Interactions between cations in modifying the binding of hexokinases I and II to mitochondria

Summary Interactions between cations in modifying the binding of hexokinases I and II to mitochondria was examined with reference to the intracellular condition. Mitochondria-binding of either of hexokinases I and II, both prepared from mouse ascites ELD cells, was markedly increased by Mg2+ as has been known well. However, even in the absence of Mg^s+, marked binding was attained by 100 mM K⁺ alone especially for hexokinase I, which seemed generally more ready to bind to mitochondria. On the other hand, the effect of Mg²⁺ to increase the binding was reduced by the addition of K⁺, and the decreasing effect of K⁺ was much more marked for hexokinase II than I. These results indicate that, in addition to Mg²⁺, monovalent cations as represented by K⁺, also have marked effect on the binding, and the effect is different for each of hexokinases I and II, which may be responsible for the difference in the intracellular distribution between these hexokinases.     

DEAD-box RNA helicase subunits of the Drosha complex are required for processing of rRNA and a subset of microRNAs

MicroRNAs (miRNAs) control cell proliferation, differentiation and fate through modulation of gene expression by partially base-pairing with target mRNA sequences. Drosha is an RNase III enzyme that is the catalytic subunit of a large complex that cleaves pri-miRNAs with distinct structures into pre-miRNAs. Here, we show that both the p68 and p72 DEAD-box RNA helicase subunits in the mouse Drosha complex are indispensable for survival in mice, and both are required for primary miRNA and rRNA processing. Gene disruption of either p68 or p72 in mice resulted in early lethality, and in both p68(-/-) and p72(-/-) embryos, expression levels of a set of, but not all, miRNAs and 5.8S rRNA were significantly lowered. In p72(-/-) MEF cells, expression of p72, but not a mutant lacking ATPase activity, restored the impaired expression of miRNAs and 5.8S rRNA. Furthermore, we purified the large complex of mouse Drosha and showed it could generate pre-miRNA and 5.8S rRNA in vitro. Thus, we suggest that DEAD-box RNA helicase subunits are required for recognition of a subset of primary miRNAs in mDrosha-mediated processing.     

Dexamethasone stimulates the expression of ghrelin and its receptor in rat hypothalamic 4B cells

Growth hormone (GH)-releasing peptides (GHRPs) are synthetic peptides that strongly induce GH release. GHRPs act via a specific receptor, the GHRP receptor (GHSR), of which ghrelin is a natural ligand. GHRPs also induce adrenocorticotropic hormone (ACTH) release in healthy subjects. GHRPs or ghrelin stimulate ACTH release via corticotropin-releasing factor (CRF) and arginin vasopressin in the hypothalamus. Stress-activated CRF neurons are suppressed by glucocorticoids in the hypothalamic paraventricular nucleus (PVN), while CRF gene is up-regulated by glucocorticoids in the PVN cells without the influence of input neurons. However, little is known about the regulation of ghrelin and GHSR type 1a (GHSR1a) genes by glucocorticoids in PVN cells. To elucidate the regulation of ghrelin and GHSR gene expression by glucocorticoids in PVN cells, here we used a homologous PVN neuronal cell line, hypothalamic 4B, because these cells show characteristics of the parvocellular neurons of the PVN. These cells also express ghrelin and GHSR1a mRNA. Dexamethasone increased ghrelin mRNA levels. A potent glucocorticoid receptor antagonist, RU-486, significantly blocked dexamethasone-induced increases in ghrelin mRNA levels. Dexamethasone also significantly stimulated GHSR1a mRNA and protein levels. Finally, ghrelin increased CRF mRNA levels, as did dexamethasone. Incubation with both dexamethasone and ghrelin had an additive effect on CRF and ghrelin mRNA levels. The ghrelin-GHSR1a system is activated by glucocorticoids in the hypothalamic cells.     

Inbreeding depression, increased phenotypic variance, and a trade-off between gonads and appendages in selfed progeny of the aphid Prociphilus oriens

