A B S T R A C T We investigated the mechanism of gastroesophageal reflux (GER) in 10 healthy volunteer subjects. Continuous recordings of intraluminal esophageal pH and pressure were obtained on two consecutive nights from 6:00 p.m. to 6:30 a.m. in each subject. During each study, the subject remained recumbent, except to eat a standardized meal after 1 h of basal recording. A manometric assembly with seven recording lumens monitored: (a) lower esophageal sphincter (LES) pressure via a sleeve device 6.5 cm in length, (b) esophageal-body motor activity, (c) swallowing activity in the pharynx, and (d) gastric pressure. An electrode 5 cm above the LES recorded esophageal pH. Sleep was monitored by electroencephalogram. All subjects showed wide variations ofbasal LES pressure. GER was not related to low steady-state basal LES pressure, but rather occurred during transient 5-30 s episodes of inappropriate complete LES relaxation. The inappropriate LES relaxations were usually either spontaneous or immediately followed appropriate sphincter relaxation induced by swallowing. The majority of GER episodes occurred within the first 3 h after eating. During the night LES relaxation and GER occurred only during transient arousals from sleep or when the subjects were fully awake, but not during stable sleep. After GER the esophagus was generally cleared of refluxed acid by primary peristalsis and less frequently by secondary peristalsis. Nonperistaltic contractions were less effective than peristalsis for clearing acid from the esophagus. We conclude that in asympto-A preliminary communication of this work was abstracted in 1978 (Gastroenterology. 74: 1119.) and was presented at the

Rag A/Gtr1p are G proteins and are known to be involved in the RCC1-Ran pathway. We employed the two-hybrid method using Rag A as the bait to identify proteins binding to Rag A, and we isolated two novel human G proteins, Rag C and Rag D. Rag C demonstrates homology with Rag D (81.1% identity) and with Gtr2p of Saccharomyces cerevisiae (46.1% identity), and it belongs to the Rag A subfamily of the Ras family. Rag C and Rag D contain conserved GTP-binding motifs (PM-1, -2, and -3) in their N-terminal regions. Recombinant glutathione S-transferase fusion protein of Rag C efficiently bound to both [ 3 H]GTP and [ 3 H]GDP. Rag A was associated with both Rag C and Rag D in their C-terminal regions where a potential leucine zipper motif and a coiled-coil structure were found. Rag C and D were associated with both the GDP and GTP forms of Rag A. Both Rag C and Rag D changed their subcellular localization, depending on the nucleotidebound state of Rag A. In a similar way, the disruption of S. cerevisiae GTR1 resulted in a change in the localization of Gtr2p.G proteins are a superfamily of regulatory GTP hydrolases and are composed of a large number of proteins. These include Ras family proteins, hetrotrimeric G protein ␣ subunits, and elongation factors TU and G, among others (1). Ras-like small G proteins such as Ras, Rab, Rho, ARF, and Ran are monomeric and bind to the guanine nucleotides, GTP or GDP, to function as molecular switches while also playing crucial roles in cell growth, differentiation, and protein traffic between different compartments within the cells (2, 3). Ras is a key regulator of cell growth and is an essential component of the signal transduction pathways initiated by receptor tyrosine kinase (4). The Rho family members consist of Rho, Rac, and Cdc42 subtypes that control the actin cytoskeleton and that play a role in the regulation of transcription (5). ADP-ribosylation factors play a role in the vesicular trafficking pathway (6). The Rab subfamily plays a role in secretory and endocytic pathways and is located within a distinct cellular compartment (7).Ran is a well characterized nuclear Ras-like small G protein that plays an essential role in the import and export of proteins and RNAs across the nuclear membrane through the nuclear pore complex (8) and also plays a role in the induction of microtubule self-organization in Xenopus egg extracts (9 -14). There are a large number of factors that interact with either the GDP-bound form or the GTP-bound form of Ran (Gsp1p), these being nucleoporin, RanBP2/NUP358 (15, 16), Prp20p interacting protein, RanBP3(Yrb2p) (17-19), the exosome involved in ribosomal RNA processing, Dis3p (20, 21), microtubule nucleation, RanBPM (22), and regulators of Ran, Ran-GAP1 (23-25), RanBP1(Yrb1p) (26, 27), RCC1/RanGEF (28), and Mog1p (29).RCC1 catalyzes guanine nucleotide exchange on Ran (30) and is located inside the nucleus, bound to chromatin (31). The concentration of GTP within the cell is ϳ30 times higher than the concentration of GDP, thus resulting in the prefer...

The tsBN7 cell line, one of the mutant lines temperature sensitive for growth which have been isolated from the BHK21 cell line, was found to die by apoptosis following a shift to the nonpermissive temperature. The induced apoptosis was inhibited by a protein synthesis inhibitor, cycloheximide, but not by the bcl-2-encoded protein. By DNA-mediated gene transfer, we cloned a cDNA that complements the tsBN7 mutation. It encodes a novel hydrophobic protein, designated DAD1, which is well conserved (100% identical amino acids between humans and hamsters). By comparing the base sequences of the parental BHK21 and tsBN7 DAD1 cDNAs, we found that the DAD1-encoding gene is mutated in tsBN7 cells. The DAD1 protein disappeared in tsBN7 cells following a shift to the nonpermissive temperature, suggesting that loss of the DAD1 protein triggers apoptosis.

