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Biomedical subjects

T Guan

Publications and source records attributed to T Guan.

At least 19 recordsLinked to original sources

RNA export mediated by tap involves NXT1-dependent interactions with the nuclear pore complex.

Nuclear export of ribonucleoprotein complexes requires cis-acting signals and recognition by receptors that mediate translocation through the nuclear pore complex. Translocation is likely to involve a series of physical interactions between the ribonucleoprotein complex and nucleoporins within the nuclear pore complex. Here, we have characterized the function of NXT1 in the context of the Tap-dependent RNA export pathway. Tap has been implicated in the nuclear export of RNA transcripts derived from Mason-Pfizer monkey virus that contain the constitutive transport element. We demonstrate that NXT1 stimulates binding of a Tap-RNA complex to nucleoporins in vitro, and we provide mutational analysis that shows these interactions are necessary for nuclear export of an intron-containing viral mRNA in vivo. Tap contains separate domains for binding to nucleoporins and NXT1, both of which are critical for its export function. RNA export is mediated by a heterodimer of Tap and NXT1, and the function of NXT1 on this pathway is to regulate the affinity of the Tap-RNA complex for nucleoporins within the nuclear pore complex. We propose that NXT1-dependent binding of the Tap-RNA complex to the nucleoporin p62, which we have reconstituted in vitro using recombinant proteins, represents a single step of the translocation reaction.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

Involvement of the lamin rod domain in heterotypic lamin interactions important for nuclear organization.

The nuclear lamina is a meshwork of intermediate-type filament proteins (lamins) that lines the inner nuclear membrane. The lamina is proposed to be an important determinant of nuclear structure, but there has been little direct testing of this idea. To investigate lamina functions, we have characterized a novel lamin B1 mutant lacking the middle approximately 4/5 of its alpha-helical rod domain. Though retaining only 10 heptads of the rod, this mutant assembles into intermediate filament-like structures in vitro. When expressed in cultured cells, it concentrates in patches at the nuclear envelope. Concurrently, endogenous lamins shift from a uniform to a patchy distribution and lose their complete colocalization, and nuclei become highly lobulated. In vitro binding studies suggest that the internal rod region is important for heterotypic associations of lamin B1, which in turn are required for proper organization of the lamina. Accompanying the changes in lamina structure induced by expression of the mutant, nuclear pore complexes and integral membrane proteins of the inner membrane cluster, principally at the patches of endogenous lamins. Considered together, these data indicate that lamins play a major role in organizing other proteins in the nuclear envelope and in determining nuclear shape.

Animals↗

[The Raman spectra study on crystal-octadecyl hydrogen succinate crystal].

Raman spectra of crystal-octadecyl hydrogen succinate (OHS), whose geometric disposition of the experiment has y(zx)y, y(zz)y, and y(xz)y,y(xx)y respectively, have been recorded and analyzed with the purpose of understanding the structure of OHS crystal. It shows that some of the Raman-active modes of alkyl chain appear polarization. The analysis for crystal structure indicates that the molecular of OHS arrange in layers along [100] and [001] by the mode of dimers, and the molecular layers of chain pile up along [010]. The other groups of OHS corresponding Raman vibration peaks have been analyzed as well.

English Abstract↗

Nuclear import of adenovirus DNA in vitro involves the nuclear protein import pathway and hsc70.

Adenovirus, a respiratory virus with a double-stranded DNA genome, replicates in the nuclei of mammalian cells. We have developed a cytosol-dependent in vitro assay utilizing adenovirus nucleocapsids to examine the requirements for adenovirus docking to the nuclear pore complex and for DNA import into the nucleus. Our assay reveals that adenovirus DNA import is blocked by a competitive excess of classical protein nuclear localization sequences and other inhibitors of nuclear protein import and indicates that this process is dependent on hsc70. Previous work revealed that the hexon (coat) protein of adenovirus is the only major protein on the surface of the adenovirus nucleocapsid that docks at the nuclear pore complex. This, together with our finding that in vitro nuclear import of hexon is inhibited by an excess of classical nuclear localization sequences, suggests a role for the hexon protein in adenovirus DNA import. However, recombinant transport factors that are sufficient for hexon import in permeabilized cells do not support DNA import, indicating that there are other as yet unidentified factors required for this process.

