Generation of transgenic dogs that conditionally express green fluorescent protein

We report the creation of a transgenic dog that conditionally expresses eGFP (enhanced green fluorescent protein) under the regulation of doxycycline. Briefly, fetal fibroblasts infected with a Tet-on eGFP vector were used for somatic cell nuclear transfer. Subsequently reconstructed oocytes were transferred to recipients. Three clones having transgenes were born and one dog was alive. The dog showed all features of inducible expression of eGFP upon doxycycline administration, and successful breeding resulted in eGFP-positive puppies, confirming stable insertion of the transgene into the genome. This inducible dog model will be useful for a variety of medical research studies.     

Proteomic analysis of pregnancy-related proteins from pig uterus endometrium during pregnancy

Many important molecular events associated with implantation and development occur within the female reproductive tract, especially within the uterus endometrium, during pregnancy periods. The endometrium includes the mucosal lining of the uterus, which provides a suitable site for implantation and development of a fertilized egg and fetus. To date, the molecular cascades in the uterus endometrium during pregnancy periods in pigs have not been elucidated fully. In this study, we compared the functional regulated proteins in the endometrium during pregnancy periods with those in non-pregnant conditions and investigated changes in expression patterns during pregnancy (days 40, 70, and 93) using two-dimensional gel electrophoresis (2-DE) and western blotting. The functional regulated proteins were identified and discovered from differentially expressed proteins in the uterus endometrium during pregnancy. We discovered 820 protein spots in a proteomic analysis of uterus endometrium tissues with 2-DE gels. We identified 63 of the 98 proteins regulated differentially among non-pregnant and pregnant tissues (matched and unmatched spots). Interestingly, 10 of these 63 proteins are development-, cytoskeleton- and chaperon-related proteins such as transferrin, protein DJ-1, transgelin, galectin-1, septin 2, stathmin 1, cofilin 1, fascin 1, heat shock protein (HSP) 90β and HSP 27. The specific expression patterns of these proteins in the endometrium during pregnancy were confirmed by western blotting. Our results suggest that the expressions of these genes involved in endometrium function and endometrium development from early to late gestation are associated with the regulation of endometrium development for maintaining pregnancy.     

Involvement of neuropeptide Y and its Y1 and Y5 receptors in maintaining self‐renewal and proliferation of human embryonic stem cells

Neuropeptide Y (NPY) and NPY receptors are widely expressed in various organs and cell types and have been shown to have pleiotropic functions. However, their presence or role in human embryonic stem cells (hESCs) remains unknown. We now show that undifferentiated hESCs primarily express NPY and its Y1 and Y5 receptors. Inhibition of NPY signalling using either the selective NPY Y1 or Y5 receptor antagonist reduces the maintenance of self‐renewal and proliferation of undifferentiated hESCs. We also provide compelling evidence that exogenous NPY supports the long‐term growth of undifferentiated hESCs in the absence of feeder cell factors using only knockout serum replacement media. Further, NPY facilitates the use of chemically defined medium made up of N2/B27 supplement and basic fibroblast growth factor (bFGF) for hESC feeder‐free culture. Our results indicate that both Y1 and Y5 receptors appear to be involved in the NPY‐mediated activation of AKT/protein kinase B and extracellular signal‐regulated kinase 1/2 (ERK1/2) in hESCs. Notably, only Y1 receptor, but not Y5 receptor, is responsible for the NPY‐induced activation of cAMP‐response element binding (CREB) in hESCs. These results provide the first evidence that NPY and its Y1 and Y5 receptors have potential role in maintaining hESC self‐renewal and pluripotency. We demonstrate the underlying importance of NPY signalling and its usefulness in the development of a defined and xeno‐free culture condition for the large‐scale propagation of undifferentiated hESCs.     

An HR-MAS MR metabolomics study on breast tissues obtained with core needle biopsy

Background

Much research has been devoted to the development of new breast cancer diagnostic measures, including those involving high-resolution magic angle spinning (HR-MAS) magnetic resonance (MR) spectroscopic techniques. Previous HR-MAS MR results have been obtained from post-surgery samples, which limits their direct clinical applicability.

