins, whereas three, called U1H, U1J and U1L, are proteins specific to S. pombe. Furthermore, we demonstrate that the homolog of U1-70K and the three proteins specific to S. pombe are essential for growth. We will discuss the differences between the U1 snRNPs with respect to the organism-specific proteins found in the two yeasts and the resulting effect it has on pre-mRNA splicing.     

Oxygen distribution and consumption in the macaque retina

The oxygen distribution in the retina of six anesthetized macaques was investigated as a model for retinal oxygenation in the human retina in and adjacent to the fovea. P(O2) was measured as a function of retinal depth under normal physiological conditions in light and dark adaptation with O(2) microelectrodes. Oxygen consumption (Q(O2)) of the photoreceptors was extracted by fitting a steady-state diffusion model to P(O2) measurements. In the perifovea, the P(O2) was 48 +/- 13 mmHg (mean and SD) at the choroid and fell to a minimum of 3.8 +/- 1.9 mmHg around the photoreceptor inner segments in dark adaptation, rising again toward the inner retina. The P(O2) in the inner half of the retina in darkness was 17.9 +/- 7.8 mmHg. When averaged over the outer retina, photoreceptor Q(O2) (called Q(av)) was 4.6 +/- 2.3 ml O(2).100 g(-1).min(-1) under dark-adapted conditions. Illumination sufficient to saturate the rods reduced Q(av) to 72 +/- 11% of the dark-adapted value. Both perifoveal and foveal photoreceptors received most of their O(2) from the choroidal circulation. While foveal photoreceptors have more mitochondria, the Q(O2) of photoreceptors in the fovea was 68% of that in the perifovea. Oxygenation in macaque retina was similar to that previously found in cats and other mammals, reinforcing the relevance of nonprimate animal models for the study of retinal oxygenation, but there was a smaller reduction in Q(O2) with light than observed in cats, which may have implications for understanding the influence of light under some clinical conditions.     

Delayed expression of cytokines after reperfused myocardial infarction: possible trigger for cardiac dysfunction and ventricular remodeling

Previous studies have shown that 1 wk after permanent coronary artery ligation in rats, some cellular mechanisms involving TNF-alpha occur and contribute to the development of cardiac dysfunction and subsequent heart failure. The aim of the present study was to determine whether similar phenomena also occur after ischemia-reperfusion and whether cytokines other than TNF-alpha can also be involved. Anesthetized male Wistar rats were subjected to 1 h coronary occlusion followed by reperfusion. Cardiac geometry and function were assessed by echocardiography at days 5, 7, 8, and 10 postligation. Before death, heart function was assessed in vivo under basal conditions, as well as after volume overload. Finally, hearts were frozen for histoenzymologic assessment of infarct size and remodeling. The profile of cardiac cytokines was determined by ELISA and ChemiArray on heart tissue extracts. As expected, ischemia-reperfusion induced a progressive remodeling of the heart, characterized by left ventricular free-wall thinning and cavity dilation. Heart function was also decreased in ischemic rats during the first week after surgery. Interestingly, a transient and marked increase in TNF-alpha, IL-1beta, IL-6, cytokine-induced neutrophil chemoattractant (CINC) 2, CINC3, and macrophage inflammatory protein-3alpha was also observed in the myocardium of myocardial ischemia (MI) animals at day 8, whereas the expression of anti-inflammatory interleukins IL-4 and IL-10 remained unchanged. These results suggest that overexpression of proinflammatory cytokines occurring during the first week after ischemia-reperfusion may play a role in the adaptative process in the myocardium and contribute to early dysfunction and remodeling.     

Impact of Type 2 diabetes and aging on cardiomyocyte function and O-linked N-acetylglucosamine levels in the heart

Increased levels of O-linked attachment of N-acetylglucosamine (O-GlcNAc) on nucleocytoplasmic proteins are implicated in the development of diabetic cardiomyopathy and are regulated by O-GlcNAc transferase (OGT) expression and its substrate UDP-GlcNAc. Therefore, the goal of this study was to determine whether the development of diabetes in the Zucker diabetic fatty (ZDF) rat, a model of Type 2 diabetes, results in defects in cardiomyocyte mechanical function and, if so, whether this is associated with increased levels of O-GlcNAc and increased OGT expression. Six-week-old ZDF rats were hyperinsulinemic but normoglycemic, and there were no differences in cardiomyocyte mechanical function, UDP-GlcNAc, O-GlcNAc, or OGT compared with age-matched lean control rats. Cardiomyocytes isolated from 22-wk-old hyperglycemic ZDF rats exhibited significantly impaired relaxation, compared with both age-matched lean control and 6-wk-old ZDF groups. There was also a significant increase in O-GlcNAc levels in high-molecular-mass proteins in the 22-wk-old ZDF group compared with age-matched lean control and 6-wk-old ZDF groups; this was associated with increased UDP-GlcNAc levels but not increased OGT expression. Surprisingly, there was a significant decrease in overall O-GlcNAc levels between 6 and 22 wk of age in lean, ZDF, and Sprague-Dawley rats that was associated with decreased OGT expression. These results support the notion that an increase in O-GlcNAc on specific proteins may contribute to impaired cardiomyocyte function in diabetes. However, this study also indicates that in the heart the level of O-GlcNAc on proteins appears to be differentially regulated by age and diabetes. hexosamine biosynthesis; protein O-glycosylation; O-linked N-acetylglucosamine transferase     

