Lipid components in the detergent-resistant membrane microdomain (DRM) obtained from the synaptic plasma membrane of rat brain

Lateral association of sphingolipids and cholesterol is considered to form membrane microdomains such as “lipid rafts” obtainable as a detergent-resistant membrane microdomain (DRM) fraction after solubilization with a non-ionic detergent and density gradient centrifugation. Since not only sphinogolipids and cholesterol, but also functional lipids such as phosphatidylinositol 4,5-bisphosphate (PIP₂) are reported to be localized in DRM prepared from several cultured cells, this domain is considered to be a platform mediating lipid-signaling. Although PIP₂ is considered to have pivotal roles in the nervous system, little information is available on the localization of PIP₂ in the DRM within the synaptic plasma membrane (SPM) obtained from matured rat brains. In this study, in order to know the localization of PIP₂ in SPM-derived DRM, we measured the amount of PIP₂ in SPM and SPM-derived DRM, by the thin-layer chromatography blotting method, using a GST-fusion protein of the pleckstrin-homology domain of phospholipase Cδ1 as a PIP₂ binding probe. About 10% of the PIP₂ in SPM was recovered in DRM. In contrast, over 40% recovery was observed for the membrane cholesterol and sphingomyelin, and about 30% recovery was observed for phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine in the DRM were detected using the thin-layer chromatography method. Since the recovery of proteins in DRM was about 10%, the result indicates that there occurs no enrichment of PIP₂ in DRM prepared from SPM.     

Nucleotide sequence of dengue type 3 virus genomic RNA encoding viral structural proteins

Complementary DNAs to the 5 proximal region of the dengue virus type 3 RNA were cloned into bacterial plasmids and the nucleotide sequence of 3,000 bases from the 5 terminus of the genome were determined by DNA and RNA sequencing methods using dideoxy chain-termination reactions. Comparison of the nucleotide sequence thus obtained with those of other flavivirus genomes revealed significant homology existing in nucleotide sequence of the flavivirus genomes. When we compared amino acid sequence deduced from the nucleotide sequence with those of other flaviviruses, this genome region was found to include sequences encoding three viral structural proteins C, M, and E and a part of the viral nonstructural protein NS1 in this order in addition to the 5-noncoding sequence. The characteristics and functions of these proteins were discussed based on the deduced amino acid sequences and their hydrophobic profiles. The genetic relationship of flaviviruses was also discussed based on the genetic variation observed in their genomes.     

Expression of BAFF-R (BR3) in normal and neoplastic lymphoid tissues characterized with a newly developed monoclonal antibody

BAFF-receptor (BAFF-R) is required for the successful maturation and survival of B-cells. We developed an anti-human BAFF-R monoclonal antibody (mAb), 8A7. The reactivity of 8A7 in normal and neoplastic tissue was examined by performing immunohistochemistry on paraffin-embedded sections. 8A7 reacted with lymphocytes in the mantle and marginal zones, but not with lymphocytes in the interfollicular area. Lymphocytes in the germinal centers were found to be negative or occasionally weakly positive for 8A7. BAFF-R expression was found only in B-cell lymphoma (44/80, positive cases/examined cases): B-lymphoblastic lymphoma 0/3, B-chronic lymphocytic leukemia/small lymphocytic lymphoma 4/4, mantle cell lymphoma 9/11, follicular lymphoma 10/14, diffuse large B-cell lymphoma (DLBCL) 11/25, marginal zone B-cell lymphoma 8/10, lymphoplasmacytic lymphoma 2/2, plasma cell myeloma 0/2, and Burkitt lymphoma 0/9, but not in T/NK cell lymphomas (0/19) or Hodgkin lymphoma (0/10). BAFF-R was expressed in most low-grade B-cell neoplasms and a small number of DLBCL, suggesting that BAFF-R may play an important role in the proliferation of neoplastic lymphoid cells. Thus, the mAb is very useful for further understanding of both healthy B-cell biology and its pathogenic neoplasms.     

Distribution and frequencies of PDS (SLC26A4) mutations in Pendred syndrome and nonsyndromic hearing loss associated with enlarged vestibular aqueduct: a unique spectrum of mutations in Japanese

Molecular diagnosis makes a substantial contribution to precise diagnosis, subclassification, prognosis, and selection of therapy. Mutations in the PDS (SLC26A4) gene are known to be responsible for both Pendred syndrome and nonsyndromic hearing loss associated with enlarged vestibular aqueduct, and the molecular confirmation of the PDS gene has become important in the diagnosis of these conditions. In the present study, PDS mutation analysis confirmed that PDS mutations were present and significantly responsible in 90% of Pendred families, and in 78.1% of families with nonsyndromic hearing loss associated with enlarged vestibular aqueduct. Furthermore, variable phenotypic expression by the same combination of mutations indicated that these two conditions are part of a continuous category of disease. Interestingly, the PDS mutation spectrum in Japanese, including the seven novel mutations revealed by this study, is very different from that found in Caucasians. Of the novel mutations detected, 53% were the H723R mutation, suggesting a possible founder effect. Ethnic background is therefore presumably important and should be noted when genetic testing is being performed. The PDS gene mutation spectrum in Japanese may be representative of those in Eastern Asian populations and its elucidation is expected to facilitate the molecular diagnosis of a variety of diseases.     