Trade-offs are potentially common among two or more traits whose development is dependent on the same resources. To detect genetic trade-offs, the techniques of quantitative genetics, pedigree analyses, and selection experiments have been used. This study demonstrates genetically based trade-offs between gonads and appendages in hatched larvae of the aphid Prociphilus oriens by focusing on enlarged variance among the families of selfed progeny. The selfed and outbred families were compared in respect to the size of morphological traits, gonad volume, and hatch dates as well as egg volume. Selfing not only increased the among-family variance component in all larval traits examined, but it also increased the mean size of all the morphological traits significantly. In contrast, gonad volume, a fitness component, was reduced with selfing. Calculation of the allometry (log-transformed regression) of larval traits to egg volume indicated that in the outbred group, morphological traits grew slowly relative to egg volume with slopes below 0.25, whereas gonads exhibited isometric growth. With selfing, most morphological traits had significantly steeper slopes, whereas the slope for gonads was greatly decreased. When the effect of egg volume was statistically removed from the means of selfed families, significant negative correlation was detected between the adjusted means of gonad volume and those of tibia length. This result suggests genetic trade-offs between gonad volume and tibia length. Thus, the evidence implies that at the loci governing the development of appendages, the dominant alleles function to canalize the development of tibiae into an optimal size, irrespective of egg volume. It is hypothesized that increased homozygosity of the deleterious recessive alleles reduced gonad volume through increasing the resource allocation to tibiae. The hypothesis of the gonad-appendage trade-off could be applied to explain the phenotypic evolution in some aphid species.     

Partial replacement of waxy cornstarch by recrystallized amylose retards the development of insulin resistance in rats

We examined in rats whether or not the prolonged ingestion of recrystallized amylose (RCA) would prevent the development of insulin resistance. Rats were fed on a diet containing waxy cornstarch (WCS) as carbohydrate or a diet containing 30% RCA in place of WCS for 18 wk. Glucose tolerance test (GTT) was conducted at every four weeks. On wk 16, the plasma insulin response as assessed by the area under the curve was lower in the RCA diet group than in the WCS diet group. The fasting plasma insulin level tended to increase over time in both groups, but was lower in the RCA diet group on wk 16. An autopsy revealed that the adipose tissue mass and serum free fatty acid concentrations were significantly higher in the WCS diet group. The results suggest that prolonged ingestion of RCA had the effect of slowing the development of insulin resistance through a lower concentration of serum free fatty acids, presumably due to the prevention of adipocyte hypertrophy.     

Vitamin B12, a chlorophyll-related analog to pheophytin a from marine brown algae, promotes neurite outgrowth and stimulates differentiation in PC12 cells

We previously isolated an analog to chlorophyll-related compounds, pheophytin a, from the marine brown alga Sargassum fulvellum and demonstrated that it is a neurodifferentiation compound. In the current study, we investigated the effects of the pheophytin a analog vitamin B₁₂ on PC12 cell differentiation. In the presence of a low level of nerve growth factor (10 ng ml⁻¹), vitamin B₁₂ demonstrated neurite outgrowth-promoting activity in PC12 cells. The effect was dose-dependent in the range of 6–100 μM. In the absence of nerve growth factor, vitamin B₁₂ did not promote differentiation. To investigate the mechanism for this effect, we conducted differentiation assays and western blot analysis with signal transduction inhibitors and found that vitamin B₁₂ did not promote PC12 cell differentiation in the presence of K252a or U0126 inhibitors. These results suggest that vitamin B₁₂ stimulates PC12 cell differentiation through enhancement of the mitogen-activated protein kinase signal transduction pathway, which is also induced by nerve growth factor. Thus, vitamin B₁₂ may be a good candidate for treatment of neurodegenerative diseases such as Alzheimer’s disease.     

Role of Glutamate and GABA Transporters in Development of Pentylenetetrazol-Kindling

Kindling is a form of epileptogenesis that can be induced with pentylenetetrazol (PTZ). We undertook this study to evaluate the contribution of glutamate and GABA transporters to the process of PTZ kindling. Rats were injected i.p. three times per week with PTZ (40 mg/kg) until they were fully kindled. In rats who achieved full kindling, measurement of hippocampal glutamate and GABA transporters within 24 h by western blot showed that GLAST, GLT-1, and EAAC1 were elevated significantly. However, fully kindled rats at 30 days after their last seizure had no change in either glutamate or GABA transporters proteins. These sequential observations suggest that glutamate transporters may contribute to the occurrence of seizures, but were not associated with maintenance of epileptogenesis. During this experiment, we collected data from animals that had kindled easily and animals who were resistant to kindling. Easily-kindled rats reached full kindling with less than five injections of PTZ. Kindling resistant animals failed to achieve full kindling even after administration of 12 consecutive injections of PTZ. Levels of EAAC1 and GAT-1 in easily-kindled rats were decreased by 30% when compared to kindling resistant animals at 30 days after the last PTZ injection. Since decreased EAAC1 and GAT-1 would diminish GABA function, less quantity of these proteins would appear to be associated with the convulsive threshold at the beginning of kindling development. We wonder if glutamate and GABA transporters might be operant in a convulsion threshold set factor or as a pace factor for kindling.