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W C Shen

Publications and source records attributed to W C Shen.

At least 55 records · Page 3Linked to original sources
PubMed1996

Nucleus-associated pools of Rna1p, the Saccharomyces cerevisiae Ran/TC4 GTPse activating protein involved in nucleus/cytosol transit.

Rna1p is the GTPase activating enzyme for Ran/TC4, a Ras-like GTPase necessary for nuclear/cytosolic exchange. Although most wild-type Rna1p is located in the cytosol, we found that the vast majority of the mutant Rna1-1p and, under appropriate physiological conditions, a small portion of the wild-type Rna1p cofractionate with yeast nuclei. Subnuclear fractionation studies show that most of the Rna1p is tightly associated with nuclear components, and that a portion of the active protein can be solubilized by treatments that fail to solubilize inactive Rna1-1p. To learn the precise nuclear locations of the Rna1 proteins, we studied their subcellular distributions in HeLa cells. By indirect immuno-fluorescence we show that wild-type Rna1p has three subcellular locations. The majority of the protein is distributed throughout the cytosol, but a portion of the protein is nucleus-associated, located at both the cytosolic surface and within the nucleoplasm. Mutant Rna1-1p is found at the outer nuclear surface and in the cytosol. We propose that a small pool of the wild-type Rna1p is located in the nuclear interior, supporting the model that the same components of the Ran/TC4 GTPase cycle exist on both sides of the nuclear membrane.

Base Sequence↗
PubMed1996

Transcellular delivery of an insulin-transferrin conjugate in enterocyte-like Caco-2 cells.

Insulin, acylated with dimethylmaleic anhydride, was conjugated to transferrin (Tf) via a disulfide linkage. The molar insulin: Tf ratio in the conjugate was 3:1. The insulin-Tf conjugate (insulin-Tf) was tested for the transport of insulin across enterocyte-like Caco-2 cell monolayers by the process of transferrin receptor (TfR)-mediated transcytosis. The uptake of insulin-Tf in Caco-2 cells was TfR-mediated but no insulin receptor-mediated. Transport studies showed that insulin-Tf transport was 5- to 15-fold higher than free insulin transport across Caco-2 cells in both apical-to-basal and basal-to-apical directions. Brefeldin A (BFA), an agent that we have previously shown to cause an increase in TfR transcytosis, further enhanced the transport of the conjugated insulin three-fold in both directions; thus, a combination of the conjugate and BFA can cause a net 45-fold increase in the apical-to-basolateral transport of insulin across Caco-2 cell monolayers. The transported conjugate was intact as indicated by elution on a Sephadex G-50 column. Insulin in the transcytosed conjugate, unlike the original dimethylmaleyl insulin, was capable of binding to anti-insulin antibodies, indicating that free amino groups of insulin were regenerated either during or after the transcytotic process. Because Caco-2 cell monolayers provide a good model for intestinal epithelium, the insulin-Tf conjugate in combination with BFA can be a rational approach to increase the oral absorption of insulin in vivo.

Biological Transport↗
PubMed1996

Comparison of pharmacokinetic parameters of a polypeptide, the Bowman-Birk protease inhibitor (BBI), and its palmitic acid conjugate.

PURPOSE: The alteration of the pharmacokinetic parameters of the polypeptide BBI through conjugation with palmitic acid was examined. METHODS: 125I-BBI or 125I-Pal-BBI was administered iv to 6 week old CF-1 mice at a dose of 3 mg/kg. The mice were sacrificed at 5, 10, 20, 60, 120, 240, 360, and 480 min and the total radioactivity was determined for blood and each organ. The blood was analyzed on a Sephadex G-50 size-exclusion column to determine the amount of intact polypeptide present in the blood. From the amount of intact polypeptide at each time point, the pharmacokinetic parameters were determined. RESULTS: By conjugating three palmitic acids to each BBI molecule, the area under the curve (AUC) and mean residence time (MRT) increase by a factor of 10.8 and 2.8, respectively. There was also a difference in the organ distribution between the two treatments; while 125I-BBI was rapidly cleared from the kidneys, 125I-Pal-BBI was predominantly to the liver. Subsequent studies suggested that the binding of the conjugate to non-albumin serum proteins was most likely the cause of the altered pharmacokinetics. CONCLUSIONS: The residence time in the blood and the lipophilicity of BBI were increased upon conjugation with palmitic acid through a reversible disulfide linkage. Pharmacokinetic studies showed an increase in the AUC and a decrease in kidney clearance in palmitic acid conjugates, indicating a potential increase of the therapeutic efficacy of the polypeptide drug.

