Aberrant regulation of the metabolism of the insulin receptor in Swarm rat chondrosarcoma chondrocytes.

Treatment of Swarm rat chondrosarcoma chondrocytes for 3 days in media containing either non-recombinant pig or recombinant human insulin (1 micrograms/ml) increased the rate of proteoglycan synthesis approximately 6-fold compared with cells cultured in the absence of insulin. The concentrations of human and pig insulin that stimulated the cells to double their rate of proteoglycan synthesis were approximately 1 ng/ml and approximately 2 ng/ml respectively. Because physiological concentrations of insulin do not influence proteoglycan synthesis in non-transformed chondrocytes, the findings indicated a possible abnormality in the insulin-dependent regulation of the insulin receptor in these tumour cells. Like most cells, chondrosarcoma chondrocytes down-regulated their insulin receptors when incubated with insulin for 30 min. However, the number of plasma-membrane and intracellular insulin receptors did not decrease when the chondrocytes were exposed to insulin chronically for 4 days. Chondrocytes were cultured in media containing 2H-, 13C- and 15N-labelled amino acids, and the heavy-isotope density-shift method was used to investigate both the rate of degradation and the rate of synthesis of the insulin receptor. Although the rate of synthesis of the receptor was slightly faster in the insulin-treated cultures, as assessed by a slightly faster rate of appearance of the 'heavy' receptor, the rate of degradation of the receptor was slower in the insulin-treated cultures. The half-lives for the 'light' receptors were approx. 18 h and 10 h for chondrocytes cultured in insulin-containing and insulin-free media respectively. These studies in vitro indicate that the apparent up-regulation of insulin receptors that occurs in this transformed cell upon long-term exposure to insulin is primarily the result of a decreased rate of receptor degradation.     

Polymorphisms and deduced amino acid substitutions in the coding sequence of the ryanodine receptor (RYR1) gene in individuals with malignant hyperthermia.

Twenty-one polymorphic sequence variants of the RYR1 gene, including 13 restriction fragment length polymorphisms (RFLPs), were identified by sequence analysis of human ryanodine receptor (RYR1) cDNAs from three individuals predisposed to malignant hyperthermia (MH). All RFLPs were detectable in PCR-amplified products, and their segregation was consistent with our initial finding of linkage to MH in the nine families previously informative for one or more intragenic markers (MacLennan et al., 1990, Nature 343:559-561). Four amino acid substitutions were identified in the study: Arg for Gly248, Cys for Arg470, Leu for Pro1785, and Cys for Gly2059. Of 45 families tested, a single family presented the Arg for Gly248 substitution where it segregated with malignant hyperthermia, making it a candidate mutation for predisposition to MH in man. The other three polymorphic substitutions failed to segregate with malignant hyperthermia in those families in which they occurred, implying that they represent polymorphisms with little or no effect on the function of the RYR1 gene.     

Endotoxin and cytokines in patients with gastrointestinal tract perforation

Plasma levels of endotoxin and various cytokines were assessed in 70 patients with gastrointestinal tract perforation. Sepsis developed in 29 of them, and eight of these (27.6%) had on admission endotoxin levels higher than 9.8 pg ml(-1). The clinical outcome correlated with the level of tumour necrosis factor alpha (TNFalpha), rather than with the endotoxin level. The high interleukin 6 (IL-6) level was shown in septic patients and no correlation was observed between the IL-6 level and the clinical outcome. Plasma TNFalpha levels tended to change independently from endotoxin levels, suggesting that TNFalpha may have been locally produced in inflammatory lesions.     

