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Biomedical subjects

H Yin

Publications and source records attributed to H Yin.

At least 91 records · Page 5Linked to original sources

Expression and regulation of the chicken Nkx-6.2 homeobox gene suggest its possible involvement in the ventral neural patterning and cell fate specification.

Rapid accumulating evidence has suggested that the homeodomain transcription factors of the Nkx family play important roles in controlling vertebrate organ patterning and differentiation. In this study, we report the cloning, expression and regulation of a novel chicken homeobox gene, cNkx-6.2, whose expression is similar, but not identical, to that of mouse Nkx-6.2. The earliest expression of cNkx-6.2 was detected at the neural plate stage in the prospective midbrain and hindbrain regions. As the neural development proceeds, cNkx-6.2 expression was restricted in the ventral region of the entire neural axis except the forebrain region. At late stages of development, cNkx-6.2 expression is downregulated in the ventricular neuroepithelial cells, but subsequently upregulated in a sub-population of cells. Tissue recombination and explant culture experiments demonstrated that expression of cNkx-6.2 can be induced by the notochord signal and purified SHH protein, and repressed by BMP-4 and -7, indicating that the cNkx-6.2 expression can be influenced by both ventral and dorsal midline signals. Taken together, these studies have suggested two different roles for the cNkx-6.2 transcription factor: participating in the Shh-initiated ventral patterning during early CNS development and controlling cell fate specification and differentiation during late development.

Amino Acid Sequence↗

The amyloidogenicity of gelsolin is controlled by proteolysis and pH.

BACKGROUND: Normally, gelsolin functions in plasma as part of the actin-scavenging system to assemble and disassemble actin filaments. The Asp 187-->Asn (D187N) Asp 187-->Tyr (D187Y) gelsolin mutations facilitate two proteolytic cuts in the parent protein generating a 71-residue fragment that forms amyloid fibrils in humans, putatively causing Finnish type familial amyloidosis (FAF). We investigated the role of the D187N mutation in amyloidogenicity using biophysical studies in vitro. RESULTS: Both the recombinant wild-type and D187N FAF-associated gelsolin fragments adopt an ensemble of largely unfolded structures that do not self-associate into amyloid at pH 7. 5. Incubation of either fragment at low pHs (6.0-4.0) leads to the formation of well-defined fibrils within 72 hours, however. CONCLUSIONS: The D187N mutation has been suggested to destabilize the structure of the gelsolin parent protein (specifically domain 2), facilitating two proteolytic cleavage events. Our studies demonstrate that generating the largely unstructured peptide is not sufficient alone for amyloid formation in vitro (on a time scale of months). A drop in pH or an analogous environmental change appears necessary to convert the unstructured fragment into amyloid fibrils, probably through an associative mechanism. The wild-type gelsolin fragment will make amyloid fibrils from pH 6 to 4 in vitro, but neither the wild-type fragment nor fibrils have been observed in vivo. It is possible that domain 2 of wild-type gelsolin is stable in the context of the whole protein and not susceptible to the proteolytic degradation that affords the 71-residue FAF-associated peptide.

Amyloidosis↗

Metaphase karyotypes and G-banding in sandflies of the Lutzomyia longipalpis complex.

Mitotic metaphase chromosomes (2n = 8) from brain cells of fourth instar sandfly larvae of four geographical strains of the Lutzomyia longipaplis complex were examined microscopically, with bright-field illumination, after staining by a new G-banding technique involving exposure of air-dried chromosome preparations to quinacrine and ultraviolet light. Differences of G-banding and/or position of the centromere on chromosome 4 (the smallest chromosome pair) distinguished four putative sibling species from Costa Rica, Colombia and Brazil (distinctive populations from Jacobina and Lapinha Caves). The karyotype of the population from Jacobina, Brazil, showed an apparently plesiomorphic pattern of G-banding. On the basis of their recognizably different mitotic karyotypes, cytogenetic identification of separate taxa in the L. longipalpis complex should be useful for specific female vector competence and ecology studies.

