Identification of three isoforms for mitochondrial adenine nucleotide translocator in the pufferfish Takifugu rubripes

Three adenine nucleotide translocator (ANT) genes were identified through in silico data mining of the Fugu genome database along with isolation of their corresponding cDNAs in vivo from the pufferfish (Takifugu rubripes). As a result of phylogenetic analysis, the ANT gene on scaffold_254 corresponded to mammalian ANT1, whereas both of those on scaffold_6 and scaffold_598 to mammalian ANT3. The ANT gene encoded by scaffold_6 was expressed ubiquitously in various tissues, whereas the ANT genes encoded by scaffold_254 and scaffold_598 were predominantly expressed in skeletal muscle and heart, respectively.     

Insulin-like Growth Factor Signaling Pathway Involved in Regulating Longevity of Rotifers

Rotifers have been used to study the mechanisms of ageing for more than a century, but the underlying molecular basis of ageing in rotifers is largely unknown. The insulin/insulin-like growth factor (IGF-1) signaling pathway has been found to regulate the lifespan of evolutionarily distinct eukaryotes from yeast to mammals. We therefore assume that the insulin/IGF-1 pathway is a candidate for regulating the rotifer’s lifespan. Accordingly, we examined the action of an inhibitor to PI3-kinase involved in the pathway for the rotifer Brachionus plicatilis O. F. Müller. This kinase was first discovered as age-1 to regulate the longevity of Caenorhabditis elegans. As expected, the inhibitor treatment resulted in the extension of lifespan by 30% compared to the reference group without the treatment, whereas reproductive characters were not apparently changed. These results were consistent with those observed in C. elegans, suggesting that the lifespan of B. plicatilis is likely to be regulated by the signaling pathway involving PI3-kinase.     

Rab1a rescues the toxicity of PRAF3

The PRA1-superfamily member PRAF3 plays pivotal roles in membrane traffic as a GDI displacement factor via physical interaction with a variety of Rab proteins, as well as in the modulation of antioxidant glutathione through its interaction with EAAC1 (SLC1A1). Overproduction of PRAF3 is known to be toxic to the host cells, although the factors capable of cancelling the toxicity remained unknown. We here show that Rab1a can rescue the cytotoxicity caused by PRAF3 possibly by “positively” regulating ER-Golgi trafficking, cancelling the “negative” modulation by PRAF3. Our results illuminate the close physiological relationship between PRAF3 and Rab proteins.     

Differences in liver homogenates from Donryu, Fischer, Sprague-Dawley and Wistar strains of rat in the drug-metabolizing enzyme assay and the salmonella/hepatic S9 activation test

Comparison studies for detecting differences between liver microsome and S9 preparations from 4 strains (Donryu, Fischer, Sprague-Dawley, Wistar) of young male rats were carried out with pretreatment of the animals by inducers such as PCBs and PB plus 5,6-BF. Each microsome fraction was assayed for the enzymic activity of metabolism of model substrates such as aniline, benzophetamine, BP, DMN and 7-ethoxycoumarin. The hepatic S9 sample was also compared, as regards its metabolizing ability to activate 9 pre-mutagens (2AA, AAF, o-AAT, BP, DAB, DMBA, DMN, m-PDA, quinoline) to directly acting mutagens in the Salmonella/hepatic S9 activation test by using TA98, TA100 and TA1537 strains with or without cytochrome P450 inhibitors (SKF-525A, metyrapone, 7,8-benzoflavone).In the enzymic assay with PCBs-induced microsomes, BP hydroxylation revealed a strain-specific difference: the microsomes from Fischer and Wistar rats were more effective for metabolizing BP than those from the other strains of rat. The effect of induction by PB plus 5,6-BF for Fischer rats showed relatively higher enzymic activity in the same induction group. Other microsomes prepared from rats with and without induction by PB plus 5,6-BF did not show a clear-cut strain dependency in the enzymic activities assayed.In the mutation experiments with hepatic S9 samples, the examination of DAB and quinoline revealed a marked strain difference when S9 samples prepared from PCBs-pretreated and PB-plus-5,6-BF-induced rats were used: the S9 sample from Fischer rats was available for activating the two pre-mutagens to directly acting mutagens. No marked difference in the metabolic activation of the remaining 7-pre-mutagens was observed on other S9 preparations.In examinations of mutagenicity activities with the use of three inhibitors, the two S9 preparations made with the two induction methods showed inhibition profiles closely similar to each other. However, there were minor differences in the profiles by these inhibitors.From these findings it was concluded that Fischer rat-liver S9 is useful for detecting mutagens in the metabolic activation test, when induction by PB plus 5,6-BF was used in the Ames Salmonella test.     

