Biomedical subjects

R Wu

Publications and source records attributed to R Wu.

At least 181 records · Page 10Linked to original sources

PubMed1997

UDP-GlcNAc:Ser-protein N-acetylglucosamine-1-phosphotransferase from Dictyostelium discoideum recognizes serine-containing peptides and eukaryotic cysteine proteinases.

Phosphoglycosylation catalyzed by UDP-GlcNAc:Ser-protein N-acetylglucosamine-1-phosphotransferase (Ser:GlcNAc phosphotransferase) adds GlcNAcalpha-1-P to peptidyl-Ser of selected Dictyostelium discoideum proteins. Lysosomal cysteine proteinase (CP), proteinase-1(CP7), is the major phosphoglycosylated protein in bacterially grown amoebae. GlcNAc-1-P is added within a Ser-rich domain containing SSS, SGSG, or SGSQ repeated motifs that are not found in other papain-like CPs. We studied the substrate specificity of the transferase using peptides containing these motifs and 12 other peptides with one or more Ser residues. Phosphoglycosylation is comparable for all three Dictyostelium CP motifs, but it is not restricted to them. Flanking residues in the other peptides strongly influence phosphoglycosylation efficiency. Dictyostelium microsomal membranes also phosphoglycosylate endogenous acceptors, and some of these acceptors occur as an 18 S complex with the transferase. CP-serine motif peptides inhibit endogenous acceptor phosphoglycosylation weakly (30-40%) at 800 microM, whereas catalytically inactive proteinase-1(CP7) and other non-phosphoglycosylated eukaryotic CPs, lacking the serine domain, inhibit transferase activity at 1-4 microM. SDS denaturation destroys the inhibitory potential of all CPs showing that transferase recognizes a conformation-dependent feature that is shared by all. Proteinase-1(CP7) expressed in Escherichia coli lacks GlcNAc-1-P, but it is a substrate for Ser:GlcNAc phosphotransferase, Km = 5.6 microM. Thus, Ser:GlcNAc phosphotransferase recognizes both acceptor peptide sequences and a conformational feature of eukaryotic CPs. This may be physiologically important for establishing or maintaining non-overlapping groups of GlcNAc-1-P- and Man-6-P-modified Dictyostelium proteins that reside in functionally distinct endo-lysosomal vesicles.

Animals↗

PubMed1997

Loss of FHIT expression in cervical carcinoma cell lines and primary tumors.

Allelic deletions involving the short arm of chromosome 3 (3p13-21.1) have been observed frequently in cervical carcinomas. Recently, a candidate tumor suppressor gene, FHIT (Fragile Histidine Triad), was cloned and mapped to this chromosomal region (3p14.2). Abnormal FHIT transcripts have been identified previously in a variety of tumor cell lines and primary carcinomas, although their significance and the molecular mechanisms underlying their origin remain incompletely defined. In addition, integration of human papillomavirus DNA has been identified at a fragile site (FRA3B) within the FHIT locus in cervical cancer. These observations motivated us to evaluate FHIT mRNA and protein expression in cervical cancer cell lines, primary cervical carcinomas, and normal tissues. Transcripts of the expected size and sequence were the predominant species identified by reverse transcription (RT)-PCR in cultured keratinocytes and all normal tissues evaluated. In contrast, aberrant FHIT transcripts were readily demonstrated in 6 of 7 cervical carcinoma cell lines and 17 of 25 (68%) primary cervical carcinomas. Northern blot analyses demonstrated reduced or absent FHIT expression in the cervical carcinoma cell lines, particularly those with aberrant RT-PCR products. Immunohistochemical analysis of Fhit expression in cervical tissues revealed strong immunoreactivity in nonneoplastic squamous and glandular cervical epithelium and marked reduction or loss of Fhit protein in 25 of 33 (76%) primary cervical carcinomas. In those cervical cancer cell lines and primary tumors with exclusively aberrant or absent FHIT transcripts by RT-PCR, Fhit protein expression was always markedly reduced or absent. The frequent alterations in FHIT expression in many cervical carcinomas, but not in normal tissues, suggest that FHIT gene alterations may play an important role in cervical tumorigenesis.