Animals↗

Membrane lipid alpha-crystallin interaction and membrane Ca2+ -ATPase activities.

PURPOSE: To determine the effect of alpha-crystallin binding on lens membrane lipid characteristics and the stability of Ca2+ -ATPase activity when challenged with H2O2 or elevated temperatures. METHODS: Alpha-Crystallin binding to muscle sarcoplasmic reticulum membranes was quantified using a centrifugation protocol. Alpha-Crystallin binding to lens epithelial lipids was measured by a fluorescence energy transfer technique. Lipid phase transition temperature and lipid order was measured using fluorescence spectroscopy. Ca2+ -ATPase activity was measured using classical biochemical assays. RESULTS: The main phase transition temperatures of multilamellar vesicles composed of sphingomyelin or lipids extracted from bovine lens were 40 degrees C and 20 degrees C, respectively. In the presence of saturating amounts of alpha-crystallin, the phase transition temperature and lipid order of both sphingomyelin and lens lipid membranes remained almost the same as that without alpha-crystallin. The interaction of alpha-crystallin and lipid is likely to be restricted to the membrane surface. The binding of alpha-crystallin did not influence the oxidative or thermal inactivation of the Ca2+ -ATPase pump. CONCLUSION: Alpha-Crystallin-lens membrane binding does not protect the Ca2+ -ATPase pump from thermal derangement or oxidation by H2O2.

Animals↗

Growth stimulation of a rat pituitary cell line MtT/E-2 by environmental estrogens in vitro and in vivo.

Endocrine disruptors are a diverse group of chemicals that alter the functions of the endocrine system. A large proportion of endocrine disruptors have estrogenic effects, thus are called environmental estrogens. In the present study, an estrogen (E2) responsive rat pituitary cell line, MtT/E-2, was employed to examine 1) the potency of several endocrine disruptors including bisphenol A (BPA), o,p'-DDD, methoxychlor, 1,2,3,4,5,6-hexachlorocyclohexane (HCH) and dibromoacetic acid (DBAA) in terms of E2 responsive pituitary cell growth; 2) whether BPA has estrogenic action in vivo causing the growth of MtT/E-2 cells grafted in rats. Binding assays showed the test chemicals were able to compete with 3H-E2 binding to the estrogen receptor (ER). The compounds also stimulated growth of MtT/E-2 cells at rates corresponding to their ER binding affinity. Their transcription activation of an (ERE)3-SV40-luciferase reporter in MtT/E-2 cells was comparable to their stimulation of cell growth, with the exception of HCH which showed little induction of cell growth but strong stimulation in ERE dependent transcription activation. MtT/E-2 cells were inoculated into ovariectomized female F344 rats treated with E2 or BPA. The first tumors were noted at day 22 in the E2 treated group, at day 25 in the highest dose of BPA group and at day 41 in the control group. These results suggest 1) that the growth assay with MtT/E-2 cells provides simple and sensitive test for detection of estrogenic activity of environmental chemicals; 2) that BPA has estrogenic potency to stimulate E2 responsive cell growth in vivo as well as in vitro.

Acetates↗

Application of recombinant human granulocyte colony stimulating factor in children with acute myeloid leukemia.