Zinc-fingers and homeoboxes (ZHX) 1 is a transcription factor that interacts with the activation domain of the A subunit of nuclear factor-Y (NF-YA). Using a yeast two-hybrid system, a novel ubiquitous transcription factor ZHX2 as a ZHX1-interacting protein was cloned. ZHX2 consists of 837 amino acid residues and contains two zinc-finger motifs and five homeodomains (HDs) as well as ZHX1. The mRNA is expressed among various tissues. ZHX2 not only forms a heterodimer with ZHX1, but also forms a homodimer. Moreover, ZHX2 interacts with the activation domain of NF-YA. Further analysis revealed that ZHX2 is a transcriptional repressor that is localized in the nuclei. Since ZHX2 shares a number of properties in common with ZHX1, we conclude that all these come under the ZHX family. The minimal functional domains of ZHX2 were then characterized. The dimerization domain with both ZHX1 and ZHX2 is the region containing HD1, the domain that interacts with NF-YA is the HD1 to HD2 region, the repressor domain is the HD1 to a proline-rich region. Lastly, using an immunoprecipitation assay, we showed that ZHX2 intrinsically interacts with NF-YA in HEK-293 cells and that ZHX2 represses the promoter activity of the cdc25C gene stimulated by NF-Y in Drosophila Schneider line 2 cells. Thus the ZHX family of proteins may participate in the expression of a number of NF-Y-regulated genes via a more organized transcription network.

The human RCCI gene was cloned after DNA-mediated gene transfer into the tsBN2 cell line, which shows premature chromosome condensation at nonpermissive temperatures (39.5-40°C). This gene codes for a 2.5-kb poly(A) + RNA that is well conserved in hamsters and humans. We isolated 15 cDNA clones from the Okayama-Berg human cDNA library, and found two that can complement the tsBN2 mutation with an efficiency comparable to that of the genomic DNA clone. The base sequences of these two active cDNA clones differ at the 5' proximal end, yet both have a common open reading frame, encoding a protein of 421 amino acids with a calculated molecular weight of 44,847 and with seven homologous repeated domains of about 60 amino acids. This human RCC1 gene was located to human chromosome 1 using sorted chromosomal fractions.

Adult stem cells from bone marrow, referred to as mesenchymal stem cells or marrow stromal cells (MSCs), are defined as pluripotent cells and have the ability to differentiate into multiple mesodermal cells. In this study, we investigated whether MSCs from rat, mouse, and human are able to differentiate into steroidogenic cells. When transplanted into immature rat testes, adherent marrow-derived cells (including MSCs) were found to be engrafted and differentiate into steroidogenic cells that were indistinguishable from Leydig cells. Isolated murine MSCs transfected with green fluorescence protein driven by the promoter of P450 side-chain cleaving enzyme gene (CYP11A), a steroidogenic cell-specific gene, were used to detect steroidogenic cell production in vitro. During in vitro differentiation, green fluorescence protein-positive cells, which had characteristics similar to those of Leydig cells, were found. Stable transfection of murine MSCs with a transcription factor, steroidogenic factor-1, followed by treatment with cAMP almost recapitulated the properties of Leydig cells, including the production of testosterone. Transfection of human MSCs with steroidogenic factor-1 also led to their conversion to steroidogenic cells, but they appeared to be glucocorticoid- rather than testosterone-producing cells. These results indicate that MSCs represent a useful source of stem cells for producing steroidogenic cells that may provide basis for their use in cell and gene therapy.

The human CCGI gene complements tsBN462, a temperature-sensitive G1 mutant of the BHK21 cell line. The previously cloned cDNA turned out to be a truncated form of the actual CCGI cDNA. The newly cloned CCGI cDNA was 6.0 kb and encoded a protein with a molecular mass of 210 kDa. Using an antibody to a predicted peptide from the CCG1 protein, a protein with a molecular mass of over 200 kDa was identified in human, monkey, and hamster cell lines. In the newly defined C-terminal region, an acidic domain was found. It contained four consensus target sequences for casein kinase II and was phosphorylated by this enzyme in vitro. However, this C-terminal region was not required to complement tsBN462 mutation since the region encoding the C-terminal part was frequently missing in complemented clones derived by DNA-mediated gene transfer. CCG1 contains a sequence similar to the putative DNA-binding domain of HMG1 in addition to the previously detected amino acid sequences common in nuclear proteins, such as a proline cluster and a nuclear translocation signal. Consistent with these predictions, CCG1 was present in nuclei, possessed DNA-binding activity, and was eluted with similar concentrations of salt, 0.3 to 0.4 M NaCl either from isolated nuclei or from a DNA-cellulose column.The cell cycle of eucaryotic cells is composed of the consecutive phases Gl, S, G2, and M. In the G, phase, depending on the stimulus of growth factors, protein and RNA molecules are produced and the new cell cycle initiates (25). Unresolved problems concerning the start of the cell cycle include how the stimulus of external growth factors reaches the nucleus and how cells recognize the accumulation of materials required to enter the new cell cycle. To investigate the initiation of the cell cycle at the molecular level, it is essential to identify the genes involved. In both budding and fission yeasts, various temperature-sensitive (ts) mutants defective in the progression of the cell cycle have been isolated and have proven to be most useful for identifying the genes required for cell cycle progression (12,22). Various ts+ cell cycle mutants have also been isolated from cultured animal cells (3). The FT210 cell line, a ts G2 mutant of FM3A, has a ts cdc2 gene product; thus, the cdc2 gene is apparently essential for the cell cycle of animal cells (36).We isolated ts cell cycle mutants from the BHK21 cell line derived from the Syrian hamster and then classified them into complementation groups (21). One of these ts mutants, tsBN462, has a defect in progression of the G1 phase, which is that after release from the G, block, it is unable to enter the S phase at a nonpermissive temperature. But once it enters the S phase, DNA replication progresses normally. By using this mutant as the recipient of the DNA-mediated gene transfer, a human gene located between qll and q13 on the X chromosome has been cloned and designated the cell cycle gene 1 (CCGI) (5,31,32). Its cDNA complements tsi3 in addition to tsBN462, both of which were independently isolated ...

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