Visualization and live imaging analysis of a mosquito saliva protein in host animal skin using a transgenic mosquito with a secreted luciferase reporter system.

Mosquitoes inject saliva into a vertebrate host during blood feeding. The analysis of mosquito saliva in host skin is important for the elucidation of the inflammatory responses to mosquito bites, the development of antithrombotic drugs, and the transmission-blocking of vector-borne diseases. We produced transgenic Anopheles stephensi mosquitoes expressing the secretory luciferase protein (MetLuc) fused to a saliva protein (AAPP) in the salivary glands. The transgene product (AAPP-MetLuc) of transgenic mosquitoes exhibited both luciferase activity as a MetLuc and binding activity to collagen as an AAPP. The detection of luminescence in the skin of mice bitten by transgenic mosquitoes showed that AAPP-MetLuc was injected into the skin as a component of saliva via blood feeding. AAPP-MetLuc remained at the mosquito bite site in host skin with luciferase activity for at least 4 h after blood feeding. AAPP was also suspected of remaining at the site of injury caused by the mosquito bite and blocking platelet aggregation by binding to collagen. These results demonstrated the establishment of visualization and time-lapse analysis of mosquito saliva in living vertebrate host skin. This technique may facilitate the analysis of mosquito saliva after its injection into host skin, and the development of new drugs and disease control strategies.

Occlusal Support, Dysphagia, Malnutrition, and Activities of Daily Living in Aged Individuals Needing Long-Term Care: A Path Analysis.

OBJECTIVES: This study aimed to examine the interrelationships among occlusal support, dysphagia, malnutrition, and activities of daily living in aged individuals needing long-term care. DESIGN: Cross-sectional study and path analysis. SETTING: Long-term health care facilities, acute care hospitals, and the community. PARTICIPANTS: Three hundred and fifty-four individuals aged ≥ 65 years with dysphagia or potential dysphagia in need of long-term care. MEASUREMENTS: The modified Eichner Index, Dysphagia Severity Scale, Mini Nutritional Assessment Short Form, and Barthel index. RESULTS: The participants included 118 males and 236 females with a mean (standard deviation) age of 83 (8) years. A total of 216 participants had functional occlusal support with or without dentures. Of the total participants, 73 were within normal limits regarding the severity of dysphagia, 119 exhibited dysphagia without aspiration, and 162 exhibited dysphagia with aspiration. Only 34 had a normal nutritional status, while 166 participants were malnourished, and 154 were at risk of malnutrition. The median Barthel index score was 30. Path analysis indicated two important findings: occlusal support had a direct effect on dysphagia (standard coefficient = 0.33), and dysphagia was associated directly with malnutrition (standard coefficient = 0.50). Dysphagia and malnutrition were associated directly with impaired activities of daily living (standard coefficient = 0.57, 0.22). CONCLUSION: In aged individuals needing long-term care, occlusal support is associated directly with dysphagia and indirectly with malnutrition and activities of daily living via dysphagia.

Semaphorin 3A Induces Odontoblastic Phenotype in Dental Pulp Stem Cells.

In cases of pulp exposure due to deep dental caries or severe traumatic injuries, existing pulp-capping materials have a limited ability to reconstruct dentin-pulp complexes and can result in pulpectomy because of their low potentials to accelerate dental pulp cell activities, such as migration, proliferation, and differentiation. Therefore, the development of more effective therapeutic agents has been anticipated for direct pulp capping. Dental pulp tissues are enriched with dental pulp stem cells (DPSCs). Here, the authors investigated the effects of semaphorin 3A (Sema3A) on various functions of human DPSCs in vitro and reparative dentin formation in vivo in a rat dental pulp exposure model. Immunofluorescence staining revealed expression of Sema3A and its receptor Nrp1 (neuropilin 1) in rat dental pulp tissue and human DPSC clones. Sema3A induced cell migration, chemotaxis, proliferation, and odontoblastic differentiation of DPSC clones. In addition, Sema3A treatment of DPSC clones increased ß-catenin nuclear accumulation, upregulated expression of the FARP2 gene (FERM, RhoGEF, and pleckstrin domain protein 2), and activated Rac1 in DPSC clones. Furthermore, in the rat dental pulp exposure model, Sema3A promoted reparative dentin formation with dentin tubules and a well-aligned odontoblast-like cell layer at the dental pulp exposure site and with novel reparative dentin almost completely covering pulp tissue at 4 wk after direct pulp capping. These findings suggest that Sema3A could play an important role in dentin regeneration via canonical Wnt/ß-catenin signaling. Sema3A might be an alternative agent for direct pulp capping, which requires further study.

