[Nursing experience of bladder function reconstruction in a patient with neobladder surgery].
Explore the source record for details and available documents.
Exercise Therapy↗
Biomedical subjects
S Lin
Publications and source records attributed to S Lin.
The biochemical status of the DNA synthesome can distinguish between permanent and temporary cell growth arrest.
We previously identified and characterized the human leukemia (HL-60) cell DNA synthetic machinery as a multiprotein form of DNA polymerase, which was designated the DNA synthesome. This multiprotein replication complex contains DNA polymerases alpha and delta, primase, replication factor C, replication protein A, helicase, poly(ADPribose) polymerase, proliferating cell nuclear antigen, DNA ligase I, and topoisomerases I and II. Recently, the HeLa cell-derived DNA synthesome was identified as a discrete high molecular weight protein band in native polyacrylamide gels. Here, we report our findings regarding the change in the organizational status of the DNA synthesome when HL-60 cells undergo either terminal differentiation or temporary G1 growth arrest. We observed that the HL-60 cell DNA synthesome also migrates as a discrete high molecular weight protein band in nondenaturing polyacrylamide gels. This high molecular weight protein band was present in nuclei derived from both actively cycling cells and aphidicolin-arrested cells but was absent in TPA-induced terminally differentiated cells. We also found that DNA polymerase delta, replication factor C, and proliferating cell nuclear antigen are absent in cells that are induced to differentiate in response to 12-O-tetradecanoyl phorbol-13-acetate treatment but are present in actively cycling cells. The level of replication protein A in differentiated cells was similar to that of cycling cells, whereas the level of annexin I, a cytoskeleton protein, is higher in differentiated cells than it is in actively cycling cells. We conclude that the DNA synthesome remains integrated and inactive in temporarily growth-arrested cells but is disassembled in differentiated cells. Furthermore, we conclude that disassembly of the organized replication complex is a specific cellular event in the process of permanent cell cycle exit and that the process leading to disassembly may be regulated, in part, at the level of gene transcription.
Aphidicolin↗
Existence of an abnormal nitric oxide metabolism seen in glomeruli and inner medullary collecting duct in diabetic rat.
OBJECTIVE: To investigate the nitric oxide (NO)-dependent 3', 5'-cyclic guanosine monophosphate (cGMP) stimulation or inhibition in isolated glomeruli and inner medullary collecting duct (IMCD) cells from normal and diabetic rats for elucidating the probable role of NO in diabetic nephropathy. METHODS: Glomeruli and IMCD cells were obtained from anesthetized diabetic rats, 2 weeks after streptozotocin-induction, and normal rats, and incubated for 10 minutes under basal state or adding different drugs. cGMP was measured by radioimmunoassay. RESULTS: Diabetic glomeruli had a higher generation of cGMP under basal state but failed to further response of L-arginine, which was contrary to the glomeruli from normal rats. NG-monomethyl-L-arginine prevented the elevation of cGMP after L-arginine stimulation in normal rats but not in diabetic rats. Acetylcholine produced a concentration-dependent of cGMP accumulation in the glomeruli of both groups, but the extent was relatively smaller in diabetic than in normal. A similar tendency was also observed in sodium nitroprusside treated glomeruli. However the cGMP increment in response to atrial natriuretic peptide was identical in the two groups. For the IMCD cells, neither the difference of basal cGMP nor those responses related to acetylcholine or sodium nitroprusside could be observed. But the attenuated response to L-arginine also existed in diabetic IMCD cells. CONCLUSION: Both diabetic glomeruli and IMCD cells have an abnormal NO metabolism, which may account in part for the disturbance of renal hemodynamics.
Animals↗
[Endometrial cytology in normal postmenopausal women and during hormone replacement therapy].
