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Biomedical subjects

G An

Publications and source records attributed to G An.

At least 19 recordsLinked to original sources
PubMed1995

Two nuclear proteins in tracheal epithelial cells are recognized by antibodies specific to a squamous differentiation marker, sprI.

In cell-free translations of RNA from primary cultures of pig trachea surface epithelial cells we observed that a mRNA encoding a 20 kDa proline-rich protein (sPRP) was dramatically induced during culturing (Tesfaigzi et al., 1990, Biochem. Biophys. Res. Commun., 172:M1304-1309). This mRNA was not detected in tracheal tissue or in epithelial cells prior to culturing. Antisera were raised to synthetic peptide sequences corresponding to 23 amino acids on the C-terminus (C23-antiserum) and 29 amino acids on the N-terminus (N29 antiserum) of sPRP. On Western blot analysis, C23 antiserum reacted with a 20 kDa protein in cytosolic extracts from pig tracheal cells maintained in culture for 4 days. The reaction with the 20 kDa protein was inhibited by adding C23 peptide. Two nuclear proteins (66 and 70 kDa) obtained by micrococcal nuclease treatment of tracheal cell nuclei were detected on Western blots with C23 antiserum. These proteins were present in cells both before and after culturing. Sucrose gradient fractionation indicated that these nuclear proteins are associated with chromatin. Small amounts of the 66 and 70 kDa proteins were obtained from nuclear matrix fractions. These nuclear proteins also reacted with N29 antiserum. Since these proteins share similar epitopes with the N- and C-termini of sPRP, it is likely that the 20 kDa protein (sPRP) is part of these proteins. However, purification of the nuclear proteins followed by an amino acid sequence analysis is necessary to clarify whether sPRP is part of these proteins.

Amino Acid Sequence↗
PubMed1995

Phenotypic alterations of petal and sepal by ectopic expression of a rice MADS box gene in tobacco.

Floral organ development is controlled by a group of regulatory factors containing the MADS domain. In this study, we have isolated and characterized a cDNA clone from rice, OsMADS3, which encodes a MADS-domain containing protein. The OsMADS3 amino acid sequence shows over 60% identity to AG of Arabidopsis, PLE of Antirrhinum majus, and AG/PLE homologues of petunia, tobacco, tomato, Brassica napus, and maize. Homology in the MADS box region is most conserved. RNA blot analysis indicated that the rice MADS gene was preferentially expressed in reproductive organs, especially in stamen and carpel. In situ localization studies showed that the transcript was present primarily in stamen and carpel. The function of the rice OsMADS3 was elucidated by ectopic expression of the gene under the control of the CaMV 35S promoter in a heterologous tobacco plant system. Transgenic plants exhibited an altered morphology and coloration of the perianth organs. Sepals were pale green and elongated. Limbs of the corolla were split into sections which in some plants became antheroid structures attached to tubes that resembled filaments. The phenotypes mimic the results of ectopic expression of dicot AG gene or AG homologues. These results indicate that the OsMADS3 gene is possibly an AG homologue and that the AG genes appear to be structurally and functionally conserved between dicot and monocot.

Amino Acid Sequence↗
PubMed1995

Calmodulin gene family in potato: developmental and touch-induced expression of the mRNA encoding a novel isoform.

Eight genomic clones of potato calmodulin (PCM1 to 8) were isolated and characterized. Sequence comparisons of different genes revealed that the deduced amino acid sequence of PCM1 had several unique substitutions, especially in the fourth Ca(2+)-binding area. The expression patterns of different genes were studied by northern analysis using the 3'-untranslated regions as probes. The expression of PCM1, 5, and 8 was highest in the stolon tip and it decreased during tuber development. The expression of PCM6 did not vary much in the tissues tested, except in the leaves, where the expression was lower; whereas, the expression of PCM4 was very low in all the tissues. The expression of PCM2 and PCM3 was not detected in any of the tissues tested. Among these genes, only PCM1 showed increased expression following touch stimulation. To study the regulation of PCM1, transgenic potato plants carrying the PCM1 promoter fused to the beta-glucuronidase (GUS) reporter gene were produced. GUS expression was found to be developmentally regulated and touch-responsive, indicating a positive correlation between the expression of PCM1 and GUS mRNAs. These results suggest that the 5'-flanking region of PCM1 controls developmental and touch-induced expression. X-Gluc staining patterns revealed that GUS localization is high in meristematic tissues such as the stem apex, stolon tip, and vascular regions.

