PV-1 Labels Trans-Cellular Openings in Mouse Endothelial Cells and is Negatively Regulated by VEGF

The PV-1 protein is endogenously expressed from a single mRNA in the mouse pancreatic MS-1 endothelial cell line as a 60-kDa N-glycosylated and 50-kDa non-glycosylated protein that form DTT sensitive oligomers. In the absence of cell permeabilization, PV-1 labels transcellular openings of variable size, many that penetrate through the cytosol with circular openings on the free and attached surface of the plasma membrane. Intracellular PV-1 is localized in perinuclear aggregates that can extend as a fibrous network through the cytosol and often surround the nuclear compartment. In some cells, PV-1 is organized as a large unipolar spindle-like structure that is often associated with severe deformation of the nucleus. The VEGF-R2 inhibitor SU5614 increased the PV-1 protein in a dose-dependent manner and inhibited MS-1 cell growth, without inducing apoptosis. This report provides compelling evidence for a functional role of PV-1 in the formation of large transendothelial channels and modulation of nuclear shape. Moreover, these data suggest the PV-1 protein is negatively regulated by VEGF.     

Developmental regulation of PV-1 in rat lung: association with the nuclear envelope and limited colocalization with Cav-1

Plasmalemma vesicle protein-1 (PV-1) is a caveolae-associated protein that is enriched in lung endothelial cells. The PV-1 protein is first detected in the lung at embryonic day 12, before that of caveolin-1 (Cav-1). There is a postnatal rise in PV-1 and Cav-1 mRNA levels, reaching a peak at the time of weaning and declining to their lowest levels in the adult lung. In contrast, the PV-1 protein progressively increases during postnatal development with its highest levels in the adult lung; the Cav-1 protein remains relatively constant throughout this period. Alveolar endothelial cells express both PV-1 and Cav-1 proteins, but PV-1, unlike Cav-1, is also detectable in some bronchial epithelial cells. Endothelial cells transfected with a rat PV-1 construct show a punctate membrane distribution of PV-1, perinuclear accumulation, and an association with the nuclear envelope. In these cells, PV-1 exhibits only partial perinuclear colocalization with Cav-1 and F-actin. In summary, PV-1 is developmentally regulated in the rat lung and shows a divergent intracellular localization, with a limited caveolae/Cav-1 colocalization in cultured endothelial cells.     

Histone Demethylase Retinoblastoma Binding Protein 2 is Overexpressed in Hepatocellular Carcinoma and Negatively Regulated by hsa-miR-212

Background

The H3K4 demethylase retinoblastoma binding protein 2 (RBP2) is involved in the pathogenesis of gastric cancer, but its role and regulation in hepatocellular carcinoma (HCC) is unknown. We determined the function of RBP2 and its regulation in HCC in vitro and in human tissues.

Methods

We analyzed gene expression in 20 specimens each of human HCC and normal liver tissue by quantitative real-time PCR and immunohistochemistry. Proliferation was analyzed by foci formation and senescence by β-galactosidase staining. Promoter activity was detected by luciferase reporter assay.

Results

The expression of RBP2 was stronger in cancerous than non-cancerous tissues, but that of its binding microRNA, Homo sapiens miR-212 (hsa-miR-212), showed an opposite pattern. SiRNA knockdown of RBP2 significantly upregulated cyclin-dependent kinase inhibitors (CDKIs), with suppression of HCC cell proliferation and induction of senescence. Overexpression of hsa-miR-212 suppressed RBP2 expression, with inhibited cell proliferation and induced cellular senescence, which coincided with upregulated CDKIs; with low hsa-miR-212 expression, CDKIs were downregulated in HCC tissue. Inhibition of hsa-miR-212 expression upregulated RBP2 expression. Luciferase reporter assay detected the direct binding of hsa-miR-212 to the RBP2 3′ UTR.

Conclusions

RBP2 is overexpressed in HCC and negatively regulated by hsa-miR-212. The hsa-miR-212–RBP2–CDKI pathway may be important in the pathogenesis of HCC.     