Post-heparin plasma hepatic triacylglycerol lipase-catalyzed tributyrin hydrolysis. Effect of trypsin treatment.

Hepatic triacylglycerol lipase (EC 3.1.1.3) hydrolyzes water-insoluble fatty acid esters, e.g., trioleoylglycerol (lipase activity) and water-soluble fatty acid esters, e.g., tributyrin (esterase activity). Esterase activity of hepatic triacylglycerol lipase is enhanced by triolein emulsion and phospholipid vesicles [1]. The catalytic mechanism and structure of human hepatic triacylglycerol lipase isolated from human post-heparin plasma and the effect of trypsin treatment on the lipase and esterase activities of the enzyme were examined. Treatment of hepatic triacylglycerol lipase with trypsin resulted in loss of its lipase activity, but had no effect on its esterase activity. Chromatography of hepatic triacylglycerol lipase on Bio-Gel A5m showed that hepatic triacylglycerol lipase binds to dipalmitoylphosphatidylcholine vesicles. However, on chromatography of the trypsin-treated enzyme after incubation with dipalmitoylphosphatidylcholine vesicles, a part of hepatic triacylglycerol lipase that retained esterase activity was eluted separately from the dipalmitoylphosphatidylcholine vesicles. Addition of vesicles of dipalmitoylphosphatidylcholine to the trypsin-treated enzyme did not enhance its esterase activity. These results are consistent with the hypothesis that hepatic triacylglycerol lipase has a catalytic site that hydrolyzes tributyrin and a lipid interface recognition site, and that these sites are different: trypsin modified the lipid interface recognition site of the hepatic triacylglycerol lipase but not the catalytic site.

Effects of phospholipids on hydrolysis of trioleoylglycerol by human serum carboxylesterase.

Human serum carboxylesterase (EC 3.1.1.1), purified by affinity chromatography on trimethylammonium anilinium-Sepharose, hydrolyzed the short-chain fatty acid ester tributyrin (40 mumol/mg protein per h), but scarcely hydrolyzed the long-chain fatty acid ester triolein (less than 0.2 mumol/mg protein per h). Phospholipids enhanced triolein hydrolysis by carboxylesterase to various extents, cardiolipin causing the most enhancement (2.5 mumol/mg protein per h). Phosphatidylserine and phosphatidylinositol also enhanced carboxylesterase-catalyzed hydrolysis of triolein (450-980 nmol/mg protein per h). The optimal pH for tributyrin hydrolysis was pH 8.0, but the pH range for triolein hydrolysis was broad, being pH 4.5-7.5. The rates of hydrolyses of monoolein, diolein and triolein by carboxylesterase in the absence and presence of 100 micrograms/ml cardiolipin were 3.9, 0.5 and 0.2 mumol/mg esterase per h and 2.0, 0.6 and 4.0 mumol/mg protein per h, respectively. Thus, on addition of cardiolipin, triolein hydrolysis was enhanced, but tributyrin hydrolysis was reciprocally decreased. Triton X-100 (0.1%) and NaCl (1.0 M) decreased triolein hydrolysis, but did not decrease tributyrin hydrolysis. Mercaptoethanol decreased triolein hydrolysis, but not tributyrin hydrolysis. These results suggest that cardiolipin modifies the interaction of carboxylesterase with substrates in such a way as to facilitate its interaction with a hydrophobic substrate, and that disulfide bonding might be involved in the substrate recognition site.

Interaction of DNA polymerases with phospholipids.

Effects of various phospholipids on the in vitro reactions of eukaryotic DNA polymerases alpha, beta and gamma were tested systematically. When phospholipids were added directly to the reaction mixture, neither stimulation nor inhibition was produced. However, when phospholipids were preincubated with enzymes in the absence of template-primer, some of them showed strong inhibition. Cardiolipin strongly inhibited the reactions of all three DNA polymerases and also of terminal deoxynucleotidyl transferase. Phosphatidylinositol selectively inhibited the reaction of DNA polymerase gamma. Phosphatidic acid moderately inhibited DNA polymerase alpha and strongly inhibited DNA polymerase gamma. The inhibition of DNA polymerase gamma by cardiolipin was nearly competitive with template-primer. Since the inhibition was reversed by the addition of 0.05% Triton-X 100 during preincubation, the phospholipid might interact with enzyme protein at the hydrophobic region in competition with template-primer. These results suggest a possible involvement of phospholipids in DNA replication in mitochondria and in nucleus through interaction with DNA polymerase.