Amino Acid Sequence↗
PubMed1995

Study of the optimal duration of preoxygenation in children.

STUDY OBJECTIVE: To determine the optimal length of preoxygenation in children. DESIGN: Random design and comparison among groups. SETTING: Operating room of a plastic surgery hospital of the Chinese Academy of Medical Sciences and the Peking Union Medical College. PATIENTS: Forty healthy, ASA status 1 children (age 2 to 7 yrs), undergoing elective plastic surgery. INTERVENTIONS: Children in Group 1 breathed 100% oxygen (O2) for 1 minute. Group 2 children breathed 100% O2 for 2 minutes. Group 3 and Group 4 children breathed 100% O2 for 3 minutes. Anesthesia was induced with midazolam 0.3 mg/kg, fentanyl 5 micrograms/kg. Muscle relaxation was achieved with vecuronium 0.1 mg/kg (Groups 1, 2, and 3) or succinylcholine 1.5 mg/kg (Group 4). MEASUREMENTS AND MAIN RESULTS: Oxygen saturation (SpO2) was measured by pulse oximeter. The oximeter probe was applied to the right big toe. After preoxygenation, the times for SpO2 to decrease to 98% (T98), 95% (T95), and 90% (T90), respectively, were recorded during the apneic period. T98, T95, and T90 were significantly shorter in Group 1 than in Group 2 or Group 3. There was no statistically significant difference among Groups 2, 3, or 4 regarding T98, T95, and T90. The times for SpO2 to decrease from 95% to 90% were similar among the four groups. CONCLUSIONS: 2 minutes of preoxygenation in children can provide the maximum benefit of denitrogenation and achieve 2 minutes of safe apea. 95% and 99% confidence intervals were 69 to 100 and 59 to 100, respectively. Succinylcholine had only a slight effect on the safe apneic period.

Anesthesia↗
PubMed1994

Early flowering and reduced apical dominance result from ectopic expression of a rice MADS box gene.

Recent studies with dicot plants reveal that floral organ development is controlled by a group of regulatory factors containing the MADS domain. In this study, we have isolated and characterized a cDNA clone from rice, OsMADS1, which encodes a MADS-domain-containing protein. The OsMADS1 amino acid sequence shows 56.2% identity to AGL2 and 44.4% identity to AP1. The MADS box region was the most homologous to other MADS-domain-containing proteins. Northern blot analysis indicated that the rice MADS gene was preferentially expressed in floral organs. In situ localization studies showed that the transcript was uniformly present in young flower primordia and later became localized in palea, lemma, and ovary. Ectopic expression of OsMAD1 with the CaMV 35S promoter in transgenic tobacco plants dramatically alters development, resulting in short, bushy, early-flowering plants with reduced apical dominance. These results suggest that the OsMADS1 gene is involved in flower induction and that it may be used for genetic manipulation of certain plant species.

Amino Acid Sequence↗
PubMed1994

Developmental and environmental regulation of two ribosomal protein genes in tobacco.

Two cDNA clones, TSC29 and TSC40, were isolated from a cDNA library prepared from three-day-old tobacco cell suspension grown to early exponential stage. DNA sequence analyses and database searches revealed that the TSC29 transcript encodes a protein which is highly homologous to eukaryotic 60S ribosomal (r)-protein L25 and that the TSC40 product is homologous to rat 60S r-protein L34. Southern blot analysis showed that the putative r-protein genes are members of multigene families. Transcript levels of both genes were most abundant in three-day-old cell suspension and declined in older cultures. Transcripts were also present in plant vegetative and reproductive organs. However, for TSC40 in particular, the mRNA levels were lower in plant organs than in three-day-old cell suspension. Stems and roots exhibited higher expression than leaves and flowers, indicating that these clones are differentially regulated in various cell types. Both genes were expressed at low levels in mature seeds but transcript levels significantly increased after one day of germination, remained at a high level until day 4, and declined after day 5. In situ localization experiments with germinating seedlings revealed that the TSC29 transcript was preferentially localized in root tips, epidermis, and endosperm. Wounding increased the steady-state mRNA amounts of these r-protein genes, and 2,4-dichlorophenoxyacetic acid and benzyladenine further increased the transcript level.