OBJECTIVE: To explore the possibility of endometrial cyopathologic examination as a method of monitoring endometrium during hormone replacement therapy (HRT) in postmenopausal women. METHODS: Endometrial cells were taken via tubal aspiration in 60 normal postmenopausal women (non-HRT group) and 41 with HRT for 3-18 months (HRT group). Their morphologic changes were observed and compared by cytopathologist. RESULTS: Atrophic endometrium was found in 51.7% of the non-HRT group. Its proportion increased with age and the time after menopause. Macrophages were seen in 68.3% of this group. However, in the HRT group the occurrence of atrophic type and macrophage (12.2%, 7.0% respectively) was significantly lower than that in the non HRT group (P < 0.05). Heterogeneity of endometrial cell type was shown both in non-HRT (38.3%) and HRT (65.8%) groups. CONCLUSIONS: Endometrial cells of postmenopausal women are not always atrophic in appearance. They change significantly during HRT. Endometrial cytological examination may be useful for monitoring during HRT.
Endometrium↗
[The shrinkage of ovarian and uterine size and the decline of serum estradiol level in post-menopausal women].
OBJECTIVE: To investigate the shrinkage of ovarian and uterine size, and the decline of serum estradiol (E2) level in normal women. METHODS: Ovarian and uterine area of 92 perimenopausal and 292 postmenopausal women, measured by transvaginal ultrasonography, were analysed retrospectively and were compared with those of the controls consisting of 41 normal women in midfollicular phase, so did the serum E2 levels. RESULTS: As compared with the control group, ovarian area and endometrium thickness shrunk on an average of 30%, 2% and 56%, 48% in early perimenopausal and postmenopausal groups respectively. Serum E2 level and uterine area increased by 11% and 38% in early perimenopausal group, but decreased by 67% and 21% in postmenopausal groups respectively. No obviously further decline of serum E2 level was found 1 year after menopause, and of ovarian, uterine area 3-5 years after menopause. CONCLUSION: These data represent a normal profile of ovarian and uterine shrinkage and decline of serum E2 levels related to the time of menopause.
Adult↗
Ligand stabilization of the beta 2 adrenergic receptor: effect of DTT on receptor conformation monitored by circular dichroism and fluorescence spectroscopy.
Treatment of the beta 2 adrenergic receptor with the reducing agent dithiothreitol (DTT) is known to abolish ligand binding to the receptor. Interestingly, the loss of binding can be prevented by preoccupation of the receptor with ligand. It is unclear, however, whether the ligand blocks access of DTT to the receptor, or the ligand stabilizes the receptor structure. In the present study, we have utilized circular dichroism (CD) and intrinsic tryptophan fluorescence to directly probe structural changes in the beta 2 adrenergic receptor in response to DTT treatment. Analysis of CD spectra of purified beta 2 receptor in the detergent micelle indicated that the receptor has an alpha-helix content of 60%, which is substantially more than what would be attributed to the seven transmembrane domains. The alpha-helix content was unchanged in the presence of DTT, suggesting that DTT treatment does not alter the secondary structure of the receptor. In contrast, the tryptophan fluorescence spectra demonstrated that DTT induces a reversible conformational change of the beta 2 receptor. Thus, DTT caused a red-shift in the maximum emission wavelength of the intrinsic tryptophan fluorescence. The change in emission spectrum correlated with a loss in the ability of the receptor to bind antagonist. Both changes in receptor binding and fluorescence emission were reversible, as removal of DTT allowed the receptor to restore 70% of ligand binding and return to the initial emission spectrum. Furthermore, we found adrenergic antagonists were able to slow the rate of the conformational change induced by DTT but not the rate of disulfide reduction, suggesting that the antagonists stabilize the structure of the reduced receptor.
Amino Acid Sequence↗
Localization of leptin receptor mRNA expression in mouse brain.
The distribution of the leptin receptor in the brain of C57 mice was investigated using a non-radioactive in situ hybridization method. Leptin receptor mRNA expression was highest in the arcuate nucleus and median eminence of the hypothalamus, but it was also abundant in hippocampus, primarily in the dentate gyrus and CA1, and was detected at low levels in piriform cortex and the medial margin of the medial habenular nucleus. The localization of leptin receptor-containing neurones in the present study indicates the possibility that the leptin receptor is expressed on neuropeptide Y-containing neurones.
