Molecular cloning and expression vector construction of bovine TRIM28.

The bovine TRIM28 gene was amplified from ovary tissue by using RT-PCR. The TRIM28 gene was inserted into the eukaryotic expression vector pIRES2-EGFP and transfected into bovine fetal fibroblasts by using Lipofectamine 3000. TRIM28 mRNA and protein were detected by fluorescence microscope and western blotting. The results showed that the full length of TRIM28 was cloned and pIRES2-EGFP-TRIM28 was constructed successfully. EGFP expression was observed, and the pIRES2-EGFP-TRIM28 transfected group expressed more TRIM28 protein than that by the pIRES2-EGFP group. The TIMR28 gene has been successfully transferred into bovine fetal fibroblasts.

Increase in the expression of human leukocyte antigen class I in human fibroblasts by soluble factors secreted from human cytomegalovirus-infected cells.

The human cytomegalovirus (HCMV) is known to downregulate the expression of the human leukocyte antigen (HLA) class I for escape from immune surveillance. In order to understand the HCMV immune evasion mechanism, expression of HLA class I on the surface of HCMV-infected cells was investigated. A decrease in the HLA class I expression was observed at higher MOI; whereas at a lower MOI a slight increase in the HLA class I expression was observed. When HCMV-infected and uninfected cells were separately prepared on coverslips and co-cultured, the increased HLA class I expression was observed in uninfected cells. Treatment of the uninfected cells with the culture supernatant from HCMV-infected cells resulted in an increase in the HLA class I expression. A biochemical analysis of the HCMV-infected cell culture supernatant revealed the presence of interferon (IFN) beta interleukin (IL)-1beta, and IL-6. The HLA class I-enhancing activity of the culture supernatant was mimicked by IFN beta, but not by IL1-beta or IL-6, and was partially reversed by pretreatment with an antibody to IFN beta. Therefore, it appears that the HCMV infection of human foreskin fibroblast cells induces interferon beta and other soluble factor(s) that are responsible for the up-regulation of the HLA class I expression.

Isolation of the Bacillus subtilis cdd downstream region and analysis of genetic structure around the cdd vicinity.

A 310 bp EcoRI/HindIII fragment downstream of the Bacillus subtilis cdd gene was isolated from pSO52 which harbours the cdd gene and its vicinity, and was inserted into the pDIA5304. The hybrid vector pSO701 was integrated into the targeted locus of the B. subtilis chromosome and the cdd downstream region was rescued by furthermost BamHI walking toward the sigA locus. By sequencing and analyzing the intergenic region between the cdd and p23-dnaG-sigA operon from the selected clone pSO702, the era genes encoding glycyl tRNA synthetase alpha and beta chains with two unknown distal genes at both terminals were identified and mapped. The cdd was separated by 6,964 bp from the p23-dnaG-sigA operon. Combined data with an additional analysis of the cdd upstream from the sequenced 280 kb stretch in GenBank database (accession No. D84432) indicates that the revised gene order like hrcA-grpE-dnaK-dnaJ-phoH-dgk-cdd-era-tRNA( gly) synthetase alpha and beta-p23-dnaG-sigA-cccA with unidentified distal genes was established genetically on a counter clockwise orientation at 225 degrees of the linkage map.

Distribution of polycyclic aromatic hydrocarbons in agricultural soils in South Korea.

The content and type of polycyclic aromatic hydrocarbons (PAH) in soils from paddy fields and upland areas in South Korea were determined using gas chromatography linked to mass spectrometry (GC-MS). The distribution map of total PAH content was obtained as a contour plot using a geographical information system. The overall distribution of PAH was found to be closely related to the pollution sources, the size of city and the type of industry. The average content of total PAH in all samples was 236 microgkg(-1), and the range was from 23.3 to 2834 microgkg(-1). The highest concentrations were found in soils sampled near iron processing plants. The concentration of PAH decreased in the order fluoroanthene>benzo(b)fluoroanthene>pyrene. Special PAH compound ratios, such as phenanthrene/anthracene and fluoroanthene/pyrene, were calculated to evaluate the origin. The collected data suggested that the pyrogenic origins such as motor vehicle exhaust and heavy industry emission were the dominant source of PAH in Korean soils.

Expression of heat shock protein 72 in rat cochlea with cisplatin-induced acute ototoxicity.

Cisplatin ototoxicity is known to involve mainly the organ of Corti. Outer hair cells (OHCs). especially in the basal turn, are preferentially involved. One possible mechanism of ototoxicity might be alteration of the antioxidant system causing an increase in free radicals. It has been demonstrated that heat shock proteins (HSPs), which are believed to protect cells by dissolving and refolding misfolded or denatured protein are induced by various form of stress. HSP is also demonstrated to be induced by free radicals. The purpose of this study was to evaluate HSP 72 induction in cochlea following cisplatin injection in the animal model. Sprague-Dawley (SD) rats were injected intraperitoneally with normal saline as control or cisplatin at a dose of 5, 10 or 20 mg/kg. Cochleae were harvested 1, 3, 6 and 12 h after injection and compared with those of controls. Immunocytochemical study with surface preparation and Western blotting were performed to investigate the expression of HSP 72. Auditory brainstem response (ABR) was also recorded to assess functional change according to the dosage of cisplatin and duration after injection. In the 5 and 10 mg/kg groups, immunostaining for HSP 72 in the OHCs reached a plateau level at 3 h, which was maintained until 12 h after injection. The amount of immunoreactive OHCs in the 20 mg/kg group was smaller than those in 5 and 10 mg/kg groups and declined after 6 h. The bands for HSP 72 became less intense as the cisplatin dosage increased from 5 to 10 and 20 mg/kg in Western blotting. The change in ABR threshold was small in the 5 and 10 mg/kg groups and a marked change in threshold was observed in the 20 mg/kg group. Detection of HSP 72 after cisplatin injection could confirm the OHCs as one of the major injured cells in the cochlea. With a lethal dosage of cisplatin (20 mg/kg), HSP 72 expression was less prominent and declined after 6 h.

