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.     

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.     

In situ measurement of cell stiffness of Arabidopsis roots growing on a glass micropillar support by atomic force microscopy

Atomic force microscopy (AFM) can measure the mechanical properties of plant tissue at the cellular level, but for in situ observations, the sample must be held in place on a rigid support and it is difficult to obtain accurate data for living plants without inhibiting their growth. To investigate the dynamics of root cell stiffness during seedling growth, we circumvented these problems by using an array of glass micropillars as a support to hold an Arabidopsis thaliana root for AFM measurements without inhibiting root growth. The root elongated in the gaps between the pillars and was supported by the pillars. The AFM cantilever could contact the root for repeated measurements over the course of root growth. The elasticity of the root epidermal cells was used as an index of the stiffness. By contrast, we were not able to reliably observe roots on a smooth glass substrate because it was difficult to retain contact between the root and the cantilever without the support of the pillars. Using adhesive to fix the root on the smooth glass plane overcame this issue, but prevented root growth. The glass micropillar support allowed reproducible measurement of the spatial and temporal changes in root cell elasticity, making it possible to perform detailed AFM observations of the dynamics of root cell stiffness.     

Proteomic analysis of kidneys from selenoprotein M transgenic rats in response to increased bioability of selenium

Background

To characterize changes in global protein expression in kidneys of transgenic rats overexpressing human selenoprotein M (SelM) in response to increased bioabivility of selenium (Sel), total proteins extracted from kidneys of 10-week-old CMV/hSelM Tg and wild-type rats were separated by 2-dimensional gel electrophoresis and measured for changes in expression.

Results

Ten and three proteins showing high antioxidant enzymatic activity were up- and down-regulated, respectively, in SelM-overexpressing CMV/hSelM Tg rats compared to controls based on an arbitrary 2-fold difference. Up-regulated proteins included LAP3, BAIAP2L1, CRP2, CD73 antigen, PDGF D, KIAA143 homolog, PRPPS-AP2, ZFP313, HSP-60, and N-WASP, whereas down-regulated proteins included ALKDH3, rMCP-3, and STC-1. After Sel treatment, five of the up-regulated proteins were significantly increased in expression in wild-type rats, whereas there were no changes in CMV/hSelM Tg rats. Only two of the down-regulated proteins showed reduced expression in wild-type and Tg rats after Sel treatment.

Conclusions

These results show the primary novel biological evidences that new functional protein groups and individual proteins in kidneys of Tg rats relate to Sel biology including the response to Sel treatment and SelM expression.     

The influence of environmental instability on the reproductive strategy of the critically endangered Acıgöl killifish (Aphanius transgrediens)

To investigate the variability in the reproductive tactics of Acıgöl killifish (Aphanius transgrediens), one of the most seriously threatened freshwater teleost species, this study compared its reproductive ecology in two contrasting habitats that differ substantially in terms of stability of environmental parameters, particularly salinity regime (stable vs. unstable). Fish were sampled monthly from October 2013 to September 2014 with the aim of testing whether the reproductive life-history response of fish to stable and unstable conditions differed. The reproductive effort (gonad weight) of both sexes did not differ significantly between the two habitats, but females in the unstable habitat had significantly lower fecundity and larger eggs. The relationship between fecundity and fish size was stronger in the stable habitat, whereas the relationship was quite variable and uncertain in the unstable habitat. Fish born in the unstable habitat reached their first maturity at a smaller size than those in the stable habitat. The gonado-somatic index and the duration of hydrated eggs showed that reproduction continued from February to May in both habitats; nonetheless, a second spawning event occurred during July and August in the unstable habitat, which included the reproductive contributions of YOY individuals and older generations. This study’s results suggest that A. transgrediens employs varying reproductive strategies against environmental instability in its restricted unique range. This may have further implications for the ways in which habitat-specific conservation methods are used.     

A Japanese child with geleophysic dysplasia caused by a novel mutation of FBN1

Geleophysic dysplasia (GD) is a rare disorder characterized by severe short stature, short hands and feet, limited joint mobility, skin thickening, characteristic facial features (e.g., a “happy” face), and cardiac valvular disorders that often result in an early death. The genes ADAMTSL2 (a disintegrin-like and metalloprotease with thrombospondin type 1 motif-like 2) and FBN1 (fibrillin 1) were recently identified as causative genes for GD. Here, we describe a 10-year-old Japanese female with GD who was born to non-consanguineous parents. At the age of 11 months, she was referred to our hospital because of very short stature for her age (− 4.4 standard deviations of the age-matched value) and a “happy” face with full cheeks, a shortened nose, hypertelorism, and a long and flat philtrum, characteristic of GD. Her hands and feet were small, her skin was thickened, and her joint mobility was generally limited. She had cardiac valvular disorders and history of recurrent respiratory failure. Mutation analysis revealed no abnormalities in ADAMTSL2. However, analysis of FBN1 revealed a novel heterozygous mutation (c.5161T > T/G) in exon 41, which encodes transforming growth factor-β-binding protein-like domain 5 (TB5). GD is an extremely rare disorder and, to our knowledge, only one case of GD with an FBN1 mutation has been reported in Japan. Similar to the previously reported cases of GD, the mutation in the current patient was located in the TB5 domain, which suggests that abnormalities in this domain of FBN1 are responsible for GD.     

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.