Adenoviridae↗

Regulation of adenovirus membrane penetration by the cytoplasmic tail of integrin beta5.

Adenovirus (Ad) cell entry involves sequential interactions with host cell receptors that mediate attachment (CAR), internalization (alphavbeta3 and alphavbeta5), and penetration (alphavbeta5) of the endosomal membrane. These events allow the virus to deliver its genome to the nucleus. While integrins alphavbeta3 and alphavbeta5 both promote Ad internalization into cells, integrin alphavbeta5 selectively facilitates Ad-mediated membrane permeabilization and endosome rupture. In the experiments reported herein, we demonstrate that the intracellular domain of the integrin beta5 subunit specifically regulates Ad-mediated membrane permeabilization and gene delivery. CS-1 melanoma cells expressing a truncated integrin beta5 or a chimeric (beta5-beta3) cytoplasmic tail (CT) supported normal levels of Ad endocytosis but had reduced Ad-mediated gene delivery and membrane permeabilization relative to cells expressing a wild-type integrin beta5. Thin-section electron microscopy revealed that virion particles were capable of being endocytosed into cells expressing a truncated beta5CT, but they failed to escape cytoplasmic vesicles and translocate to the nucleus. Site-specific mutagenesis studies suggest that a C-terminal TVD motif in the beta5CT plays a major role in Ad membrane penetration.

Adenoviruses, Human↗

Nup50, a nucleoplasmically oriented nucleoporin with a role in nuclear protein export.

We present here a detailed analysis of a rat polypeptide termed Nup50 (formerly NPAP60) that was previously found to be associated with the nuclear pore complex (F. Fan et al., Genomics 40:444-453, 1997). We have found that Nup50 (and/or a related 70-kDa polypeptide) is present in numerous rat cells and tissues. By immunofluorescence microscopy, Nup50 was found to be highly concentrated at the nuclear envelope of rat liver nuclei, whereas in cultured NRK cells it also is abundant in intranuclear regions. On the basis of immunogold electron microscopy of both rat liver nuclear envelopes and NRK cells, we determined that Nup50 is specifically localized in the nucleoplasmic fibrils of the pore complex. Microinjection of anti-Nup50 antibodies into the nucleus of NRK cells resulted in strong inhibition of nuclear export of a protein containing a leucine-rich nuclear export sequence, whereas nuclear import of a protein containing a classical nuclear localization sequence was unaffected. Correspondingly, CRM1, the export receptor for leucine-rich export sequences, directly bound to a fragment of Nup50 in vitro, whereas several other import and export receptors did not significantly interact with this fragment. Taken together, our data indicate that Nup50 has a direct role in nuclear protein export and probably serves as a binding site on the nuclear side of the pore complex for export receptor-cargo complexes.

Animals↗

[The relationship between clinical types of postinfarction angina and stenosis of ischemia-related artery].

OBJECTIVE: To approach the pathogenesis of postinfarction angina and set up its treatment guidelines. METHODS: 67 patients with postinfarction angina undergoing coronary angiography during the hospitalization were studied. RESULTS: (1) According to the clinical classification of angina pectoris, spontaneous angina is the most common type (43.9%); mixed angina is second (25.7%), effort angina and variant angina are relatively low frequency (16.7% & 13.6%). (2) Stenosis >or= 90% in the ischemia-related artery accounted for the anginal symptoms constituted 100%, 82.8%, 72.7% and 44.4% of the cases with mixed angina, spontaneous angina, effort angina and variant angina respectively. (3) Postinfarction angina caused by non infarction-related artery was relatively less frequent (8.9%). CONCLUSIONS: Severe residual stenosis of infarction-related artery is the main pathologic factor on the pathogenesis of postinfarction angina, so early interventional therapy or bypass surgery should be taken.