Methodology/Principal Findings

In the present study, we performed HR-MAS MR spectroscopic studies on 31 breast tissue samples (13 cancer and 18 non-cancer) obtained by percutaneous core needle biopsy. We showed that cancer and non-cancer samples can be discriminated very well with Orthogonal Projections to Latent Structure-Discriminant Analysis (OPLS-DA) multivariate model on the MR spectra. A subsequent blind test showed 69% sensitivity and 94% specificity in the prediction of the cancer status. A spectral analysis showed that in cancer cells, taurine- and choline-containing compounds are elevated. Our approach, additionally, could predict the progesterone receptor statuses of the cancer patients.

Conclusions/Significance

HR-MAS MR metabolomics on intact breast tissues obtained by core needle biopsy may have a potential to be used as a complement to the current diagnostic and prognostic measures for breast cancers.     

Gene Expression Pattern in Transmitochondrial Cytoplasmic Hybrid Cells Harboring Type 2 Diabetes-Associated Mitochondrial DNA Haplogroups

Decreased mitochondrial function plays a pivotal role in the pathogenesis of type 2 diabetes mellitus (T2DM). Recently, it was reported that mitochondrial DNA (mtDNA) haplogroups confer genetic susceptibility to T2DM in Koreans and Japanese. Particularly, mtDNA haplogroup N9a is associated with a decreased risk of T2DM, whereas haplogroups D5 and F are associated with an increased risk. To examine functional consequences of these haplogroups without being confounded by the heterogeneous nuclear genomic backgrounds of different subjects, we constructed transmitochondrial cytoplasmic hybrid (cybrid) cells harboring each of the three haplogroups (N9a, D5, and F) in a background of a shared nuclear genome. We compared the functional consequences of the three haplogroups using cell-based assays and gene expression microarrays. Cell-based assays did not detect differences in mitochondrial functions among the haplogroups in terms of ATP generation, reactive oxygen species production, mitochondrial membrane potential, and cellular dehydrogenase activity. However, differential expression and clustering analyses of microarray data revealed that the three haplogroups exhibit a distinctive nuclear gene expression pattern that correlates with their susceptibility to T2DM. Pathway analysis of microarray data identified several differentially regulated metabolic pathways. Notably, compared to the T2DM-resistant haplogroup N9a, the T2DM-susceptible haplogroup F showed down-regulation of oxidative phosphorylation and up-regulation of glycolysis. These results suggest that variations in mtDNA can affect the expression of nuclear genes regulating mitochondrial functions or cellular energetics. Given that impaired mitochondrial function caused by T2DM-associated mtDNA haplogroups is compensated by the nuclear genome, we speculate that defective nuclear compensation, under certain circumstances, might lead to the development of T2DM.     

Formation of Pmel17 Amyloid Is Regulated by Juxtamembrane Metalloproteinase Cleavage, and the Resulting C-terminal Fragment Is a Substrate for ��-Secretase