Acid Anhydride Hydrolases↗

PubMed1997

Mutations in PTEN are frequent in endometrial carcinoma but rare in other common gynecological malignancies.

Loss of heterozygosity of chromosome 10q has been reported in approximately 40% of endometrial carcinomas. PTEN, a candidate tumor suppressor gene located at chromosome 10q23.3, was recently identified and found to be homozygously deleted or mutated in several different types of human tumors. To determine if PTEN is a target of 10q loss of heterozygosity in carcinomas of the endometrium, we examined 32 primary endometrial carcinomas for mutations in PTEN. The tumors included the two major histopathological types of endometrial carcinoma: endometrioid (n = 26; 14 microsatellite instability (MI)-positive and 12 MI-negative) and serous (n = 6). Overall, mutations were detected in 50% of the endometrial carcinomas we analyzed. Mutations were present in 12 of 14 (86%) MI-positive and 4 of 12 (33%) MI-negative endometrioid tumors. Furthermore, mutations were found in all three histological grades of MI-positive endometrioid carcinoma. All six serous endometrial carcinomas lacked detectable mutations. To evaluate the role of PTEN in other common malignancies of the female genital tract, 12 serous ovarian carcinomas and 10 squamous cervical carcinomas were analyzed and were negative for mutations. Our results support PTEN as a tumor suppressor gene and suggest that mutations in PTEN play a significant role in the pathogenesis of the endometrioid type of endometrial carcinoma.

Carcinoma↗

PubMed1997

Elucidating mechanisms of thermostabilization of poliovirus by D2O and MgCl2.

To understand a significant reduction in the loss of poliovirus infectivity by D2O and a combination of D2O and MgCl2 at 37-45 degrees C, this paper attempts to elucidate the mechanisms underlying the thermostabilization of poliovirus. Three serotypes of Sabin oral poliovirus vaccine strains were investigated. Temperature-dependent fluorescence emission intensity studies showed that the effects of D2O and MgCl2 on the stability and conformation of poliovirus are correlated with those of the infectivity of poliovirus. Fluorescence steady-state polarization revealed that the conformation of poliovirus capsid is sensitive to D2O medium and MgCl2 salt, and that the rigidity of poliovirus conformation is increased in their presence. The exposure of poliovirus tryptophan residues to water is modified by D2O and MgCl2, as evidenced by changes in fluorescence emission intensity excited at 295 nm. The involvement of hydrogen bonding in the D2O effect was demonstrated by the greatly increased value of relative fluorescence intensity. Conformational alteration was also shown by changes in the positive band (193-230 nm) of circular dichroism spectra. D2O and MgCl2 were also found to reduce the interaction of virus with water as examined by differential scanning microcalorimetry, leading to a decline in the extent of water penetration into the poliovirus capsid. All these observations were found to be more profound in a combination of D2O with MgCl2 than D2O or MgCl2 alone. By inducing a conformation favorable to maintaining the poliovirus assembly and by reducing virus-water interaction to decrease water penetration into the poliovirus capsid, D2O, MgCl2, or D2O-MgCl2 is able to exert its thermostabilization effect. Thus, to maintain the virus assembly and conformation of the virus and to reduce the swelling of the virus capsid are key factors in increasing the thermostability of poliovirus. These two factors are mutually complementary. The latter can provide a favorable environment for the formers and the formers, in turn, lead to the latter.

Calorimetry, Differential Scanning↗

PubMed1997

Direct amplification of unknown genes and fragments by Uneven polymerase chain reaction.

Gene cloning is a time-consuming task for molecular biologists, because it often takes weeks or months to construct, screen and finally clone a gene from a DNA library. Thus, more effective methods are needed for gene cloning. This paper describes a modified polymerase chain reaction (PCR) cycling condition. Uneven PCR, to generate specific unknown fragments or genes directly from total DNA instead of cloning fragments from DNA libraries. The essence of the method is to use two different annealing temperatures in consecutive PCR cycles to effectively amplify the target products while inhibiting the synthesis of non-specific products. Under favorable conditions, a desired DNA fragment or gene in the size range up to approximately 4 kb can be obtained and ready for cloning within a day or two.

Cloning, Molecular↗

PubMed1997

Improvements for measuring 1H-1H coupling constants in DNA via new stripe-COSY and superstripe-COSY pulse sequences combined with a novel strategy of selective deuteration.