Amino Acid Sequence↗
PubMed1994

A 20 nucleotide upstream element is essential for the nopaline synthase (nos) promoter activity.

The nopaline synthase (nos) promoter is expressed in a wide range of plant cell types and regulated by various developmental and environmental factors. The nos upstream control region essential for this regulation was studied by means of synthetic oligomers using transient and stable transformation systems. Insertion of a 20 nucleotide sequence containing two hexamer motifs and a spacer region into deletion mutants lacking the upstream control region was essential for promoter activity. Mutation of one or more nucleotides of either hexamer sequence significantly altered the strength of expression of the nos promoter. Point mutations within the spacer region also strongly influenced promoter strength. Insertion of multiple copies of the 20 nucleotide sequence into the nonfunctional deletion mutants proportionally increased the promoter activity. These results suggest that this twenty nucleotide sequence is essential for the nos promoter to function. Substitution of the nos element with the ocs or 35S as-1 which contain similar hexamer motifs restored not only promoter activity but also responses to wounding, auxin, methyl jasmonate, and salicylic acid.

Amino Acid Oxidoreductases↗
PubMed1994

Expression of MUC2 gene is down-regulated by vitamin A at the transcriptional level in vitro in tracheobronchial epithelial cells.

The functional role of airway mucin in the respiratory system is well recognized. The isolation of mucin cDNA clones, MUC genes, introduces new information regarding the structure of the mucin core protein; however, the nature of the authentic core protein of airway mucin is still unresolved. In this communication, the effects of vitamin A on the regulation of MUC2 gene expression in primary tracheobronchial epithelial (TBE) cells of human and nonhuman primates were examined. Vitamin A has been recognized as one of the most important nutrients in the regulation of airway mucous cell differentiation. The expression of the MUC2 gene has been demonstrated in both rat and human tracheal tissues. The monkey cDNA clone MT80 was isolated from a cDNA library derived from vitamin A-depleted cultures of monkey TBE cells using a synthetic oligonucleotide probe corresponding to the 69 nucleotides of a tandemly repeated sequence in human MUC2 cDNA. DNA sequencing revealed a similar tandemly repeated sequence, except that 72 oligonucleotide repeats were observed in the monkey cDNA clone. Using the MT80 cDNA as a probe, the expression of the MUC2 gene was studied in vitro. The corresponding MUC2 message level in primary cultures of monkey TBE cells was down-regulated by vitamin A. This result was consistently demonstrated in primary human and hamster TBE cultures. The down-regulation was both time- and dosage-dependent on vitamin A. A nuclear run-on assay demonstrated a decrease in the transcriptional rate of the MUC2 gene in nuclei isolated from vitamin A-treated cultures. These results suggest that MUC2 gene expression in TBE cells is transcriptionally down-regulated by vitamin A.

Amino Acid Sequence↗
PubMed1994

A PCR differential screening method for rapid isolation of clones from a cDNA library.

We have developed a new two-step differential screening method that allows the rapid isolation of induced or suppressed pure gene clones from a lambda phage cDNA library. This method involves a primary differential screening step and a PCR differential screening step. From the primary screening step, impure pools of positive cDNA clones are obtained. Each pool of clones is then amplified directly by PCR using two primers flanking the cloning site in the vector. The PCR products are run on two duplicate agarose gels and blotted onto two filter strips. These two filters are then subjected to differential Southern hybridization with different cDNA probes. Each pure positive cDNA band on the gel is identified and selected for further subcloning. This method has three advantages over the traditional differential screening method. First, several rounds of plaque rescreening are replaced by a single PCR screening. Second, plaque hybridization is replaced by a more reliable and accurate PCR DNA Southern hybridization. Third, the time-consuming and tedious phage DNA isolation step is eliminated, and subcloning is facilitated by direct cloning of the pure PCR product to a plasmid vector. We have successfully used this method to isolate ozone-responsive genes from a cDNA library of monkey respiratory airways.

Animals↗
PubMed1993

Isolation and characterization of the human spr1 gene and its regulation of expression by phorbol ester and cyclic AMP.