Animals↗
PubMed1996

Paclitaxel and nocodazole differentially alter endocytosis in cultured cells.

PURPOSE: Microtubule-based transport facilitates the endocytosis of exogenous macromolecules. We have determined how microtubule accumulation and disassembly alter endocytosis. METHODS: The effects of paclitaxel, which promotes microtubule assembly, and nocodazole, which promotes microtubule disassembly, on fluid-phase and receptor-mediated endocytosis were measured using uptake of horseradish peroxidase and 125I-transferrin, respectively. Changes in membrane and microtubule organization were examined by fluorescence microscopy. RESULTS: Neither paclitaxel (4 microM, 60 min pretreatment) nor nocodazole (1 microgram/ml, 60 min pretreatment) significantly inhibited fluid-phase endocytosis. However, paclitaxel caused a redistribution of fluorescent fluid-phase marker to the periphery. Both paclitaxel and nocodazole treatment significantly (p < or = 0.05) reduced the initial uptake of 125I-transferrin at 5 min to approximately 50% of control. Despite the similarity of the effects on initial endocytic uptake, the effects on steady state accumulation of 125I-transferrin were quite distinct. Exposure of CV-1 cells to paclitaxel for an additional 30, 60 or 90 min also showed reduced accumulation of 125I-transferrin up to a maximum significant (p < or = 0.05) inhibition of 48% +/- 10% of control at 90 min. In contrast, nocodazole caused an initial significant (p < or = 0.05) increase in 125I-transferrin accumulation after 30 min (159% +/- 13% of control), while by 90 min 125I-transferrin accumulation had returned to control levels. Microtubule content, particularly of stable microtubules, was increased in CV-1 cells by paclitaxel, but abolished by nocodazole treatment. CONCLUSIONS: Our data show that changes in the microtubule array can alter the dynamics of receptor movement through the endosomal pathway. However, microtubule assembly versus disassembly have different effects.

Animals↗
PubMed1996

Los1p, involved in yeast pre-tRNA splicing, positively regulates members of the SOL gene family.

To understand the role of Los1p in pre-tRNA splicing, we sought los1 multicopy suppressors. We found SOL1 that suppresses both point and null LOS1 mutations. Since, when fused to the Ga14p DNA-binding domain, Los1p activates transcription, we tested whether Los1p regulates SOL1. We found that las1 mutants have depleted levels of SOL1 mRNA and Sol1p. Thus, LOS1 appears to positively regulate SOL1. SOL1 belongs to a multigene family with at least two additional members, SOL2 and SOL3. Sol proteins have extensive similarity to an unusual group of glucose-6-phosphate dehydrogenases. As the similarities are restricted to areas separate from the catalytic domain, these G6PDs may have more than one function. The SOL family appears to be unessential since cells with a triple disruption of all three SOL genes are viable. SOL gene disruptions negatively affect tRNA-mediated nonsense suppression and the severity increases with the number of mutant SOL genes. However, tRNA levels do not vary with either multicopy SOL genes or with SOL disruptions. Therefore, the Sol proteins affect tRNA expression/ function at steps other than transcription or splicing. We propose that LOS1 regulates gene products involved in tRNA expression/function as well as pre-tRNA splicing.

Amino Acid Sequence↗
PubMed1996

Malignant soft tissue sarcoma of the hypopharynx successfully treated by radiotherapy alone.