Chromosomal location, cloning and nucleotide sequence of the Bacillus subtilis cdd gene encoding cytidine/deoxycytidine deaminase.

The Bacillus subtilis cdd gene encoding cytidine/2'-deoxycytidine deaminase has been located by transduction at approximately 225 degrees on the chromosome, and the gene order trpC-lys-cdd-aroD was established. The gene was isolated from a library of B. subtilis DNA cloned in lambda D69 by complementation of an Escherichia coli cdd mutation. Minicell experiments revealed a molecular mass of 14,000 dalton for the cytidine deaminase subunit encoded by the cloned DNA fragment. The molecular weight of the native enzyme was determined to be 58,000, suggesting that it consists of four identical subunits. The nucleotide sequence of 1170 bp, including the cdd gene, was determined. An open reading frame encoding a polypeptide with a calculated molecular mass of 14,800 dalton was deduced to be the coding region for cdd. The deduced amino acid composition of the 136-amino acid-long subunit shows that it contains six cysteine residues. A computer search in the GenBank DNA sequence library revealed that the 476 bp HindIII fragment containing the putative promoter region and the first ten codons of cdd is identical to the P43 promoter-containing fragment previously isolated by Wang and Doi (1984). They showed that the fragment contained overlapping promoters transcribed by B. subtilis sigma 43 and sigma 37 RNA polymerase holoenzymes during growth and stationary phase.

A rearrangement of the CDD gene at the 5' UTR produces two types of transcripts that contain a natural antisense region.

In order to study the regulatory region for transcription, the genomic DNA of the human CDD gene was cloned and analyzed. In contrast to previously reported CDD cDNA sequence, the sequence of the isolated genomic clone was rearranged at the 5' untranslated region (UTR) by a 30 bp inversion and a 57 bp insertion. Polymerase chain reaction (PCR) of chromosomal DNA and mRNA, and sequencing analysis of the PCR products revealed that sequences corresponding to both the genomic clone and the cDNA clone were present in the chromosomal DNA and were also transcribed into mRNA. Because of the 30 base inversion, the two types of CDD transcripts contain antisense sequences in their 5' UTR. Their role in the regulation of CDD expression is discussed.

Effects of adenotonsillar hypertrophy on snoring in children.

A prospective study was performed to evaluate the effects of adenotonsillar hypertrophy on snoring in children. Thirty male patients were grouped with respect to the severity of snoring and were evaluated in terms of the noise level of the snoring sound, the lowest arterial oxygen saturation, degree of palatine tonsillar hypertrophy, body mass index and cephalometric findings that included the adenoidal-nasopharyngeal ratio, the angle between the lines sella turcica/nasion and most posterior point of anterior maxilla/nasion, the angle between the lines sella turcica/nasion and most posterior point of anterior mandible/nasion, the posterior airway space, the distance from the sella to the nasion, lower face height and the distance from the basion to the posterior nasal spine. The noise level of the snoring sound, the lowest arterial oxygen saturation and the adenoidal-nasopharyngeal ratio showed a significant correlation with the severity of snoring, but the degree of palatine tonsillar hypertrophy and the body mass index failed to disclose any significant relationship.

Recovery of nasal physiology after functional endoscopic sinus surgery: olfaction and mucociliary transport.

We assessed the changes in olfaction and mucociliary transport after functional endoscopic sinus surgery (FESS) in 80 patients with chronic paranasal sinusitis. Olfaction was evaluated with the butanol threshold test and mucociliary transport was assessed by saccharin transit time (STT). Postoperative butanol threshold scores were significantly reduced (p < 0.01), and the changes were more profound in severer forms of paranasal sinusitis as graded by ostiomeatal-unit computed tomography. The mean preoperative STT (27 min) which was significantly longer than that of controls (12 min) was significantly reduced 1, 6 and 12 months following FESS (p < 0.01). The results suggest that impairment of olfaction and mucociliary transport in chronic paranasal sinusitis may be significantly improved following FESS.

The Bacillus subtilis genome from gerBC (311 degrees) to licR (334 degrees).

As part of the international project to sequence the Bacillus subtilis genome, the DNA region located between gerBC (311 degrees) and licR (334 degrees) was assigned to the institut Pasteur. In this paper, the cloning and sequencing of 176 kb of DNA and the analysis of the sequence of the entire 271 kb region (6.5% of the B. subtilis chromosome) is described; 273 putative coding sequences were identified. Although the complete genome sequences of seven other organisms (five bacteria, one archaeon and the yeast Saccharomyces cerevisiae) are available in public database, 65 genes from this region of the B. subtilis chromosome encode proteins without significant similarities to other known protein sequences. Among the 208 other genes, 115 have paralogues in the currently known B. subtilis DNA sequences and the products of 178 genes were found to display similarities to protein sequences from public databases for which a function is known. Classification of these genes shows a high proportion of them to be involved in the adaptation to various growth conditions (non-essential cell wall constituents, catabolic and bioenergetic pathways); a small number of the genes are essential or encode anabolic enzymes.