The small proline-rich protein gene (spr1) is a marker whose expression is frequently associated with squamous cell differentiation. We observed that the expression of the spr1 gene is strongly induced by phorbol 12-myristate 13-acetate (PMA). Both the time course result and the nuclear run-on transcriptional assay suggested that the regulation of spr1 expression by PMA is controlled at the transcriptional level. To understand the nature of this regulation, human genomic clones of the spr1 gene were isolated. DNA sequence analysis revealed that the human spr1 gene contains two exons and a single intron located within the 5'-untranslated region. An AP-1 binding site (TGAGTCA) is found at -142, and a putative cyclic AMP-responsive element (TGAGGTCA) at -597 base pairs upstream of the transcription start site. A chimeric construct containing the 5'-flanking region of the spr1 gene and the chloramphenicol acetyltransferase (CAT) reporter gene was used to transfect HeLa cells or monkey primary TBE cells. The CAT activity in transfected cells is stimulated 7.5-11-fold by PMA, and the stimulation is inhibited by a protein kinase C inhibitor or by pretreating cells with PMA to down-regulate the protein kinase C activity. The CAT activity is also stimulated 3.5-fold by dibutyryl cyclic AMP, a protein kinase A activator. The stimulations by PMA and cAMP are additive. These results suggest that protein kinase C and probably protein kinase A play important roles in regulating the transcription of the spr1 gene.

Amino Acid Sequence↗
PubMed1993

Expression of c-fos and c-jun family genes after focal cerebral ischemia.

The expression of the protooncogenes, c-fos, jun B, c-jun, and jun D was investigated in a rat focal cerebral ischemia model by Northern analysis and in situ hybridization. Severe ischemia (reduction of regional blood flow by 88-92%) in this model is confined to cerebral cortex irrigated by the right middle cerebral artery. Ischemia for 30 minutes, which caused only slight cortical damage (infarct size, < 10 mm3), induced both jun B and c-fos mRNAs exclusively in the right cerebral cortex. Ischemia for 90 minutes, which led to large cortical infarction (infarct size, > 140 mm3), also induced the expression of these two genes in the right cerebral cortex as well as the ipsilateral hippocampus. The latter sustained very mild ischemia (reduction of regional blood flow by 10-20%). The coinduction of jun B and c-fos expression occurred immediately after reperfusion and peaked at 60 minutes after reperfusion. The expression of c-jun was enhanced in a similar pattern, but at a much lower magnitude. In contrast, no change in jun D expression was observed. Nuclear run-on assays indicated that the increase in c-fos, jun B, and c-jun mRNA levels was due to the increase of transcription rate in these genes. Mobility shift assays showed a basal DNA binding activity of transcription factor AP-1 in the right cerebral cortex. Ischemia for 30 or 90 minutes followed by reperfusion for 4 hours resulted in a four- to sixfold increase of AP-1 binding activity. The enhanced DNA binding activity persisted for as long as 24 hours.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Expression of a squamous cell marker, the spr1 gene, is posttranscriptionally down-regulated by retinol in airway epithelium.

Vitamin A (retinol) is required for the normal mucociliary differentiation of respiratory epithelium. A depletion of vitamin A promotes squamous cell metaplasia. To understand how vitamin A suppresses squamous cell differentiation, the expression of a squamous cell differentiation marker, the small proline-rich protein gene (spr1), was studied in cultured monkey tracheobronchial epithelial (TBE) cells. The expression of the spr1 gene was inhibited about 40 fold by retinol. The mRNA levels of the spr1 gene started to decline within 6 h of retinol treatment and reached a minimum level after 7 days. The inhibition by retinol was concentration dependent and did not require concurrent protein synthesis. The inhibition of the spr1 mRNA by retinol was not due to a decrease in the transcription rate of its gene but due to a decrease in its stability, as determined by nuclear run-on assays and mRNA half-life measurement, respectively. This result was further supported by a DNA transfection study using a chimeric construct containing the spr1 promoter region and the chloramphenicol acetyltransferase (CAT) reporter gene. The CAT activity in transfected cells was not inhibited by retinol. These results suggest that spr1 gene expression is posttranscriptionally down-regulated by retinol.

Animals↗
PubMed1993

Molecular cloning and characterization of anther-preferential cDNA encoding a putative actin-depolymerizing factor.

