[The progress of anorectal surgery in China].
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China↗
Biomedical subjects
D Yu
Publications and source records attributed to D Yu.
A new system to place single copies of genes, sites and lacZ fusions on the Escherichia coli chromosome.
To place a single-copy lacZ fusion on the E. coli chromosome, a method was developed based on in vivo homologous DNA recombination through P1 transduction. The fusions, initially constructed on plasmids, are crossed to lambdalacZ fusion vectors which are then lysogenized at the chromosomal lambda att site. The features of the new system are: (1) lambda lysogens carrying the fusion are made without regard for copy number; (2) P1 transduction from the lysogenic strain into an appropriate recipient generates the single-copy fusion; (3) The lacZ fusion has no prophage associated with it; (4) the lacZ fusion can be transferred by P1 transduction to other strains, simply by selecting for an antibiotic marker; (5) the system can be widely applied to construct single copies of any gene or site placed between bla and lacZ on the standard lacZ fusion plasmid vectors; and (6) the single-copy construct flanked by prophage att sites can be excised by site-specific recombination to generate non-replicating circular DNA of the clone or a cell cured of the construct.
Bacteriophage lambda↗
Solid-phase stereoselective synthesis of 2'-O-methyl-oligoribonucleoside phosphorothioates using nucleoside bicyclic oxazaphospholidines.
The use of 2'-OMe-ribonucleoside bicyclic oxazaphospholidines derived from (R)- or (S)-2-pyrrolidinemethanol has enabled the stereoselective synthesis of (Rp)-, and (Sp)-2'-O-methyloligoribonucleoside phosphorothioates. Interestingly, higher stereoselectivity (96-98%) was observed in the synthesis of (Sp)-2'-O-methyl-oligoribonucleoside phosphorothioates compared to that in the case of (Sp)-oligodeoxyribonucleoside phosphorothioates (90%).
Chromatography, High Pressure Liquid↗
Variable and tissue-specific hormone resistance in heterotrimeric Gs protein alpha-subunit (Gsalpha) knockout mice is due to tissue-specific imprinting of the gsalpha gene.
Albright hereditary osteodystrophy (AHO), an autosomal dominant disorder characterized by short stature, obesity, and skeletal defects, is associated with heterozygous inactivating mutations of GNAS1, the gene encoding the heterotrimeric G protein alpha-subunit (Gsalpha) that couples multiple receptors to the stimulation of adenylyl cyclase. It has remained unclear why only some AHO patients present with multihormone resistance and why AHO patients demonstrate resistance to some hormones [e.g., parathyroid hormone (PTH)] but not to others (e.g., vasopressin), even though all activate adenylyl cyclase. We generated mice with a null allele of the mouse homolog Gnas. Homozygous Gs deficiency is embryonically lethal. Heterozygotes with maternal (m-/+) and paternal (+/p-) inheritance of the Gnas null allele have distinct phenotypes, suggesting that Gnas is an imprinted gene. PTH resistance is present in m-/+, but not +/p-, mice. Gsalpha expression in the renal cortex (the site of PTH action) is markedly reduced in m-/+ but not in +/p- mice, demonstrating that the Gnas paternal allele is imprinted in this tissue. Gnas is also imprinted in brown and white adipose tissue. The maximal physiological response to vasopressin (urinary concentrating ability) is normal in both m-/+ and +/p- mice and Gnas is not imprinted in the renal inner medulla (the site of vasopressin action). Tissue-specific imprinting of Gnas is likely the mechanism for variable and tissue-specific hormone resistance in these mice and a similar mechanism might explain the variable phenotype in AHO.
Alleles↗
Effect of laminin on the nuclear localization of nucleolin in rat intestinal epithelial IEC-6 cells.
Laminin is a major component of extracellular matrix. The mechanism of action of laminin on cell proliferation, differentiation, and migration is not fully understood. In this study, we investigated the role of extracellular matrix, especially laminin, on the cellular localization of the nuclear protein, nucleolin, and on cell proliferation. Immunofluorescent and western blot analysis indicated that nucleolin was translocated most efficiently to the nucleus in the small intestinal rat epithelial cell line (IEC-6) when cultured on laminin-coated plates. Specifically, nucleolin was observed predominantly in cytoplasm in the cells cultured without laminin. In contrast, nuclear localization was observed in the cells cultured on laminin. This effect of laminin on nucleolin translocation was time-dependent. Laminin was also observed to stimulate proliferation of IEC-6 cells in serum free medium. Our results suggest that laminin alters the distribution of nucleolin which may be an early signal for cell proliferation.
