To determine the role of the cardiovascular-restricted, hairy-related bHLH transcription factor, CHF1/Hey2, in the biological response to vascular injury. We investigated the response of CHF1/Hey2-def Show more
To determine the role of the cardiovascular-restricted, hairy-related bHLH transcription factor, CHF1/Hey2, in the biological response to vascular injury. We investigated the response of CHF1/Hey2-deficient mice to vascular injury in vivo and the response of primary cultured vascular smooth muscle cells (VSMCs) from these mice to growth factors in vitro. Neointima formation after arterial wire injury is decreased in knockout (KO) compared with wild-type (WT) mice (0.025+/-0.011 mm2 in WT [n=13]) versus 0.016+/-0.008 mm2 in KO (n=12; P<0.05) and is accompanied by reduced cellular proliferation. CHF1/Hey2-deficient VSMCs proliferate slowly compared with WT VSMCs and also show decreased migration in response to platelet-derived growth factor (PDGF) (62.6+/-10.3 CPF versus 37.2+/-13.5 CPF; P<0.01) and heparin-binding epidermal growth factor-like growth factor (HB-EGF) (27.4+/-7.7 CPF versus 6.4+/-3.7 CPF, P<0.05). Furthermore, lamellipodia formation and membrane ruffling induced by these chemoattractants are diminished in KO VSMCs, which is correlated with decreased activation of the small GTPase Rac1. Although total Rac1 protein was not changed in KO VSMCs, the level of the Rac guanine exchange factor (GEF), Sos1, was decreased. CHF1/Hey2 is an important regulator of vascular smooth muscle cell (VSMC) accumulation during vascular remodeling and responsiveness to growth factors in vitro. Show less
To investigate the expression of RDH10, an all-trans retinol dehydrogenase identified in the retinal pigment epithelium (RPE), in retinal Muller cells. The RDH10 protein levels in mouse eyecups and bo Show more
To investigate the expression of RDH10, an all-trans retinol dehydrogenase identified in the retinal pigment epithelium (RPE), in retinal Muller cells. The RDH10 protein levels in mouse eyecups and bovine tissues were examined by Western blot analysis using a polyclonal antibody against RDH10. The cellular localization in the retina was determined by immunohistochemistry. Expression of RDH10 in rMC-1, a cell line derived from rat Muller cells, was determined by RT-PCR and Western blot analysis. All-trans retinol dehydrogenase activity assays were performed using lysates from rMC-1 cells. The generation of all-trans retinal from tritiated all-trans retinol was analyzed by HPLC. RDH10, retinal G protein-coupled receptor (RGR), and RPE65 all had higher expression levels in the eyecups of BALB/c than in C57Bl/6 mice. In addition to the RPE, RDH10 was also detected at lower levels in the retina and liver. Immunohistochemistry showed that RDH10 was localized in Muller cells in retinal sections. RDH10 was detected in rMC-1 cells, at both the RNA and protein levels. The rat RDH10 cDNA containing the full-length coding region was cloned from rMC-1 cells. The rat RDH10 cDNA encodes a protein of 341 amino acids and shares 99% sequence identity with human, bovine, and mouse RDH10 at the amino acid level. In rMC-1 cells, all-trans retinol dehydrogenase activity was detected in the microsomal fraction. NADP was shown to be the preferred cofactor, which is identical with the cofactor preference of the recombinant RDH10. RDH10 was expressed in retinal Muller cells, in addition to the RPE. RDH10 generates all-trans retinal, which is the substrate for the photoisomerase RGR in Muller cells. Show less
The components of the Wnt-signaling pathway are mutated in tumors, but the relationship between these components and cervical cancer has not been elucidated. In this study, we used immunohistochemistr Show more
The components of the Wnt-signaling pathway are mutated in tumors, but the relationship between these components and cervical cancer has not been elucidated. In this study, we used immunohistochemistry, single strand confirmation polymorphism (SSCP) and direct sequencing methods to analyze the mutation and protein expressions of both CTNNB1 and AXIN1 in cervical cancer. Among the 30 tested cervical cancers, no mutation of CTNNB1 but 3 polymorphisms were found. Mutation analysis of AXIN1 revealed that one specimen had a heterozygous mutation at codon 740 (GCC right curved arrow ACC) and six polymorphisms were also found. Immunohistochemistry showed no relationship between the protein expression patterns and mutation of AXIN1 and CTNNB1. Mutations of CTNNB1 may not be a factor, whereas mutations of AXIN1 may play a limited role in tumorigenesis of cervical cancer. In addition, aberrant expression patterns are not mutation related, so that other factors may be responsible for these changes. Show less
Complementary DNA microarray and quantitative polymerase chain reaction were used as tools for discovering genes that are differentially expressed in the mouse under normal physiological conditions at Show more