STUDIES ON SHELL FORMATION : IX. An Electron Microscope Study of Crystal Layer Formation in the Oyster

Details of crystal growth in the calcitostracum of Crassostrea virginica have been studied with the purpose of analyzing the formation of the overlapping rows of oriented tabular crystals characteristic of this part of the shell. Crystal elongation, orientation, and dendritic growth suggest the presence of strong concentration gradients in a thin layer of solution in which crystallization occurs. Formation of the overlapping rows can be explained by three processes observed in the shell: a two-dimensional tree-like dendritic growth in which one set of crystal branchings creeps over an adjacent set of branchings; three-dimensional dendritic growth; and growth by dislocation of crystal surfaces. Multilayers of crystals may thus be formed at one time. This is favored by infrequent secretion of a covering organic matrix which would inhibit crystal growth. The transitional zone covering the outer part of the calcitostracum and the inner part of the prismatic region is generally characterized by aggregates of small crystals with definite orientation. Growth in this zone appears to take place in a relatively homogeneous state of solution without strong concentration gradients. Thin membranes and bands of organic matrix were commonly observed in the transitional zone bordering the prismatic region. The membrane showed a very fine oriented network pattern.     

Determination of antifungal activities in serum samples from mice treated with different antifungal drugs allows detection of an active metabolite of itraconazole

To establish an in vitro method of predicting in vivo efficacy of antifungal drugs against Candida albicans and Aspergillus fumigatus, the antifungal activities of fluconazole, itraconazole, and amphotericin B were determined in mouse serum. The minimum inhibitory concentration (MIC) of each drug was measured using mouse serum as a diluent. For C. albicans, the assay endpoint of azoles was defined as inhibition of mycelial extension (mMIC) and for A. fumigatus, as no growth (MIC). The MICs of amphotericin B for both pathogens were defined as the MIC at which no mycelial growth occurred. Serum MIC or mMIC determinations were then used to estimate the concentration of the drugs in serum of mice treated with antifungal drugs by multiplying the antifungal titer of the serum samples by the serum (m)MIC. The serum drug concentrations were also determined by HPLC. The serum concentrations estimated microbiologically showed good agreement with those determined by HPLC, except for itraconazole. Analysis of the serum samples from itraconazole-treated mice by a sensitive bioautography revealed the presence of additional spots, not seen in control samples of itraconazole. The bioautography assay demonstrated that the additional material detected in serum from mice treated with itraconazole was an active metabolite of itraconazole. The data showed that the apparent reduction in the itraconazole serum concentration as determined by HPLC was the result of the formation of an active metabolite, and that the use of a microbiological method to measure serum concentrations of drugs can provide a method for prediction of in vivo efficacy of antifungal drugs.     