Animals↗
Importance of the N-glycan in the V3 loop of HIV-1 envelope protein for CXCR-4- but not CCR-5-dependent fusion.
The V3 region of HIV-1 envelope protein possesses a single N-linked sugar chain, which is conserved in most HIV-1 strains. We studied its role in the life cycle of HIV-1 strains with different co-receptor usage. Removal of the glycan appeared to cause a marked reduction of CXCR-4- but not CCR-5-dependent virus entry. A basic amino acid substitution at the 11th position of V3 markedly compensated for the removal of the N-glycan. These results indicate that the N-glycan plays an important role for CXCR-4-dependent virus entry and that this role is exerted in a particular context of the peptide backbone.
Amino Acid Sequence↗
Overexpression of both p185c-erbB2 and p170mdr-1 renders breast cancer cells highly resistant to taxol.
We recently found that overexpression of p185c-erbB2 in c-erbB2 transfected MDA-MB-435 breast cancer cells (435.eB transfectants) confers a 5-9-fold increase in Taxol resistance. To examine whether Taxol resistance is a common phenomenon in other c-erbB2 overexpressing breast cancer cell lines, we tested a panel of human breast cancer cell lines established from different patients and expressing pl85c-erbB2 at different levels for their sensitivity to Taxol and Taxotere, a synthetic taxoid. Higher expression of p185c-erbB2 in these breast cancer cell lines indeed correlated well with resistance to Taxol and Taxotere, and the degree of resistance was about 100-fold that in c-erbB2-overexpressing 435.eB transfectants, demonstrating that these breast cancer cells are highly resistant to Taxol. Since mdr-1-encoded p-glycoprotein (p170mdr-1) has been implicated in Taxol resistance, we next examined the p170mdr-1 levels in these breast cancer cell lines that are highly resistant to Taxol. Higher levels of p170mdr-1 expression were found in several breast cancer cell lines that are highly resistant to Taxol. Since these same breast cancer cell lines also expressed higher levels of p185c-erbB2, we sought to determine the relative contribution of p185c-erbB2 and p170mdr-1 overexpression to Taxol resistance. We first specifically down-regulated cell surface p185c-erbB2 using anti-p185c-erbB2 monoclonal antibodies and assayed sensitivity of these cells to Taxol. We next specifically inactivated p170mdr-1 function using p170mdr-1 blockers (thioridazine or verapamil) and again assayed Taxol sensitivity. Both p185c-erbB2 down-regulation and p170mdr-1 blockade significantly sensitized the breast cancer cell lines to Taxol. The results indicate that overexpression of either p185c-erbB2 or p170mdr-1 renders human breast cancer cells resistant to Taxol. Furthermore, p185c-erbB2 synergizes with p170mdr-1 conferring higher degrees of Taxol resistance. Finally, combination therapy (down-regulation of p185c-erbB2 plus blocking p170mdr-1 plus administration of Taxol) may be beneficial to breast cancer patients whose tumors express high levels of both p185c-erbB2 and p170mdr-1.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Genetic analysis of the UL 15 gene locus for the putative terminase of herpes simplex virus type 1.
The herpes simplex virus (HSV-1) UL15 gene encodes one of the six viral gene products required for viral DNA cleavage and packaging. UL15 is a spliced gene and encodes two separately translated proteins, UL15 and UL15.5. Sequence analysis reveals that UL15 shares homology with gp 17, the large catalytic subunit of the bacteriophage T4 terminase, a protein which cleaves the polymeric T4 DNA into monomers. Both proteins contain a putative ATP binding motif known as the Walker A and B boxes. In this report, immunofluorescence was used to show that UL15 localizes to the nucleus in the absence of any other viral proteins; this indicates that UL15 contains its own nuclear localization signal. In addition, we found that UL15 colocalizes with replication compartments at early times (6 h postinfection). Since, at this time, preformed capsids as well as other cleavage and packaging proteins are also recruited to replication compartments, it seems likely that cleavage and packaging occurs in the same compartments in which DNA synthesis occurs. Also in this report, we have investigated UL15.5, the N-terminally truncated gene product of the UL15 open reading frame (ORF). The start codon has been mapped to Met443 within the UL15 ORF. Furthermore, we have shown that plasmids containing a UL15.5 knockout mutation still complement the growth of UL15 insertion mutant viruses, indicating that UL15.5 is not required for viral growth in cell culture. Last, we constructed a UL15 mutant, UL15C(G263A), in which the invariant Gly263 in the Walker box A of the ATP binding motif (GKT) was substituted with an alanine. We show that the mutant gene fails to support the growth of UL15 insertion mutant viruses, indicating that the putative ATP binding motif of UL15 is indispensable for its function.
