Expression and functional analyses of novel mutations of ATP-binding cassette transporter-1 in Japanese patients with high-density lipoprotein deficiency.

ATP-binding cassette transporter-1 (ABCA1) gene is mutated in patients with familial high-density lipoprotein deficiency (FHD). In order to know the molecular basis for FHD, we characterized three different ABCA1 mutations associated with FHD (G1158A/A255T, C5946T/R1851X, and A5226G/N1611D) with respect to their expression in the passaged fibroblasts from the patients and in the cells transfected with the mutated cDNAs. Fibroblasts from the all patients showed markedly decreased cholesterol efflux to apolipoprotein (apo)-Al. In the fibroblasts homozygous for G1158A/A255T, the immunoreactive mass of ABCA1 could not be detected, even when stimulated by 9-cis-retinoic acid and 22-R-hydroxycholesterol. In the fibroblasts homozygous for C5946T/R1851X, ABCA1 mRNA was comparable. Because the mutant ABCA1 protein (R1851X) was predicted to lack the epitope for the antibody used, we transfected FLAG-tagged truncated mutant (R1851X/ABCA1-FLAG) cDNA into Cos-7 cells, showing that the mutant protein expression was markedly reduced. The expression of N1611D ABCA1 protein was comparable in both fibroblasts and overexpressing cells, although cholesterol efflux from the cells was markedly reduced. These data indicated that, in the three patients investigated, the abnormalities and dysfunction of ABCA1 occurred at the different levels, providing important information about the expression, regulation, and function of ABCA1.     

Distribution of the Parathyroid Hormone-Related Peptide and Its Receptor in the Saccus Vasculosus and Choroid Plexus in the Red Stingray (Dasyatis akajei: Elasmobranch)

1. The exact role of the parathyroid hormone-related peptide (PTHrP) is not fully understood. We used immunohistochemistry to localize the PTHrP and its receptor in the brain of the red stingray, particularly in the saccus vasculosus (SV) and choroid plexus.2. Immunoreactive PTHrP and its receptor were detected in the epithelial cells of the SV and the choroid plexus. In addition, the neuronal perikarya in the nucleus of the SV located in the hypothalamus is positive for the PTHrP.3. No PTHrP-containing neurons were detected in the choroid plexus. Extracts of SV and choroid plexus showed positive reactions against the PTHrP and its receptor antibody in Western blot analysis.4. High levels of immunoreactive PTHrP were detected in the plasma equivalent to those present in human humoral malignant hypercalcemia. In contrast, the immunoreactive PTHrP concentration in the cerebrospinal fluid was below detectable levels.5. Our results suggest that the regulation of the PTHrP in the SV differs from that in the choroid plexus in the red stingray.     

Intergranular bridges in the anterior pituitary cell and their possible involvement in Ca2+-induced granule-granule fusion

Quick-freeze deep-etch electron microscopy showed the presence of bridge-like structures between adjacent secretory granules in rat anterior pituitary secretory cells. These intergranular bridges were variable in length and thickness. The finest bridges were 7–8 nm in length, while the longest ones were as long as 80 nm. Annexin II, one of the Ca²⁺-dependent phospholipid-binding proteins, is known to interlink between two membranes and induce aggregation of liposomes and chromaffin granules under the presence of Ca²⁺. In anterior pituitary cells, annexin II was detected by immunoelectron microscopy at the contact sites of secretory granules with other granules. The anterior pituitary cells treated under the presence of extracellular Ca²⁺ with Clostridium perfringens enterotoxin which induces Ca²⁺ influx showed multigranular exocytosis, i.e., multiple fusions of secretory granules with each other and with the plasma membrane. The granule-granule fusion in progress could be captured by the quick-freeze deep-etch technique. The membranes of adjacent secretory granules were partially fused at their contact sites where intergranular strands were no longer seen, while there existed intergranular strands between unfused portions of the granule membranes. From these results, we consider that the intergranular bridges, some of which may be composed of annexin II, are involved in Ca²⁺-induced granule-granule fusion in anterior pituitary cells.     