Post-heparin plasma hepatic triacylglycerol lipase-catalyzed hydrolysis of tributyrin. Effect of lipid interface.

The mechanism of action of hepatic triacylglycerol lipase (EC 3.1.1.3) was examined by comparing the hydrolysis of a water-soluble substrate, tributyrin, with that of triolein by hepatic triacylglycerol lipase purified from human post-heparin plasma. The hydrolyzing activities toward tributyrin and triolein were coeluted from heparin-Sepharose at an NaCl concentration of 0.7 M. The maximal velocity of hepatic triacylglycerol lipase (Vmax) for tributyrin was 17.9 mumol/mg protein per h and the Michaelis constant (Km) value was 0.12 mM, whereas the Vmax for triolein was 76 mumol/mg per h and the Km value was 2.5 mM. The hydrolyses of tributyrin and triolein by hepatic triacylglycerol lipase were inhibited to similar extends by procainamide, NaF, Zn2+, Cu2+, Mn2+, SDS and sodium deoxycholate. Triolein hydrolysis was inhibited by the addition of tributyrin. Triolein hydrolysis was also inhibited by the addition of dipalmitoylphosphaidylcholine vesicles. In contrast, the additions of triolein emulsified with Triton X-100 and dipalmitoylphosphatidylcholine vesicles enhanced the rate of tributyrin hydrolysis by hepatic triacylglycerol lipase. In the presence of dipalmitoylphosphatidylcholine, the Vmax and Km values of hepatic triacylglycerol lipase for tributyrin were 41 mumol/mg protein per h and 0.12 mM, respectively, indicating that the enhancement of hepatic triacylglycerol lipase activity for tributyrin by dipalmitoylphosphatidycholine vesicles was mainly due to increase in the Vmax. The enhancement of hepatic triacylglycerol lipase activity for tributyrin by phospholipid was not correlated with the amount of tributyrin associated with the phospholipid vesicles. On Bio-Gel A5m column chromatography, glycerol tri[1-14C]butyrate was not coeluted with triolein emulsion, and hepatic triacylglycerol lipase activity was associated with triolein emulsion even in the presence of 2 mM tributyrin. These results suggest that hepatic triacylglycerol lipase has a catalytic site for esterase activity and a separate site for lipid interface recognition, and that on binding to a lipid interface the conformation of the enzyme changes, resulting in enhancement of the esterase activity.

Nanosecond time-resolved fluorescence investigations of temperature-induced conformational changes in cytochrome oxidase in phosphatidylcholine vesicles and solubilized systems.

Intrinsic and lipid phase transition-induced conformational changes in cytochrome oxidase in phosphatidylcholine vesicle and solubilized systems were examined by the fluorescence lifetime of N-(1-anilinonaphthyl-4)-maleimide conjugated with the enzyme. The time-dependent fluorescence intensity of N-(1-anilinonaphthyl-4)-maleimide attached to cytochrome oxidase was described as a triple exponential decay. Both the intrinsic and lipid phase transition-induced conformational changes were detectable in plots of the average lifetime against temperature. In most cases a peak occurred at the temperature of the conformational change. The time-dependent emission anisotropy showed that N-(1-anilinonaphthyl-4)-maleimide embedded in cytochrome oxidase in phosphatidylcholine vesicles underwent a rapid restricted wobbling within a cone. The half-angle of the cone was around 30 degrees for cytochrome oxidase in dimyristoyl phosphatidylcholine vesicles.

Fatty acids selectively inhibit eukaryotic DNA polymerase activities in vitro.

The in vitro relationship between eukaryotic DNA polymerases and fatty acids was investigated. Some fatty acids strongly inhibited the activities of DNA polymerase alpha and/or beta in vitro. The kinetics of inhibition by linoleic acid showed that DNA polymerase alpha was non-competitively inhibited with respect to the DNA template and substrate (dTTP), while DNA polymerase beta was inhibited competitively with both DNA and substrate.

