[Site-specific microbiological tests and the diagnostic significance-- central nervous system infections and meningitis].
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Encephalitis↗
Biomedical subjects
T Haruta
Publications and source records attributed to T Haruta.
[Anterior pituitary hormones--mainly about growth hormone].
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Acromegaly↗
Evidence for an insulin receptor substrate 1 independent insulin signaling pathway that mediates insulin-responsive glucose transporter (GLUT4) translocation.
Interaction of the activated insulin receptor (IR) with its substrate, insulin receptor substrate 1 (IRS-1), via the phosphotyrosine binding domain of IRS-1 and the NPXY motif centered at phosphotyrosine 960 of the IR, is important for IRS-1 phosphorylation. We investigated the role of this interaction in the insulin signaling pathway that stimulates glucose transport. Utilizing microinjection of competitive inhibitory reagents in 3T3-L1 adipocytes, we have found that disruption of the IR/IRS-1 interaction has no effect upon translocation of the insulin-responsive glucose transporter (GLUT4). The activity of these reagents was demonstrated by their ability to block insulin stimulation of two distinct insulin bioeffects, membrane ruffling and mitogenesis, in 3T3-L1 adipocytes and insulin-responsive rat 1 fibroblasts. These data suggest that phosphorylated IRS-1 is not an essential component of the metabolic insulin signaling pathway that leads to GLUT4 translocation, yet it appears to be required for other insulin bioeffects.
3T3 Cells↗
Activated phosphatidylinositol 3-kinase is sufficient to mediate actin rearrangement and GLUT4 translocation in 3T3-L1 adipocytes.
Insulin stimulation of 3T3-L1 adipocytes causes rapid translocation of actin and the GLUT4 glucose transporter to the plasma membrane. Both processes depend on the activity of phosphatidylinositol 3-kinase. Using single cell microinjection, we have transiently expressed a constitutively activated mutant of phosphatidylinositol 3-kinase, p110*, in 3T3-L1 adipocytes. Fluorescent detection of GLUT4 protein and actin within these cells demonstrates that expression of p110* is sufficient to cause translocation of GLUT4 to the plasma membrane and the formation of actin membrane ruffles. These effects are inhibited by wortmannin in the p110*-expressing cells, indicating that the phosphatidylinositol 3-kinase activity of the protein is required. Overexpression of an identical protein containing a point mutation in the kinase domain, p110*Deltakin, was incapable of mediating either action, confirming that neither the microinjection process nor a nonspecific effect of the protein was responsible for the observed effects. These data suggest that although insulin is capable of inducing numerous signaling pathways, the isolated activation of phosphatidylinositol 3-kinase can initiate the signaling cascade leading to both actin rearrangement and GLUT4 translocation in the absence of insulin stimulation.
3T3 Cells↗
Hsp70 family molecular chaperones and mutant insulin receptor: differential binding specificities of BiP and Hsp70/Hsc70 determines accumulation or degradation of insulin receptor.
We have examined the binding specificities of Hsp70 family molecular chaperones, BiP and Hsp70/Hsc70, to wild-type or mutant insulin receptors. BiP bound to proreceptor of wild-type insulin receptor, but not to mature receptor. A mutant insulin receptor, which lacked 47 amino acid residues (delta Ex13 IR) corresponding to exon 13 of insulin receptor gene, accumulated in the endoplasmic reticulum as uncleaved proreceptor with immature oligosaccharide chains. This deletion mutant bound to BiP more tightly than wild type. Introduction of two types of mutations, Asp1179 or Leu1193, into delta Ex13 IR led to accelerated degradation, and these double mutants bound weakly to BiP. In contrast, Ser735 insulin receptor was normally transported to the plasma membrane and normally bound to BiP. Furthermore, Asp1179, Leu1193 insulin receptors and delta Ex13 IR combination mutant with either Asp1179 or Leu1193 bound more tightly to Hsp70/Hsc70 compared with wild-type, Ser735, and delta Ex13 IR. These results suggest that the binding specificity of mutant insulin receptors to two molecular chaperones, i.e., BiP and Hsp70/Hsc70, plays an important role for their posttranslational processing that may lead to the accumulation in the endoplasmic reticulum or the degradation of insulin receptors.
