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Biomedical subjects

W Hong

Publications and source records attributed to W Hong.

At least 127 records · Page 7Linked to original sources
PubMed1995

Segregation of ERGIC53 and the mammalian KDEL receptor upon exit from the 15 degrees C compartment.

Protein trafficking along the exocytotic pathway occurs by vesicular transport between successive membranous compartments. Transport from the endoplasmic reticulum (ER) to the Golgi apparatus has been proposed to be bridged by a morphologically defined ER-Golgi intermediate compartment (ERGIC). Using the subcellular dynamics of two markers for the ERGIC, the 53 kDa protein ERGIC53 and the mammalian KDEL receptor (KDEL-R), we have investigated the biochemical and physiological characteristics of ER-Golgi anterograde and retrograde transport. The KDEL-R at steady state is mainly confined to the perinuclear Golgi region while the ERGIC53 has a more elaborate distribution, including the ER. Both proteins can be colocalized to spotty structures distributed throughout the cytoplasm by incubating the cells at 15 degrees C. Upon returning the cells to 37 degrees C, the direction of transport for the two proteins diverged. KDEL-R was seen to emanate into tubular structures which eventually culminated in a focused, perinuclear staining. These dynamic changes are consistent with the anterograde transport process from the ER to the Golgi apparatus. ERGIC53, on the other hand, was distributed into an extended reticular network as well as the nuclear envelope, a staining pattern characteristic of the ER. With time, ERGIC53 was seen to return to the spotty structures again. The ER retrieval of ERGIC53 is consistent with the fact that the protein contains a dilysine motif which may function as an ER retrieval signal. The movement of ERGIC53 into the ER is not affected by microtubule disrupting agents, which inhibit the movement of KDEL-R to the Golgi. Both the processes are, however, sensitive to the alkylating agent N-ethylmaleimide. When reconstituted in vitro using digitonin permeabilized cells, the movement of ERGIC53 into the ER has a requirement for metabolic energy, is partially inhibited by the nonhydrolyzable guanine nucleotide analog GTP gamma S but could not be made to be cytosol dependent. These results documented the convergence of anterograde transport and retrograde transport at the 15 degrees C compartment and implied the existence of a segregation or a sorting process that would result in the segregation of proteins with different targeting signals in the structure.

Animals↗
PubMed1994

Overexpression of E2F-1 in rat embryo fibroblasts leads to neoplastic transformation.

The transcription factor E2F has been implicated in controlling the activation of multiple genes associated with cell proliferation. E2F-1, which is a component of E2F, can promote oncogenesis when transfected into REF cells. The transformation caused by E2F-1 correlates with constitutive overexpression of the transgene, increased transcription of E2F-dependent genes and the enhancement of two E2F DNA binding complexes containing the retinoblastoma susceptibility gene product (Rb) and E2F-1. The oncogenic potential of E2F-1 is dependent on functional DNA binding and transactivation domains but does not require the ability to interact directly with Rb. These findings provide the first direct evidence that sustained unregulated expression of E2F-1 can lead to the loss of cell proliferation control and that E2F-1 is a key component in cell cycle control.

Animals↗
PubMed1994

Golgi retardation in Madin-Darby canine kidney and Chinese hamster ovary cells of a transmembrane chimera of two surface proteins.

Dipeptidyl peptidase IV (DDD) is a type II plasma membrane protein. Replacement of its transmembrane domain with that of another surface protein, aminopeptidase N, resulted in accumulation in the Golgi apparatus of Madin-Darby canine kidney cells and a delayed Golgi to surface transport in Chinese hamster ovary (CHO) cells. The compartment of retardation was identified as post medial-Golgi, most likely to be the trans-Golgi/trans Golgi network (TGN). Compared to native DDD, the rate of endoplasmic reticulum to Golgi transport for the chimera was largely unchanged in both cell types. On the other hand, Golgi to surface transport was delayed by more than 2 h in CHO cells and essentially undetectable up to 22 h of chase in Madin-Darby canine kidney cells. The decrease in the rate of Golgi to surface transport in CHO cells resulted in a significant accumulation of the fusion protein in the trans-Golgi/TGN. This phenomena is very unlikely to be due to any drastic conformational changes, as neither the enzyme activity nor the dimerization of the constructed molecule was affected. The findings of this study indicate that the transmembrane domain, in the context of its flanking sequences, is important for efficient Golgi to cell surface transport.

