Skin autofluorescence is associated with severity of vascular complications in Japanese patients with Type 2 diabetes.

AIMS: Skin autofluorescence, a non-invasive measure of the accumulation for advanced glycation end products, has been reported to be a useful marker for diabetic vascular risks in the Caucasian population. The aim of this study was to evaluate associations between skin autofluorescence and vascular complications in non-Caucasian patients with Type 2 diabetes. METHODS: Subjects in this cross-sectional study comprised 130 Japanese patients with Type 2 diabetes. Skin advanced glycation end products were assessed by skin autofluorescence using an autofluorescence reader. Association between skin autofluorescence and severity of vascular complications was evaluated. RESULTS: Of the 130 patients, 60 (46.2%) had microvascular complications such as diabetic retinopathy, neuropathy and nephropathy, 10 (7.7%) had macrovascular complications and 63 (48.5%) had micro- and/or macrovascular complications. Skin autofluorescence increased with severity of vascular complications. Independent determinants of skin autofluorescence were age (ß = 0.24, P < 0.01), mean HbA(1c) in previous year (ß = 0.17, P = 0.03), microvascular complications (ß = 0.44, P < 0.01) and macrovascular complications (ß = 0.27, P < 0.01). Multiple logistic regression analysis revealed that diabetes duration (odds ratio 1.15, P < 0.01), systolic blood pressure (odds ratio 1.04, P = 0.01), skin autofluorescence (odds ratio 3.62, P = 0.01) and serum albumin (odds ratio 0.84, P < 0.01) were independent factors for the presence of vascular complications in these patients. CONCLUSIONS: Skin autofluorescence had independent effects on vascular complications in Japanese patients with Type 2 diabetes. This indicates that skin advanced glycation end products are a surrogate marker for vascular risk and a non-invasive autofluorescence reader may be a useful tool to detect high-risk cases in non-Caucasian patients with diabetes.

Gene expression of neurotrophins and their receptors in lead nitrate-induced rat liver hyperplasia.

Neurotrophins including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) are known to play important roles in the survival, proliferation, differentiation, and/or maintenance of function in several tissues including neuronal tissues. The role of neurotrophins in liver tissue, however, has not yet been clarified. In the present study, we assessed the temporal change in gene expression of neurotrophins, NGF, BDNF, and NT-3, and their receptors, low affinity neurotrophin receptor (p75NTR) and Trks A, B, and C, by RT-PCR technique in the liver of rats treated with lead nitrate (LN; 0.1 mmol/kg body weight), an inducer of liver hyperplasia. The mRNAs for NGF, BDNF with exon 4, NT-3, p75NTR, and all Trk members were detected in the LN-untreated liver. LN treatment resulted in increases in the levels of NGF, BDNF with exon 4, NT-3, p75NTR, and TrkA mRNAs and further led to expression of BDNF mRNA with exon 3. The increase in gene expression of neurotrophins and their receptors was closely correlated with those in liver weight. In this report, we propose for the first time that neurotrophins may play crucial roles in LN-induced liver hyperplasia.

Separation and characterization of octylphenol ethoxylate surfactants used by reversed-phase high-performance liquid chromatography on branched fluorinated silica gel columns.

The separation and characterization of octylphenol ethoxylate surfactants were carried out by reversed-phase high-performance liquid chromatography on branched fluorinated silica gel columns. For Triton X-100, simultaneous separation of octylphenol ethoxylate oligomers, positional isomers of octylphenyl group and butylphenol ethoxylate oligomers was achieved. These oligomers were completely separated and identified by means of MS spectra. Ethoxylated oligomers are eluted in the sequence from small to large oligomers. Fifty-five oligomers of Triton X-405 could be separated by using gradient elution. To separate octylphenol ethoxylate surfactant, non-end-capped branched fluorinated silica gel columns were superior to end-capped columns. The relationship between ln k' and methanol concentration was linear, indicating that branched fluorinated silica gel columns were operating in the reversed-phase mode. As Van 't Hoff plots of capacity factor for all oligomers gave straight lines, the equilibrium of conformation for the ethylene oxide chain might lay to one side of either zigzag or meander conformers.