Three bond proton-proton vicinal coupling constants are of potential value for analyzing sugar conformations in DNA. However, self-cancellation in antiphase cross peaks and modulation of peak splittings by transverse cross relaxation can alter the apparent coupling constants such that they do not accurately reflect the sugar conformations. Transverse cross relaxation is most effective between strongly coupled geminal proton pairs. Here we report the use of stereospecific deuteration at the H2" position in the A5 and A6 residues in the 12 base pair DNA sequence [d(CGCGAATTCGCG)2] as a means of investigating the effect of transverse cross relaxation on P.E.COSY type cross peaks. Deuteration of the H2" proton is expected to reduce the transverse cross relaxation rate by the square of ratio of the proton to deuteron gyromagnetic ratios, i.e., by a factor of 42. Additionally, a striking eight- to ninefold increase in the signal intensity was observed for cross peaks involving the remaining H2' proton resulting from diminished dipolar relaxation. Further improvements in signal-to-noise ratio were realized by collecting P.E.COSY spectra in strips, using an experiment referred to as stripe-COSY, employing selective excitation pulses which reduced the number of required t1 increments by a factor of four. A final improvement was achieved by employing selective time-shared homonuclear decoupling during the acquisition period, in an experiment referred to as superstripe-COSY, to collapse splittings due to passive couplings. Collectively, these approaches provide P.E. COSY-type spectra with two to three orders of magnitude increased sensitivity per unit time and that are relatively free from artifacts.

DNA↗

PubMed1997

Ultrastructural findings of the macula utriculi in a case of a petrous apex cholesteatoma: a comparison with findings in a patient with an acoustic neuroma.

The morphological characteristics of the vestibular sensory cells of the macula utriculi obtained during surgery in a patient with a petrous apex cholesteatoma were examined using scanning and transmission electron microscopy. Findings were compared to cells studied in a patient with acoustic neuroma. Scanning electron microscopy showed that compared to the apparently normal cells in the acoustic neuroma case, most sensory cells in the cholesteatoma case had large cuticular plates, irregular locations of cilia and no clear polarizations. Supporting cells showed profuse short microvilli on the whole surface. With transmission electron photomicrographs, type I hair cells were not seen and certain morphological changes were observed in type-II-like cells and supporting cells. We presume that the degenerative changes in the vestibular epithelia were due to circulatory disturbances and/or direct pressure applied to the vestibular nerve at the internal auditory canal, with subsequent involvement of the macula utriculi.

Acoustic Maculae↗

PubMed1997

Focal microcirculation disorder induced by photochemical reaction in the guinea pig cochlea.

A small region of microcirculation disorder in the cochlea of the guinea pig could be induced by a photochemical reaction. Photoillumination to the cochlea was done after systemic infusion of Rose Bengal (RB). The lateral wall of the second or third turn of the cochlea was illuminated for 10 min with a 1 mm diameter focused green light supplied by a xenon lamp. Degeneration of the stria vascularis (SV) was observed by a scanning electron microscope at 60-300 min after illumination. The range of length of degenerated area in the SV was from 111 to 1800 microns, with a mean of 760 microns. The organ of Corti along the illuminated lesion of the SV was well preserved in all animals at 60-300 min. In contrast, degeneration of sensory hair cells and scar formation in the SV were observed in the focal lesions of the three animals killed 1 week after illumination. The increase of diameter in the vessel of the SV from the radiating arteriole, the vessel of basilar membrane (VSBM) and limbus vessel (LVS) were observed in the illuminated area with diaminobenzidine (DAB) staining. These findings suggest that segmental microcirculation damage occurred in the SV and modiolus. In physiological studies, compound action potentials (CAP) were evaluated. Endocochlear potentials (EP) were also measured at the second turn under three different situations (groups A, B and C). A photochemically induced lesion was created at the site of EP measurement (group A), a site in the second turn 1 mm from the EP measurement site (group B) and a site in the third turn adjacent to the EP measurement site (group C). Threshold shift of CAP (up to 5.6 +/- 1.8 dB SPL) and reduction of EP (down to 11.4 +/- 10.7 mV) in the photochemically injured location were detected during about 15 min. EP did not recover to the predamaged level (79.9 +/- 3.7 mV) during 20 min. The morphological and physiological changes were not observed in the control group with illumination only. There were no significant decreases in EP values at the sites 1 mm from the lesion (group B) and at the inferior turn adjacent to the lesion (group C) compared to the marked decrease at the site of the photochemically induced lesion (group A). These findings suggest that CAP and EP are significantly affected by the interruption of segmental blood supply in the cochlea and remarkable decrease of EP occurs in the focal region of the guinea pig cochlea. We conclude that a localized blood circulation disorder induced by the photochemical reaction can make a focal lesion in guinea pig cochlea morphologically and physiologically.