Sarcoma of the hypopharynx has been reported very rarely in the literature, only six cases having been found among all head and neck malignancies reported to SEER (Surveillance, Epidemiology, and End Results) during 1973-1987. We report a 14-year-old boy with a huge malignant soft tissue sarcoma arising from the hypopharynx. Tracheostomy and feeding gastrostomy were performed as emergency life-saving procedures. Surgical resection had been attempted, but abandoned. Because of the rapidity of tumor growth, we gave the patient a course of accelerated radiotherapy (170 cGy/fraction, two fractions per day) with a total dose of 7140 cGy within one month. A series of endoscopy and imaging studies demonstrated complete regression of the tumor, and the patient is currently alive without evidence of disease 3.5 years after treatment. We conclude that for an unresectable tumor without distant metastasis, radiation therapy may be tried. The time, dose, and fractionation of radiotherapy should be carefully designed and individualized.

Adolescent↗
PubMed1995

Water-soluble fatty acid derivatives as acylating agents for reversible lipidization of polypeptides.

A novel method allowing the conjugation of a fatty acid to a peptide or protein in aqueous buffer is described in this paper. L-Cysteinyl 2-pyridyl disulfide (CPD) (III), which was obtained by reacting L-cysteine (I) with 2,2-dithiopyridine (II), was reacted with the N-hydroxysuccinimide ester of palmitic acid (IV) to yield a water-soluble derivative of palmitic acid, termed Pal-CPD (V). Pal-CPD (V) could be reacted with a sulfhydryl-containing peptide or protein in aqueous buffer to yield the palmitic acid-derivatized conjugate (VI). The palmitic acid-derivatized Bowman-Birk protease inhibitor (BBI), synthesized using this conjugation method, was demonstrated to have 140-fold higher uptake into Caco-2 cell monolayers compared to native-BBI. The biological activity of the conjugate, as assessed using an in vitro transformation assay, was retained.

Acylation↗
PubMed1995

Disposition of positively charged Bowman-Birk protease inhibitor conjugates in mice: influence of protein conjugate charge density and size on lung targeting.

The influence of conjugate charge density and size on the targeting of cationic Bowman-Birk inhibitor (BBI) conjugates to the lungs was studied in mice. The biodistribution of BBI, either as the native protein or in the conjugated form (conjugated to a dicationic, tetracationic, or polycationic carrier), indicated that by increasing the charge density of BBI conjugates, the lung accumulation of the conjugates administered intravenously (i.v.) can be increased. The order of lung accumulation in these studies was as follows: polycationic- > tetracationic- > dicationic-conjugated BBI > BBI. The influence of conjugate size on lung accumulation was studied in three experiments. First, the biodistribution of poly(D-lysine) carriers of equal charge density but different molecular weight demonstrated that lung accumulation of polycationic carriers increases with an increase in carrier size. Second, the biodistributions of BBI, tyramine-derivatized poly(D-lysine)3 kDa, and poly(D-lysine)3 kDa conjugated to BBI indicated that an increase in conjugate size alone is not sufficient to promote the lung accumulation of cationic BBI conjugates. Finally, the biodistribution poly(D-lysine) complexed with heparin showed that targeting of a conjugate to the lungs can be abolished by neutralizing the charge on the carrier. Collectively, data in this paper demonstrate that the carrier-mediated targeting of BBI to the lungs is dependent on (a) cationization of BBI, (b) the conjugate positive charge density, and (c) the size of the cationic conjugate if the charge density is maintained. Also, the data show the size of the conjugate alone does not make a significant impact on lung accumulation.

Aniline Compounds↗
PubMed1995

Serial MRI changes in radiation myelopathy.

Using MRI we assessed the changes in signal, size, and contrast enhancement characteristics of the cervical spinal cord in radiation myelopathy developing after radiotherapy for nasopharyngeal carcinoma. We studied two men and five women, aged 40-77 years. The first MRI study was performed 1-4 months after the initial clinical manifestations of myelopathy, and follow-up MRI 2-22 months after the onset of symptoms. On the first study, all patients showed low signal intensity in a long segment of the cervical spinal cord on T1-weighted images, high signal on T2*-weighted images, and focal contrast enhancement at C1-2. In five patients there was also swelling of the spinal cord. The site of eccentric focal contrast enhancement correlated with the clinical manifestations. Follow-up imaging less than 10 months after the onset of symptoms showed no significant changes in signal intensity. Focal contrast enhancement at C1-2 remained the same in three patients, was more dense and larger in one, and less dense in another. Subsidence of swelling was seen in two patients. Atrophy of the spinal cord at C1-2, without abnormal signal and with faint contrast enhancement at C1-2 was revealed as early as 10 months after the onset of symptoms, but the contrast enhancement disappeared by 22 months. There was no correlation between clinical manifestations and spinal cord atrophy on MRI.