Animals↗
Head group analogs of arachidonylethanolamide, the endogenous cannabinoid ligand.
Several analogs of an endogenous cannabimimetic, arachidonylethanolamide (anandamide), were synthesized to study the structural requirements of the ethanolamide head group. CB1 receptor affinities of the analogs were evaluated by a standard receptor binding assay using tritiated CP-55,940 as the radioligand and compared to anandamide which was shown to have a Ki of 78 nM. Replacement of the amide carbonyl oxygen by a sulfur atom had a detrimental effect on the CB1 affinity. The thio analogs of both anandamide and (R)-methanandamide showed very weak affinity for CB1. The secondary nature of the amidic nitrogen was also shown to be important for affinity, indicating a possible hydrogen-bonding interaction between the amide NH and the receptor. Introduction of a phenolic moiety in the head group resulted in the loss of receptor affinity except when a methylene spacer was introduced between the amidic nitrogen and the phenol. A select group of analogs were also tested for their affinity for the CB2 receptor using a mouse spleen preparation and were found to possess low affinities for the CB2 sites. Notably, anandamide and (R)-methanandamide demonstrated high selectivity for the CB1 receptor. Overall, the data presented here show that structural requirements of the head group of anandamide are rather stringent.
Adjuvants, Immunologic↗
Structure-reactivity relationships for beta-galactosidase (Escherichia coli, lac Z). 3. Evidence that Glu-461 participates in Brønsted acid-base catalysis of beta-D-galactopyranosyl group transfer.
Experiments are reported to determine the role of Glu-461 in the beta-D-galactopyranosyl group transfer reaction catalyzed by beta-galactosidase. E461G beta-galactosidase catalyzes the hydrolysis of 4-nitrophenyl beta-D-galactopyranoside through a galactosyl-enzyme intermediate that shows a high reactivity toward the anionic nucleophile azide ion, but no detectable reactivity toward the neutral nucleophile trifluoroethanol. By contrast, the galactosylated wild type enzyme is reactive toward trifluoroethanol but not anions. The change in specificity observed for the E461G mutant can be rationalized by a mechanism in which Glu-461 participates in general acid-base catalysis at the leaving group/nucleophile. The observed low activity of E461G beta-galactosidase for hydrolysis of 2,2,2-trifluoroethyl beta-D-galactopyranoside is due entirely to a wild type enzyme contaminant in our preparation of the mutant enzyme, and the mutant enzyme itself has essentially no catalytic activity for cleavage of this substrate. The substitution of glutamate at position 461 by glycine leads to a more than 500 000-fold reduction in the rate constant for enzymatic cleavage of the glycosidic bond to the strongly basic trifluoroethoxide leaving group (pKa = 12.4), but to a smaller 1300-fold reduction in the rate constant for cleavage of the bond to the more weakly basic 4-nitrophenoxide leaving group (pKa = 7.1). This corresponds to a more than 3.5 kcal/mol greater stabilization by Glu-461 of the transition state for the reaction of the substrate with the more basic trifluoroethoxide leaving group. These data are consistent with the conclusion that Glu-461 provides general acid catalysis of leaving group departure, which is most effective for cleavage of the relatively strong bonds to basic alkoxide leaving groups.
Azides↗
Structure-reactivity relationships for beta-galactosidase (Escherichia coli, lac Z). 4. Mechanism for reaction of nucleophiles with the galactosyl-enzyme intermediates of E461G and E461Q beta-galactosidases.