Adult↗

[The Raman spectra study on crystal-octadecyl hydrogen maleate (OHM)].

Raman spectra of crystal-octadecyl hydrogen maleate (OHM), whose geometric disposition of the experiment has y (zx) y y(zz)y, and y(xz)y, y(xx)y respectively, have been recorded and analyzed with the purpose of understanding the structure of OHM crystal. It shows that some of the Raman-active modes of alkyl chain appear polarization. The analysis for crystal structure indicates that the molecular of OHM arrange in layers along [100] and [001] by the mode of dimers, and the molecular layers of chain pile up along [010]. The other groups of OHM corresponding Raman vibration peaks have been analysed as well.

English Abstract↗

A role for RanBP1 in the release of CRM1 from the nuclear pore complex in a terminal step of nuclear export.

We recently developed an assay in which nuclear export of the shuttling transcription factor NFAT (nuclear factor of activated T cells) can be reconstituted in permeabilized cells with the GTPase Ran and the nuclear export receptor CRM1. We have now used this assay to identify another export factor. After preincubation of permeabilized cells with a Ran mutant that cannot hydrolyze GTP (RanQ69L), cytosol supports NFAT export, but CRM1 and Ran alone do not. The RanQ69L preincubation leads to accumulation of CRM1 at the cytoplasmic periphery of the nuclear pore complex (NPC) in association with the p62 complex and Can/Nup214. RanGTP-dependent association of CRM1 with these nucleoporins was reconstituted in vitro. By biochemical fractionation and reconstitution, we showed that RanBP1 restores nuclear export after the RanQ69L preincubation. It also stimulates nuclear export in cells that have not been preincubated with RanQ69L. RanBP1 as well as Ran-binding domains of the cytoplasmic nucleoporin RanBP2 promote the release of CRM1 from the NPC. Taken together, our results indicate that RanGTP is important for the targeting of export complexes to the cytoplasmic side of the NPC and that RanBP1 and probably RanBP2 are involved in the dissociation of nuclear export complexes from the NPC in a terminal step of transport.

Binding Sites↗

[Measurement of boundary line of urinary bladder and peritoneum in abdominal wall and the application in performing cesarean section with urinary bladder reversal method].

OBJECTIVE: To provide the basic research data and clinical application in performing extraperitoneal cesarean section (CS) with urinary bladder reversal method. METHODS: The position and the surface projection of the vesico-peritoneal fold and the apex of the urinary bladder were measured in 107 cases of term pregnant women (37-42 weeks, aged 20-40). RESULTS: The distance from the bladder apex to the umbilicus and to the symphysis pubis were (14.1 +/- 3.4) cm and (6.8 +/- 1.4) cm respectively, while from the utero-vesical pouch to umbilicus and symphysis pubis were (15.8 +/- 3.9) cm and (4.9 +/- 1.5) cm respectively. During late pregnancy the projection of bladder apex was located at the point between the middle and the lower 1/3 of the line from umbilicus to symphysis pubis; while the utero-vesical pouch was located at the point between upper 3/4 and the lower 1/4 of the same line. The time interval from the start of operation to the delivery of baby was (14.56 +/- 6.3) min, and the total operation period was (45.5 +/- 9.3) min. The largest baby weighed 5,050 gm and there was no bladder injury. CONCLUSION: To define the position of vesical peritoneal fold would give benefit to the extraperitoneal CS with bladder reversal method which was performed under direct visualization and could avoid injury of bladder.

Adult↗

[Insulin receptor substrate-1 and glucose transporter gene polymorphisms in noninsulin-dependent diabetes mellitus].