The formation of insoluble cross β-sheet amyloid is pathologically associated with disorders such as Alzheimer, Parkinson, and Huntington diseases. One exception is the nonpathological amyloid derived from the protein Pmel17 within melanosomes to generate melanin pigment. Here we show that the formation of insoluble MαC intracellular fragments of Pmel17, which are the direct precursors to Pmel17 amyloid, depends on a novel juxtamembrane cleavage at amino acid position 583 between the furin-like proprotein convertase cleavage site and the transmembrane domain. The resulting Pmel17 C-terminal fragment is then processed by the γ-secretase complex to release a short-lived intracellular domain fragment. Thus, by analogy to the Notch receptor, we designate this cleavage the S2 cleavage site, whereas γ-secretase mediates proteolysis at the intramembrane S3 site. Substitutions or deletions at this S2 cleavage site, the use of the metalloproteinase inhibitor TAPI-2, as well as small interfering RNA-mediated knock-down of the metalloproteinases ADAM10 and 17 reduced the formation of insoluble Pmel17 fragments. These results demonstrate that the release of the Pmel17 ectodomain, which is critical for melanin amyloidogenesis, is initiated by S2 cleavage at a juxtamembrane position.Folding of proteins is a highly regulated process ensuring their correct three-dimensional structure. Under pathological circumstances, a soluble protein can be folded into highly stable cross β-sheet amyloid structures, which are believed to play pathological roles in disorders such as Alzheimer, Parkinson, and Huntington diseases. An exception to this general concept is the physiological amyloid structure of the melanosomal matrix formed by the protein Pmel17. Melanosomes are lysosome-related organelles that contain pigment granules (melanin) in melanocytes and retinal epithelial cells (reviewed in Ref. 1). Melanogenesis is believed to proceed through several sequential maturation steps, classified by melanosomes from stage I to stage IV. Maturation of stage II melanosomes requires the formation of Pmel17 intralumenal fibers (2, 3).Pmel17 (also called gp100, ME20, RPE1, or silver) is a type I transmembrane glycoprotein of up to 668 amino acids in humans (reviewed in Ref. 4). The requirement of Pmel17 for the generation of functional melanin has been shown in a number of different organisms, because, for example, certain point mutations in the Pmel17/silver gene result in hypopigmentation phenotypes (5–7). The most characteristic domain within Pmel17 is a specific lumenal proline/serine/threonine rich repeat domain (see Fig. 1A), that is imperfectly repeated 13 times in the Mα fragment. Importantly, deletion of the rich repeat domain results in a complete loss of fibril formation, pointing to the requirement of Pmel17, and especially the rich repeat domain, in melanin formation (8). Pmel17 exists in different isoforms generated by alternative splicing. Pmel17-i² is the most abundant isoform, whereas the Pmel17-l isoform contains a 7-amino acid insertion close to the transmembrane domain (9, 10).Open in a separate windowFIGURE 1.Effect of the γ-secretase inhibitor DAPT on Pmel17 processing. A, schematic diagram of Pmel17 and epitopes of antibodies. Pmel17 contains five potential N-glycosylation sites indicated by branched structures. The long form of Pmel17, Pmel17-l, is characterized by a seven amino acid insertion (VPGILLT) within the lumenal domain close to the transmembrane domain (TM), which is absent in Pmel17-i. NVS marks a potential N-glycosylation site near this insertion. The epitopes of antibodies αPep13h and HMB45 are indicated. Cleavage by a furin-like PC results in the formation of the Mα and the membrane-bound 26-kDa Mβ fragment, which are connected via disulfide bonds. Release and further processing of the Mα fragment into MαN and MαC fragments results in the formation of fibrils and marks the transition of stage I to stage II melanosomes (dashed line). B, human MNT-1 cells were incubated with increasing amounts of DAPT for 18 h, and then the lysates were separated by SDS-PAGE and analyzed by immunoblotting with αPep13h antibody. DAPT treatment resulted in the accumulation of a C-terminal fragment of Pmel17 (CTF), whereas Pmel17 P1 and Mβ fragment were unchanged. C, probing the Triton-soluble fraction with HMB45 revealed increased amounts of the highly glycosylated P2 form of Pmel17 after DAPT incubation. D, detection of Pmel17 amyloidogenic fragments (MαC) in the SDS-extracted insoluble pellet using antibody HMB45. E, murine B16-FO cells treated with increasing concentrations of DAPT. Immunoblotting using antibodyαPep13h revealed the formation of CTF of similar size as in MNT-1 cells. F, time course analysis of Pmel17, Mβ, and Pmel17-CTF after DAPT treatment. The cell lysates were immunoblotted using αPep13h. Pmel17-CTF was detectable after 10 min of incubation with 1 μm DAPT. G, the size of the Pmel17-CTF was determined using an unstained low molecular range peptide standard. The marker peptides were detected by Ponceau S staining and Pmel17-CTF were detected by immunoblot using αPep13h.Pmel17 traffics through the secretory pathway as a 100-kDa protein (called P1). In the late Golgi compartment it undergoes further glycosylation, resulting in a short lived 120-kDa protein (called P2). P2 is rapidly cleaved within the post-Golgi by a furin-like proprotein convertase (PC) to generate two fragments that remain tethered to each other by disulfide bonds: a C-terminal polypeptide containing the transmembrane domain (Mβ) and a large N-terminal ectodomain (Mα) (2) (Fig. 1A). Consequently, inhibition of this furin-like activity not only prevents the generation of Mα and Mβ fragments but also inhibits the formation of melanosomal striation in HeLa cells (3). These findings suggest that Mα must first be dissociated from the Mβ for melanogenesis to proceed. It is unclear how Mα is released from the membrane. Reduction of disulfide bonds would release Mα from Mβ; alternatively, proteolytic digestion of Mβ should also free Mα from the membrane tether. It has been speculated that, given the presence of lysosomal hydrolases in melanosomes and proteolytic maturation of Pmel17, proteolysis is the more likely mechanism (4). Recently, it was shown that recombinant Mα is able to form amyloid structures in vitro in an unprecedented rapidity, and furthermore, Pmel17 amyloid also accelerated melanin formation (11). These findings demonstrate that mammalian amyloid formed by Pmel17 is functional and physiological.The insoluble pool of Pmel17 in cells consists mostly of truncated Mα C-terminal fragments (MαC) of heterogeneous sizes, indicating that further processing of Mα occurs after its release from the membrane (8, 12). MαC fragments are found in the insoluble fraction of melanocytes as well as in nonmelanotic cells, the latter after overexpression of Pmel17 (8), and are reduced or absent in amelanotic cells (8, 13, 14). Meanwhile, the C-terminal fragment derived from the Mβ fragment and recognized by a C-terminal specific epitope antibody is less stable, indicating rapid turnover (2).The presenilin (PS) family of proteins consists of two homologous integral transmembrane proteins, PS1 and PS2, which are part of the γ-secretase complex. The latter consists of presenilin 1 or 2, nicastrin, APH-1, and PEN-2 (15) and catalyzes the cleavage of the hydrophobic transmembrane domain of a burgeoning list of proteins, also called regulated intramembrane cleavage. Other substrates for the γ-secretase-mediated intramembrane cleavage include Notch, amyloid precursor protein (APP), cadherin (E-cadherin), nectin-1, the low density lipoprotein-related receptor, CD44, ErbB-4, the voltage-gated sodium channel β2-subunit, and the Notch ligands Delta and Jagged. Importantly, in Alzheimer disease, the presenilin-mediated γ-secretase cleavage of APP releases the amyloid β-protein fragment, a peptide believed to play a key role in Alzheimer disease pathogenesis. Interestingly, a recent report described the absence of melanin pigment in presenilin-deficient animals, an observation confirmed by the lack of melanin formation in cells treated with γ-secretase inhibitors (16). The mechanism responsible for this finding is unclear, leading us to ask whether Pmel17 processing is a presenilin-dependent process and, if so, whether this cleavage is involved in melanogenesis.In this study, we show the presence of an endoproteolytic activity that cleaves the extracellular domain of Pmel17-i at a juxtamembrane position between the known PC cleavage site and the transmembrane domain, which we term the S2 cleavage site, by a TAPI-sensitive ADAM (a disintegrin and metalloproteinase protein) protease. This intracellular shedding of Pmel17 after S2 cleavage results in the liberation of the Mα N-terminal ectodomain, the precursor to Pmel17 amyloid, which is able to form insoluble Pmel17 aggregates. The C-terminal transmembrane fragment generated by S2 cleavage is further processed by γ-secretase (S3 cleavage) to release the Pmel17 intracellular domain, which is then rapidly degraded.     