OBJECTIVE: To evaluate the effect of recombinant human granulocyte colony stimulating factor (rhG-CSF) on accelerating neutrophil recovery and decrease fatal infections for childhood acute myeloid leukemia (AML). METHODS: From November 1992 to March 1997, 45 patients were enrolled into our study and 15 were newly diagnosed. All were treated with high dose chemotherapy combined with rhG-CSF. RESULTS: Of 15 newly diagnosed patients, 13 achieved complete remission (CR) after one course of therapy and 2 achieved CR after two courses of therapy. For newly diagnosed patients, the durations of absolute neutrophil counts (ANC) < 0.5 x 10(9)/L were 5 days and 10 days in rhG-CSF group and control group respectively (P < 0.05). The incidences of infection of these two groups were 40% and 60% respectively (P < 0.05). As for patients who received intensive therapy, the durations of ANC < 0.5 x 10(9)/L were 5 days and 8 days in rhG-CSF group and control group, respectively (P < 0.05), and the incidences of infection were 25% and 44.4% respectively (P < 0.05). CONCLUSIONS: The application of rhG-CSF in children with AML after chemotherapy may hasten the hematopoietic recovery. The duration of neutropenia was shortened by 3-4 days, and the incidence of fatal infection was reduced. rhG-CSF does not stimulate AML growth in vivo.

Antineoplastic Agents↗

[Diagnosis and treatment of pulmonary hamartoma].

OBJECTIVE: To summarize the characteristics of pathogenesis, image, treatment and prognosis of pulmonary hamartoma. METHODS: 39 cases with hamartoma confirmed by pathology received surgical operation during 1971-1998 period. Their clinical data including symptoms, disease courses, image characteristics and surgery types were investigated. The prognostic results of 31 cases were collected by a follow-up study. RESULTS: The average age of morbidity was 44 years old and the mean disease course of hamartoma was 13 months. 21 cases without symptoms were determinated by chance and 18 cases with some extent of cough, expectoration, fever, chest distress, and short of breath. The hamartomas of 21 cases were in right lung, 18 in the left. Image characteristics presented as popcom-shaped tumor with clear border and without satellite focus. 31 cases underwent enucleation, 4 wedge-shaped resection, 3 lobectomy and 1 pneumonectomy. No recurrence or malignant change was demonstrated by follow-up study. CONCLUSIONS: Pulmonary hamartoma is a kind of neoplasms with satisfactory prognosis. Intraluminal hamartoma should be differentiated from hilar lung cancer. Localized resection should be performed for the treatment of the disease.

Adolescent↗

[Effects of short veno-venous hemofiltration on severe acute pancreatitis].

OBJECTIVE: To estimate the effects of therapy of short veno-venous hemofiltration (SVVH, 4hr) on severe acute pancreatitis (SAP). METHODS: Twenty patients were divided randomly into hemofiltration group (HF, 10 pts) and non-hemofiltration group (NHF, 10 pts). The local, systemic manifestations were compared between two groups; and the concentration of serum pro-inflammatory cytokines (TNFalpha, IL-1beta, IL-6, IL-8, sIL-2R) and anti-inflammatory cytokines (IL-2, IL-10) were determined at different observation points. RESULTS: In the HF and NHF group, the duration for disappearance of abdominal pain and tenderness, and amelioration for abdominal distension was (8.5 +/- 6.3) d vs. (90.0 +/- 49.0) d and (16.9 +/- 5.2) d vs. (63.4 +/- 36.0) d. CT scores at the 14th day and APACHE II scores at the 10th day was (5.6 +/- 1.3) vs. (7.7 +/- 0.8) and (4.5 +/- 1.5) vs. (7.4 +/- 2.1). The average hospital stay and cost of therapy were (33.0 +/- 20.7) d vs. (56.4 +/- 16.0) d and (53,200 +/- 16,000) vs. (89,100 +/- 25,000) RMB (P < 0.05). The concentration of 5 serum pro-inflammatory cytokines at each observation points after hemofiltration was decreased significantly (P < 0.05) in the HF group. But the concentration of the two serum anti-inflammatory cytokines was increased significantly (P < 0.05) as compared with that of the NHF group. CONCLUSIONS: Through SVVH, the imbalance of pro-inflammatory and anti-inflammatory cytokines has been corrected at early stage; thus, the severity of the disease is ameliorated significantly and the efficacy of therapy is increased.

Female↗

[Low-tensioned and "b"-like ileocystoplasty].