OBJECTIVE: To evaluate the role of insulin receptor substrate-1 (IRS-1) and glucose transporter-1 (GLUT1) gene polymorphisms in Chinese patients with noninsulin-dependent diabetes mellitus (NIDDM) and diabetic neophropathy (DN). METHODS: Aminoacid polymorphism in codon 972 of IRS-1 gene and the polymorphic Xba I site of GLUT1 gene were analyzed by PCR-RFLP in 131 patients with NIDDM and 124 normal subjects. DN was defined as persistent albuminuria and/or impaired renal function, without known cause of renal diseases other than diabetes. Insulin sensitivity index (ISI) and body mass index (BMI) were also calculated. RESULTS: The distribution of IRS-1 gene polymorphism showed no difference between patients with NIDDM and normal controls. The frequencies of Xba I (+/-) genotype (59% vs. 33%, P < 0.01) and Xba I (-) allele (37% vs. 21%, P < 0.01) were significantly higher in NIDDM patients than in normal subjects. To further explore the linkage of GLUT1 gene polymorphism with DN, we examined the GLUT1 genotype of NIDDM patients with or without renal damage. The frequency of Xba I (+/-) genotype (75% vs. 44%, P < 0.01) and Xba I (-) allele (44% vs. 29%, P < 0.05) was significantly higher in NIDDM patients with diabetic nephropathy than either those without nephropathy or normal subjects. However, there were no significant differences of GLUT1 genotype and allele frequency in NIDDM patients without nephropathy and normal controls. The presence of Xba I (-) allele appeared to have a strong association with the development of diabetic nephropathy. The odds ratio was 1.915, and the 95% confidence interval was 1.044-3.514. The association of Xba I (-) allele of GLUT1 gene with NIDDM partly reflected their close association with DN. Although there was no correlation between the gene polymorphism of GLUT1 and BMI, patients carrying the Xba I (-) allele showed a lower ISI. CONCLUSION: No association was found between the gene polymorphism of IRS-1 and NIDDM. THe Xba I (-) allele of GLUT1 gene might be taken as a genetic marker of NIDDM with diabetic nephropathy and this genetic susceptibility appears to be associated with the insulin resistance in patients with NIDDM.

Adult↗

Lamin-binding fragment of LAP2 inhibits increase in nuclear volume during the cell cycle and progression into S phase.

Lamina-associated polypeptide 2 (LAP2) is an integral membrane protein of the inner nuclear membrane that binds to both lamin B and chromatin and has a putative role in nuclear envelope (NE) organization. We found that microinjection of a recombinant polypeptide comprising the nucleoplasmic domain of rat LAP2 (residues 1-398) into metaphase HeLa cells does not affect the reassembly of transport-competent nuclei containing NEs and lamina, but strongly inhibits nuclear volume increase. This effect appears to be specifically due to lamin binding, because it also is caused by microinjection of the minimal lamin-binding region of LAP2 (residues 298-373) but not by the chromatin-binding domain (residues 1-88). Injection of the lamin-binding region of rat LAP2 into early G1 phase HeLa cells also strongly affects nuclear growth; it almost completely prevents the threefold nuclear volume increase that normally occurs during the ensuing 10 h. Moreover, injection of the fragment during early G1 phase strongly inhibits entry of cells into S phase, whereas injection during S phase has no apparent effect on ongoing DNA replication. Since the lamin-binding fragment of LAP2 most likely acts by inhibiting dynamics of the nuclear lamina, our results suggest that a normal function of LAP2 involves regulation of nuclear lamina growth. These data also suggest that lamina dynamics are required for growth of the NE and for nuclear volume increase during the cell cycle, and that progression into S phase is dependent on the acquisition of a certain nuclear volume.

Animals↗

High levels of the GTPase Ran/TC4 relieve the requirement for nuclear protein transport factor 2.