Dissection of recognition determinants of Escherichia coli σ32 suggests a composite −10 region with an 'extended −10' motif and a core −10 element

σ³² controls expression of heat shock genes in Escherichia coli and is widely distributed in proteobacteria. The distinguishing feature of σ³² promoters is a long −10 region (CCCCATNT) whose tetra-C motif is important for promoter activity. Using alanine-scanning mutagenesis of σ³² and in vivo and in vitro assays, we identified promoter recognition determinants of this motif. The most downstream C (−13) is part of the −10 motif; our work confirms and extends recognition determinants of −13C. Most importantly, our work suggests that the two upstream Cs (−16, −15) constitute an 'extended −10' recognition motif that is recognized by K130, a residue universally conserved in β- and γ-proteobacteria. This residue is located in the α-helix of σDomain 3 that mediates recognition of the extended −10 promoter motif in other σs. K130 is not conserved in α- and δ-/ε-proteobacteria and we found that σ³² from the α-proteobacterium Caulobacter crescentus does not need the extended −10 motif for high promoter activity. This result supports the idea that K130 mediates extended −10 recognition. σ³² is the first Group 3 σ shown to use the 'extended −10' recognition motif.     

QSAR analysis of pyrazolidine-3,5-diones derivatives as Dyrk1A inhibitors

Individuals with Down syndrome (DS) suffer from mental retardation. Overexpression and the resulting increased specific activity of Dyrk1A kinase located on chromosome 21 cause a learning and memory deficit in Dyrk1A transgenic mice. To search for therapeutic agents with Dyrk1A inhibition activity, previously we obtained HCD160 as a new hit compound for Dyrk1A inhibition. In the present study, we synthesized 34 HCD160 derivatives to investigate the quantitative structure–activity relationship (QSAR). This analysis could provide important information for novel drug discovery for treatment of DS related learning and memory deficits.