Regulation of Histamine Turnover via Muscarinic and Nicotinic Receptors in the Brain

To clarify the regulation of central histaminergic (HAergic) activity by cholinergic receptors, the effects of drugs that stimulate the cholinergic system on brain histamine (HA) turnover were examined, in vivo, in mice and rats. The HA turnover was estimated from the accumulation of tele-methylhistamine (t-MH) during the 90-min period after administration of pargyline (65 mg/kg, i.p.). In the whole brain of mice, oxotremorine, at doses higher than 0.05 mg/kg, s.c., significantly inhibited the HA turnover, this effect being completely antagonized by atropine but not by methylatropine. A large dose of nicotine (10 mg/kg, s.c.) also significantly inhibited the HA turnover. This inhibitory effect was antagonized by mecamylamine but not by atropine or hexamethonium. A cholinesterase inhibitor, physostigmine, at doses higher than 0.1 mg/kg, s.c., significantly inhibited the HA turnover. This effect was antagonized by atropine but not at all by mecamylamine. None of these cholinergic antagonists used affected the steady-state t-MH level or HA turnover by themselves. In the rat brain, physostigmine (0.1 and 0.3 mg/kg, s.c.) also decreased the HA turnover. This inhibitory effect of physostigmine was especially marked in the striatum and cerebral cortex where muscarinic receptors are present in high density. Oxotremorine (0.2 mg/kg, s.c.) and nicotine (1 mg/kg, s.c.) also decreased the HA turnover in the rat brain. However, these effects showed no marked regional differences. These results suggest that the stimulation of central muscarinic receptors potently inhibits the HAergic activity in the brain and that strong stimulation of central nicotinic receptors can also induce a similar effect.     

Alteration of rat liver proteoglycans during regeneration.

Sepharose CL-6B column chromatography of crude extracts from the slices of regenerating rat livers after partial hepatectomy and sham-operated controls labeled with [35S]sulfuric acid revealed an enhancement of [35S]sulfate incorporation into proteoglycan fractions during regeneration. The 35S-labeled proteoglycans contained heparan sulfate (more than 80% of the total) and chondroitin/dermatan sulfate. The 35S-incorporation into both glycosaminoglycans increased to maxima 3-5 days after partial hepatectomy and decreased thereafter toward the respective control levels. When [35S]sulfuric acid was replaced by [3H]glucosamine, similar results were obtained. These results suggest that the maximal stimulation of proteoglycan synthesis in regenerating rat liver follows the maximal mitosis of hepatic cells 1-2 days after partial hepatectomy. The 35S-labeled proteoglycans from regenerating liver 3 days after partial hepatectomy and control were analyzed further. They were similar in chromatographic behavior on a gel filtration or an anion-exchange column and in glycosaminoglycan composition. Their glycosaminoglycans were indistinguishable in electrophoretic mobility. However, these proteoglycans were slightly but significantly different in their affinity to octyl-Sepharose and in the molecular-weight distribution of their glycosaminoglycans.     

Molecular cloning of two tandemly arranged peroxidase genes from Populus kitakamiensis and their differential regulation in the stem

A genomic library was prepared from Populus kitakamiensis and screened with the cDNA for an anionic peroxidase from P. kitakamiensis. One genomic clone was isolated that contained two tandemly oriented genes for anionic peroxidases, prxA3a and prxA4a. Both genes consisted of four exons and three introns; the introns had consensus nucleotides, namely, GT and AG, at their 5 and 3 ends, respectively. The prxA3a and prxA4a genes encoded 347 and 343 amino acid residues, respectively, including putative signal sequences at the amino-termini. Putative promoters and polyadenylation signals were found in the flanking regions of both genes. The sequence of the coding region of prxA3a was completely identical to that of the cDNA clone pA3, whereas the sequence of the coding region of prxA4a was only 73% identical to that of the cDNA clone pA3. Northern blot analysis showed that the patterns of expression of the mRNAs that corresponded to prxA3a and prxA4a differed in stems of P. kitakamiensis.     

Effect of furosemide on urinary excretion of prostaglandin E in normal volunteers and pateints with essential hypertension

Urinary excretion of prostaglandin E was measured radioimmunologically in 19 healthy persons ( 15 men and 4 women ) and in 16 patients ( 10 men and 6 women ) with essential hypertension before and after the administration of furosemide. The excretion rates were increased from 26.3±3.0 to 64.5±11.3 ng/hr in the former and from 11.9±2.7 to 26.9±85 ng/hr in the latter. There was a significant difference between them, healthy subjects showing a greater increase than patients with essential hypertension.There was an obvious sexual difference in urinary excretion of prostaglandin. In men, greater increase in the excretion rates was found than in the women. Greater increases were also obtained in healthy men than in hypertensive men and in healthy women than in hypertensive women. The present results suggest that furosemide enhances urinary excretion of prostaglandin E by mechanisms which entails either an increase in prostaglandin synthesis or a decrease in renal metabolism.     