Analgesic action of nicotine on tibial nerve transection (TNT)-induced mechanical allodynia through enhancement of the glycinergic inhibitory system in spinal cord

The activation of cholinergic pathways by nicotine elicits various physiological and pharmacological effects in mammals. For example, the stimulation of nicotinic acetylcholine receptors (nAChRs) leads to an antinociceptive effect. However, it remains to be elucidated which subtypes of nAChR are involved in the antinociceptive effect of nicotine on nerve injury-induced allodynia and the underlying cascades of the nAChR-mediated antiallodynic effect. In this study, we attempted to characterize the actions of nicotine at the spinal level against mechanical allodynia in an animal model of neuropathic pain, tibial nerve transection (TNT) in rats. It was found that the intrathecal injection of nicotine, RJR-2403, a selective alpha4beta2 nAChR agonist, and choline, a selective alpha7 nAChR agonist, produced an antinociceptive effect on the TNT-induced allodynia. The actions of nicotine were almost completely suppressed by pretreatment with mecamylamine, a non-selective nicotinic antagonist, or dihydro-beta-erythroidine, a selective alpha4beta2 nAChR antagonist, and partially reversed by pretreatment with methyllycaconitine, a selective alpha7 nAChR antagonist. Furthermore, pretreatment with strychnine, a glycine receptor antagonist, blocked the antinociception induced by nicotine, RJR-2403, and choline. On the other hand, the GABAA antagonist bicuculline did not reverse the antiallodynic effect of nicotine. Together, these results indicate that the alpha4beta2 and alpha7 nAChR system, by enhancing the activities of glycinergic neurons at the spinal level, exerts a suppressive effect on the nociceptive transduction in neuropathic pain.     

Studies on Shell Formation : VIII. Electron Microscopy of Crystal Growth of the Nacreous Layer of the Oyster Crassostrea virginica

Electron microscope observations have been made by means of the replica method on growth processes of calcite crystals of the nacreous layer of the shell of the oyster, Crassostrea virginica. Layer formation is initiated by the secretion of a conchiolin matrix and the deposition of rounded crystal seeds on or in this material. In some areas crystal seeds are elongate and within a given area show a similar orientation, probably due to slower deposition. The seeds appear to increase in size by dendritic growth, and smaller seeds become incorporated into larger ones which come into contact to form a single layer. With further growth, crystals overlap, forming a step-like arrangement. The direction of growth is frequently different in neighboring regions. Crystal seeds deposited on crystal surfaces are usually elongate and oriented. Well developed crystals have a tabular idiomorphic form and are parallel in their growth. Rounded and irregular crystals were also observed. The crystals show reticular structure with units of the order of 100 A and striations corresponding with the rhombohedral axes of the crystals. The role of the mantle is discussed in relation to the growth patterns of crystals and shell structure.     

H-Ferritin Is Preferentially Incorporated by Human Erythroid Cells through Transferrin Receptor 1 in a Threshold-Dependent Manner

Ferritin is an iron-storage protein composed of different ratios of 24 light (L) and heavy (H) subunits. The serum level of ferritin is a clinical marker of the body’s iron level. Transferrin receptor (TFR)1 is the receptor not only for transferrin but also for H-ferritin, but how it binds two different ligands and the blood cell types that preferentially incorporate H-ferritin remain unknown. To address these questions, we investigated hematopoietic cell-specific ferritin uptake by flow cytometry. Alexa Fluor 488-labeled H-ferritin was preferentially incorporated by erythroid cells among various hematopoietic cell lines examined, and was almost exclusively incorporated by bone marrow erythroblasts among human primary hematopoietic cells of various lineages. H-ferritin uptake by erythroid cells was strongly inhibited by unlabeled H-ferritin but was only partially inhibited by a large excess of holo-transferrin. On the other hand, internalization of labeled holo-transferrin by these cells was not inhibited by H-ferritin. Chinese hamster ovary cells lacking functional endogenous TFR1 but expressing human TFR1 with a mutated RGD sequence, which is required for transferrin binding, efficiently incorporated H-ferritin, indicating that TFR1 has distinct binding sites for H-ferritin and holo-transferrin. H-ferritin uptake by these cells required a threshold level of cell surface TFR1 expression, whereas there was no threshold for holo-transferrin uptake. The requirement for a threshold level of TFR1 expression can explain why among primary human hematopoietic cells, only erythroblasts efficiently take up H-ferritin.