Three Kinds of Foamy Cells in the Spleen: Comparative Histochemical and Ultrastructural Studies

By light and electron microscopy, we observed foamy cells in the spleens from a patient with hemolytic anemia due to red cell adenosine deaminase (ADA) overproduction, a patient with rheumatoid arthritis (RA) treated with gold, and patients with idiopathic thrombocytopenic purpura (ITP)

The foamy cells associated with red cell ADA overproduction were essentially similar to Gaucher-like cells described in patients with thalassemia, and it was suggested that the accelerated destruction of red cells was one of the factors responsible for the development of foamy cells. Foamy cells in ITP and RA were closely associated with an increased destruction of platelets in the spleen. Morphologic transitions between phagocytosed platelets and myelinlike materials were traced in these disorders. In RA, however, foamy cells were heterogeneous from an ultrastructural standpoint, with different cytoplasmic inclusions. In addition to myelinlike materials, dense bodies, vacuoles with flocculent materials, and gold were noted in most of foamy cells. As gold compounds are known to inhibit lysosomal enzymes, we surmise that an acquired disturbance in lysosomal digestion is partially responsible for the accumulation of intermediate metabolites.

In the pathogenesis of foamy cells associated with blood cell dyscrasia, the accelerated destruction of blood cells and/or acquired disorders in catabolic pathways within the macrophages are suggested to be the underlying mechanism of an intralysosomal accumulation of incompletely degraded cellular debris.     

De novo interstitial deletion of 4q[46,XX,del(4)(q27q28.2)] with intact blood group-MN locus,confining its locus to 4q28.2–4q31.1

We report a malformed female infant withde novo interstitial deletion of 4q[46,XX,del(4)(q27q28.2)]. The MN blood type analysis of the family members showed that the patient had an intact blood group-MN locus. The locus of the gene responsible for the MN antigen activity is confined to a 4q28.2–4q31.1 segment on the basis of the result of this patient and the previous mapping data.     

Adult Still's disease reflects a Th2 rather than a Th1 cytokine profile

Adult Still's disease (ASD) is a chronic multisystemic disease. Extraordinarily high serum levels of IL-18 in ASD patients have been described, whereas the mechanism remains to be clarified. This study aimed to evaluate proinflammatory cytokines and to consider their pathological roles. In patients with rheumatic diseases (n = 151), blood samples were taken at the active phase and the serum levels of IL-18 and other proinflammatory cytokines were measured by ELISA. The extra-high levels of IL-18 were confirmed selectively in ASD patients (n = 10). In the active phase of ASD patients, the levels of IL-6 were elevated accordingly, but IL-1beta and TNF-alpha were undetectable. As to Th1-Th2 cytokines, the levels of IL-4 and IL-13, but not INF-gamma, IL-12, or IL-2, were elevated in all ASD patients examined. Moreover, the serum levels of IL-18 showed a good correlation with those of IL-4, suggesting that ASD reflects a Th2 rather than a Th1 cytokine profile.     

Aldosterone

Aldosterone is one the representative cardiovascular hormones involved in the blood pressure and body-fluid homeostasis. Elevation of aldosterone leads to systemic hypertension through its action on the mineralocorticoid receptor (MR) in the kidney. More recent studies demonstrated that aldosterone may produce target organ damage through its direct actions on the non-epithelial MR of the heart in addition to its systemic effects. Clinical experience in primary aldosteronism supports the concept that aldosterone is a risk factor of cardiovascular complications, since concentric type of cardiac hypertrophy is most common in primary aldosteronism among various types of endocrine hypertension. Clinical mega-trial in congestive heart failure (RALES study, EPHESUS study) demonstrated blocking angiotensin II action is not sufficient for cardioprotection unless aldosterone action is equally blocked. An important phenomenon related to this issue is the aldosterone breakthrough which implies a reelevation of plasma aldosterone during chronic administration of ACE inhibitors and Angiotensin receptor antagonists. Normal level of aldosterone could still be a risk factor. Combination of ACE inhibitor or ARB with aldosterone antagonist could result in a better cardioprotection in cardiovascular diseases. Although spironolactone has been the only one aldosterone antagonist, a new antagonist eplerenone has been developed. Eplerenone is specific to MR and is practically devoid of the major side effect gynecomastia of spironolactone. Another topic of aldosterone is its very quick cardiovascular effect presumably via a non-genomic action. All these recent findings support that this adrenocortical steroid hormone is as important as angiotensin II. Determining aldosterone levels is therefore much morel important than before in the diagnosis and treatment of cardiovascular diseases.     

Widespread lipoplex-mediated gene transfer to vascular endothelial cells and hemangioblasts in the vertebrate embryo.

We report here a method that allows fast, efficient, and low-cost screening for gene function in the vascular system of the vertebrate embryo. Through intracardiac delivery of nucleic acids optimally compacted by a specific cationic lipid, we are able to induce in vivo endothelial cell-specific gain-of-function during development of the vascular network in the chick embryo. When the nucleic acids are delivered during the period of intraembryonic hematopoiesis, aortic hemangioblasts, the forerunners of the hematopoietic stem cells known to derive from the aortic endothelium, are also labeled. Similarly, we show that siRNA could be used to induce loss-of-function in vascular endothelial cells. This gene transfer technique was also applied to the mouse embryo with a high efficiency. The present method allows large-scale analysis and may represent a new and versatile tool for functional genomics.