The spectrophotometrical analysis of rhododendrol-induced leucoderma using a novel multispectral camera.

BACKGROUND: Many users in Japan of skin brightening/lightening cosmetics containing rhododendrol (RD) have developed leucoderma. Leucoderma appears on skin areas repeatedly treated with RD-containing cosmetics. RD-induced leucoderma (RDIL) presents different degrees of well-defined hypopigmentation. It is crucial to determine the degree of hypopigmentation to differentiate RDIL from vitiligo vulgaris (VV). OBJECTIVES: To quantitatively evaluate hypopigmentation of RDIL lesions and the recovery of pigmentation, and to compare the hypopigmentation with VV and normal skin. MATERIALS AND METHODS: Sixteen cases of RDIL, nine cases of VV and 15 healthy controls were examined using a novel multispectral camera (MSC) that can simultaneously obtain the reflection intensity at 10-nm wavelength intervals from 400 to 760 nm of the photographed area. ∆Absorbance was calculated by subtracting the log of reflection intensity of the target area from that of a white reflection standard. RESULTS: Most RDIL lesions showed lower ∆Absorbance than healthy skin and higher ∆Absorbance than VV lesions between 400 and 550 nm. Statistical comparison of the maximum ∆Absorbance from 420 to 460 nm (Max∆Absorbance) for VV, RDIL and control skin showed that the Max∆Absorbance of RDIL was significantly higher than that of VV and lower than that of control skin. The comparison of ∆Absorbance of the same sites in RDIL lesions between the initial visit and 6 months later showed significant improvement after 6 months. CONCLUSIONS: These studies demonstrated quantitative changes in RDIL and its recovery phase and suggested the utility of a MSC in obtaining objective colour information of skin disorders.

Functional screening of 2 Mb of human chromosome 21q22.2 in transgenic mice implicates minibrain in learning defects associated with Down syndrome.

Using Down syndrome as a model for complex trait analysis, we sought to identify loci from chromosome 21q22.2 which, when present in an extra dose, contribute to learning abnormalities. We generated low-copy-number transgenic mice, containing four different yeast artificial chromosomes (YACs) that together cover approximately 2 megabases (Mb) of contiguous DNA from 21q22.2. We subjected independent lines derived from each of these YAC transgenes to a series of behavioural and learning assays. Two of the four YACs caused defects in learning and memory in the transgenic animals, while the other two YACs had no effect. The most severe defects were caused by a 570-kb YAC; the interval responsible for these defects was narrowed to a 180-kb critical region as a consequence of YAC fragmentation. This region contains the human homologue of a Drosophila gene, minibrain, and strongly implicates it in learning defects associated with Down syndrome.

Characterization of the signal transduction of prostaglandin E receptor EP1 subtype in cDNA-transfected Chinese hamster ovary cells.

We examined the signal transduction of mouse prostaglandin E receptor EP1 subtype using Chinese hamster ovary cells stably expressing the cloned EP1. Sulprostone, an EP1 agonist, induced a rapid increase in intracellular Ca2+ concentration in the EP1-expressing cells. Most of the increase was abolished by removal of extracellular Ca2+, and was insensitive to U-73122, a phospholipase C inhibitor. Sulprostone stimulated phosphatidylinositol hydrolysis, but this stimulation was abolished by removal of extracellular Ca2+, indicating that EP1-stimulated phosphatidylinositol hydrolysis is the result of extracellular Ca2+ influx. Thus, the signal transduction of EP1 is extracellular Ca2+ entry through a pathway independent of phospholipase C activation. We further examined the regulation of the signal transduction of EP1 having potential phosphorylation sites for either protein kinase C or protein kinase A. Short-term exposure of the cells to 12-O-tetradecanoylphorbol 13-acetate (TPA) completely suppressed the sulprostone-induced increase in intracellular Ca2+ concentration, while forskolin or dibutyryl cAMP did not affect it, suggesting that protein kinase C but not protein kinase A is involved in the regulation of the EP1 signal transduction. Furthermore, long-term exposure to TPA decreased PGE2 protein kinase A is involved in the regulation of the EP1 signal transduction. Furthermore, long-term exposure to TPA decreased PGE2 binding activity of EP1 due to the reduction of the EP1 mRNA level. Protein kinase C induces short- and long-term desensitization of EP1.

Functional interaction of prostaglandin E receptor EP3 subtype with guanine nucleotide-binding proteins, showing low-affinity ligand binding.