Second-order rate constants for transfer of the beta-D-galactopyranosyl group from the galactosyl-enzyme intermediates of the galactosyl transfer reactions catalyzed by E461G and E461Q beta-galactosidases to anionic nucleophiles have been determined. The second-order rate constant for reaction of the galactosylated E461G enzyme with azide ion is 4900 M-1 s-1. By contrast, there is no detectable reaction of the galactosylated wild type enzyme with azide ion (Richard et al., 1995b), and the E461G mutation leads to a large decrease in the second-order rate constant kcat/Km for catalysis of cleavage of beta-D-galactopyranosyl azide, which is the microscopic reverse of the reaction of azide ion with the galactosyl-enzyme intermediate. These data show that the E461G mutation causes a more than 8000-fold increase in the equilibrium constant for transfer of the beta-D-galactopyranosyl group from beta-galactosidase to azide ion. We propose that this change represents the requirement for the coupling of galactosyl transfer from the native enzyme to the thermodynamically unfavorable protonation of the carboxylate group of Glu-461, but the expression of the full chemical affinity of azide ion for galactosyl transfer from the mutant enzyme which lacks this ionizable side chain at position 461. The reactions of acetate, butyrate and methoxyacetate ions with the galactosylated E461G enzyme and of acetate with the galactosylated E461Q enzyme give both the corresponding beta-galactopyranosyl derivatives and D-galactose, and the formation of the latter represents formal catalysis of the reaction of water with the galactosylated enzyme. However, the reaction of formate ion with the galactosylated E461G enzyme gives only D-galactose. These results suggest that carboxylate anions can take the place of the excised propionate side chain of Glu-461 to provide general base catalysis of the reaction of water with the galactosyl-enzyme intermediates. The relative reactivity of anionic nucleophiles toward the covalent galactosyl-enzyme intermediate of the reactions catalyzed by the E461G enzyme is similar to that observed for partitioning of stable carbocations in water. This suggests that replacement of the anionic side chain of Glu-461 by a hydrogen exposes an enzyme-stabilized oxocarbenium ion intermediate to reaction with external nucleophilic reagents.
Acetates↗
Structural requirements for targeting of surfactant protein B (SP-B) to secretory granules in vitro and in vivo.
Human surfactant protein B (SP-B) is synthesized by type II cells as a 381-residue preproprotein which is proteolytically processed to a 79-residue mature peptide and targeted to lamellar bodies for secretion. To identify secretory granule targeting determinants, constructs encoding the SP-B preproprotein (SP-B), COOH-terminally deleted SP-B (SP-BDeltaC), the NH2-terminal propeptide (SP-BN), and a chimeric molecule consisting of albumin and the mature peptide (ALB/SP-BM) were transfected into AtT-20 and PC12 cells. Pulse-chase studies demonstrated that 10-30% of SP-B and SP-BDeltaC remained in cells in an endoglycosidase H-resistant form. Secretion of stored SP-B was stimulated by forskolin/12-O-tetradecanoylphorbol-13-acetate and intracellular SP-B was localized to secretory granules by immunoelectron microscopy. In contrast, SP-BN and ALB/SP-BM were constitutively secreted and not detected in secretory granules. Specific processing of SP-B was not detected in either AtT-20 or PC12 cells. Expression of SP-BDeltaC in transgenic mice resulted in secretion of fully processed mature SP-B, indicating correct processing and targeting of this construct in vivo. We conclude that 1) SP-B processing occurs in a cell-specific manner, 2) the proprotein contains secretory granule targeting determinants that are not cell-specific, 3) the NH2-terminal propeptide and the mature peptide are required for targeting SP-B to lamellar body, and 4) the COOH-terminal propeptide is not required for processing or sorting of SP-B.
Animals↗
Novel azacyclic ureas that are potent inhibitors of HIV-1 protease.
A series of novel, azacyclic ureas which are highly potent inhibitors of the HIV-1 protease (IC50 = 4.1 to < 0.5 nM) were synthesized. Aqueous solubilities of this series of compounds were improved by incorporating polar functional groups at the P1' P2 and P2' positions. These compounds also possess good anti-viral activity by inhibition of the cytopathic effect of HIV-13B in MT-4 cells in vitro.