Complementary DNA microarray and quantitative polymerase chain reaction were used as tools for discovering genes that are differentially expressed in the mouse under normal physiological conditions at distinctive stages of male germ cell development, that is, type A spermatogonia, pachytene spermatocytes, and round spermatids. By using this strategy, we identified a set of genes exhibiting differential expression patterns in spermatogenesis, suggesting that specific functions of the encoded products occurred during the developmental process. Among them were several genes previously not known to be active in testis, which signified undiscovered functional roles of these genes during spermatogenesis. Many of the genes identified were not previously characterized. This study highlights new targets for manipulation to unravel the molecular mechanism of spermatogenesis. Show less
In order to characterize the genetic diversity and phylogenetic affiliations of marine cyanophage isolates and natural cyanophage assemblages, oligonucleotide primers CPS1 and CPS8 were designed to sp Show more
In order to characterize the genetic diversity and phylogenetic affiliations of marine cyanophage isolates and natural cyanophage assemblages, oligonucleotide primers CPS1 and CPS8 were designed to specifically amplify ca. 592-bp fragments of the gene for viral capsid assembly protein g20. Phylogenetic analysis of isolated cyanophages revealed that the marine cyanophages were highly diverse yet more closely related to each other than to enteric coliphage T4. Genetically related marine cyanophage isolates were widely distributed without significant geographic segregation (i.e., no correlation between genetic variation and geographic distance). Cloning and sequencing analysis of six natural virus concentrates from estuarine and oligotrophic offshore environments revealed nine phylogenetic groups in a total of 114 different g20 homologs, with up to six clusters and 29 genotypes encountered in a single sample. The composition and structure of natural cyanophage communities in the estuary and open-ocean samples were different from each other, with unique phylogenetic clusters found for each environment. Changes in clonal diversity were also observed from the surface waters to the deep chlorophyll maximum layer in the open ocean. Only three clusters contained known cyanophage isolates, while the identities of the other six clusters remain unknown. Whether or not these unidentified groups are composed of bacteriophages that infect different Synechococcus groups or other closely related cyanobacteria remains to be determined. The high genetic diversity of marine cyanophage assemblages revealed by the g20 sequences suggests that marine viruses can potentially play important roles in regulating microbial genetic diversity. Show less
Spermatogenesis is the process by which spermatogonial stem cells divide and differentiate to produce sperm. In vitro sperm production has been difficult to achieve because of the lack of a culture sy Show more
Spermatogenesis is the process by which spermatogonial stem cells divide and differentiate to produce sperm. In vitro sperm production has been difficult to achieve because of the lack of a culture system to maintain viable spermatogonia for long periods of time. Here we report the in vitro generation of spermatocytes and spermatids from telomerase-immortalized mouse type A spermatogonial cells in the presence of stem cell factor. This differentiation can occur in the absence of supportive cells. The immortalized spermatogonial cell line may serve as a powerful tool in elucidating the molecular mechanisms of spermatogenesis. Furthermore, through genomic modification and transplantation techniques, this male germ cell line may be used to generate transgenic mice and to develop germ cell gene therapy. Show less
The t(10;11)(p12;q23) chromosomal translocation in human acute myeloid leukemia results in the fusion of the MLL and AF10 genes. The latter codes for a novel leucine zipper protein, one of many MLL fu Show more
The t(10;11)(p12;q23) chromosomal translocation in human acute myeloid leukemia results in the fusion of the MLL and AF10 genes. The latter codes for a novel leucine zipper protein, one of many MLL fusion partners of unknown function. In this report, we demonstrate that retroviral-mediated transduction of an MLL-AF10 complementary DNA into primary murine myeloid progenitors enhanced their clonogenic potential in serial replating assays and led to their efficient immortalization at a primitive stage of myeloid differentiation. Furthermore, MLL-AF10-transduced cells rapidly induced acute myeloid leukemia in syngeneic or severe combined immunodeficiency recipient mice. Structure/function analysis showed that a highly conserved 82-amino acid portion of AF10, comprising 2 adjacent alpha-helical domains, was sufficient for immortalizing activity when fused to MLL. Neither helical domain alone mediated immortalization, and deletion of the 29-amino acid leucine zipper within this region completely abrogated transforming activity. Similarly, the minimal oncogenic domain of AF10 exhibited transcriptional activation properties when fused to the MLL or GAL4 DNA-binding domains, while neither helical domain alone did. However, transcriptional activation per se was not sufficient because a second activation domain of AF10 was neither required nor competent for transformation. The requirement for alpha-helical transcriptional effector domains is similar to the oncogenic contributions of unrelated MLL partners ENL and ELL, suggesting a general mechanism of myeloid leukemogenesis by a subset of MLL fusion proteins, possibly through specific recruitment of the transcriptional machinery. Show less