The functional interaction of prostaglandin E (PGE) receptor EP3 subtype with GTP-binding proteins (G proteins) was characterized in the membranes prepared from mouse EP3 receptor cDNA-transfected Chinese hamster ovary cells. PGE2 inhibited forskolin-stimulated adenylate cyclase activity in CHO cells expressing EP3 receptor and this inhibition was abolished by pertussis toxin (PT) treatment. The PGE2 binding to the membranes was increased by GTP gamma S, and PT treatment also increased the binding activity to the same level as that increased by GTP gamma S, but the sensitivity of GTP gamma S was lost. Reconstitution with PT-sensitive G proteins into the ADP-ribosylated membranes reduced the PGE2 binding activity with the following preference: Gi1 = Gi2 > Gi3 > GO, but GTP gamma S completely blocked the reduction by G proteins. The G-protein-induced reduction of the binding was due to the increase in Kd without the change of Bmax, and due to suppression of association rate. [3H]PGE2-bound EP3 receptor solubilized from the ADP-ribosylated membranes in the presence or absence of GTP gamma S was eluted at the position of M(r) = approx. 60 kDa, similar to the relative molecular mass of EP3 receptor deduced from its amino acid sequence. In contrast, [3H]PGE2-bound receptor solubilized from Gi2-reconstituted membranes was eluted at the position of M(r) = approx. 130 kDa, corresponding to the M(r) of the complex of EP3 receptor and Gi2, but GTP gamma S shifted the position of its elution from M(r) = 130 to 60 kDa. Furthermore, addition of PGE2 stimulated the GDP release from G proteins reconstituted into the ADP-ribosylated membranes, and PGE2 inhibited forskolin-stimulated adenylate cyclase activity in G-protein-reconstituted membranes with a selectivity order of Gi1 = Gi2 > Gi3 > GO. These results indicate that EP3 receptor can functionally couple to PT-sensitive G proteins and unusually the complex form with G proteins has low affinity for the ligand but the form not associated with G proteins has high affinity.

Target-cell specificity of fusogenic liposomes: membrane fusion-mediated macromolecule delivery into human blood mononuclear cells.

Fusogenic liposome, a unique vector prepared by fusing ultraviolet-inactivated Sendai virus and liposome, is known to efficiently deliver content into various animal cells through membrane fusion. In this study, we examined the target-cell specificity of fusogenic liposome (FL)-mediated macromolecule delivery into human blood cells using diphtheria toxin fragment A (DTA) as a probe. Among the peripheral blood mononuclear cells (PBMC), FL was able to deliver its encapsulates into CD14+ monocytes and CD4-/CD8- T-cells, but not into CD19+ B-lymphocytes, CD4+ T-cells or CD8+ T-cells. The susceptibility of human leukemia cell lines to FL was similar to that of PBMC; the order of the reactivity was U937 (monoblastic leukemia)>MOLT4, Jurkat (T-lymphoma)>Daudi, BALL1 (B-lymphoma)>K562 (erythroblastic leukemia). Interestingly, FL showed similar binding activity to all of these leukemia cell lines. These findings indicate that, among blood cells, monocytes, monoblastic leukemia cells, CD4-/CD8- T-cells and T-lymphoma cells are preferable targets for FL-mediated macromolecule delivery. This is the first demonstration of the existence of non-permissive cells against FL. Our results also suggest that some molecules on target-cells other than the binding targets of SV-derived protein may participate in fusion between FL and cells.

Efficient gene transfer by fiber-mutant adenoviral vectors containing RGD peptide.

One of the hurdles to adenovirus (Ad)-mediated gene transfer is that Ad vectors mediate inefficient gene transfer into cells lacking in the primary receptors, Coxsackievirus and adenovirus receptor (CAR). We previously developed a fiber-mutant Ad vector containing the Arg-Gly-Asp (RGD)-containing peptide motif on the HI loop of the fiber knob, and showed that the mutant vector had enhanced gene transfer activity to human glioma cells, which showed little CAR expression, compared to the vector containing wild type fiber. In this study, the feasibility of the Ad vector containing RGD peptide on the fiber knob was examined in a wide variety of cell types: CAR-positive or -negative human tumor cells, mouse cells, and leukemia cells. The mutant vector infected the cells, which lacked CAR expression but showed alpha(v) integrin expression, about 10-1000 times more efficiently than the vector containing wild type fiber via an RGD-integrin (alpha(v)beta3 and alpha(v)beta5)-dependent, CAR-independent cell entry pathway. The results of this study indicate that Ad vector containing RGD peptide on the fiber knob could be of great utility for gene therapy and gene transfer experiments.