Action Potentials↗

PubMed1997

Comparison of Colombian and Costa Rican strains of rice hoja blanca tenuivirus.

The sequences of RNA-3 and RNA-4 of rice hoja blanca tenuivirus isolates from Colombia and from Costa Rica were determined and analyzed. These isolates were 98.9% and 98.6% identical in the coding and non-coding regions of RNA-3, and 96.9 and 91.5% identical in the coding and non-coding regions of RNA-4, and are therefore strains of the same virus. There is about three times as much variation between isolates (based on consensus sequences) as there is within isolates (based on sequences of individual clones). There is also considerably more variation for RNA-4 (both between and within isolates) than there is for RNA-3, even though between tenivirus species RNA-3 has diverged more than RNA-4, implying that the evolution of the tenuivirus RNAs is not necessarily dependent on the amount of variation found for these RNAs.

Colombia↗

PubMed1997

The duration of delirium in medical and postoperative patients referred for psychiatric consultation.

OBJECTIVE: We wished to produce a complete frequency distribution of the duration of delirium in a large number of patients referred for psychiatric consultation, plot the data as a "survivorship" curve for delirium, and examine differences between postoperative and medical patients and between demented and nondemented patients. METHODS: The senior author entered into the study 94 consecutive patients referred to him because of confusion and agitation and diagnosed by him as having a delirium and followed them closely throughout their hospital course. Patients were telephoned for follow-up after discharge. RESULTS: The rate of disappearance of delirium appeared log linear for approximately 2 weeks, but rate of resolution for medical patients was slower than for postoperative patients. The mean and median duration of delirium for medical patients were, respectively, 13.2 and 8 days, and for postoperative patients, 7.6 days and 6 days. Combined mortality over 3 1/2 years was 46.8%. Demented patients had longer average durations of delirium than nondemented patients, but differences were not statistically significant because of large variance. CONCLUSIONS: In a large heterogeneous group of hospitalized patients referred for psychiatric consultation, the mean duration of delirium was shorter for postoperative than for medical patients. The difference could not be explained by different criteria for referral. Mortality was high for both groups.

Aged↗

PubMed1997

A sensitive GC-MS procedure for the analysis of flunitrazepam and its metabolites in urine.

Analysis of urine specimens collected from individuals ingesting 2 and 4 mg flunitrazepam (FN) showed positive results by OnLine and OnTrak immunoassays for up to 60 h. Gas chromatographic-mass spectrometric (GC-MS) analysis of these specimens for FN, 3-OH-FN, 7-amino-FN, 7-amino-3-OH-FN, desmethyl-FN, and 3-OH-desmethyl-FN after glucuronidase treatment showed only low levels of 7-amino-FN with almost none of the other metabolites. These levels were far below the expected results based on the immunoassay data. This study reports on a GC-MS procedure for FN and the previously listed metabolites. The method is based on acid hydrolysis of the urine specimens, which converts FN and all its metabolites described previously to one of four amino-benzophenone derivatives (1-4) with oxazepam-d5 as the internal standard. Under the experimental conditions, the internal standard is converted to 2-amino-5-chloro-benzophenone-d5. The limit of detection for 7-amino-FN and 7-amino-desmethyl-FN and their 3-OH derivatives was less than 1 ng/mL. Analysis of urine specimens collected for 72-h postingestion of 1, 2, or 4 mg FN showed appreciable levels of benzophenone 3 (product of 7-amino-FN and 7-amino-3-OH-FN) and lower levels of benzophenone 4 (product of 7-amino-desmethyl-FN and 7-amino-3-OH-desmethyl-FN) with no detectable levels of benzophenones 1 and 2. The method makes it possible to confirm the presence of FN metabolites in urine at least 72-h postingestion of small doses of the drug.