The GTPase Ran/TC4 and the 14-kDa protein nuclear transport factor 2 (NTF2) are two of the cytosolic factors that mediate nuclear protein import in vertebrates. Previous biochemical studies have shown that NTF2 binds directly to the GDP-bound form of Ran/TC4 and to proteins of the nuclear pore complex that contain phenylalanine-glycine repeats. In the present study we have used molecular genetic approaches to study the Saccharomyces cerevisiae homologue of NTF2. The scNTF2 gene encodes a protein that is 44% identical to the human protein. We found that deletion of the scNTF2 gene is lethal and that repression of NTF2p expression by a regulatable promoter results in gross structural distortions of the nuclear envelope. In a screen for high copy number suppressors of a scNTF2 deletion, the only gene we isolated other than scNTF2 itself was GSP1, the S. cerevisiae homologue of Ran/TC4. Furthermore, we found that high levels of Ran/TC4 can relieve the requirement for NTF2 in a mammalian-permeabilized cell assay for nuclear protein import. These data suggest that certain of the nuclear protein import functions of NTF2 and Ran/TC4 are closely linked and that NTF2 may serve to modulate a transport step involving Ran/TC4.

Biological Transport↗

Integral membrane proteins of the nuclear envelope are dispersed throughout the endoplasmic reticulum during mitosis.

We have analyzed the fate of several integral membrane proteins of the nuclear envelope during mitosis in cultured mammalian cells to determine whether nuclear membrane proteins are present in a vesicle population distinct from bulk ER membranes after mitotic nuclear envelope disassembly or are dispersed throughout the ER. Using immunofluorescence staining and confocal microscopy, we compared the localization of two inner nuclear membrane proteins (laminaassociated polypeptides 1 and 2 [LAP1 and LAP2]) and a nuclear pore membrane protein (gp210) to the distribution of bulk ER membranes, which was determined with lipid dyes (DiOC6 and R6) and polyclonal antibodies. We found that at the resolution of this technique, the three nuclear envelope markers become completely dispersed throughout ER membranes during mitosis. In agreement with these results, we detected LAP1 in most membranes containing ER markers by immunogold electron microscopy of metaphase cells. Together, these findings indicate that nuclear membranes lose their identity as a subcompartment of the ER during mitosis. We found that nuclear lamins begin to reassemble around chromosomes at the end of mitosis at the same time as LAP1 and LAP2 and propose that reassembly of the nuclear envelope at the end of mitosis involves sorting of integral membrane proteins to chromosome surfaces by binding interactions with lamins and chromatin.

3T3 Cells↗

A small ubiquitin-related polypeptide involved in targeting RanGAP1 to nuclear pore complex protein RanBP2.

We have found that the mammalian Ran GTPase-activating protein RanGAP1 is highly concentrated at the cytoplasmic periphery of the nuclear pore complex (NPC), where it associates with the 358-kDa Ran-GTP-binding protein RanBP2. This interaction requires the ATP-dependent posttranslational conjugation of RanGAP1 with SUMO-1 (for small ubiquitin-related modifier), a novel protein of 101 amino acids that contains low but significant homology to ubiquitin. SUMO-1 appears to represent the prototype for a novel family of ubiquitin-related protein modifiers. Inhibition of nuclear protein import resulting from antibodies directed at NPC-associated RanGAP1 cannot be overcome by soluble cytosolic RanGAP1, indicating that GTP hydrolysis by Ran at RanBP2 is required for nuclear protein import.

3T3 Cells↗

RanGTP targets p97 to RanBP2, a filamentous protein localized at the cytoplasmic periphery of the nuclear pore complex.

RanBP2, a protein containing FG repeat motifs and four binding sites for the guanosine triphosphatase Ran, is localized at the cytoplasmic periphery of the nuclear pore complex (NPC) and is believed to play a critical role in nuclear protein import. We purified RanBP2 from rat liver nuclear envelopes and examined its structural and biochemical properties. Electron microscopy showed that RanBP2 forms a flexible filamentous molecule with a length of approximately 36 nm, suggesting that it comprises a major portion of the cytoplasmic fibrils implicated in initial binding of import substrates to the NPC. Using in vitro assays, we characterized the ability of RanBP2 to bind p97, a cytosolic factor implicated in the association of the nuclear localization signal receptor with the NPC. We found that RanGTP promotes the binding of p97 to RanBP2, whereas it inhibits the binding of p97 to other FG repeat nucleoporins. These data suggest that RanGTP acts to specifically target p97 to RanBP2, where p97 may support the binding of an nuclear localization signal receptor/substrate complex to RanBP2 in an early step of nuclear import.