A possible mechanism of TPA-mediated downregulation of neurotrophin-3 gene expression in rat cultured vascular smooth muscle cells.

We have previously reported that in cultured rat vascular smooth muscle cells (VSMCs), neurotrophin-3 (NT-3) gene expression was suppressed by TPA (12-O-tetradecanoyl phorbol-13-acetate), which induces an AP-1 transcription factor. In the present study, to clarify the mechanism for TPA-mediated downregulation of NT-3 gene expression, effects of cycloheximide and dexamethasone (Dex) on the TPA-mediated downregulation were examined in VSMCs. Pretreatment with cycloheximide, an inhibitor of protein synthesis, or simultaneous treatment with Dex, an inhibitor of AP-1, suppressed the TPA-mediated downregulation of NT-3 gene expression. Furthermore, co-transfection of c-fos and c-jun expression vectors into VSMCs resulted in decrease in the NT-3 gene expression. The present findings suggest that TPA-induced AP-1 de novo synthesis causes the downregulation of NT-3 gene expression in VSMCs.

Novel alternative splicing in the 5' exon of the neurotrophin-3 gene.

The neurotrophin-3 (NT-3) gene has previously been reported to consist of three exons including two 5' short untranslated exons and a 3' long exon encoding the entire protein, and to give rise to two classes of transcripts by alternative splicing of the 5' exons to the 3' coding exon. In the present study, we demonstrated the presence of at least four new classes of transcripts of the NT-3 gene, in addition to the two known transcripts. The present finding proposes the further complexity of the regulational mechanism for NT-3 expression.

Gene expression of neurotrophins and their receptors in cultured rat vascular smooth muscle cells.

Most previous researches on neurotrophins including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) have focused on the nervous system, because their receptors are widely distributed in neuronal tissues. Recently, however, the participation of neurotrophins in inflammation and atherosclerosis has been proposed. Therefore, the gene expression of neurotrophins is now an urgent issue is to be investigated in nonneuronal tissues. Here, we evaluated the gene expression of neurotrophins and their receptors in rat cultured vascular smooth muscle cells (VSMCs) by the reverse transcriptase-polymerase chain reaction method. The transcripts of NGF, NT-3, and TrkC (high-affinity receptor for NT-3), and two BDNF alternative spliced transcript variants with exons 3 and 4 were clearly detected in VSMCs cultured under conventional culture conditions. The upregulation of mRNA levels for NGF, two BDNF variants with exons 1 and 2, low-affinity neurotrophin receptor, and high-affinity receptors, TrkA (for NGF) and TrkB (for BDNF), was observed in response to the treatment with serum and phorbol-ester following the serum-starvation. In contrast, the expression of NT-3 and TrkC genes was downregulated under these conditions. Co-expression of these factors and their receptors and the characteristic regulation of their gene transcriptions suggest that these factors play crucial roles in the function of VSMCs through an autocrine mechanism.

Mutation of the trkB gene encoding the high-affinity receptor for brain-derived neurotrophic factor in stroke-prone spontaneously hypertensive rats.

Brain-derived neurotrophic factor and its receptor, trkB, are thought to play a crucial role for protection against neuronal death induced by brain ischemia, such as in stroke. In the present study we found a missense mutation in the trkB gene from all of the five substrains of stroke-prone spontaneously hypertensive rats (SHRSP) that were examined. This mutation was not found in six out of seven hypertensive but stroke-resistant ancestral strains (SHR) of SHRSP, nor in any of seven strains of normotensive, non-stroke-prone strains. Hippocampal neurons, which are particularly vulnerable to damage in stroke, were shown to be more susceptible to ischemic damage in SHRSP than in either SHR or normotensive, stroke-resistant controls. The association of a mutated trkB gene with the stroke-prone genotype found in this study suggests that the trkB gene merits further study as a promising candidate gene for stroke.

Characterization and transcriptional regulation of rat glutamine tRNA-encoding genes: repression of tRNA(UUGGln) synthesis.