The molecular machinery of cell cycle control is known in more detail for budding yeast, Saccharomyces cerevisiae, than for any other eukaryotic organism. In recent years, many elegant experiments on Show more
The molecular machinery of cell cycle control is known in more detail for budding yeast, Saccharomyces cerevisiae, than for any other eukaryotic organism. In recent years, many elegant experiments on budding yeast have dissected the roles of cyclin molecules (Cln1-3 and Clb1-6) in coordinating the events of DNA synthesis, bud emergence, spindle formation, nuclear division, and cell separation. These experimental clues suggest a mechanism for the principal molecular interactions controlling cyclin synthesis and degradation. Using standard techniques of biochemical kinetics, we convert the mechanism into a set of differential equations, which describe the time courses of three major classes of cyclin-dependent kinase activities. Model in hand, we examine the molecular events controlling "Start" (the commitment step to a new round of chromosome replication, bud formation, and mitosis) and "Finish" (the transition from metaphase to anaphase, when sister chromatids are pulled apart and the bud separates from the mother cell) in wild-type cells and 50 mutants. The model accounts for many details of the physiology, biochemistry, and genetics of cell cycle control in budding yeast. Show less
Y Sun, J Zhang, S K Kraeft+8 more · 1999 · The Journal of biological chemistry · American Society for Biochemistry and Molecular Biology · added 2026-04-24
We describe the molecular cloning and characterization of a novel giant human cytoplasmic protein, trabeculin-alpha (M(r) = 614,000). Analysis of the deduced amino acid sequence reveals homologies wit Show more
We describe the molecular cloning and characterization of a novel giant human cytoplasmic protein, trabeculin-alpha (M(r) = 614,000). Analysis of the deduced amino acid sequence reveals homologies with several putative functional domains, including a pair of alpha-actinin-like actin binding domains; regions of homology to plakins at either end of the giant polypeptide; 29 copies of a spectrin-like motif in the central region of the protein; two potential Ca(2+)-binding EF-hand motifs; and a Ser-rich region containing a repeated GSRX motif. With similarities to both plakins and spectrins, trabeculin-alpha appears to have evolved as a hybrid of these two families of proteins. The functionality of the actin binding domains located near the N terminus was confirmed with an F-actin binding assay using glutathione S-transferase fusion proteins comprising amino acids 9-486 of the deduced peptide. Northern and Western blotting and immunofluorescence studies suggest that trabeculin is ubiquitously expressed and is distributed throughout the cytoplasm, though the protein was found to be greatly up-regulated upon differentiation of myoblasts into myotubes. Finally, the presence of cDNAs similar to, yet distinct from, trabeculin-alpha in both human and mouse suggests that trabeculins may form a new subfamily of giant actin-binding/cytoskeletal cross-linking proteins. Show less
C Y Chen, T R Graham · 1998 · Genetics · Oxford University Press · added 2026-04-24
ADP-ribosylation factor (ARF) is a small GTP-binding protein that is thought to regulate the assembly of coat proteins on transport vesicles. To identify factors that functionally interact with ARF, w Show more
ADP-ribosylation factor (ARF) is a small GTP-binding protein that is thought to regulate the assembly of coat proteins on transport vesicles. To identify factors that functionally interact with ARF, we have performed a genetic screen in Saccharomyces cerevisiae for mutations that exhibit synthetic lethality with an arf1Delta allele and defined seven genes by complementation tests (SWA1-7 for synthetically lethal with arf1Delta). Most of the swa mutants exhibit phenotypes comparable to arf1Delta mutants such as temperature-conditional growth, hypersensitivity to fluoride ions, and partial protein transport and glycosylation defects. Here, we report that swa5-1 is a new temperature-sensitive allele of the clathrin heavy chain gene (chc1-5), which carries a frameshift mutation near the 3' end of the CHC1 open reading frame. This genetic interaction between arf1 and chc1 provides in vivo evidence for a role for ARF in clathrin coat assembly. Surprisingly, strains harboring chc1-5 exhibited a significant defect in transport of carboxypeptidase Y or carboxypeptidase S to the vacuole that was not observed in other chc1 ts mutants. The kinetics of invertase secretion or transport of alkaline phosphatase to the vacuole were not significantly affected in the chc1-5 mutant, further implicating clathrin specifically in the Golgi to vacuole transport pathway for carboxypeptidase Y. Show less