Animals↗
The dominant negative effect of a kinase-defective insulin receptor on insulin-like growth factor-I-stimulated signaling in Rat-1 fibroblasts.
To study the interaction between insulin receptor (IR) and insulin-like growth factor-I (IGF-I) receptor (IGF-IR) tyrosine kinases, we examined IGF-I action in Rat-1 cells expressing a naturally occurring tyrosine kinase-deficient mutant IR (Asp 1048 IR). IGF-I normally stimulated receptor autophosphorylation, IRS-I phosphorylation, and glycogen synthesis in cells expressing Asp 1048 IR. However, the Asp 1048 IR inhibited IGF-I-stimulated thymidine uptake by 45% to 52% and amino acid uptake (aminoisobutyric acid [AIB]) by 58% in Asp 1048 IR cells. Furthermore, IGF-I-stimulated tyrosine kinase activity toward synthetic polymers, Shc phosphorylation, and mitogen-activated protein (MAP) kinase activity was inhibited. The inhibition of mitogenesis and AIB uptake was restored with the amelioration of the impaired tyrosine kinase activity and Shc phosphorylation by the introduction of abundant wild-type IGF-IR in Asp 1048 IR cells. These results suggest that the Asp 1048 IR causes a dominant negative effect on IGF-IR in transmitting signals to Shc and MAP kinase activation, which leads to decreased IGF-I-stimulated DNA synthesis, and that the kinase-defective insulin receptor does not affect IGF-I-stimulated IRS-I phosphorylation, which leads to the normal IGF-I-stimulated glycogen synthesis.
Animals↗
Enhancing effects of unsaturated fatty acids with various structures on the permeation of indomethacin through rat skin.
Effects of straight-chain, cis-type, unsaturated fatty acids with various structures (alkyl chain lengths, numbers of double bonds, position of double bonds, and cis- and trans-positional isomers) on the skin permeation of indomethacin were examined by using rat skins in-vitro. Furthermore, the disordering degrees of the intercellular lipid domain in the stratum corneum, which were treated with preparations of unsaturated fatty acids, were measured by the Fourier transform infrared (FT-IR) method using excised rabbit ear skins. Unsaturated fatty acids enhanced the permeation of indomethacin through rat skins. These permeation-enhancing effects by unsaturated fatty acids were affected by changes of their alkyl chain length from C14 to C22. The lag-times on the permeation of indomethacin were shortened by unsaturated fatty acids in the following order: C20 = C18 = C22 < C16 < C14. These fluxes were increased by unsaturated fatty acids in the following order: C20 > C22 = C18 = C16 > C14. Therefore, gondoic acid (cis-11-eicosenoic acid; C20H38O2) mostly enhanced the skin permeation of indomethacin. However, the enhancing effects of unsaturated fatty acids (C18 chain) were not affected by their differences of position and numbers of double bonds. These permeation-enhancing effects which were evaluated by flux were related to the degrees of wave-number shift in the frequency of the antisymmetric CH bond stretching absorbance (near 2920 cm-1) on FT-IR spectra of the fatty acid-treated stratum corneum. Therefore, the perturbation increase of lipid domain in the stratum corneum by these fatty acids probably was the cause of the enhancing effects of permeation of indomethacin.
Animals↗
[Transferability of cefozopran to cerebrospinal fluid in rabbits with meningitis caused by Staphylococcus aureus].
The transferability of cefozopran (CZOP) to cerebrospinal fluid (CSF) was studied employing rabbits with experimental meningitis caused by Staphylococcus aureus. The mean plasma concentration was 293 +/- 17.6 micrograms/ml at 15 minutes after intravenous administration of CZOP at a dose level of 100 mg/kg. The mean concentration in CSF reached its maximum, 16.5 +/- 2.74 micrograms/ml at 60 minutes after administration. Pharmacokinetic parameters calculated from these values were as follows: Cmax (CSF/plasma) 5.72%, AUC (CSF/plasma) 6.61% between 15 and 60 minutes, 9.38% between 15 and 120 minutes and 11.2% between 15 and 180 minutes, T 1/2 for CZOP in CSF: 138 minutes, T 1/2 (CSF/plasma): 2.81. In comparison to those of beta-lactams that were obtained in the same way, the transferability of CZOP to CSF was moderate but concentration in CSF was high, hence, in consideration of the antimicrobial potency against the main pathogens of meningitis, it appears worthwhile of running clinical trials for CZOP.