A Novel Nuclear Protein Phosphatase 2C Negatively Regulated by ABL1 is Involved in Abiotic Stress and Panicle Development in Rice

Type 2C protein phosphatase plays an important role in the signal transduction of stress response in plants. In this paper, we identified a novel stress-induced type 2C protein phosphatase gene OsSIPP2C1 from rice. OsSIPP2C1 contains a complete open reading frame of 1,074 bp, encoding a protein with 357 amino acids. OsSIPP2C1 expression was up-regulated by high salt, PEG6000 and exogenous ABA, and enhanced in the abl1 mutant under normal, salt, or drought condition. Interestingly, OsSIPP2C1 expression was increased during the early panicle development. Subcellular localization assay using rice protoplast cells indicated that OsSIPP2C1 was predominantly located in the nucleus. Together, it is suggested that a nuclear PP2C protein OsSIPP2C1 negatively regulated by ABL1 is involved in abiotic stress and panicle development in rice.     

Sgt1 Dimerization Is Negatively Regulated by Protein Kinase CK2-mediated Phosphorylation at Ser361

The kinetochore, which consists of centromere DNA and structural proteins, is essential for proper chromosome segregation in eukaryotes. In budding yeast, Sgt1 and Hsp90 are required for the binding of Skp1 to Ctf13 (a component of the core kinetochore complex CBF3) and therefore for the assembly of CBF3. We have previously shown that Sgt1 dimerization is important for this kinetochore assembly mechanism. In this study, we report that protein kinase CK2 phosphorylates Ser³⁶¹ on Sgt1, and this phosphorylation inhibits Sgt1 dimerization.The kinetochore is a structural protein complex located in the centromeric region of the chromosome coupled to spindle microtubules (1, 2). The kinetochore generates a signal to arrest cells during mitosis when it is not properly attached to microtubules, thereby preventing chromosome missegregation, which can lead to aneuploidy (3, 4). The molecular structure of the kinetochore complex of the budding yeast Saccharomyces cerevisiae has been well characterized; it is composed of more than 70 proteins, many of which are conserved in mammals (2).The centromere DNA in the budding yeast is a 125-bp region that contains three conserved regions, CDEI, CDEII, and CDEIII (5, 6). CDEIII (25 bp) is essential for centromere function (7) and is bound to a key component of the centromere, the CBF3 complex. The CBF3 complex contains four proteins, Ndc10, Cep3, Ctf13 (8–15), and Skp1 (14, 15), all essential for viability. Mutations in any of the CBF3 proteins abolish the ability of CDEIII to bind to CBF3 (16, 17). All of the kinetochore proteins, except the CDEI-binding Cbf1 (18–20), localize to the kinetochores in a CBF3-dependent manner (2). Thus, CBF3 is a fundamental kinetochore complex, and its mechanism of assembly is of great interest.We have previously found that Sgt1 and Skp1 activate Ctf13; thus, they are required for assembly of the CBF3 complex (21). The molecular chaperone Hsp90 is also required to form the active Ctf13-Skp1 complex (22). Sgt1 has two highly conserved motifs that are required for protein-protein interaction: the tetratricopeptide repeat (21) and the CHORD protein and Sgt1-specific motif. We and others have found that both domains are important for the interaction of Sgt1 with Hsp90 (23–26), which is required for assembly of the core kinetochore complex. This interaction is an initial step in kinetochore activation (24, 26, 27), which is conserved between yeast and humans (28, 29).We have recently shown that Sgt1 dimerization is important for Sgt1-Skp1 binding and therefore for kinetochore assembly (30). In this study, we have found that protein kinase CK2 phosphorylates Sgt1 at Ser³⁶¹, and this phosphorylation inhibits Sgt1 dimerization. Therefore, CK2 appears to regulate kinetochore assembly negatively in budding yeast.     

Uncoupling protein 2, but not uncoupling protein 1, is expressed in the female mouse reproductive tract