Adult↗
PubMed1995

MRI in internal cerebral vein thrombosis: case note.

A 40-year-old man developed general malaise, dizziness and progressive headache 1 week after acute haemorrhagic conjunctivitis. Bizarre behaviour, confusion and adversive seizures occurred later. MRI showed haemorrhagic infarct in the bilateral basal ganglia and thalamus and abnormal signal density in the internal cerebral veins. Bilateral carotid angiography showed no filling of superior sagittal sinus and internal cerebral veins. With MRI, cerebral venous thrombosis can be suspected and diagnosed earlier than before, obviating any invasive investigation.

Adult↗
PubMed1995

Location of N2,N2-dimethylguanosine-specific tRNA methyltransferase.

Most steps in the maturation of nuclear coded tRNAs occur in the nucleus in eukaryotic cells, but little is known as to the intranuclear location of this RNA maturation pathway. Indirect immunofluorescence experiments using antibody to N2,N2 dimethylguanosine-specific tRNA methyltransferase, a tRNA processing enzyme, and to Nup1p, a nuclear pore protein, show that both locate to the nuclear periphery in wild type cells. Staining of the nuclear membrane is more uniform with anti-Trm1p than the punctate staining observed with antibodies recognizing Nup1p. Biochemical fractionation experiments comparing fractionation of Trm1p with Nup1p, tRNA splicing ligase, and tRNA splicing endonuclease show that Trm1p behaves more like the known peripheral nuclear membrane proteins, Nup1p and tRNA splicing ligase, than like the integral membrane protein, tRNA splicing endonuclease. Cells overproducing Trm1p also concentrate it to the nuclear periphery. Thus, the site(s) of interaction of Trm1p are not easily saturable and are likely to be in excess to Trm1p. Trm1p is shared by mitochondria and the nucleus. Cells transformed with a gene coding Trm1p with a mutant nuclear targeting signal display cytoplasmic staining and an enzyme with increased solubility when compared to the solubility of wild type enzyme. Thus, mutations that prevent the enzyme from entering the nucleus result in an increase in its cytosolic but not mitochondrial concentration suggesting that the mitochondrial/nuclear distribution of Trm1p is not due solely to competition of mitochondrial and nuclear targeting information.

Amino Acid Sequence↗
PubMed1995

Embolization of carotid cavernous fistula by means of direct puncture through the superior orbital fissure.

PURPOSE: To embolize carotid cavernous fistulas (CCFs) by means of transorbital puncture of the cavernous sinus (CS) and the cavernous segment of the internal carotid artery (ICA) through the superior orbital fissure. MATERIALS AND METHODS: Five patients with CCF were treated with embolization of the CS, and six were treated with embolization of both the CS and the cavernous portion of the ICA after transorbital puncture. All except one had previously undergone occlusion of the ipsilateral proximal ICA before direct transorbital puncture. RESULTS: The fistulas in these cases were all obliterated completely. Only two patients had temporary ptosis. No other remarkable complications were noted. The patent ICA on the side of the fistula in one patient remained patent after embolization. CONCLUSION: Direct transorbital puncture through the superior orbital fissure is an alternate treatment for CCF, especially when the ipsilateral ICA has been occluded.

Adult↗
PubMed1995

Carbamylation decreases the cytotoxicity but not the drug-carrier properties of polylysines.

The charge density of Poly(D-lysine) was reduced by the carbamylation of the lysyl residues with potassium cyanate. A decrease in the charge density of poly(D-lysine) by 25% and 50% reduced the cytotoxicity of the ligand to cultured L929 cells by a 5-, and a 20 to 25-fold level, respectively, as estimated by using either the viability or the protein assay. The uptake of cyanate-modified poly(D-lysine) ligands in cultured L929 cells was not reduced, while the uptake of poly(D-lysine)/Heparin complex was reduced by 80%, as compared to that of unmodified poly(D-lysine). The in vivo biodistribution of cyanate-modified poly(D-lysine) ligands in the lungs and the liver of mice was not altered in comparison to that of unmodified poly(D-lysine), whereas the poly(D-lysine)/Heparin complex was only accumulated in the liver but not in the lungs. The data in this paper indicate that a 50% decrease in the positive charge density of poly(D-lysine) reduces the toxicity, but not the carrier potential of this polycationic ligand.