Amino Acid Sequence↗
PubMed1994

Localization of the Lys, Asp, Glu, Leu tetrapeptide receptor to the Golgi complex and the intermediate compartment in mammalian cells.

The carboxyl-terminal Lys-Asp-Glu-Leu (KDEL), or a closely-related sequence, is important for ER localization of both lumenal as well as type II membrane proteins. This sequence functions as a retrieval signal at post-ER compartment(s), but the exact compartment(s) where the retrieval occurs remains unresolved. With an affinity-purified antibody against the carboxyl-terminal sequence of the mammalian KDEL receptor, we have investigated its subcellular localization using immunogold labeling on thawed cryosections of different tissues, such as mouse spermatids and rat pancreas, as well as HeLa, Vero, NRK, and mouse L cells. We show that rab1 is an excellent marker of the intermediate compartment, and we use this marker, as well as budding profiles of the mouse hepatitis virus (MHV) in cells infected with this virus, to identify this compartment. Our results demonstrate that the KDEL receptor is concentrated in the intermediate compartment, as well as in the Golgi stack. Lower but significant labeling was detected in the rough ER. In general, only small amounts of the receptor were detected on the trans side of the Golgi stack, including the trans-Golgi network (TGN) of normal cells and tissues. However, some stress conditions, such as infection with vaccinia virus or vesicular stomatitis virus, as well as 20 degrees C or 43 degrees C treatment, resulted in a significant shift of the distribution towards the trans-TGN side of the Golgi stack. This shift could be quantified in HeLa cells stably expressing a TGN marker. No significant labeling was detected in structures distal to the TGN under all conditions tested. After GTP gamma S treatment of permeabilized cells, the receptor was detected in the beta-COP-containing buds/vesicles that accumulate after this treatment, suggesting that these vesicles may transport the receptor between compartments. We propose that retrieval of KDEL-containing proteins occurs at multiple post-ER compartments up to the TGN along the exocytotic pathway, and that within this pathway, the amounts of the receptor in different compartments varies according to physiological conditions.

Animals↗
PubMed1994

Childhood-onset schizophrenia: an NIMH study in progress.

An ongoing study of the phenomenology, genetics, neuropsychology, physiology (eye tracking, autonomic responsivity), neuroimaging, biochemistry, and pharmacology of childhood-onset schizophrenia is described, and pilot data are presented for the first 22 subjects. Differentiation from autism "spectrum" disorders and other poorly defined, severe neurodevelopmental disorders is needed. Eye tracking and autonomic results are similar to patterns seen in later-onset schizophrenia and possibly more striking. Magnetic resonance imaging showed larger left frontal ventricular horn area for the schizophrenia subjects, larger left caudate, and lack of normal caudate asymmetry. Fluorodeoxyglucose positron emission tomography during an auditory continuous performance task revealed decreased right parietal/occipital glucose metabolic rate in the schizophrenia subjects, which may be secondary to poor attentional performance, and increased glucose metabolic rate in three left frontal regions, a left parietal region, and the right putamen. Clozapine has been effective and well tolerated in an open trial with 12 adolescents who responded poorly to typical neuroleptics; 16 subjects have been enrolled in a double-blind comparison of haloperidol and clozapine. Longitudinal study of this narrowly defined and possibly more homogeneous group of very early-onset schizophrenia subjects will be relevant to current neurodevelopmental theories addressing the role of puberty, progression of pathology, and continuity or discontinuity with later-onset schizophrenia.