Amino Acid Sequence↗
Interdigitation of bilayers from ether lipid analogs: (R)-PAF, (R)-Lyso-PAF and the antineoplastic (R)-ET-18-OMe.
The ether lipid analogs (R)-PAF, (R)-ET-18-OMe and (R)-Lyso-PAF can form bilayers. Using differential scanning calorimetry (DSC) and small angle X-ray diffraction, we have investigated bilayers formed entirely of these molecules. Small angle X-ray diffraction experiments strongly indicated that these bilayers are interdigitated at 98% relative humidity in the gel phase. The slight differences in the structures of the ether lipids affect their thermal behavior and packing properties. Of the three, (R)-Lyso-PAF shows the highest phase transition temperature, broadest phase transition peak and smallest enthalpy change. These characteristics may be attributed to hydrogen bonding of the -OH group at sn-2 position of its glycerol backbone to water or to other (R)-Lyso-PAF molecules. The interdigitated structure shows that these bilayers are similar to lipid bilayers and suggests that these molecules could easily insert themselves into membranes to reach their respective active sites or to perturb the membranes. The packing and thermal characteristics of (R)-Lyso-PAF may help explain its biological inactivity.
Antineoplastic Agents↗
Characterization of the structure and function of a new mitogen-activated protein kinase (p38beta).
Mitogen-activated protein (MAP) kinase cascades represent one of the major signal systems used by eukaryotic cells to transduce extracellular signals into cellular responses. Four MAP kinase subgroups have been identified in humans: ERK, JNK (SAPK), ERK5 (BMK), and p38. Here we characterize a new MAP kinase, p38beta. p38beta is a 372-amino acid protein most closely related to p38. It contains a TGY dual phosphorylation site, which is required for its kinase activity. Like p38, p38beta is activated by proinflammatory cytokines and environmental stress. A comparison of events associated with the activation of p38beta and p38 revealed differences, most notably in the preferred activation of p38beta by MAP kinase kinase 6 (MKK6), whereas p38 was activated nearly equally by MKK3, MKK4, and MKK6. Moreover, in vitro and in vivo experiments showed a strong substrate preference by p38beta for activating transcription factor 2 (ATF2). Enhancement of ATF2-dependent gene expression by p38beta was approximately20-fold greater than that of p38 and other MAP kinases tested. The data reported here suggest that while closely related, p38beta and p38 may be regulated by differing mechanisms and may exert their actions on separate downstream targets.
Activating Transcription Factor 2↗
Structural requirements for intracellular transport of pulmonary surfactant protein B (SP-B).
Human SP-B is synthesized by the alveolar Type II epithelial cell as a 381 amino acid preproprotein. The 79 residue mature SP-B peptide is extremely hydrophobic and flanked by propeptides of 200 and 102 amino acids at its NH2- and COOH-termini, respectively. The purpose of this study was to identify peptide domains of the SP-B proprotein necessary for trafficking of the mature peptide in the secretory pathway. To this end several constructs were generated, by subcloning the full length human SP-B (SP-B), COOH-terminally truncated SP-B (SP-B delta C, in which residues 201-381 were deleted), NH2-terminally deleted SP-B (SP-B delta N, in which residues 28-200 were deleted), NH2-terminal propeptide (SP-BN), mature SP-B (SP-BM) and COOH-terminal propeptide (SP-BC), into the mammalian expression vector pcDNA3. The resulting expression constructs were characterized by DNA sequencing and in vitro transcription/translation and subsequently transfected into Chinese hamster ovary cells. 48 h after transfection, cells were labeled with [35S]-met/cys and analyzed by immunoprecipitation, SDS-PAGE and autoradiography. Proteins encoded by SP-B, SP-B delta C, SP-BN and SP-BC constructs were secreted into media; in contrast, SP-B constructs lacking the NH2-terminal propeptide (SP-B delta N) remained in the endoplasmic reticulum (as assessed by endoglycosidase H sensitivity) and were rapidly degraded. We conclude that (1) 27 amino acids at the NH2-terminus of SP-B contain a functional signal peptide and (2) the NH2-terminal propeptide of the SP-B precursor is necessary and sufficient for intracellular trafficking of the mature peptide.