Animals↗
PubMed1995

Angiographic findings in Tolosa-Hunt syndrome: a case report.

A 71-year-old man suffered from left-side throbbing headache over the region of the fifth cranial nerve first division, followed by left ptosis and ophthalmoplegia. Under tentative diagnosis of Tolosa-Hunt syndrome (THS), oral prednisolone 60 mg was given daily. His headache and ophthalmoplegia dramatically improved within 24 hours. Magnetic resonance (MR) imaging and computed tomography (CT) of the head were negative. However, irregularity in the siphon of the left internal carotid artery was disclosed by angiography. This case illustrates that angiography may be useful in diagnosing THS, when CT and MR imaging are negative.

Aged↗
PubMed1995

[Structure and biological properties of a conjugate of Bowman-Birk type soy proteinase inhibitor with a block copolymer of ethylene oxide and propylene oxide].

The structure of the conjugate of Bowman-Birk soybean proteinase inhibitor (BBI) with the block copolymer of ethylene oxide and propylene oxide (proxanol) containing five moles of proxanol per mole of protein, has been studied. Data from reverse phase hydrophobic HPLC suggest that the conjugate is less hydrophobic compared to native BBI. A shift of the second derivative UV absorption spectrum for the conjugate towards the shortwave region indicates a greater accessibility of the Tyr-59 residue localized in the interdomain region of the BBI molecule for the solvent. It has been assumed that the conjugate-induced increase in Ki for chymotrypsin may be due to both disturbances in the intact structure of the interdomain region of BBI and screening of the anti-chymotrypsin reactive center as a result of hydrophobic interactions of propylene oxide blocks of proxanol with exposed hydrophobic groups around the reactive center. Supporting evidence in favour of BBI molecule hydrophilization as a result of modification by proxanol can be derived from decreased conjugate penetration into intestinal epithelial cells as well as from the slow elimination of the conjugate from mouse blood stream.

Animals↗
PubMed1995

Somatosensory evoked potential study in cervical radiation myelopathy.

BACKGROUND: To correlate the findings of median nerves somatosensory evoked potentials (SEPs) with clinical manifestations and magnetic resonance (MR) findings in cervical radiation myelopathy after therapeutic irradiation for nasopharyngeal carcinoma. METHODS: Median nerve somatosensory evoked potentials (SEPs) were recorded in 10 patients with cervical radiation myelopathy. Seven patients were studied by SEP once, two patients received SEP twice, and one patient, three times. RESULTS: On the basis of the median nerve SEP findings, three groups were identified: Group I, normal SEP responses (N9, N13 and N20); Group II, normal N13 with abnormal N20; Group III, Normal or abnormal N9 with both abnormal N13 and N20. CONCLUSIONS: The size, eccentricity and extension to the dorsal columns of the lesions correlated well with SEP findings. When the lesions were confined mainly to the lateral column without much involvement of the dorsal columns, SEP findings were normal; with more extensive lesions involving the dorsal columns, SEPs became abnormal.

Adult↗
PubMed1994

MRI of Joubert's syndrome.

OBJECTIVE: To report five cases of the rare Joubert's syndrome. SUBJECTS: All five cases were studied by 1.0-Tesla MRI. All the patients showed typical clinical manifestations of Joubert's syndrome including neonatal respiratory abnormalities, developmental delay, ataxia, retinal atrophy and nystagmus. RESULTS: The T1WI of MRI showed characteristic MRI features of Joubert's syndrome including dilatation of the fourth ventricle with some appearing bat-wing shaped, elongation and stretching of the superior cerebellar peduncles, dysphasia of the vermis, widening of the foramen of Magendie and the posterior cistern. One case was associated with encephalomeningocele. CONCLUSIONS: MRI can provide characteristic findings of Joubert's syndrome and confirm the clinical diagnosis.

Brain↗