Animals↗
Generation and characterization of mice deficient in hepsin, a hepatic transmembrane serine protease.
Hepsin is a type II transmembrane serine protease highly expressed on the surface of hepatocytes. The physiological function of hepsin is not known, although in vitro studies indicate that hepsin plays a role in the initiation of blood coagulation and in hepatocyte growth. To determine the functional importance of hepsin, we generated hepsin-deficient mice by homologous recombination. Homozygous hepsin-/- mice were viable and fertile, and grew normally. In functional assays including tail bleeding time, plasma clotting times, and tissue factor- or LPS-induced disseminated intravascular coagulation models, no significant difference was found between hepsin-/- and wild-type litter mates. Liver weight and serum concentrations of liver-derived proteins or enzymes were similar in hepsin-/- and wild-type mice. Interestingly, serum concentrations of bone-derived alkaline phosphatase were approximately twofold higher in hepsin-/- mice of both sexes when compared with wild-type litter mates. No obvious abnormalities were found in major organs in hepsin-/- mice in histological examinations. Our results indicate that hepsin is not essential for embryonic development and normal hemostasis. Hepsin-/- mice will help to evaluate the long-term effects of hepsin deficiency in these animals.
Alkaline Phosphatase↗
Study on the relationship between the Bcl-2/Bax ratio and the growth types of leukemic cells and drug resistance in acute myelogenous leukemia.
The ratio of Bcl-2 to Bax (Bcl-2/Bax) in 40 patients with acute myelogenous leukemia (AML) were determined. At the same time, the CFU-L of AML patients and drug resistance were detected by cell culture and MTT assay. The results showed that Bcl-2/Bax in AML was significantly higher than that in normal control (P < 0.001). Bcl-2/Bax ratio in colony growth group was higher than that in no-growth group (P < 0.05). Between drug resistance group and drug sensitivity group there was a significant difference in Bcl-2/Bax ratio (P < 0.05). There were more cases of drug resistance in high ratio group (H) than in low ratio group (L) (P < 0.05). Meanwhile, Complete Remission(CR) rate in group H was obviously lower than that in group L (P < 0.05) and patients in group H tended to have poor response. The above results indicated that the alteration of Bcl-2 and Bax is an important mechanism in the pathogenesis, progress and the development of drug resistance in AML. The determination of Bcl-2/Bax ratio has far-reaching implication in the treatment, choice of chemotherapeutic agents, and prediction of prognosis.
Adult↗
Hepatocyte growth factor up-regulates SGLT1 and GLUT5 gene expression after massive small bowel resection.
BACKGROUND/PURPOSE: Hepatocyte growth factor (HGF), originally known to stimulate hepatocyte DNA synthesis, also has been shown to stimulate growth of intestinal epithelial cells in vitro. The authors recently demonstrated that HGF can dramatically increase substrate absorption beyond the normal adaptive response after massive small bowel resection in the rat. However, the mechanism for this enhanced substrate absorption is unknown. This study was designed to determine if up-regulation of gene expression by HGF of the Na+/glucose cotransporter SGLT1 and the facilitative glucose transporter GLUT5 is a possible mechanism of action. METHODS: Young adult Sprague-Dawley rats underwent an 80% small bowel resection and jejunostomy tube placement. Seven days later, an osmotic minipump was connected to the subcutaneously placed jejunostomy tube. The rats were divided into two groups based on the contents in the minipumps: group 1, saline (control, n = 5); and group 2, HGF at 75 microg/kg/d (n = 5). After a 14-day infusion, biopsy specimens of the small bowel mucosa were obtained. After total RNA extraction, Northern blot analysis was performed with SGLT1 and GLUT5 cDNA probes. Auto radiographs were quantitated by image analysis. RESULTS: SGLT1 mRNA levels were significantly up-regulated in the HGF-treated animals (121% increase, P<.01) when compared with the control. Up-regulation of GLUT5 mRNA levels was also seen in the HGF-treated animals (96% increase, P < .01). CONCLUSIONS: These data, demonstrating that HGF upregulates intestine epithelial glucose transporter gene expression after massive small bowel resection, may elucidate a mechanism of action for the enhanced carbohydrate absorption after HGF administration. This growth factor may be useful for patients with short bowel syndrome.
Animals↗
Enhancement of intestinal adaptation by hepatocyte growth factor.