Optimization of transcriptional regulatory elements for constructing plasmid vectors.

In studies regarding both gene therapy and gene function, transgene expression by plasmid vectors benefits from the use of transcriptional regulatory elements which permit high-level gene expression. Therefore, with respect to transgene (luciferase) expression activity both in vitro (using HeLa, HepG2, and ECV304 cells) and in vivo (mouse liver and skeletal muscle), we investigated the effective combination of commonly-used regulatory elements, such as the promoter/enhancer, intron, and polyadenylation signal (P(A)) sequence by constructing a series of plasmids that differed only in the particular sequence element being evaluated. Of the several promoter/enhancers that were tested, hybrid CA promoter/enhancer containing human cytomegalovirus immediate-early 1 gene (CMV) enhancer and chicken beta-actin promoter with the beta-actin intron sequence, and the improved CMV promoter/enhancer containing the largest intron of CMV (intron A) produced the highest levels of expression both in vitro and in vivo. P(A) sequences were found to have significant effects on transgene expression. The effect of a multiple enhancer was also examined. Optimized plasmids of this study were pCASL3 (composed of CMV enhancer, beta-actin promoter, beta-actin intron, Simian virus (SV40) P(A) sequence and SV40 enhancer) and pCMVSL3 (composed of CMV enhancer, CMV promoter, intron A, SV40 P(A) sequence and SV40 enhancer). These comparative analyses could provide a systematic reference for the development of vector construction for gene therapy, vaccine development, and gene transfer experiments.

Purification and properties of L-histidine decarboxylase from mouse stomach.

L-Histidine decarboxylase was purified to electrophoretic homogeneity from mouse stomach according to a procedure described previously [Ohmori E, Fukui T, Imanishi N, Yatsunami K and Ichikawa A, J Biochem (Tokyo) 107: 834-839, 1990]. The purified enzyme exhibited a specific activity of 750 nmol histamine formed per min per mg protein, which constituted a 37,500-fold purification compared to the crude extract, with a 1.6% yield. The molecular mass of the enzyme was estimated to be 54 kDa by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and 100 kDa by gel filtration. The isoelectric point of the enzyme was determined to be pH 5.4. The Km value for L-histidine was estimated to be 0.29 mM. The single mRNA encoding the amino acid sequence of the mouse stomach enzyme was examined and its size was found to be 2.7 kb. These molecular and catalytic property values of the L-histidine decarboxylase of mouse stomach are quite similar to those of the enzyme from mouse mastocytoma P-815 cells.

On the mechanism of plasmin-induced platelet aggregation. Implications of the dual role of granule ADP.

Plasmin-induced platelet aggregation has been considered to be a cause of reocclusion after thrombolytic treatment with plasminogen activators. However, little is known regarding the mechanism and regulation of plasmin-induced platelet aggregation. In this study, we demonstrated that plasmin causes the degranulation of platelets, and that ADP released from granules plays a crucial role in the induction of platelet aggregation. This conclusion is supported by results showing that both ADP antagonists and ADPase can inhibit the effect of plasmin on platelets. We also demonstrated that pretreatment of platelets with ADP makes the platelets more sensitive to plasmin, and plasmin-induced platelet aggregation is, therefore, observed at lower concentrations where no aggregation occurs in quiescent platelets. In other words, it is thought that ADP potentiates the plasmin-induced aggregation. The effect of ADP was inhibited by N(6)-[2-(methylthio)-ethyl]-2-(3,3, 3-trifluoropropyl)thio-5'-adenylic acid, monoanhydride with dichloromethylenebisphosphonic acid (AR-C69931), a selective antagonist for the P2T(AC) subtype of P2 receptor, but not by the P2Y1 receptor-selective antagonist adenosine 3'-phosphate 5'-phosphosulfate (A3P5PS). The P2X1 receptor agonist alpha, beta-methylene adenosine 5'-triphosphate (alpha,beta-MeATP) did not mimic the action of ADP. These data indicate that ADP potentiates plasmin-induced platelet aggregation via the P2T(AC) receptor. In addition, epinephrine, a typical G(i) agonist against platelets, could potentiate the plasmin-induced platelet aggregation, suggesting that the signal via the G(i) protein is involved in potentiating the plasmin-induced platelet aggregation, ADP is secreted from platelet granules, and concomitantly works in conjunction with plasmin in a P2T(AC) receptor-mediated manner.