OBJECTIVE: To perfectly solve the urine-kept and urinary problems of the vesical cancer patients after vesicoectomy. METHODS: We performed low-tensioned and "b"-like ileocystoplasty in 16 patients. RESULTS: After the operation, new vesica urinaria worked well in keeping and emptying urine-urine through the urethra. Residual urine test, chemical test and urography showed that there were no disturbance of body water and electrolyte metetabolism, no reterograde urine in the ureter, no uracratia, and no damage to renal function. CONCLUSIONS: Ileocystoplasty can be used clinically.

Aged↗

[Effect of anti-vascular endothelial growth factor antibody on the progression of human gastric cancer orthotopic xenotransplanted into nude mice].

OBJECTIVE: To study the inhibiting effect of anti-vascular endothelial growth factor (VEGF) on the growth and metastasis of gastric cancer. METHODS: The anti-tumor and anti-metastasis effect of anti-VEGF antibody, mitomycin C (MMC) were investigated by means of an orthotopic xenotransplanted model of human gastric cancer SGC-7901 in nude mice which had been randomly divided into 4 groups: control group receiving PBS, group receiving 50 microg/mice anti-VEGF antibody, group receiving 2 mg/kg MMC, and group receiving 50 microg/mice anti-VEGF antibody combined with 2 mg/kg MMC. Anti-VEGF antibody was given i.p. twice a week and MMC was administered i.p. once a week for 8 weeks from day 7 after transplantation. All animals were sacrificed at the end of 10 weeks. The tumor was weighted and the intra-tumoral microvessel density (MVD) was recorded, and the liver was histologically examined in order to discover micrometastasis. RESULTS: Both anti-VEGF antibody and MMC showed a significant inhibitory effect on the growth of primary tumors; in the combination treatment group the inhibitory effect was more significant than single agent. Liver metastasis developed in 19 of 28 mice (67.9%) of the controls and in 6 of 11 mice (54.5%) receiving MMC. In contrast, liver metastasis occurred in 2 of 10 mice (20%) receiving anti-VEGF antibody and none receiving combination treatment. In addition, the MVD was less significant in the anti-VEGF antibody group and combination treatment group than other groups. CONCLUSIONS: Anti-VEGF may provide a new approach to the treatment of gastric cancer by inhibiting tumor angiogenesis, and combination of anti-VEGF antibody with MMC could be more effective.

Animals↗

[Fluorescence enhancement of the dysprosium-sulfosalicylic acid system by lanthanum and its application].

A fluorescence enhancement produced by adding lanthanum(III) to dysprosium-sulfosalicylic acid system was observed. La3+ enhanced the fluorescence intensity by two orders of magnitude compared with the system without La3+. The system was used for the determination of trace amount of dysprosium in the range of 3.0 x 10(-7)-1.0 x 10(-5) mol/dm3, and the detection limit was 8.0 x 10(-8) mol/dm3. The procedure was applied to the determination of dysprosium in synthetic rare earth samples and standard rare earth samples with satisfactory results.

English Abstract↗

Identification of the heme-modified peptides from cumene hydroperoxide-inactivated cytochrome P450 3A4.

Cumene hydroperoxide-mediated (CuOOH-mediated) inactivation of cytochromes P450 (CYPs) results in destruction of their prosthetic heme to reactive fragments that irreversibly bind to the protein. We have attempted to characterize this process structurally, using purified, 14C-heme labeled, recombinant human liver P450 3A4 as the target of CuOOH-mediated inactivation, and a battery of protein characterization approaches [chemical (CNBr) and proteolytic (lysylendopeptidase-C) digestion, HPLC-peptide mapping, microEdman sequencing, and mass spectrometric analyses]. The heme-peptide adducts isolated after CNBr/lysylendopeptidase-C digestion of the CuOOH-inactivated P450 3A4 pertain to two distinct P450 3A4 active site domains. One of the peptides isolated corresponds to the proximal helix L/Cys-region peptide 429-450 domain and the others to the K-region (peptide 359-386 domain). Although the precise residue(s) targeted remain to be identified, we have narrowed down the region of attack to within a 17 amino acid peptide (429-445) stretch of the 55-amino acid proximal helix L/Cys domain. Furthermore, although the exact structures of the heme-modifying fragments and the nature of the adduction remain to be established conclusively, the incremental masses of approximately 302 and 314 Da detected by electrospray mass spectrometric analyses of the heme-modified peptides are consistent with a dipyrrolic heme fragment comprised of either pyrrole ring A-D or B-C, a known soluble product of peroxidative heme degradation, as a modifying species.