BACKGROUND/PURPOSE: Hepatocyte growth factor (HGF) was originally shown to enhance hepatocyte DNA synthesis. Recently, the expression of HGF and its receptor (c-met) were observed in the intestinal tract. In a previous study, we demonstrated that HGF can increase normal rat intestinal epithelial mass and function in vivo. This study was designed to determine if HGF, given either systemically or luminally, can enhance intestinal epithelial mass and function beyond the normal adaptive response after massive small bowel resection. METHODS: Twenty young adult male Sprague-Dawley rats underwent an 80% small bowel resection. Seven days after resection, systemic infusion (via the jugular vein) or luminal perfusion (via the jejunum) were performed using subcutaneously placed osmotic minipumps. The rats were divided into four groups: group 1, systemic saline, (control, n=5); group 2, systemic HGF at 150 microg/kg/d (n=5); group 3, luminal saline, (control, n=5); and group 4, luminal HGF at 75 microg/kg/d (n=5). After a 14-day infusion, [C14] galactose and [C14] glycine absorption (micromol/cm2 intestine), mucosal DNA content (microg/mg mucosa) and protein content (microg/mg mucosa) were measured in the remaining small intestine of each rat. RESULTS: Systemic infusion of HGF increased galactose absorption 68% (P<.05), glycine absorption 57% (P<.05), DNA content 17% (P<.01), and protein content 57% (P<.01), when compared with the appropriate control. Luminal perfusion of HGF also increased galactose absorption 114% (P<.01), glycine absorption 126% (P<.01), DNA content 32% (P<.01), and protein content 45% (P<.01), when compared with the appropriate control. CONCLUSIONS: These data demonstrate for the first time that HGF can significantly enhance intestinal epithelial cell function and mucosal mass beyond the normal adaptive response. Luminal administration appeared to produce a greater response when compared with systemic administration but was significant only for galactose absorption (P<.05). HGF may be clinically useful in patients with short bowel syndrome.
Adaptation, Physiological↗
Enteral glutamine does not enhance the effects of hepatocyte growth factor in short bowel syndrome.
PURPOSE: This study was designed to determine if luminally administered glutamine alone functions as a growth factor or is synergistic with hepatocyte growth factor (HGF) after massive small bowel resection (MSBR). METHODS: Twenty Sprague-Dawley rats underwent an 80% small bowel resection and jejunostomy tube placement. Seven days later the rats were divided into four groups: group 1, control, no further treatment (n = 5); group 2 received glutamine (4% of total food intake per day) via an orogastric tube (n = 5); group 3 received intraluminal HGF via a jejunostomy tube at 75 microg/kg/d (n = 5); and group 4 received glutamine and HGF at the same doses, respectively. After a 14-day HGF infusion, glutamine feeding, or both combined, [C14] glycine absorption (micromol/L/cm2 intestine) and mucosal DNA and protein content (microg/mg mucosa) were measured in the remaining small bowel. RESULTS: Glutamine alone had no effect on substrate absorption and protein or DNA content. HGF increased galactose absorption (106% increase over control, P<.01), glycine absorption (95% increase over control, P<.05), protein content (44% increase over control, P<.01), and DNA content (32% increase over control, P<.01). The combination of glutamine and HGF did not prove to be synergistic. CONCLUSIONS: These data demonstrate that in this short bowel model, glutamine alone did not enhance intestinal function. Furthermore, glutamine is not synergistic with HGF. This study suggests that glutamine alone may not be useful clinically in patients with inadequate intestinal function.
Adaptation, Physiological↗
Overexpression of ErbB2 blocks Taxol-induced apoptosis by upregulation of p21Cip1, which inhibits p34Cdc2 kinase.
Overexpression of the receptor tyrosine kinase p185ErbB2 confers Taxol resistance in breast cancers. Here, we investigated the underlying mechanisms and found that overexpression of p185ErbB2 inhibits Taxol-induced apoptosis. Taxol activates p34Cdc2 kinase in MDA-MB-435 breast cancer cells, leading to cell cycle arrest at the G2/M phase and, subsequently, apoptosis. A chemical inhibitor of p34Cdc2 and a dominant-negative mutant of p34Cdc2 blocked Taxol-induced apoptosis in these cells. Overexpression of p185ErbB2 in MDA-MB-435 cells by transfection transcriptionally upregulates p21Cip1, which associates with p34Cdc2, inhibits Taxol-mediated p34Cdc2 activation, delays cell entrance to G2/M phase, and thereby inhibits Taxol-induced apoptosis. In p21Cip1 antisense-transfected MDA-MB-435 cells or in p21-/- MEF cells, p185ErbB2 was unable to inhibit Taxol-induced apoptosis. Therefore, p21Cip1 participates in the regulation of a G2/M checkpoint that contributes to resistance to Taxol-induced apoptosis in p185ErbB2-overexpressing breast cancer cells.