Animals↗
Stability of free Mg2+ concentration and increased concentration of free Ca2+ in vascular smooth muscle cells during dietary magnesium deficiency in rat.
Intracellular free calcium and magnesium ion concentrations ([Ca2+]i and [Mg2+]i respectively) were estimated in thoracic aorta smooth muscle strips isolated from magnesium-deficient and control rats using fura-2/AM and mag-fura-2/AM, respectively. Adult male Wistar rats were fed a magnesium-deficient diet (10 mg Mg/kg diet) or a control diet (700 mg Mg/kg diet) for 30 days. Plasma magnesium level in magnesium-deficient rats was half of that in control rats at 30th day. Therefore, thoracic aorta strips, denuded of endothelium, were loaded with fura-2/AM or mag-fura-2/AM in the presence of 0.5 or 1.0 mM Mg2+, and [Ca2+]i or [Mg2+]i was measured under the same Mg2+ conditions. The [Ca2+]i in the aorta strips isolated from magnesium-deficient rats in the presence of 0.5 mM Mg2+ (254.9 +/- 13.1 nM) was approximately three times greater than in those from control rats in the presence of 1.0 mM Mg2+ (86.5 +/- 9.2 nM). The [Mg2+]i was not significantly different between the two groups at either Mg2+ level. The muscle tension and [Ca2+]i increased after [Mg2+]o was exchanged from 1.0 to 0.5 mM; however, [Mg2+]i showed no change. The total calcium content increased and total magnesium content decreased in thoracic aorta strips isolated from magnesium-deficient rats. These results suggest that [Mg2+]i is stable, but that [Ca2+]i increases in vascular smooth muscle cells of thoracic aortas isolated from dietary magnesium-deficient rats.
Animals↗
[Therapeutic efficacy of azithromycin in pediatrics].
Azithromycin (AZM), a newly developed azalide antibiotic, was administered at a standard dose of 10 mg/kg once daily for 3 days to pediatric patients with bacterial infections and the therapeutic efficacy of AZM was investigated. 1. A total of 12 patients with the following diseases was evaluated: pharyngitis in two, tonsillitis in four, bronchitis in one, Mycoplasma pneumonia in one, scarlet fever in two and enteritis in two. The drug was rated "excellent" in eight cases and "good" in four. 2. Eleven strains were isolated from patients: five strains of Streptococcus pyogenes, four strains of Haemophilus influenzae, and two strains of Haemophilus parainfluenzae. Isolated bacteria were eradicated in eight strains and persisting in one, resulting in 88.9% in eradication rate. No follow-up examinations in post-treatment were performed in two cases. 3. No adverse reaction was reported, while one case of eosinophilia was noted as an abnormal laboratory test value. 4. As far as compliance is concerned, patients claimed that the formulation of the drug is "easy to take" or "ordinary". With the results presented as above, we have concluded that AZM is a useful antibiotic in pediatric patients with bacterial infections.
Administration, Oral↗
Insulin-stimulated GLUT4 translocation is mediated by a divergent intracellular signaling pathway.
Insulin stimulates glucose transport largely by mediating translocation of the insulin-sensitive glucose transporter (GLUT4) from an intracellular compartment to the plasma membrane. Using single cell microinjection of 3T3-L1 adipocytes, coupled with immunofluorescence detection of GLUT4 proteins, we have determined that inhibition of endogenous p21ras or injection of oncogenic p21ras has no effect on insulin-stimulated GLUT4 translocation. On the other hand, microinjection of anti-phosphotyrosine antibodies or inhibition of endogenous phosphatidylinositol 3-kinase by microinjection of a GST-p85 SH2 fusion protein markedly inhibits this biologic effect of insulin. These data suggest that the p21ras/mitogen-activated protein kinase pathway is not involved in this metabolic effect of insulin, whereas tyrosine phosphorylation and stimulation of phosphatidylinositol 3-kinase activity are critical components of this signaling pathway.