Metabolism of PG in man: Effect of furosemide on the excretion of the main metabolite of PG F2α

The excretion rates of main urinary metabolite of PG F₂α were measured radioimmunologically in 4 healthy persons and in 13 essential hypertensives. The resting values were 9.3±0.73 in the former and 10.4±2.17 ng/min in the latter. There was no significant differences between them. The excretion of the metabolite decresed prominently after the administration of furosemide. The percent decrease was 57% in healthy persons and 70% in essential hypertension. The percent result supports that furosemide inhibit the catabolism of PG F₂α.     

Unique T Cells with Unconventional Cytokine Profiles Induced in the Livers of Mice during Schistosoma mansoni Infection

During infection with Schistosoma, serious hepatic disorders are induced in the host. The liver possesses unique immune systems composed of specialized cells that differ from those of other immune competent organs or tissues. Host immune responses change dramatically during Schistosoma mansoni infection; in the early phase, Th1-related responses are induced, whereas during the late phase Th2 reactions dominate. Here, we describe unique T cell populations induced in the liver of mice during the period between Th1- and Th2-phases, which we term the transition phase. During this phase, varieties of immune cells including T lymphocytes increase in the liver. Subsets of CD4⁺ T cells exhibit unique cytokine production profiles, simultaneously producing both IFN-γ and IL-13 or both IFN-γ and IL-4. Furthermore, cells triply positive for IFN-γ, IL-13 and IL-4 also expand in the S. mansoni-infected liver. The induction of these unique cell populations does not occur in the spleen, indicating it is a phenomenon specific to the liver. In single hepatic CD4⁺ T cells showing the unique cytokine profiles, both T-bet and GATA-3 are expressed. Thus, our studies show that S. mansoni infection triggers the induction of hepatic T cell subsets with unique cytokine profiles.     

Rheb (Ras Homologue Enriched in Brain)-dependent Mammalian Target of Rapamycin Complex 1 (mTORC1) Activation Becomes Indispensable for Cardiac Hypertrophic Growth after Early Postnatal Period

Cardiomyocytes proliferate during fetal life but lose their ability to proliferate soon after birth and further increases in cardiac mass are achieved through an increase in cell size or hypertrophy. Mammalian target of rapamycin complex 1 (mTORC1) is critical for cell growth and proliferation. Rheb (Ras homologue enriched in brain) is one of the most important upstream regulators of mTORC1. Here, we attempted to clarify the role of Rheb in the heart using cardiac-specific Rheb-deficient mice (Rheb^−/−). Rheb^−/− mice died from postnatal day 8 to 10. The heart-to-body weight ratio, an index of cardiomyocyte hypertrophy, in Rheb^−/− was lower than that in the control (Rheb^+/+) at postnatal day 8. The cell surface area of cardiomyocytes isolated from the mouse hearts increased from postnatal days 5 to 8 in Rheb^+/+ mice but not in Rheb^−/− mice. Ultrastructural analysis indicated that sarcomere maturation was impaired in Rheb^−/− hearts during the neonatal period. Rheb^−/− hearts exhibited no difference in the phosphorylation level of S6 or 4E-BP1, downstream of mTORC1 at postnatal day 3 but showed attenuation at postnatal day 5 or 8 compared with the control. Polysome analysis revealed that the mRNA translation activity decreased in Rheb^−/− hearts at postnatal day 8. Furthermore, ablation of eukaryotic initiation factor 4E-binding protein 1 in Rheb^−/− mice improved mRNA translation, cardiac hypertrophic growth, sarcomere maturation, and survival. Thus, Rheb-dependent mTORC1 activation becomes essential for cardiomyocyte hypertrophic growth after early postnatal period.