Gene transfer vectors based on Sendai virus.

A gene delivery system is a fundamental technology used in human gene therapy. In order to treat patients suffering from incurable metabolic diseases, we must be able to deliver genes efficiently in situ and induce stable gene expression in non-dividing tissue cells. However, none of the current gene transfer systems (both viral and non-viral) satisfies this goal. In order to develop a novel gene delivery system that is free from the defects of existing gene transfer vectors, we analyzed natural biological phenomena that involve gene transfer and expression, and made artificial components that mimic the functioning of these systems. Our recent results shed light on three major aspects of gene transfer and expression: (1) the direct delivery of DNA into cytoplasm using fusogenic liposomes, (2) the transfer of DNA from cytoplasm to nucleus with a nuclear localization signal, and (3) the stabilization of DNA in the nucleus as an independent replicon. The possible development of a hybrid vector by combining these components is discussed.

Involvement of a calcium-independent pathway in plasmin-induced platelet shape change.

Plasmin-induced platelet activation is considered to be a cause of reocclusion after thrombolytic therapy with plasminogen activators. However, little is known regarding its mechanism and regulation, particularly with respect to the initial step shape change. We here demonstrate that a Ca2+-independent pathway is involved in plasmin-induced human platelet shape change, and that Rho-kinase plays an important role in this pathway. When the increase in cytosolic Ca2+ was prevented by an intracellular Ca2+ chelator, 5,5'-dimethyl-BAPTA, plasmin-induced platelet shape change was partially inhibited but still occurred. In the presence of 5,5'-dimethyl-BAPTA, a specific Rho-kinase inhibitor, Y-27632, completely inhibited the shape change. Phosphorylation of myosin light chain, a key regulator of platelet shape change, was completely inhibited by Y-27632 in 5,5'-dimethyl-BAPTA-treated platelets. Although plasmin caused tyrosine phosphorylation of the 80 kDa protein during the shape change, it did not seem to have a critical role. cAMP-elevating agents inhibited plasmin-induced shape change in 5,5'-dimethyl-BAPTA- or Y-27632-treated platelets with similar efficiency. These results indicated that plasmin causes platelet shape change by activating Ca2+-dependent and Ca2+-independent-Rho-kinase-dependent pathways, both of which are sensitive to cAMP.

Iodine content of various meals currently consumed by urban Japanese.

Various meals being currently consumed by urban Japanese were determined for iodine. The meal samples were collected in 1982 and 1984. The habitual daily home meals of 4 middle-aged Japanese living in urban areas contained 45-1,921 micrograms (mean; 362, 361, 429 and 1,023 micrograms, respectively) of iodine per day. The regular meals served in two university hospitals contained 95-287 micrograms (mean; 195 micrograms) and 89-4,746 micrograms (mean; 1,290 micrograms) of iodine per day, respectively, and the diets for diabetes mellitus contained 59-144 micrograms (mean; 96 micrograms) of iodine per day. In the daily meals containing iodine exceeding ca. 300 micrograms, some kinds of seaweeds and, in some cases, several foods containing a red food color with low iodine bioavailability, erythrosine, provided a large portion of iodine. The iodine contents of refectory meals in a university were 47-203 micrograms (mean; 113 micrograms) per meal and those of lunches in two elementary schools were 25-31 micrograms (mean; 27 micrograms) and 18-43 micrograms (mean; 36 micrograms) per lunch, respectively. These results suggest that the current daily iodine intake of urban Japanese is not great and that erythrosine elevates the iodine content of meals.

Effects of transgalactosylated disaccharides on the human intestinal microflora and their metabolism.