Amino Acid Sequence↗

Force and velocity measured for single molecules of RNA polymerase.

RNA polymerase (RNAP) moves along DNA while carrying out transcription, acting as a molecular motor. Transcriptional velocities for single molecules of Escherichia coli RNAP were measured as progressively larger forces were applied by a feedback-controlled optical trap. The shapes of RNAP force-velocity curves are distinct from those of the motor enzymes myosin or kinesin, and indicate that biochemical steps limiting transcription rates at low loads do not generate movement. Modeling the data suggests that high loads may halt RNAP by promoting a structural change which moves all or part of the enzyme backwards through a comparatively large distance, corresponding to 5 to 10 base pairs. This contrasts with previous models that assumed force acts directly upon a single-base translocation step.

DNA, Bacterial↗

Structure-function relationships of human liver cytochromes P450 3A: aflatoxin B1 metabolism as a probe.

Cytochromes P450 3A4 and 3A5, the dominant drug-metabolizing enzymes in the human liver, share >85% primary amino acid sequence identity yet exhibit different regioselectivity toward aflatoxin B1 (AFB1) biotransformation [Gillam et al., (1995) Arch. Biochem. Biophys. 317, 374-384]. P450 3A4 apparently prefers AFB1 3alpha-hydroxylation, which results in detoxification and subsequent elimination of the hepatotoxin, over AFB1 exo-8,9-oxidation. In contrast, P450 3A5 is incapable of appreciable AFB1 3alpha-hydroxylation and converts it predominantly to the exo-8,9-oxide which is genotoxic. To elucidate the structural features that govern the regioselectivity of the human liver 3A enzymes in AFB1 metabolism and bioactivation, a combination of approaches including sequence alignment, homology modeling, and site-directed mutagenesis was employed. Specifically, the switch in AFB1 regioselectivity was examined after individual substitution of the divergent amino acids in each of the six putative substrate recognition sites (SRSs) of P450 3A4 with the corresponding amino acid of P450 3A5. Of the P450 3A4 mutants examined, P107S, F108L, N206S, L210F, V376T, S478D, and L479T mutations resulted in a significant switch of P450 3A4 regioselectivity toward that of P450 3A5. The results confirmed the importance of some of these residues in substrate contact in the active site, with residue N206 (SRS-2) being critical for AFB1 detoxification via 3alpha-hydroxylation. Moreover, the P450 3A4 mutant N206S most closely mimicked P450 3A5, not only in its regioselectivity of AFB1 metabolism but also in its overall functional capacity. Furthermore, the other SRS-2 mutant, L210F, also resembled P450 3A5 in its overall AFB1 metabolism and regioselectivity. These findings reveal that a single P450 3A5 SRS domain (SRS-2) is capable of conferring the P450 3A5 phenotype on P450 3A4. In addition, some of these P450 3A4 mutations that affected AFB1 regioselectivity had little influence on testosterone 6beta-hydroxylation, thereby confirming that each substrate-P450 active site fit is indeed unique.

Aflatoxin B1↗

Design of a novel P450: a functional bacterial-human cytochrome P450 chimera.