Animals↗

[Angiotensin-converting enzyme gene polymorphism and the clinical pathological features and progression in lupus nephritis].

The renin-angiotensin system (RAS) is now well established as an important determinant in the progression of renal damage. The level of plasma angiotensin converting enzyme (ACE) is genetically determined and this gene effect is associated with insertion (I)/deletion (D) polymorphism in intron 16 of ACE gene. It has been reported that DD genotype of ACE gene is associated with a poor outcome in patients with IgA nephropathy and trend to develop renal damage in diabetic mellitus. In this study, the correlation between ACE gene I/D polymorphism and the clinical features of lupus nephritis (LN) was determined in 144 LN patients and 150 normal controls by PCR. 72 LN patients were followed up for more than two years. It was found that the DD genotype was significantly higher in LN patients than in normal controls (P < 0.01), while the II genotype was much less in LN (P < 0.01). There was no significant difference in DI genotype frequency between LN patients and normal control (P > 0.05). Patients with hematuria, type IV LN, activation of LN, crescentic formation and severe tubulo-interstitial lesions showed an excess of the DI genotype. However, the DD genotype was not associated with the clinical and pathological characteristics of LN. To assess whether ACE genotype influence the progression of LN, we compared the clinical and pathological findings in patients of different genotype subgroups. There was no significant difference in blood pressure, proteinuria, serum creatinine, active index of disease and crescentic formation at presentation among these groups. However, as patients were divided into two groups according to their rate of decline in renal function. We found that patients with progressive renal function damage had a higher frequency of DI genotype than those with stable renal function (P < 0.01). It is conclude that patients with LN the DI genotype was associated with the severity and the poor prognosis in patients with LN.

Adolescent↗

Molecular and functional characterization of the p62 complex, an assembly of nuclear pore complex glycoproteins.

Macromolecular trafficking across the nuclear envelope involves interactions between cytosolic transport factors and nuclear pore complex proteins. The p62 complex, an assembly of 62, 58, 54, and 45-kD O-linked glycoproteins-localized near the central gated channel of the nuclear pore complex, has been directly implicated in nuclear protein import. The cDNA cloning of rat p62 was reported previously. We have now carried out cDNA cloning of rat p58, p54, and p45. We found that p58 contains regions with FG (Phe, Gly) and PA (Pro, Ala) repeats at both its NH2 and COOH termini separated by a predicted alpha-helical coiled-coil region, while p54 has an NH2-terminal FG and PA repeat region and a COOH-terminal predicted coiled-coil region. p45 and p58 appear to be generated by alternative splicing, with p45 containing the NH2-terminal FG repeat region and the coiled-coil region of p58. Using immunogold electron microscopy, we found that p58/p45 and p54 are localized on both sides of the nuclear pore complex, like p62. Previous studies have shown that immobilized recombinant p62 can bind the cytosolic nuclear import factor NTF2 and thereby deplete transport activity from cytosol. We have now found that immobilized recombinant p58 and p54 also can deplete nuclear transport activity from cytosol, and that p62, p58, and p54 bind directly to the cytosolic nuclear import factors p97 and NTF2. At least in the case of p58, this involves FG repeat regions. Moreover, p58 can bind to a complex containing transport ligand, the nuclear localization sequence receptor (Srp1 alpha) and p97. These data support a model in which the p62 complex binds to a multicomponent particle consisting of transport ligand and cytosolic factors to achieve accumulation of ligand near the central gated channel of the nuclear pore complex.

Amino Acid Sequence↗