In rat liver, the amount of tRNA(UUGGln) (RG-2) was found to be approx. 20% of that of tRNA(CUGGln) (RG-1). The independent RG-1 and RG-2 genes were isolated from a rat genomic library together with four RG-related genes containing two to five alterations in their coding regions. Sequence analysis demonstrated that there was no difference between the internal promoter sequences of RG-1 and RG-2. However, interestingly, the transcriptional activity of RG-1 was approximately four-times higher than that of RG-2 in an in vitro transcription reaction. Replacement of the 5'-flanking sequence of RG-2 by the corresponding sequence of RG-1 or by a plasmid DNA sequence caused activation of RG-2 transcription. Gel retardation assay demonstrated that the 5'-flanking region of RG-2 contained a unique sequence specifically recognized by a nuclear protein. Taken together, these results strongly suggest that the transcriptional activity of RG-2 might be negatively regulated by the binding of a nuclear protein at a specific site in the 5'-flanking region of the gene.

Retrovirus infection and aging: Increase in UAG suppressor tRNA expression in aged mice.

The effect of aging on expression of a natural glutamine suppressor tRNA (tRNA(Gln(UmUG))) was studied in different tissues of mice; this tRNA recognizes UAG and inserts glutamine at the site of the termination codon. The level of tRNA(Gln(UmUG)) was found to be strongly increased in aged mice, compared to newborn and mature animals. An elevated expression of tRNA(Gln(UmUG)) has also been found in retrovirus-infected cells; in Moloney virus-infected cells the suppressor tRNA allows to read-through the UAG codon within the retroviral protease gene. We suggest that the increase in the level of tRNA(Gln(UmUG)) may influence retroviral gene expression with age.

Insertion (sufB) in the anticodon loop or base substitution (sufC) in the anticodon stem of tRNA(Pro)2 from Salmonella typhimurium induces suppression of frameshift mutations.

The dominant +1 frameshift suppressors sufA6, sufB1 and sufB2, in Salmonella typhimurium act at runs of C and affect tRNA(Pro)1, tRNA(Pro)2 and tRNA(Pro)2, respectively. A recessive +1 frameshift suppressor, sufC, has a similar suppressor specificity (Riddle, D.L., and Roth, J.R., Mol. Biol. 66, 483 and 495, 1972). We show that sufC strains harbour two frameshift suppressors of which one, sufX201, is allelic to sufB. We cloned the sufB+ wild type allele and by recombination in vivo the mutations sufB1, sufB2 and sufX201. Determination of the DNA sequence revealed that the sufB1 and sufB2 mutations result in an extra G in the anticodon loop of the minor tRNA(Pro)2. The sufX201 mutation results in a base substitution (G43 to A43) in the anticodon stem of this tRNA. Although the sufB1 and sufB2 mutations were earlier shown to be dominant, the sufB+ wild type allele on multi copy plasmid inhibited the chromosomal sufB1, sufB2 and sufX201 mediated frameshift suppression but not that mediated by the dominant sufA6 mutation. These results are discussed in view of the possible coding specificity of these mutated tRNAs. The DNA sequence showed a potential consensus promoter sequence upstream of the structural gene for tRNA(Pro)2 and downstream a dyad symmetrical structure followed by a T cluster, a possible rho-independent termination signal. The Salmonella tRNA(Pro)2 gene is identical to the Escherichia coli counterpart reported by Komine, Y. et al. (J. Mol. Biol. 212, 579-598, 1990). While the 5' flanking sequence similarity between the two species is about 83%, the similarity of the 3' flanking sequence is only 42%. Still, the Salmonella tRNA(Pro)2 gene has a rho-independent transcriptional termination signal similar to the one present in E. coli tRNA(Pro)2 gene.

Analysis of Moloney murine leukemia virus revertants mutated at the gag-pol junction.

Among Moloney murine leukemia viruses (Mo-MuLVs) having stop codons other than UAG at the gag-pol junction, Mo-MuLV with UAA, but not with UGA, had a replication disadvantage. Mo-MuLV with a glutamine codon (CAG) at the junction did not replicate. A revertant of this virus consisted of the original virus and a virus with a deletion of the pol region. Protease and Pr65gag encoded by their respective genomes complemented each other.

Isolation and characterization of s-myc, a member of the rat myc gene family.