Anti-Anxiety Agents↗

PubMed1997

Antibodies against cardiolipin and oxidatively modified LDL in 50-year-old men predict myocardial infarction.

Autoantibodies against oxidatively modified low-density lipoproteins (oxLDL) and cardiolipin occur in patients with vascular diseases, including atherosclerosis. The ability of such antibodies to predict myocardial infarction (MI) was investigated in a prospective nested case-control study in which healthy 50-year-old men were followed up for 20 years. Raised levels of antibodies against oxLDL and cardiolipin at 50 years of age correlated positively with the incidence of MI and mortality related to MI 10 to 20 years later. IgG and IgA antibodies against cardiolipin were associated with MI between 50 to 60 years of age and IgG and IgA antibodies against oxLDL with MI at 60 to 70 years of age. Moreover, higher antibody levels were noted in those who died from acute MI in comparison to those who survived. The predictive power of IgA and IgG antibodies was strong and largely independent of that of other strong risk factors. In conclusion, raised levels of antibodies against oxLDL and cardiolipin may predict MI and MI-related death.

Aged↗

PubMed1997

Elevated serum levels of mucin-associated antigen in patients with acute respiratory distress syndrome.

Increased serum levels of mucin-associated antigen have been previously demonstrated in patients with cystic fibrosis (CF) and interstitial pneumonia, and in lung-transplant recipients. The present study assessed the serum airway mucin levels in patients with acute respiratory distress syndrome (ARDS). An enzyme-linked immunosorbent assay (ELISA) method with a human-airway-mucin-specific monoclonal antibody (17Q2) was used to measure serum mucin levels in normal subjects, chronic smokers, patients with chronic bronchitis and other pulmonary diseases, patients with acute cardiogenic lung edema, and patients with ARDS. The serum mucin levels measured 9.9 +/- 0.8 ng/ml (mean +/- SEM, n = 59) in normal subjects, 12.7 +/- 1.6 ng/ml (n = 29) in chronic smokers, 21.8 +/- 1.9 ng/ml (n = 28) in patients with chronic bronchitis and other pulmonary diseases, 9.0 +/- 3.1 ng/ml (n = 5) in patients with acute cardiogenic lung edema. The serum mucin level was 53.8 +/- 6.6 ng/ml (n = 13) in patients with ARDS (p < 0.05, as compared with the four other groups). Serial measurements of serum mucin levels were obtained in patients with ARDS. Statistical analysis showed an inverse correlation of serial measurements of serum mucin with static respiratory-system compliance (p = 0.021), an inverse correlation of sequential serum mucin levels and log(Pa(O2)/Fl(O2)) (p = 0.016), and a positive correlation of sequential serum mucin levels and lung injury score (LIS) (p = 0.019). Gel-filtration analysis showed that mucin-associated antigens in ARDS sera were polydispersed and smaller than the antigens in normal sera. This study indicates that an increasing amount of degraded mucin occurs in patients with ARDS.

Acute Disease↗

PubMed1997

Selection of invasive and metastatic subpopulations from a human lung adenocarcinoma cell line.

To better understand the mechanism(s) underlying lung cancer invasion and metastasis, a Transwell invasion chamber was used to select progressively more invasive cancer cell populations from a clonal cell line of human lung adenocarcinoma, CL1. Five sublines with progressive invasiveness, designated CL1-1, CL1-2, CL1-3, CL1-4, and CL1-5, were obtained through this in vitro selection process. Their invasive abilities through basement membrane matrix showed a 4- to 6-fold increase over that of the parental cells. Moreover, the sublines manifested an increase in their colony-forming ability on soft agar, tumorigenicity, and metastatic potency in severe combined immunodeficiency (SCID) mice. Examining the phenotypes of the cell lines revealed increased expression of 92 kD gelatinase and an increase in the cell population stained with anti-keratin-8 and -18 antibodies. Clonal isolation of anti-keratin-18-antibody-positive and -negative cell populations demonstrated a correlated enhancement of the invasiveness of these cells and their expression of keratin-18. These results support the notion that the metastatic behavior of lung cancer cells can be characterized with this in vitro system, and that the properties of these progressively invasive cancer cells can be clonally studied.