Inducible nitric oxide synthase augments injury elicited by oxidative stress in rat cardiac myocytes

The effects of nitric oxide (NO) produced by cardiac inducibleNO synthase (iNOS) on myocardial injury after oxidative stress wereexamined. Interleukin-1 induced cultured rat neonatal cardiac myocytes to express iNOS. After induction of iNOS,L-arginine enhanced NOproduction in a concentration-dependent manner. Glutathione peroxidase(GPX) activity in myocytes was attenuated by elevated iNOS activity andby an NO donor,S-nitroso-N-acetyl-penicillamine (SNAP). Although NO production by iNOS did not induce myocardial injury, NO augmented release of lactate dehydrogenase from myocyte cultures after addition ofH₂O₂(0.1 mM, 1 h). Inhibition of iNOS withN-nitro-L-argininemethyl ester ameliorated the effects of NO-enhancing treatments onmyocardial injury and GPX activity. SNAP augmented the myocardialinjury induced byH₂O₂.Inhibition of GPX activity with antisense oligodeoxyribonucleotide forGPX mRNA increased myocardial injury byH₂O₂.Results suggest that the induction of cardiac iNOS promotes myocardialinjury due to oxidative stress via inactivation of the intrinsicantioxidant enzyme, GPX.

     

Reduction of glycogen in eggs of the silkworm,Bombyx mori,by use of a trehalase inhibitor,trehazolin, and diapause induction in glycogen-reduced eggs

A new trehalase inhibitor, trehazolin, caused a potent inhibition of ovary trehalase in the silkworm, Bombyx mori. A single injection of trehazolin into pupae (40&mgr;g/animal) did not interfere with the accumulation of proteins and lipids, but markedly reduced glycogen content in eggs accompanied by a remarkable increase in hemolymph trehalose levels. The most potent effect of trehazolin was expressed in eggs that developed at the mid-stage of pupal-adult development. In these eggs glycogen content was reduced to a trace level, less than 3% of that of the control. The reduced glycogen content was almost restored to the control level by injection of glucose but not by trehalose. Trehazolin treatment influenced oviposition and larval hatching, whereas embryogenesis went on normally in glycogen-reduced eggs. Injection of synthetic diapause hormone into non-diapause type hosts induced an incidence of 45% diapause in the eggs and increased their glycogen content. Surprisingly, injection of trehazolin never affected diapause induction by the hormone, despite considerably reduced glycogen content in these eggs. Thus, our findings provide a new method for production of eggs containing various amounts of glycogen, and a novel system for analyzing diapause-associated metabolism besides the well-known glycogen-sorbitol metabolism.     

Pituitary Adenylate Cyclase-Activating Polypeptide Causes Ca2+ Release from Ryanodine/Caffeine Stores Through a Novel Pathway Independent of Both Inositol Trisphosphates and Cyclic AMP in Bovine Adrenal Medullary Cells