T Chen, J Kurjan · 1997 · Molecular and cellular biology · added 2026-04-24
SST2 plays an important role in the sensitivity of yeast cells to pheromone and in recovery from pheromone-induced G1 arrest. Recently, a family of Sst2p homologs that act as GTPase-activating protein Show more
SST2 plays an important role in the sensitivity of yeast cells to pheromone and in recovery from pheromone-induced G1 arrest. Recently, a family of Sst2p homologs that act as GTPase-activating proteins (GAPs) for G alpha subunits has been identified. We have identified an interaction between Sst2p and the previously identified Mpt5p by using the two-hybrid system. Loss of Mpt5p function resulted in a temperature-sensitive growth phenotype, an increase in pheromone sensitivity, and a defect in recovery from pheromone-induced G1 arrest, although the effects on pheromone response and recovery were mild in comparison to those of sst2 mutants. Overexpression of either Sst2p or Mpt5p promoted recovery from G1 arrest. Promotion of recovery by overexpression of Mpt5p required Sst2p, but the effect of overexpression of Sst2p was only partially dependent on Mpt5p. Mpt5p was also found to interact with the mitogen-activated protein kinase homologs Fus3p and Kss1p, and an mpt5 mutation was able to suppress the pheromone arrest and mating defects of a fus3 mutant. Because either mpt5 or cln3 mutations suppressed the fus3 phenotypes, interactions of Mpt5p with the G1 cyclins and Cdc28p were tested. An interaction between Mpt5p and Cdc28p was detected. We discuss these results with respect to a model in which Sst2p plays a role in pheromone sensitivity and recovery that acts through Mpt5p in addition to a role as a G alpha GAP suggested by the analysis of the Sst2p homologs. Show less
Y Chen, B G Atkinson · 1997 · Developmental genetics · Wiley · added 2026-04-24
During the spontaneous or thyroid hormone (TH)-induced metamorphosis of Rana catesbeiana, developmental changes occur in its liver that are necessary for the transition of this organism from an ammono Show more
During the spontaneous or thyroid hormone (TH)-induced metamorphosis of Rana catesbeiana, developmental changes occur in its liver that are necessary for the transition of this organism from an ammonotelic larva to a ureotelic adult. These changes include the coordinated expression of genes encoding the urea cycle enzymes carbamyl phosphate synthetase (CPS-I) and arnithine transcarbamylase (OTC). Although the expression of these genes is dependent on TH, the mechanisms(s) by which TH initiates this tissue-specific response is thought to be indirect and to involve early TH-induced upregulation of a gene(s), which, in turn, upregulates the coordinated expression of these urea-cycle enzyme genes. Herein, we demonstrate that mRNAs encoding the Rana homologue of the mammalian transcription factor C/EBP alpha (designated RcC/EBP-1) accumulate early in response to TH and that the product of these mRNAs can bind to and transactivate the promoters of both the Rana CPS-1 and OTC genes. These results support the contention that the reprogramming of gene expression in the liver of metamorphosing tadpoles involves a TH-induced cascade of gene activity in which RcC/EBP-1 and, perhaps, other transcription factors coordinate the expression of genes, such as those encoding CPS-I and OTC, whose products are characteristic of the adult liver phenotype. Show less
no PDFDOI: 10.1002/(SICI)1520-6408(1997)20:2<152::AID-DVG8>3.0.CO;2-6
We have constructed a physical map covering over 4 Mb of human chromosome 8q24.1 and used this map to refine the locations of the genes responsible for Langer-Giedion syndrome. The map is composed of Show more
We have constructed a physical map covering over 4 Mb of human chromosome 8q24.1 and used this map to refine the locations of the genes responsible for Langer-Giedion syndrome. The map is composed of overlapping YAC clones that were identified and ordered in relation to sequence tagged sites mapped to the Langer-Giedion chromosomal region on somatic cell hybrids. The minimal region of overlap of Langer-Giedion syndrome deletions, previously identified by analysis of 15 patients, was placed on the map by analysis of 2 patients whose deletions define the endpoints. The chromosome 8 breakpoint of a balanced t(8;9)(q24.11;q33.3) translocation from a patient with trichorhinophalangeal syndrome (TRPS I) was found to be located just within the proximal end of the minimal deletion region. A deletion of 8q24.11-q24.3 in a patient with multiple exostoses was found to overlap the distal end of the LGS deletion region, indicating that the EXT1 gene is distal to the TRPS1 gene and supporting the hypothesis that Langer-Giedion syndrome is due to loss of functional copies of both the TRPS1 and the EXT1 genes. Show less