Uncoupling proteins (UCPs) are transporters of the inner mitochondrial membrane. Whereas UCP1 is uniquely present in brown adipose tissue where it uncouples respiration from ATP synthesis and activates respiration and heat production, UCP2 is present in numerous tissues, and its exact function remains to be clarified. Two sets of data provided the rationale for this study: (i) the intriguing report that UCP1 is present in uterus of mice (Nibbelink, M., Moulin, K., Arnaud, E., Duval, C., Penicaud, L., and Casteilla, L. (2001) J. Biol. Chem. 276, 47291-47295); and (ii) an observation that Ucp2(-/-) female mice (homozygous matings) have smaller litters compared with Ucp2(+/+) animals (S. Rousset and A.-M. Cassard-Doulcier, unpublished observations). These data prompted us to examine the expression of UCP1 and UCP2 in the reproductive tract of female mice. Using wild type, Ucp1(-/-) mice, and Ucp2(-/-) mice, we were unable to detect UCP1 in uterus of mice with appropriate antibodies, and we conclude that the signal assigned to UCP1 by others was neither UCP1 nor UCP2. Using a polyclonal antibody against UCP2 and tissues from Ucp2(-/-) mice as controls, UCP2 was detected in ovary, oviduct, and uterus. Expression of Ucp2 mRNA was also observed in ovary and uterus using in situ hybridization analysis. Bone marrow transplantation experiments revealed that the UCP2 signal of the ovary was restricted to ovarian cells. UCP2 level in ovary decreased during follicular growth and increased during the pre-ovulatory period, during which aspects of an inflammatory process are known to exist. Because UCP2 down-regulates reactive oxygen species, a role in the regulation of inflammatory events linked to the preparation of ovulation is suggested.     

Notch2, but not Notch1, is required for proximal fate acquisition in the mammalian nephron

The Notch pathway regulates cell fate determination in numerous developmental processes. Here we report that Notch2 acts non-redundantly to control the processes of nephron segmentation through an Rbp-J-dependent process. Notch1 and Notch2 are detected in the early renal vesicle. Genetic analysis reveals that only Notch2 is required for the differentiation of proximal nephron structures (podocytes and proximal convoluted tubules) despite the presence of activated Notch1 in the nuclei of putative proximal progenitors. The inability of endogenous Notch1 to compensate for Notch2 deficiency may reflect sub-threshold Notch1 levels in the nucleus. In line with this view, forced expression of a gamma-secretase-independent form of Notch1 intracellular domain drives the specification of proximal fates where all endogenous, ligand-dependent Notch signaling is blocked by a gamma-secretase inhibitor. These results establish distinct (non-redundant), instructive roles for Notch receptors in nephron segmentation.     

Many but not all Genes in Chlamydomonas reinhardtii are Regulated by the Circadian Clock

Abstract: Total RNA from autotrophic Chlamydomonas reinhardtii cultures grown in constant dim light and 17 °C constant temperature was subjected to Northern blot analyses. The mRNAs for cytochrome c , β-tubulin, HSP70B (a chloroplastic heat shock protein of the 70 kD family), chloroplastic fructose-bisphosphate aldolase, and GAS3 (a "gamete-specific" protein of unknown function with high expression in gametes but low expression in vegetative cells) each exhibit a clear circadian rhythm in abundance. The rhythms differ significantly in phase and amplitude. The findings show that the genes for cytochrome c and β-tubulin indeed are regulated by the circadian clock, as previously suggested. Experiments with cultures grown at 27 °C instead of 17 °C further revealed that the rhythms in mRNA abundance for HSP70B, chloroplastic aldolase, and GAS3 also occur with a similar period at the higher temperature. Thus, the rhythms conform to the criterion of temperature compensation for the period and therefore represent true circadian rhythms. In contrast, the combined amount of mRNA for ubiquitin 52 amino acid fusion protein and ubiquitin 78 to 81 amino acid fusion protein stays constant under both temperature conditions. Because the combined amount of mRNA for the ubiquitin fusion proteins was previously shown to cycle under diurnal conditions when cell division activity is high, our data suggest a regulation of these genes by the cell division cycle and not the circadian clock. In summary, our data, together with several other reports, suggest that the circadian clock regulates many but not all genes in Chlamydomonas reinhardtii.     

Glucose disposal by insulin, but not IGF-1, is dependent on the hepatic parasympathetic nerves

Insulin-like growth factor-1 (IGF-1) has many insulin-like activities, including stimulation of glucose uptake in skeletal muscle. However, those with diabetes or chronic liver disease are insulin resistant but show a normal hypoglycemic response to IGF-1. We have previously shown that insulin sensitivity depends on a hepatic parasympathetic reflex release of a hormone from the liver. The hypothesis was tested that insulin action, but not IGF-1 action, is dependent on the hepatic parasympathetic reflex. Glucose disposal in response to three doses of IGF-1 (25, 100, 200 microg/kg) was determined in rats. IGF-1 at 200 microg/kg had similar effect on glucose disposal as did 50 mU/kg of insulin. Interruption of the hepatic parasympathetic reflex either by surgical ablation of the anterior nerve plexus or by atropine (1.0 mg/kg) resulted in insulin, but not IGF-1, resistance. Sixteen hours of fasting resulted in insulin, but not IGF-1, resistance. In conclusion, insulin, but not IGF-1, triggers the hepatic parasympathetic dependent release of a putative hepatic insulin sensitizing substance (HISS) that stimulates glucose uptake in skeletal muscle.     