Safe and rapid contouring of fibro-osseous lesions in the orbital area using navigation with minimally invasive cranial bone registration.

BACKGROUND: Bone contouring is currently the best treatment for fibro-osseous lesions after bone growth arrest. Navigation systems available for this surgery allow intra-operative visualisation with improved cosmetic outcomes. However, conventional navigation systems using superficial skin registration cannot prevent subtle discrepancies. METHOD: To address this problem, we used a non-invasive cranial bone registration that uses patient-specific dental templates to maintain exact registration. We created the preset goal using the mirror image of the unaffected side for unilateral lesions, and using images obtained before the onset of symptoms for bilateral lesions. This system achieved precise pre-operative simulation. A sound aid in the navigation system provided information regarding proximity to critical structures and to the preset goal. RESULTS: We used this system to contour fibro-osseous lesions in three patients. All patients achieved good facial contours and improvement in symptoms. CONCLUSIONS: This method offers a safe, rapid surgical aid in treating orbital fibro-osseous lesions.

Expression of neuropsin in the keratinizing epithelial tissue-immunohistochemical analysis of wild-type and nude mice.

Neuropsin is a trypsin-type serine protease that was first cloned from the mouse brain as a factor related to neural plasticity. Subsequent in situ hybridization histochemical analysis indicated a broad localization of its mRNA throughout the whole body, although the details remain obscure. In this study, we showed that neuropsin immunoreactivity is localized in the keratinized stratified epithelia of the mouse epidermis, hair, tongue, palate, nasal cavity, pharynges, esophagus, and forestomach. In the skin and mucous membranes, neuropsin immunoreactivity was found in the stratum spinosum and the stratum granulosum. The immunoreactivity in the former sublayer was mainly present in the cytoplasm, but that in the latter sublayer was exclusively present in the intercellular space or on the outer surface of the cell membrane and thus exhibited a lamellar-like peripheral distribution. During development, the appearance of neuropsin immunoreactivity in the various epithelia was found at embryonic days 14.5-15.5, prior to formation of the stratum corneum. More extensive expression of neuropsin immunoreactivity was found in the nude mouse skin and mucous membranes than in wild-type mice. Because the nude mouse is characterized by genetic impairment of keratinization, such abnormal neuropsin expression might be caused or affected by this impairment. Therefore, neuropsin, an extracellular serine protease, is suggested to be involved in keratinization in the stratified epithelia.

Characteristics of the thyroid iodide translocator and of iodide-accumulating phospholipid vesicles.

Some biochemical characteristics of the thyroid I- translocator and of I- accumulating phospholipid vesicles (P-vesicles) were studied. P-vesicles were made from thyroid plasma membranes (PM) and soybean phospholipids by sonication. The optimal incubation temperature for Na+-dependent I- accumulation in P-vesicles was from 18-26 C. Only a small amount of Na+-independent I- accumulation was observed at various incubation temperatures, but it increased in proportion with the temperature up to 36 C. The optimal incubation pH (7.0-7.5) was near the physiological extracellular pH. When PM were heated at 55 C for 30 min before preparation of P-vesicles, Na+-dependent I- accumulation in the vesicles decreased by 35%. When they were heated at 65 C for 30 min, the I- -accumulating activity was almost completely lost. The translocator was also inactivated when PM were sonicated at 37 C in the presence of trypsin. The internal and external administration of ouabain to the vesicles did not affect the activity of Na+-dependent I- accumulation. When PM were treated with sodium dodecyl sulfate at a final concentration of 0.2-0.6 mg/ml, the I- translocator was inactivated or detached from PM, whereas the ouabain-sensitive Na+, K+-ATPase activity was preserved in the PM fragments. These observations suggest that the thyroid I- translocator consists of a protein component that is bound to PM at a site separate from Na+,K+-ATPase.

Valinomycin-induced iodide leakage without impairment in sodium-dependent iodide transport in the thyroid.