Animals↗
The Fc(epsilon)RIbeta subunit functions as an amplifier of Fc(epsilon)RIgamma-mediated cell activation signals.
The high affinity IgE receptor (Fc(epsilon)RI) plays a central role in the initiation of allergic responses. Fc(epsilon)RI is multimeric and is expressed as either (alpha)(gamma2) trimers or (alpha)(beta)(gamma2) tetramers. Recently, polymorphisms of the beta chain gene have been associated with the development of various allergic phenotypes. Until now, the role of beta in Fc(epsilon)RI-dependent signaling was largely unknown. For that reason, we compared the signaling characteristics of various wild-type and mutant (alpha)(gamma2) and (alpha)(beta)(gamma2) Fc(epsilon)RI complexes. These studies demonstrate that the gamma dimer functions as an autonomous activation module, while beta functions as an amplifier that provides a gain of 5- to 7-fold, as measured by Syk activation and calcium mobilization.
Calcium↗
High-performance liquid chromatographic determination of anandamide amidase activity in rat brain microsomes.
A rapid, sensitive, and reliable method for measuring anandamide amidase activity in rat brain microsomes by reversed-phase high-performance liquid chromatography (RP-HPLC) and its applications are described. Enzymatic activity was assayed by the determination of the rates of hydrolysis of anandamide or its analogs at 37 degrees C. The reaction products were separated using an ODS guard column eluted with aqueous phosphoric acid-acetonitrile and quantitated with uv detection at 204 nm and an external standard method. Baseline separation of the acid products from their substrates was completed in less than 2 min. The detection limits were 1.4 pmol for arachidonic acid and 0.22 pmol for anandamide at a signal to noise ratio of 4:1. The stability of anandamide in the acidic mobile phase was tested, and no significant decomposition was observed up to 1 h. The method was successfully applied to the examination of substrate specificity as well as for testing the ability of amidase inhibitors to block its hydrolysis. Kinetic constants obtained for (S)-methanandamide were an apparent Km of 8.6 +/- 1.3 microM and a Vmax of 362 +/- 16 pmol/min/mg of protein. A highly potent inhibitor, palmitylsulfonyl fluoride (PSF), was found to have an IC50 of 50 nM. PSF is 210 times as potent as phenylmethylsulfonyl fluoride. The method offers several advantages over existing methodology using radioisotopes or a solvent extraction procedure.
Amidohydrolases↗
Low-temperature femtosecond-resolution transient absorption spectroscopy of large-scale symmetry mutants of bacterial reaction centers.
Reaction centers isolated from three large-scale symmetry mutants sym0, sym2-1, and sym5-2 described in the previous article of this issue [Taguchi, A. K. W., Eastman, J. E., Gallo, D. M., Jr., Sheagley, E.. Xiao, W., & Woodbury, N. W. (1996) Biochemistry 35, 3175-3186] have been investigated by low-temperature ground state and ferntosecond-resolution transient absorption spectroscopy. All three of these large-scale symmetry mutants undergo electron transfer at 20 K. The mutants sym0 and sym5-2 have yields and dominant rates of charge separation comparable to wild type. However. the sym2-mutant shows a roughly 35%, quantum yield at this temperature, and the major kinetic component of the initial electron transfer is slower than wild type by nearly a factor of 100. The sym0 mutant showed substantial changes in the monomer bacteriochiorophyll ground state and transient spectra, and both sym0 sym2-1 showed changes in the bacteriopheophyll ground state and transient spectra. In particular, sym2-1 shows a small absorbance decrease in the region of the Qx band of the B side bacteriopheophytin which could be attributed to 10%-20% electron transfer along the B pathway.
Cold Temperature↗