Abstract: Pituitary adenylate cyclase-activating polypeptide (PACAP) causes both Ca²⁺ release and Ca²⁺ influx in bovine adrenal chromaffin cells. To elucidate the mechanisms of PACAP-induced Ca²⁺ release, we investigated expression of PACAP receptors and measured inositol trisphosphates (IP₃), cyclic AMP, and the intracellular Ca²⁺ concentration in bovine adrenal medullary cells maintained in primary culture. RT-PCR analysis revealed that bovine adrenal medullary cells express the PACAP receptor hop, which is known to couple with both IP₃ and cyclic AMP pathways. The two naturally occurring forms of PACAP, PACAP38 and PACAP27, both increased cyclic AMP and IP₃, and PACAP38 was more potent than PACAP27 in both effects. Despite the effects of PACAP on IP₃ production, the Ca²⁺ release induced by PACAP38 or by PACAP27 was unaffected by cinnarizine, a blocker of IP₃ channels. The potencies of the peptides to cause Ca²⁺ release in the presence of cinnarizine were similar. The Ca²⁺ release induced by PACAP38 or by PACAP27 was strongly inhibited by ryanodine and caffeine. In the presence of ryanodine and caffeine, PACAP38 was more potent than PACAP27. PACAP-induced Ca²⁺ release was unaffected by Rp-adenosine 3′,5′-cyclic monophosphothioate, an inhibitor of protein kinase A. Ca²⁺ release induced by bradykinin and angiotensin II was also inhibited by ryanodine and caffeine, but unaffected by cinnarizine. Although IP₃ production stimulated by PACAP38 or bradykinin was abolished by the phospholipase C inhibitor, U-73122, Ca²⁺ release in response to the peptides was unaffected by U-73122. These results suggest that PACAP induces Ca²⁺ release from ryanodine/caffeine stores through a novel intracellular mechanism independent of both IP₃ and cyclic AMP and that the mechanism may be the common pathway through which peptides release Ca²⁺ in adrenal chromaffin cells.     

Samui, a novel cold-inducible gene, encoding a protein with a BAG domain similar to silencer of death domains (SODD/BAG-4), isolated from Bombyx diapause eggs.

Cellular responses to cold-acclimation have not yet been studied in depth. To explore this field, we focussed on insect diapause development. Although embryonic diapause of Bombyx mori is sustained at 25 degrees C, chilling at 5 degrees C for 2 months causes diapause termination, a transition that is marked when the sorbitol dehydrogenase gene (SDH) is activated. To clarify the relationship between this activation and incubation at 5 degrees C, we isolated a novel cold-inducible gene, Samui. Expression of Samui mRNA and protein was activated after incubation at 5 degrees C for 5-6 days, lasted for another 30 days and then weakened. Exposure to 25 degrees C suppressed both mRNA and protein expression. In nondiapause eggs incubated at 5 degrees C, Samui was also up-regulated, although the expression was weaker. Samui contained nuclear localization-signals, a ssDNA-binding motif and a BAG domain similar to that of SODD/BAG-4. Because Samui could bind to HSP70, it is a member of BAG protein family. It is proposed that Samui serves to transmit the '5 degrees C signal' for SDH expression in diapause eggs, while also protecting against cold-injures in nondiapause eggs, through binding to respective partners. This is the first report that a member of BAG protein family is up-regulated by cold.     

Standardization of fine needle aspiration cytology of the breast – comparison of Auto Cyto Fix and conventional smears

In Japan, there are some problems with fine needle aspiration (FNA) cytology of the breast, such as insufficient smeared cells, air-drying artefact and excessive erythrocytes. Liquid-based cytology has been found to solve these problems. Equipment for such preparations has been developed, but can be expensive to purchase and operate. We developed Auto Cyto Fix 1000 (ACF), which is inexpensive and automatically smears and fixes cells. The purpose of this study was to compare the various cytological features of conventional and ACF specimens. We evaluated whether the ACF method would be able to replace the conventional method. Forty-eight FNA specimens of breast were studied. All specimens were prepared by the direct smeared (DS) and ACF methods and evaluated for unsatisfactory cell collection, air-drying artefacts, background findings and epithelial cell findings. Although ACF specimens were prepared using the cells remaining in the needle and syringe after preparing DS specimens, the cellularity of two of the ACF specimens was better than that of the corresponding DS specimens. ACF specimens never showed air-drying artefact. Unlike DS specimens, which have many erythrocytes in the background, erythrocytes were filtered out and the background of ACF specimens was clean. We believe that many problems attributable to conventional FNA specimen preparation have been solved in this study. Preparation using the ACF apparatus can reduce running costs and can be used to prepare FNA specimens of the breast for cytological examination as an alternative to the conventional method.