Animals↗
The erbB2 gene as a cancer therapeutic target and the tumor- and metastasis-suppressing function of E1A.
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Adenovirus E1A Proteins↗
99mTc-ethylenedicysteine-folate: a new tumor imaging agent. Synthesis, labeling and evaluation in animals.
It is known that membrane folic acid receptors are responsible for cellular accumulation of folate and folate analogs such as methotrexate and overexpressed on various tumor cells. However, these receptors are highly restricted in normal differentiated tissues. Results of limited in vitro and in vivo animal studies suggest that folate receptors could be a potential target for tumor imaging. This study aimed to develop a 99mTc-labeled folic acid using ethylenedicysteine (EC) as a chelator and evaluate its labeling efficiency and potential use as a tumor seeking agent. Tissue distribution of 99mTc-EC-folate was determined in breast tumor-bearing rats at 20 min, 1, 2, and 4 h (n = 3/time interval, 370 KBq/rat, i.v.). Blocking study was employed to determine receptor-mediated process; 99mTc-EC-folate was co-administrated with 50 and 150 mumol/kg of cold folic acid to tumor-bearing rats. Planar imaging and whole-body autoradiograms were performed. The data was compared to that using 99mTc-EC (control). In animal studies, tumor/blood count density ratios at 20 min-4 h increased from 0.81 +/- 0.09 to 1.23 +/- 0.13 with 99mTc-EC-folate. Conversely, these values showed time-dependent decrease from 0.77 +/- 0.32 to 0.65 +/- 0.01 with 99mTc-EC in the same time period. Tumor/muscle and tumor/blood count density ratios significantly decreased with folic acid co-administrations. Planar images and autoradiograms confirmed that the tumors could be visualized clearly with 99mTc-EC-folate.
Animals↗
Herpes simplex virus type 1 cleavage and packaging proteins UL15 and UL28 are associated with B but not C capsids during packaging.
At least seven viral genes encode proteins (UL6, UL15, UL17, UL25, UL28, UL32, and UL33) that are required for DNA cleavage and packaging of herpes simplex virus type 1 (HSV-1) DNA. Sequence analysis reveals that UL15 shares homology with gp17, the large catalytic subunit of the bacteriophage T4 terminase. Thus, UL15 may play a direct role in the cleavage of viral DNA replication intermediates into monomers. In this study, we asked whether UL15 and other cleavage and packaging proteins could be detected in capsids isolated from infected cells. Consistent with previous studies showing that UL6 and UL25 are minor protein constituents of the capsids, we detected these proteins in both B and C capsids. In contrast, the previously identified full-length version (81 kDa) of UL15 was found predominantly in B capsids and in much smaller amounts in C capsids. In addition, the UL28 protein was found predominantly in B but not C capsids in a distribution similar to that of the 81-kDa version of UL15. These results suggest that UL28 and the 81-kDa form of UL15 are transiently associated with capsid intermediates during the packaging process. Surprisingly, however, a previously unidentified 87-kDa form of UL15 was found in the B and C capsids and in virions. Analysis of cells infected with mutants individually lacking UL6, UL15, UL25, UL28, or UL32 demonstrates that the lack of one cleavage and packaging protein does not affect the expression of the others. Furthermore, this analysis, together with guanidine HCl extraction analysis of purified capsids, indicates that UL6, UL25, and UL28 are able to associate with B capsids in the absence of other DNA cleavage and packaging proteins. On the other hand, the two UL15-related proteins (81 and 87 kDa) do not associate efficiently with B capsids in cells infected with UL6 and UL28 mutants. These results suggest that the ability of the UL15-related proteins to bind to B capsids may be mediated through interactions with UL6 and UL28.
Animals↗
Stereotyped behavior in developmentally delayed or autistic populations. Rhythmic or nonrhythmic?
Stereotypies are high-frequency, highly repetitive, nonfunctional behaviors that are also often characterized as rhythmic. Rhythmicity suggests that the behavior is periodic, occurring at fixed intervals. Few studies, however, have rigorously demonstrated periodicity in stereotypy. This study examined various topographies of stereotypy in 9 participants and used spectral methods to detect existence of periodicties. Two general patterns emerged in the spectral analysis. Participants who engaged in stereotypic rocking showed peaks in their power spectra; participants who engaged in other topographies of stereotypy did not show peaks. Thus, it appears that although some stereotypies--notably, rocking--have a periodic component, rhythmicity does not appear to be a characteristic of stereotypy in general.
Adolescent↗