The effects of transgalactosylated disaccharide (TD) intake on human fecal microflora and their metabolism were investigated in 12 Japanese males. TD is a mixture of sugars, galactosyl galactose, and galactosyl glucose, synthesized from lactose through the transgalactosylation reaction of Streptococcus thermophilus beta-galactosidase. Volunteers took 15 g of the test sugar daily for 6 days. The TD ingestion increased the number of bifidobacteria and lactobacilli, but decreased the number of Bacteroidaceae and Candida spp. in the feces. The ratio of bifidobacteria to total bacteria increased from 0.28 to 0.51. TD decreased the fecal concentrations of propionic acid, isobutyric acid, isovaleric acid, and valeric acid. This sugar also lowered the fecal pH, and the concentrations of fecal ammonia, p-cresol, and indole. Moreover, a positive correlation was found between the concentration of ammonia, and that of branched-chain fatty acids (isobutyric acid and isovaleric acid), p-cresol, and indole. All of these compounds are produced from amino acids through deamination by the intestinal bacteria. The depression of amino acid fermentation by intestinal bacteria may be involved in the reduction of fecal ammonia. These results suggest that a part of the transgalactosylated disaccharides passes into the colon, inducing changes in the colonic microflora composition, hastening carbohydrate fermentation, and depressing amino acid fermentation in the human gut.

[A 10-year field surveillance in Hinohara Village of Tokyo Prefecture from 1981 to 1990].

We conducted a surveillance to clarify the relationship between risk factors for diseases of adulthood and lifestyle in a Japanese rural community, Hinohara Village, a small village outside of Tokyo. The survey, carried out from 1981 to 1990 among residents aged 40 and over, comprised physical examination and blood chemistry with a questionnaire about dietary intake. Mean systolic blood pressure significantly decreased (p < 0.0001) from 140.9 mmHg in 1981 to 132.3 mmHg in 1990, whereas mean serum total cholesterol, mainly of male examinees, increased (p < 0.0001) from 181.4 mg/dl in 1981 to 191.7 mg/dl in 1990. Dietary salt intake significantly decreased (p < 0.0001) from 14.3 g/day in 1981 to 12.1 g/day in 1990. Adjusted mortality rate per 1,000 residents from cerebrovascular disease in this village decreased from 1.80 in 1981 to 0.50 in 1990. In contrast to its decline, the mortality rates from heart disease, bronchitis/pneumonia and neoplasms were 0.40, 0.35 and 0.55 in 1981 and increased to 1.25, 1.10 and 0.64 in 1990. The prevailing practice of maintaining a low-salt diet might cause the decrease of systolic blood pressure, which in turn was thought to decrease the mortality rate from cerebrovascular diseases. Although our previous study before 1981 suggested that total cholesterol was one of the preventive factors against cerebrovascular disease, in the present study a preventive effect of cholesterol was not substantiated. In contrast, cholesterol is a possible risk factor for ischemic heart disease. Thus, a changing pattern of risk factors of diseases of adulthood was observed in this village.

Isolation and characterization of kar2-404 mutation in Saccharomyces cerevisiae.

We have devised a direct screening method to isolate mutations in the KAR2 gene, and have isolated a BiP/KAR2 mutant, kar2-404, from Saccharomyces cerevisiae as a small halo-forming mutant of secreted mouse alpha-amylase. The mutation site was identified as a point mutation at t1337 to c1337 resulting in the Ile-404Thr mutation of mature Kar2-404p, located at the most NH2-terminal first beta-sheet structure (beta 1) of the putative peptide-binding domain. This isoleucine is highly conserved in the Hsp70 family. By pulse-chase experiments, no obvious difference was detected in the intracellular secretion rate of MF alpha 1-prepro-signal-mouse-alpha-amylase between the wild type and the kar2-404 mutant. However, only about half the amount of secreted heterologous protein, mouse alpha-amylase, was detected in the mutant culture medium compared with wild type. A smaller amount of homologous protein, alpha-factor, was also detected and decreased faster in the mutant culture medium than in wild type. Kar2-404p was expressed about 3-fold more than wild type Kar2p, probably to cover its defective functions, and the turnover rates of Kar2p and Kar2-404p were about the same in vivo. The purified Kar2-404p was slightly more sensitive to chymotryptic digestion than Kar2p in vitro.

Gas transport in serpentine microporous tubes under steady and pulsatile blood flow conditions.

A serpentine gas exchange unit was built with cylindrical tubular microporous membranes featuring periodic arcs with a fixed curvature ratio (ratio of tube radius to radius of curvature) of 1/14 and circular angles between 30 and 360 deg. Oxygen transfer was measured under steady and pulsatile blood flow conditions in vitro and ex vivo to assess the design features which most effectively augment gas transfer. Under steady blood flow conditions, oxygen transfer increased with circular angles beyond 70 deg. Under pulsatile conditions, a wide range of geometrical and fluid mechanical parameters could be combined to enhance gas transfer performance, which eventually depended upon the secondary Reynolds number and the Womersley parameter.