Adolescent↗
PubMed1994

[Study on pregnancy & embryology in Tibetan medicine].

The author has studied the materials on menstruation, gestational course, embryonic development and hygiene during pregnancy recorded in ancient Tibetan medical literatures, and compared them with that in Han medicine, generally known as TCM. The article points out that Tibetan and Han medicine share some common views in pregnancy and embryology, but, in the aspects of observation and knowledge in menstruation and embryology, Tibetan medicine seems more unique. Tibetan medicine not only recorded foetal shape week by week for 38 weeks, but also presented them in color pictures to illustrate the "fish-tortois-pig" stages in its course of embryonic development which is absent in Han medicine. Tibetan medicine is very creative in its knowledge on pregnant physiology and embryology.

China↗
PubMed1994

Cytosolic factors block antibody binding to the C-terminal cytoplasmic tail of the KDEL receptor.

The mammalian KDEL receptor is an extremely hydrophobic membrane protein. One of the longest stretches of hydrophilic sequence resides at the C-terminus. Various antibodies against a synthetic peptide corresponding to this region confirmed that the C-terminus is exposed to the cytoplasm. It was observed that antibody binding to the C-terminus of the KDEL receptor was diminished during immunofluorescence microscopy procedures which involved fixation prior to permeabilization as compared to when cells were permeabilized before fixation. Binding of both polyclonal and monoclonal antibodies, as assessed by indirect immunofluorescence microscopy in digitonin permeabilized cells, was inhibited by preincubation with rat liver cytosol. This inhibition was not observed with antibody against another membrane protein (p28) with a cytoplasmically exposed epitope also residing in the Golgi/intermediate compartment. Rabbit reticulocyte lysate had a similar effect while Schizosaccharomyces pombe cytosol inhibited binding to a greater degree than Saccharomyces cerevisiae cytosol. This inhibition by cytosol was prevented by coincubation with the antibody and was dose-dependent on the cytosol. Inhibition did not occur on ice or at 15 degrees C, or when the cytosol was energy-depleted by apyrase treatment. Interestingly, pretreatment of permeabilized cells with N-ethylmaleimide or its addition into the incubation mixture abolished inhibition. N-ethylmaleimide-treated cytosol, however, remained inhibitory. The findings suggest the existence of cytosolic factor (s) which interacts specifically with the cytoplasmic C-terminus of the KDEL receptor, which are likely to be components of the KDEL protein retrieval machinery.

Animals↗
PubMed1994

[Trial vaginal delivery for women with previous cesarean section].

A retrospective analysis was made on 85 cases of trial labor after previous cesarean section (CS) and compared with 100 primiparous vaginal deliveries (as control) during the last 10 years. Oxytocin infusion has been used in 61 trial cases to induce and/or to accelerate labor, and valium, procaine and atropine administered during the labor process in 23 of them. 65 of the 85 cases (76.5%) successfully delivered through the vagina (VD group), while 20 cases (23.5%) had repeat CS following failure in trial labor (CS group), and 3 women had reatened uterine rupture. There was 1 neonatal death in the VD trial labor group. No obvious difference in the Apgar scores of newborns was found between the CS group and the controls, and the duration of the labor process was much shorter in the VD trial group than that in the primiparae (7.51 +/- 2.44 hrs vs. 9.10 +/- 3.75 hrs) (P < 0.01). This study indicated that following a previous uneventful transverse lower segment cesarean section, trial labor should be encouraged under supervision.

Adult↗
PubMed1994

[Influences of lipid peroxidative injury to endothelial cells on adherence and migration of monocyte].

The influences of lipid peroxidative injury to endothelial cells (EC) on adherence to EC and migration into subendothelial space of human monocytes were studied by using a modified vascular endothelial model in vitro. Lipid peroxidative injury to cultured human umbilical vein EC was initiated by treating the EC with diamide. The results showed that injury to EC could enhance adherence to EC and migration into subendothelial space of monocytes. Monocytes mainly adhered to the intercellular space and surface of injured EC. In the area of EC denudation, the number of monocytes which migrate into the subendothelial space was more than that in the area without denudation. These results suggested that enhanced adherence to EC and migration into subendothelial space of monocytes caused by lipid peroxidative injury to EC may play an important role in atherogenesis.