A 9-kilobase-pair clone containing a myc-related gene that we have designated s-myc was isolated from a rat genomic library. The entire nucleotide sequence of the cloned DNA was determined, showing that the s-myc gene contains an open reading frame consisting of 1287 base pairs without introns. In vitro transcription-translation analysis of s-myc indicated that this gene produces a protein of approximately 50 kDa. The amino acid sequence predicted from the DNA sequence showed that the s-Myc protein is closely related to the murine N-Myc protein but lacks an acidic amino acid-rich sequence commonly present in the Myc-family proteins. Studies on transfection of s-myc into rat RT4-AC tumor cells revealed that the gene produces a polyadenylylated transcript of approximately 4.7 kilobases and that its high-level expression suppresses the tumor-igenicity of RT4-AC tumor cells in nude mice.

[Studies related to the discoloration in the surrounding gingiva of fixed prosthesis. 2. Detection period of silver-sulfate and the effects of metal alloys].

The purpose of this study is to investigate the relationship between gingival discoloration and Ag2S. Minute granules of dental alloys were embedded in the gingiva during a 5-11 months span, and their histological and colorimetric analyses were undertaken. Results showed the following; 1. Cases of low melting silver alloy, 12% Au-Ag-Pd alloy, and Ni-Cr alloy all resulted in gingival discoloration, but it did not occur during the specified experimental period. 2. Regardless of the size of the granules or time, the electron microanalyser showed no signs of sulfer detection in cases of low melting silver alloy. However, in the case of 12% Au-Ag-Pd alloy, sulfer was detected 5 months later for only those granules having the size of 0.2-0.3 microns. No sulfer was detected in cases of Ni-Cr alloy. 3. Although no proliferation of inflammatory cells were observed around those areas surrounding the 12% Au-Ag-Pd alloy granules, they appeared in cases of the low melting silver alloy and Ni-Cr alloy.

Detection of reactivating pseudorabies virus in tissue by immunoperoxidase technique.

Reactivation of pseudorabies virus (PRV) was induced in pigs by prednisolone treatment. The virus was re-isolated from nasal secretions of four, brain cortex of two and mandibular lymph node of three out of 12 pigs, respectively. The characteristic lesion of recurrently infected pigs was focal necrosis in the brain cortex and mandibular lymph node, accompanied by reactivating virus particles in the degenerating cells. Coincident with the pathological lesions, PRV antigen was detected in two of the brain cortex and six of the mandibular lymph node specimens by immunoperoxidase staining. These results suggest that the immunoperoxidase technique was more sensitive than virus isolation for demonstrating the reactivity of PRV in recurrently infected pigs.

The effect of specific mutations at and around the gag-pol gene junction of Moloney murine leukaemia virus.

By carrying out oligonucleotide-directed mutagenesis, in vitro, on a 3.3 kb XhoI-HindIII fragment from Moloney murine leukaemia virus Mo-MuLV proviral DNA, inserted into the phagemid pTZ19R, nine separate fragments have been prepared in which mutations have been inserted at and around the gag-pol gene junction. Using these mutant fragments Mo-MuLV proviral DNA has been reassembled and cloned into pBR322. Examination of the mutant proviral DNAs in mouse culture cells indicates that a terminator codon at the gag-pol junction is essential for function, but any of the three chain terminator codons gives an active virus. Also the region of secondary structure surrounding the terminator codon must be preserved.

Codon and amino-acid specificities of a transfer RNA are both converted by a single post-transcriptional modification.

An Escherichia coli isoleucine transfer RNA specific for the codon AUA (tRNA(2Ile) or tRNA(minorIle] has a novel modified nucleoside, lysidine in the first position of the anticodon (position 34), which is essential for the specific recognition of the codon AUA. We isolated the gene for tRNA(2Ile) (ileX) and found that the anticodon is CAT, which is characteristic of the methionine tRNA gene. Replacement of L(34) of tRNA(2Ile) molecule enzymatically with unmodified C(34) resulted in a marked reduction of the isoleucine-accepting activity and, surprisingly, in the appearance of methionine-accepting activity. Thus, both the codon and amino-acid specificity of this tRNA are converted by a single post-transcriptional modification of the first position of the anticodon during tRNA maturation.