A cDNA clone, LMP131A, which is preferentially expressed in mature anther was isolated from a lily cDNA library. Northern blot analysis and plaque hybridization experiments showed that the LMP131A mRNA is present at ca. 0.3% of the mRNA in mature pollen and is not detectable in carpel, petal, floral bud, leaf, or root. The clone contains an open reading frame of 139 amino acid residues which shows greater than 40% sequence identity in a 91 amino acid overlap to animal actin-depolymerizing factors (ADF), cofilin and destrin. The sequences at and near the actin-binding site are most conserved. Using the lily clone as a probe, a cDNA clone, BMP1, was isolated from a mature anther library of Brassica napus. The expression pattern of the BMP1 clone was the same as that of the lily clone. The Brassica anther-preferential clone contains an open reading frame which is 79% identical to the lily LMP131A protein. Southern blot analysis showed that there are one or a few copies of the putative ADF genes in B. napus and Arabidopsis thaliana.

Actin Depolymerizing Factors↗
PubMed1993

Identification of methyl jasmonate and salicylic acid response elements from the nopaline synthase (nos) promoter.

Transgenic tobacco plants carrying a fusion between the nopaline synthase (nos) promoter and chloramphenicol acetyltransferase (CAT) reporter gene (cat) were studied for their inducibility by salicylic acid (SA) or methyl jasmonate (MJ) treatments. Either chemical significantly increased CAT activity to a level much higher than that achieved by wounding. Northern blot analysis showed a corresponding increase in mRNA levels. After 20 h of induction of flowering plants, the response to MJ treatment was weaker in old leaves compared with young leaves, whereas the SA response was stronger in old leaves. Kinetic experiments showed that the SA response was much faster than the MJ response, suggesting that the induction mechanism of the nos promoter by these chemicals may differ. Deletion analysis showed that both SA and MJ responses require the DNA sequence between -119 and -112 from the transcription initiation site. This region contains the hexamer sequence (TGACGT) that has been found to be an important regulatory element for several promoters. The MJ response was also reduced by deletions of the CAAT box region or the sequence between -112 and -101, whereas the SA response was not significantly affected by these deletions. This suggests that the nos upstream region containing the hexamer motif is essential for the SA or MJ response and that the CAAT box region and the sequence immediately downstream from the hexamer motif are required for maximum induction by MJ.

2,4-Dichlorophenoxyacetic Acid↗
PubMed1993

A small proline-rich protein regulated by vitamin A in tracheal epithelial cells is induced in lung tumors.

In cell-free translations of RNA from primary cultures of pig trachea surface epithelial cells, we observed that a 20 kD proline-rich protein (sPRP) is induced during culturing (Biochem. Biophys. Res. Commun. 1990; 172:1304-1309). Subsequently, a cDNA encoding sPRP has been cloned from pig tracheal cell mRNA and sequenced. This cDNA shows a high similarity to cDNAs cloned from monkey tracheal cells cultured in vitamin A-free medium and from UV-irradiated human epidermal keratinocytes. Amino acid sequences from these cDNAs are exceptionally rich in proline, glutamine, cysteine, and lysine but contain no aromatic amino acids. Two repeats of 12 amino acids on the N-terminus are followed by multiple 8 amino acid repeats. When compared with monkey trachea and human keratinocyte cDNAs, the sPRP cDNA from pig trachea has an additional 24 bp nucleotide repeat. Antiserum raised to a synthetic peptide (23 amino acids) on the C-terminus of sPRP (C23-antiserum) reacted with the 20 kD sPRP in immunoprecipitations from cell-free translations. On Northern blot analysis, sPRP cDNA hybridized to RNAs of similar sizes in tracheal cells from cat, rabbit, and lamb. sPRP was not detected in tracheal cells that were cultured with 10(-9) M arotinoid. Since sPRP is considered a putative squamous cell differentiation marker, experiments using lung tumors were performed. sPRP mRNA levels were dramatically increased in squamous lung tumors that were induced by injecting hamsters with 4-(methylnitrosamino)- 1-(3-pyridyl)-1-butanone, a tobacco-specific nitrosamine. In situ hybridization with tissue sections prepared from these lung tumors revealed that cells around the keratin pearls contained high levels of sPRP mRNA.

Amino Acid Sequence↗