1-Methyl-3-isobutylxanthine↗
Signal transduction pathways leading to insulin-induced early gene induction.
We examined the signal transduction pathway leading to insulin stimulation of two immediate early genes, c-fos, and the early growth response gene, Egr-1. In Rat 1 fibroblasts overexpressing normal human insulin receptors (HIRc-B), insulin and IGF-I rapidly and transiently induced the expression of both c-fos and Egr-1 mRNA with maximum accumulation at 30 min, declining to basal levels at 120 min. Insulin (100 ng/mL) increased c-fos and Egr-1 mRNA expression 10-fold (EC50 = 20 ng/mL), whereas IGF-I (100 ng/mL) and serum (20%) led to a 3- and 11.5-fold increase, respectively. Insulin-stimulated c-fos protein expression was maximal at 1 h postinduction and undetectable at 4 h. The effects of insulin and IGF-I on both c-fos mRNA and protein expression were absent in Rat 1 fibroblasts expressing tyrosine kinase-defective human insulin receptors (A/K1018). In cells expressing insulin receptors in which the two C-terminal tyrosines are mutated to phenylalanine (Y/F2 cells), the insulin stimulated increase in Egr-1 and c-fos mRNA was comparable to that of HIRc cells, whereas, in cells expressing C-terminal truncated receptors (delta CT cells), the insulin induced increase in Egr-1 mRNA was normal, but the c-fos mRNA response was severely blunted. As expected, the insulin effect to increase ras GTP formation and MAP kinase activity was negligible in A/K1018 cells but normal, or supernormal, in Y/F2 cells. Importantly, stimulation of ras GTP was increased in delta CT cells, whereas stimulation of MAP kinase activity was almost absent.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
An extracellular domain of the beta subunit is essential for processing, transport and kinase activity of insulin receptor.
The extracellular portion of the insulin receptor (IR) beta-subunit has four cysteine and four asparagine residues which are potentially involved in disulphide bond formation between the alpha- and beta-subunits and N-linked glycosylation respectively. However, the function of this portion is not fully understood. In order to investigate the role of the extracellular domain of beta-subunit, we created a deletion mutant of IR cDNA which lacked 47 amino acid residues encoded by 141 bp corresponding to exon 13 of the IR gene. Insulin binding and surface labelling of COS 7 cells transiently expressing the mutant insulin receptors (IR delta Ex13) showed that the mutated receptors were not expressed on the cell surface. However, immunoblot analysis showed that uncleaved form (190 kDa) of the mutant receptors were intracellularly expressed. Deglycosylation with endoglycosidase H showed that the mutant receptors had mainly high-mannose oligosaccharide chains. The mutant IRs bound with high affinity to lentil lectin but with low affinity to wheat germ agglutinin. Therefore, it is suggested that misfolding of the mutant receptors inhibits transport to the Golgi apparatus where processing of oligosaccharide chains, as well as proteolytic cleavage into subunits, takes place. The binding affinity of the mutant receptors for insulin was 50% of normal. Furthermore, insulin-stimulated autophosphorylation of IR delta Ex13 was markedly impaired. These data provide the evidence for a critical role of the extracellular domain of IR beta-subunit for processing and transport as well as the intramolecular signal transduction to activate IR tyrosine kinase.
Base Sequence↗
Amplification and analysis of promoter region of insulin receptor gene in a patient with leprechaunism associated with severe insulin resistance.