We report the construction of a functional chimera from approximately 50% bacterial (cytosolic) cytochrome P450cam and 50% mammalian (membrane-bound) cytochrome P450 2C9. The chimeric protein shows a reduced CO-difference spectrum absorption at 446 nm, and circular dichroism spectra indicate that the protein is globular. The protein is soluble and catalyzes the oxidation of 4-chlorotoluene using molecular oxygen and reducing equivalents from bacterial putidaredoxin and putidaredoxin reductase. This chimera provides a novel method for addressing structure-function issues and may prove useful in the design of oxidants for benign and stereospecific synthesis, as well as catalysts for bioremediation of polluted areas. Furthermore, these results provide the first evidence that bacterial P450 enzymes and mammalian P450 enzymes are likely to share a common tertiary structure.

Bacterial Proteins↗

The yeast spindle pole body component Spc72p interacts with Stu2p and is required for proper microtubule assembly.

We have previously shown that Stu2p is a microtubule-binding protein and a component of the Saccharomyces cerevisiae spindle pole body (SPB). Here we report the identification of Spc72p, a protein that interacts with Stu2p. Stu2p and Spc72p associate in the two-hybrid system and can be coimmunoprecipitated from yeast extracts. Stu2p and Spc72p also interact with themselves, suggesting the possibility of a multimeric Stu2p-Spc72p complex. Spc72p is an essential component of the SPB and is able to associate with a preexisting SPB, indicating that there is a dynamic exchange between soluble and SPB forms of Spc72p. Unlike Stu2p, Spc72p does not bind microtubules in vitro, and was not observed to localize along microtubules in vivo. A temperature-sensitive spc72 mutation causes defects in SPB morphology. In addition, most spc72 mutant cells lack cytoplasmic microtubules; the few cytoplasmic microtubules that are observed are excessively long, and some of these are unattached to the SPB. spc72 cells are able to duplicate and separate their SPBs to form a bipolar spindle, but spindle elongation and chromosome segregation rarely occur. The chromosome segregation block does not arrest the cell cycle; instead, spc72 cells undergo cytokinesis, producing aploid cells and polyploid cells that contain multiple SPBs.

Cell Nucleus↗

Phenothiazine inhibitors of trypanothione reductase as potential antitrypanosomal and antileishmanial drugs.

Given the role of trypanothione in the redox defenses of pathogenic trypanosomal and leishmanial parasites, in contrast to glutathione for their mammalian hosts, selective inhibitors of trypanothione reductase are potential drug leads against trypanosomiasis and leishmaniasis. In the present study, the rational drug design approach was used to discover tricyclic neuroleptic molecular frameworks as lead structures for the development of inhibitors, selective for trypanothione reductase over host glutathione reductase. From a homology-modeled structure for trypanothione reductase, replaced in the later stages of the study by the X-ray coordinates for the enzyme from Crithidia fasciculata, a series of inhibitors based on phenothiazine was designed. These were shown to be reversible inhibitors of trypanothione reductase from Trypanosoma cruzi, linearly competitive with trypanothione as substrate and noncompetitive with NADPH, consistent with ping-pong bi bi kinetics. Analogues, synthesized to define structure-activity relationships for the active site, included N-acylpromazines, 2-substituted phenothiazines, and trisubstituted promazines. Analysis of Ki and I50 data, on the basis of calculated log P and molar refractivity values, provided evidence of a specially favored fit of small 2-substituents (especially 2-chloro and 2-trifluoromethyl), with a remote hydrophobic patch on the enzyme accessible for larger, hydrophobic 2-substituents. There was also evidence of an additional hydrophobic enzymic region available to suitable N-substituents of the promazine nucleus. Ki data also indicated that the phenothiazine nucleus can adopt more than one inhibitory orientation in its binding site. Selected compounds were tested for in vitro activity against Trypanosoma brucei, T. cruzi, and Leishmania donovani, with selective activities in the micromolar range being determined for a number of them.

Animals↗