Cell Adhesion↗
PubMed1993

The SXYQRL sequence in the cytoplasmic domain of TGN38 plays a major role in trans-Golgi network localization.

The trans-Golgi network (TGN) plays a central role in protein sorting/targeting. TGN38 is an integral membrane protein confined mainly to the TGN. When the cytoplasmic 32-residue sequence of TGN38 was fused to the ecto- and transmembrane domains of glycophorin A (a surface protein), the resulting chimeric protein was localized to the TGN. Detailed mutagenesis of the 32-residue sequence revealed that the Ser, Tyr, and Leu residues at positions 23, 25, and 28, respectively, are essential for TGN localization. Further studies demonstrate that the sequence SXYQRL can by itself confer significant TGN localization.

Amino Acid Sequence↗
PubMed1993

Protein trafficking along the exocytotic pathway.

Proteins of the exocytotic (secretory) pathway are initially targeted to the endoplasmic reticulum (ER) and then translocated across and/or inserted into the membrane of the ER. During their anterograde transport with the bulk of the membrane flow along the exocytotic pathway, some proteins are selectively retained in various intracellular compartments, while others are sorted to different branches of the pathway. The signals or structural motifs that are involved in these selective targeting processes are being revealed and investigations into the mechanistic nature of these processes are actively underway.

Amino Acid Sequence↗
PubMed1993

Molecular cloning, characterization, subcellular localization and dynamics of p23, the mammalian KDEL receptor.

We have isolated a cDNA clone (mERD2) for the mammalian (bovine) homologue of the yeast ERD2 gene, which codes for the yeast HDEL receptor. The deduced amino acid sequence bears extensive homology to its yeast counterpart and is almost identical to a previously described human sequence. The sequence predicts a very hydrophobic protein with multiple membrane spanning domains, as confirmed by analysis of the in vitro translation product. The protein encoded by mERD2 (p23) has widespread occurrence, being present in all the cell types examined. p23 was localized to the cis-side of the Golgi apparatus and to a spotty intermediate compartment which mediates ER to Golgi transport. A majority of the intracellular staining could be accumulated in the intermediate compartment by a low temperature (15 degrees C) or brefeldin A. During recovery from these treatments, the spotty intermediate compartment staining of p23 was shifted to the perinuclear staining of the Golgi apparatus and tubular structures marked by p23 were observed. These tubular structures may serve to mediate transport between the intermediate compartment and the Golgi apparatus.

Amino Acid Sequence↗
PubMed1993

Hypnotic and anesthetic interactions between ketamine and midazolam in female patients.

BACKGROUND: Midazolam, if used with ketamine for induction and maintenance of anesthesia, may attenuate hyperdynamic circulatory effects and prevent undesirable emergenic reactions. The nature of the interaction between midazolam and ketamine used for anesthesia induction was studied in female patients. METHODS: Quantal dose-response curves were determined in 170 female patients for the drugs, individually and in combination. Two endpoints were assessed, loss of response to verbal command (hypnosis) and loss of response to a 5-s transcutaneous tetanus (anesthesia). At the hypnotic endpoint, interactions were analyzed by fitting the data to a mathematical model in which the response was analyzed in terms of the doses of the two drugs, and an additional term was included to describe nonadditive interactions. At the anesthetic endpoint, the decrease in ED50 of ketamine in the presence of midazolam was assessed because dose-related effects could not be demonstrated for midazolam alone. RESULTS: At the hypnotic endpoint, the ED50s were: 0.15 mg/kg midazolam (95% CIs 0.11-0.38 mg/kg), 0.37 mg/kg ketamine (95% CIs 0.08-0.44 mg/kg), and the combination of 0.086 mg/kg midazolam and 0.27 mg/kg ketamine (95% CIs 0.07/0.22-0.10/0.31 mg/kg), respectively. The hypnotic effects were found to be additive, and there was no evidence of an interaction. At the anesthetic endpoint, the ED50 of ketamine alone was 0.57 mg/kg (95% CIs 0.47-0.69) and the ED50 for ketamine in the presence of midazolam was also 0.57 mg/kg (95% CIs 0.48-0.79); 0.18 mg/kg midazolam was given at this point. Midazolam had no influence on the anesthetic dose of ketamine. CONCLUSIONS: When using the combination, doses employed should be adjusted according to the depth of central nervous system depression that is required.