Rap2, but not Rap1 GTPase is expressed in human red blood cells and is involved in vesiculation

Recent studies have suggested that Rap1 and Rap2 small GTP-binding proteins are both expressed in human red blood cells (RBCs). In this work, we carefully examined the expression of Rap proteins in leukocytes- and platelets-depleted RBCs, whose purity was established on the basis of the selective expression of the beta2 subunit of the Na+/K+ -ATPase, as verified according to the recently proposed "beta-profiling test" [J.F. Hoffman, A. Wickrema, O. Potapova, M. Milanick, D.R. Yingst, Na pump isoforms in human erythroid progenitor cells and mature erythrocytes, Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 14572-14577]. In pure RBCs preparations, Rap2, but not Rap1 was detected immunologically. RT-PCR analysis of mRNA extracted from highly purified reticulocytes confirmed the expression of Rap2b, but not Rap2a, Rap2c, Rap1a or Rap1b. In RBCs, Rap2 was membrane-associated and was rapidly activated upon treatment with Ca2+/Ca2+ -ionophore. In addition, Rap2 segregated and was selectively enriched into microvesicles released by Ca2+ -activated RBCs, suggesting a possible role for this GTPase in membrane shedding.     

Arabidopsis phosphomannose isomerase 1, but not phosphomannose isomerase 2, is essential for ascorbic acid biosynthesis

We studied molecular and functional properties of Arabidopsis phosphomannose isomerase isoenzymes (PMI1 and PMI2) that catalyze reversible isomerization between D-fructose 6-phosphate and D-mannose 6-phosphate (Man-6P). The apparent K(m) and V(max) values for Man-6P of purified recombinant PMI1 were 41.3+/-4.2 microm and 1.89 micromol/min/mg protein, respectively, whereas those of purified recombinant PMI2 were 372+/-13 microm and 22.5 micromol/min/mg protein, respectively. Both PMI1 and PMI2 were inhibited by incubation with EDTA, Zn(2+), Cd(2+), and L-ascorbic acid (AsA). Arabidopsis PMI1 protein was constitutively expressed in both vegetative and reproductive organs under normal growth conditions, whereas the PMI2 protein was not expressed in any organs under light. The induction of PMI1 expression and an increase in the AsA level were observed in leaves under continuous light, whereas the induction of PMI2 expression and a decrease in the AsA level were observed under long term darkness. PMI1 showed a diurnal expression pattern in parallel with the total PMI activity and the total AsA content in leaves. Moreover, a reduction of PMI1 expression through RNA interference resulted in a substantial decrease in the total AsA content of leaves of knockdown PMI1 plants, whereas the complete inhibition of PMI2 expression did not affect the total AsA levels in leaves of knock-out PMI2 plants. Consequently, this study improves our understanding of the molecular and functional properties of Arabidopsis PMI isoenzymes and provides genetic evidence of the involvement of PMI1, but not PMI2, in the biosynthesis of AsA in Arabidopsis plants.     

UvrD2 is essential in Mycobacterium tuberculosis, but its helicase activity is not required

UvrD is an SF1 family helicase involved in DNA repair that is widely conserved in bacteria. Mycobacterium tuberculosis has two annotated UvrD homologues; here we investigate the role of UvrD2. The uvrD2 gene at its native locus could be knocked out only in the presence of a second copy of the gene, demonstrating that uvrD2 is essential. Analysis of the putative protein domain structure of UvrD2 shows a distinctive domain architecture, with an extended C terminus containing an HRDC domain normally found in SF2 family helicases and a linking domain carrying a tetracysteine motif. Truncated constructs lacking the C-terminal domains of UvrD2 were able to compensate for the loss of the chromosomal copy, showing that these C-terminal domains are not essential. Although UvrD2 is a functional helicase, a mutant form of the protein lacking helicase activity was able to permit deletion of uvrD2 at its native locus. However, a mutant protein unable to hydrolyze ATP or translocate along DNA was not able to compensate for lack of the wild-type protein. Therefore, we concluded that the essential role played by UvrD2 is unlikely to involve its DNA unwinding activity and is more likely to involve DNA translocation and, possibly, protein displacement.