Adenocarcinoma↗

PubMed1997

Growth and differentiation of conducting airway epithelial cells in culture.

The development of routine techniques for the isolation and in vitro maintenance of conducting airway epithelial cells in a differentiated state provides an ideal model to study the factors involved in the regulation of the expression of mucociliary differentiation. Several key factors and conditions have been identified. These factors and conditions include the use of biphasic culture technique to achieve mucociliary differentiation, the use of such stimulators as the thickness of collagen gel substratum, the calcium level, and vitamin A, and such inhibitors as the growth factors, epidermal growth factor and insulin, and steroid hormones, for mucous cell differentiation. Using the defined culture medium, the life cycle of the mucous cell population in vitro has been investigated. It was demonstrated that the majority of the mucous cell population in primary cultures is not involved in the replication of deoxyribonucleic acid (DNA). However, the mucous cell type is capable of self-renewal in culture, and this reproduction is vitamin A dependent. Furthermore, differentiation from nonmucous to mucous cell type can be demonstrated by adding back a positive regulator such as vitamin A to the "starved" culture. Cell kinetics data suggest that vitamin A-dependent mucous cell differentiation in culture is a DNA replication-independent process, and the process is inhibited by transforming growth factor-beta1.

Cell Differentiation↗

PubMed1997

Dictyostelium lysosomal proteins with different sugar modifications sort to functionally distinct compartments.

Many Dictyostelium lysosomal enzymes contain mannose-6-phosphate (Man-6-P) in their N-linked oligosaccharide chains. We have now characterized a new group of lysosomal proteins that contain N-acetylglucosamine-1-phosphate (GlcNAc-1-P) linked to serine residues. GlcNAc-1-P-containing proteins, which include papain-like cysteine proteinases, cofractionate with the lysosomal markers and are in functional vesicles of the endosomal/lysosomal pathway. Immunoblots probed with reagents specific for each carbohydrate modification indicate that the lysosomal proteins are modified either by Man-6-P or GlcNAc-1-P, but not by both. Confocal microscopy shows that the two sets of proteins reside in physically and functionally distinct compartments. Vesicles with GlcNAc-1-P fuse with nascent bacteria-loaded phagosomes less than 3 minutes after ingestion, while those with Man-6-P do not participate in bacterial digestion until about 15 minutes after phagocytosis. Even though both types of vesicles fuse with phagosomes, GlcNAc-1-P- and Man-6-P-bearing proteins rarely colocalize. Since both lysosomal enzymes and their bound carbohydrate modifications are stable in lysosomes, a targeting or retrieval mechanism based on these carbohydrate modifications probably establishes and/or maintains segregation.

Acetylglucosamine↗

PubMed1997

[Effect of catecholaminergic agonists and antagonists on adaptive gastric mucosal protection in rats].

The gastric mucosal damage caused by 70% ethanol significantly decreased in rats pretreated with chronic mild restraint stress (CMRS, P < 0.001). This adaptive mucosal protection induced by CMRS was absent in sympathectomized rats, while the administration of isoprenaline or dopamine could partially, and norepinephrine could not, restore the protection. In rats with rntact sympathetic nerve, preinjection of haloperidol or propranolol inhibited CMRS-induced protection which, however, was not affected by phentolamine. No change in plasma somatostatin level was observed either in stress or sympathectomized rats alone or in stress plus sympathectomized rats. The results suggest that the sympathetic nerve or its mimetic agent ang this is are involved in the adaptive protection of the gastric mucosa possibly mediated through beta and dopamine receptors.

Adaptation, Physiological↗

PubMed1997

[Determination of airway reactivity in the conscious and unrestrained guinea pigs].

A simple, stable determining system in the conscious and unrestrained guinea pigs was established that can quantitatively analyse the airway reactivity (AR). In this model, the response to histamine (His) or acetylcholine (ACh) aerosol was concentration-dependent with EC50 effective concentration causing 50% animals wheezing) of 53.3 mumol/m3 and 269 mumol/m3 respectively.

Acetylcholine↗