Adolescent↗
PubMed1993

Transmembrane topology of the mammalian KDEL receptor.

The mammalian KDEL receptor is an integral membrane protein with seven hydrophobic regions. Fusion proteins comprising a 37-kDa N-glycosylation reporter fused downstream of amino-terminal fragments of the KDEL receptor with varying numbers of hydrophobic regions were synthesized in an in vitro translation system containing canine pancreatic microsomes. The luminal or cytosolic orientation of the reporter, and hence of the hydrophilic region to which it is fused, was inferred from the presence or absence of glycosylation, which occurs only in the lumen of the microsomes. The cytosolic orientation of the N and C termini was also confirmed immunocytochemically. Our results suggest that the KDEL receptor is inserted into the membrane with only six transmembrane domains and that both the amino and carboxy termini are located in the cytoplasm.

Amino Acid Sequence↗
PubMed1993

[Effect of lipid peroxidation injury to endothelial cells (EC) on adherence of monocytes].

Inducing lipid peroxidation injury to cultured EC from human umbilical vein was achieved by treating EC with diamide. When the concentration of diamide reached 0.1 x 10(-4) mol/l, intracellular accumulation of lipid peroxide and EC damage were induced and then increased adherence of monocyte to EC was observed. Monocytes appeared to prefer to adhere onto the surfaces and intercellular spaces of the injured EC. These results suggest that the increased adherence of monocytes to EC injured by lipid peroxidation may play an important role in atherogenesis.

Arteriosclerosis↗
PubMed1992

Biochemical fractionation and characterization of proteins from Golgi-enriched membranes.

Fractions enriched in Golgi membranes were prepared from rat liver by sucrose gradient ultracentrifugation. These enriched membranes were further subfractionated on the basis of their solubilities in EGTA, 150 mM sodium carbonate, pH 11.5, sodium deoxycholate, Triton X-100, or sodium dodecyl sulfate. This led to isolation of peripheral, luminal, and integral membrane proteins of the Golgi-enriched membranes. Luminal and membrane proteins were further purified by wheat germ agglutinin and concanavalin A lectin affinity chromatographies. Some proteins from these lectin columns were resolved by preparative gel electrophoresis and microsequenced. Subsequently, antibodies were produced for two proteins by immunization of either mice or rabbits. Immunofluorescence microscopy suggests that these proteins are confined to Golgi apparatus-like structures. The protocol described is well suited for the study of organelle structure and function.

Amino Acid Sequence↗
PubMed1992

The transmembrane domain of N-glucosaminyltransferase I contains a Golgi retention signal.

The enzyme N-acetylglucosaminyltransferase I (NT, EC 2.4.1.101) is a resident type II transmembrane protein of the Golgi apparatus. To delineate the portion of its primary sequence that is responsible for the Golgi retention of this protein, we constructed chimeras containing different N-terminal portions of NT joined to a reporter sequence, the ectodomain of a type II surface membrane protein. These chimeric proteins were found to be retained in the Golgi apparatus as assessed by cell surface biotinylation and immunofluorescence. We found that the transmembrane domain of NT is sufficient to confer Golgi retention of the fusion proteins and propose that it contains the Golgi retention signal of the parent molecule.

Animals↗