A patient with leprechaunism associated with severe insulin resistance was studied to identify the molecular and genetic basis for insulin resistance. Insulin binding and surface labeling of transformed lymphocytes prepared from the patient showed a significantly decreased insulin receptor number on the cell surface. Southern blot analysis of the insulin receptor gene showed no evidence of large insertions or deletions. Furthermore, direct sequencing of all 22 exons and exon-intron junctions of the insulin receptor gene failed to show any missense mutations, nonsense mutations, or mutations at exon-intron junctions. However, Northern blot analysis indicated significantly decreased insulin receptor mRNA expression in the patient's cells. Moreover, restriction endonuclease digestion of the amplified cDNA suggested that the expression levels of one allele were less efficient than the other. These findings suggested that the regulatory region of the insulin receptor gene might have abnormalities. Therefore, we examined the 5' flanking region of the insulin receptor gene. Southern blot analysis showed no major deletions or insertions between positions -1,823 and -2 relative to the translation initiation site. A 5' flanking region of the insulin receptor gene spanning positions -881 approximately +7 was amplified by polymerase chain reaction (PCR) and introduced into a reporter plasmid carrying the human growth hormone (hGH) gene. The nucleotide sequence of the amplified fragment showed two polymorphic sites at positions -603 and -500 in the patient, as well as in normal subjects. No other abnormal sequence was found in the patient. Promoter activity measured by hGH expression in transfected mouse L cells was not influenced by the polymorphism at position -603 located in a cluster of GC boxes.(ABSTRACT TRUNCATED AT 250 WORDS)
Alleles↗
Effects of the recent great Hanshin-Awaji earthquake on emergency pediatric care.
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Child↗
[Clinical study of SY5555 dry syrup in the pediatric field].
SY5555 dry syrup (powder to be dissolved before use) was clinically used in pediatric patients. The following results were obtained: 1. The subjects were 6 pediatric patients including 1 case each with pharyngitis, tonsillitis, lacunar tonsillitis and impetigo contagiosa and 2 cases with scarlet fever. The drug was administered at a daily dose of 14.5-29.0 mg/kg divided into 3 dosages. The clinical results were excellent in 5 cases and good in 1 case with an efficacy rate of 100%. 2. Identified bacteria included 3 strains of Haemophilus influenzae, 2 strains of Streptococcus pyogenes and 1 strain of Staphylococcus aureus. Five strains were eradicated but 1 strain still remained with an bacteriological eradication rate of 83.3%. 3. No side effects were observed throughout the study period. In laboratory test results, elevated eosinophil count was observed in only 1 case. 4. Patients' compliance was good in general. 5. Based on the results mentioned above, the drug was considered to be a useful new oral antibiotic in the pediatric field.
Administration, Oral↗
Two naturally occurring mutations in the kinase domain of insulin receptor accelerate degradation of the insulin receptor and impair the kinase activity.
We identified two novel heterozygous missense mutations of the insulin receptor gene: the Asp1179 mutation in one family and the Leu1193 mutation in two unrelated families with extreme insulin resistance. In these patients, the number of insulin receptors on the cell surface was found to be markedly decreased by insulin binding and surface labeling studies in transformed lymphocytes. Insulin binding to the transfected COS 7 cells and Rat-1 cells with both mutant cDNAs was also decreased to 5-31% of normal, and the mutant insulin receptors showed a markedly decreased kinase activity. Although biosynthetic labeling studies revealed that both mutant receptors were synthesized as 190-kDa proreceptors, the degradation of the mutant proreceptors was 2-fold faster than that of the wild type proreceptors. However, the degradation rate of the mutant receptors on the cell surface was comparable to that of wild type insulin receptor. These results suggest that the Asp1179 and Leu1193 mutations in the kinase domain are unique in causing decreased insulin receptor number on the cell surface by accelerated intracellular degradation, and that insulin resistance in these patients is mainly due to the decreased receptor number rather than impaired kinase activity.
Adult↗
Leu 193 mutation in the cysteine rich region of the insulin receptor inhibits the cleavage of the insulin receptor precursor but not insulin binding.
To characterize the Leu 193 mutant insulin receptor, which was found in a patient with extreme insulin resistance, the mutant insulin receptor was overexpressed in Rat-1 fibroblasts by transfection of mutated insulin receptor cDNA. In the pulse-chase experiment, the cleavage of the proreceptors to the matured receptor subunits was impaired in the cells expressing Leu 193 insulin receptor and therefore, the mutant proreceptors were accumulated in the cell. Insulin bound to the Leu 193 insulin receptor on the cell surface with normal affinity, although the mutation was in the alpha-subunit of the insulin receptor. Therefore, the Leu 193 mutation impaired proreceptor cleavage but not insulin binding.
Animals↗