VEGF receptor inhibition blocks liver cyst growth in pkd2(WS25/-) mice

Proliferation of cyst-lining epithelial cells is an integral part of autosomal dominant polycystic kidney disease (ADPKD) cyst growth. Cytokines and growth factors within cyst fluids are positioned to induce cyst growth. Vascular endothelial growth factor (VEGF) is a pleiotropic growth factor present in ADPKD liver cyst fluids (human 1,128 ± 78, mouse 2,787 ± 136 pg/ml) and, to a lesser extent, in ADPKD renal cyst fluids (human 294 ± 41, mouse 191 ± 90 pg/ml). Western blotting showed that receptors for VEGF (VEGFR1 and VEGFR2) were present in both normal mouse bile ducts and pkd2(WS25/–) liver cyst epithelial cells. Treatment of pkd2(WS25/–) liver cyst epithelial cells with VEGF (50–50,000 pg/ml) or liver cyst fluid induced a proliferative response. The effect on proliferation of liver cyst fluid was inhibited by SU-5416, a potent VEGF receptor inhibitor. Treatment of pkd2(WS25/–) mice between 4 and 8 mo of age with SU-5416 markedly reduced the cyst volume density of the liver (vehicle 9.9 ± 4.3%, SU-5416 1.8 ± 0.7% of liver). SU-5416 treatment between 4 and 12 mo of age markedly protected against increases in liver weight [pkd2(+/+) 4.8 ± 0.2%, pkd2(WS25/–)-vehicle 10.8 ± 1.9%, pkd2(WS25/–)-SU-5416 4.8 ± 0.4% body wt]. The capacity of VEGF signaling to induce in vitro proliferation of pkd2(WS25/–) liver cyst epithelial cells and inhibition of in vivo VEGF signaling to retard liver cyst growth in pkd2(WS25/–) mice indicates that the VEGF signaling pathway is a potentially important therapeutic target in the treatment of ADPKD liver cyst disease. autosomal dominant polycystic kidney disease; SU-5416; growth factors; cytokines     

Impact of nutritional supplements and monosaccharides on growth, oxalate accumulation, and culture pH by Sclerotinia sclerotiorum

Sclerotinia sclerotiorum D-E7 was studied to determine the impact of nutritional supplements and monosaccharides on growth, oxalate accumulation, and culture pH in broth media (initial pH c. 5). Cultures with 0.1% nutritional supplement (tryptone, yeast extract, or soytone) yielded minimal growth, 2-3 mM oxalate, and a final culture pH of 4.2-4.8. In contrast, cultures with 0.1% nutritional supplement and 25 mM glucose yielded significant growth, minimal oxalate (<1 mM), and a final culture pH of 2.8-3.7. Similar trends were observed when glucose in 0.1% soytone cultures was replaced with 25 mM d-mannose, l-arabinose, or d-xylose. With 1% soytone-25 mM glucose cultures, growth and oxalate accumulation ( approximately 21 mM) occurred with little change in initial pH. This was not the case with 1% soytone-250 mM glucose cultures; increased glucose levels resulted in a decrease in oxalate accumulation ( approximately 7 mM) and in final culture pH (3.4). Time-course studies with these cultures revealed that oxalate accumulation was suppressed during growth when the culture pH dropped to <4. Overall, these results indicate that (1) the decrease in external pH (i.e. acidification) was independent of oxalate accumulation and (2) acidification coupled to glucose-dependent growth regulated oxalate accumulation by Sclerotinia sclerotiorum.     

Plasticity of human adipose stem cells to perform adipogenic and endothelial differentiation

Recent research findings postulate that adipocytes and endothelial cells (EC) may share a common progenitor. However, the interlinking pathways between adipose tissue and endothelium, and the differentiation potential of cells to convert from one tissue into the other via progenitor cells have not been elucidated and are therefore the focus of this study. Stromal vascular fraction (SVF) cells were isolated from liposuction aspirates or excised adipose tissue and separated into CD31+ and CD31- populations by magnet-assisted cell sorting. Differentiation to fat tissue was induced in both CD31 fractions after expansion by insulin, dexamethasone, isobutylmethylxanthine, triiodothyronine, pioglitazone, and transferrin. Differentiation was assayed enzymatically and by cell counting. Maturation to endothelium was performed with vascular endothelial growth factor (VEGF), insulin-like growth factor-1 plus 2% fetal calf serum, and confirmed by flow cytometry and tube formation assays on Matrigel. Our results show that the SVF contains a CD31-, S100+ cell type that can differentiate into adipocytes and EC. The SVF also comprises CD31+ cells that, although they have an endothelial phenotype, can be converted into mature adipocytes. These findings demonstrate the potency of SVF cells to perform both adipogenic and endothelial differentiation. Further, they reveal the plasticity of mature cells of mesenchymal origin to undergo conversion from endothelium to adipose tissue and vice versa.     

Role of P/Q calcium channel in familial hemiplegic migraine

Voltage-dependent calcium channels constitute one of the main pathways of calcium entry into neurons. They are the principal actors of synaptic transmission by controlling the release of neurotransmitters. They also contribute to numerous other cell functions, such as gene expression or synaptogenesis. These channels, by their essential cell functions, are at the origin of numerous channelopathies resulting from mutations of the genes encoding their different subunits. Familial Hemiplegic Migraine (FHM) represents one such example of these channelopathies. In this human disease, genetic studies have demonstrated the implication of the CACNA1A gene in a type 1 form of FHM. This gene encodes for the Ca(v)2.1 subunit of P/Q calcium channels and is the target of numerous mutations affecting the properties of channel activity. The question on how discrete mutations of this gene are able to alter the activity of the channel and contribute to the physiopathology of FHM remains an open question. The functional characterization of mutated channels in various heterologous expression systems, as well as in vivo in an animal model, provides a molecular scheme of the physiopathology of FHM in which neurons, astrocytes and blood circulation act in concert.