The mechanism of inhibition of I- accumulation in the thyroid by valinomycin was investigated on a biological model. Phospholipid vesicles (P-vesicles) capable of Na+-dependent I- accumulation were reconstituted with thyroid plasma membrane (PM). In P-vesicles internally loaded with K+, Na+-dependant I- accumulation was observed in the presence of external Na+. The accumulated I- was discharged from the vesicles by addition of valinomycin. In P-vesicles with internal choline+, Na+-dependent I- accumulation also occurred, but I- discharge was not induced by the addition of valinomycin. P- vesicles without a PM component readily accumulated I- in the presence of both external K+ and valinomycin. P-vesicles with thyroid PM also accumulated I- rapidly in the presence of external K+ upon the addition of valinomycin; however, the accumulated I- was significantly discharged when the vesicles were loaded internally with Na+. A significant I- discharge was not observed in the vesicles with or without thyroid PM when they were loaded with choline+ instead of Na+. Valinomycin-induced I- uptake without leakage was observed in both Na+- and choline+-loaded P-vesicles containing liver PM instead of thyroid PM. These results indicate that valinomycin may inhibit I- accumulation in thyroid cells according to the following mechanism: 1) valinomycin, a K+ carrier, induces K+ efflux to form a gradient of electrical potential with a negative charge within the cells, and 2) I- is subsequently driven out down the gradient through the phospholipid bilayer. Na+-dependent I- transport may not be impaired by valinomycin.

STT3, a novel essential gene related to the PKC1/STT1 protein kinase pathway, is involved in protein glycosylation in yeast.

Mutations of genes involved in the STT1/PKC1 pathway in yeast show staurosporine and temperature sensitivities (stt) which are suppressed by the addition of 1 M sorbitol [Yoshida et al., Mol. Gen. Genet. 242 (1994) 631-640]. Among the stt mutants, stt3-2 shares this phenotype. The STT3 gene encodes a novel 718-amino-acid protein with significant homology to potential transmembrane proteins of Caenorhabditis elegans and mouse mandibular condyle (about 80% homologous and 60% identical). Unlike the STT1/PKC1 gene, STT3 is essential for cell growth irrespective of osmotic support. Pulse-chase experiments show that the sst3 mutants are defective in protein glycosylation. The stt3 mutants are sensitive to hygromycin B and resistant to sodium orthovanadate, whose phenotypes are common to those defective in protein glycosylation.

Mitoxantrone and cyclophosphamide in advanced breast cancer: a pilot study.

A trial of combination chemotherapy using mitoxantrone-cyclophosphamide was started in 1983. Sixteen patients with widely metastatic cancer of the breast, including one man, received mitoxantrone, 10 mg/m2 intravenously (IV) over 30 minutes on day 1, followed by cyclophosphamide, 200 mg/m2 by mouth (PO) daily in divided doses on days 3 to 6. It is too early to evaluate four patients at present. The remaining 12 received three or more courses of treatment, and three of these patients achieved a complete response. Another four patients went into partial remission, amounting to an overall response rate of 58%. The other evaluable patients showed stable disease with improved symptoms. Hematologic toxicity was mainly granulocytopenia, but thrombocytopenia occurred in two patients. Alopecia, nausea, and vomiting were attributed to the cyclophosphamide component of the therapy. Mitoxantrone appeared to have no cardiac toxicity. It was concluded that mitoxantrone-cyclophosphamide is an effective chemotherapeutic combination with minimal toxicity and should be further studied in larger controlled trials.

Hypersensitivity to thromboxane A2 in cholesterol-rich human platelets.

The effect of changes in platelet membrane cholesterol content on thromboxane A2 (TXA2)-induced platelet activation was studied. Concentrations of 9,11-epithio-11,12-methano-TXA2 (STA2), a stable analogue of TXA2 which can cause half-maximal aggregation and release of [14C]serotonin in cholesterol-rich platelets were significantly lower than those in cholesterol-normal platelets. STA2-induced increase in cytosolic calcium concentration and [32P]phosphatidic acid formation in cholesterol-rich platelets were significantly greater than those in cholesterol-normal platelets. The maximal concentration of binding site (Bmax) for SQ29548 was significantly increased in cholesterol-rich platelets compared with cholesterol-normal platelets, while the equilibrium dissociation rate constant (Kd) for SQ29548 did not differ between cholesterol-rich and cholesterol-normal platelets. The present study suggested that sensitivity to TXA2 was increased by the incorporation of cholesterol into platelet membrane and that the cause of hypersensitivity to TXA2 in cholesterol-rich platelets may be partly explained by an increase in binding capacity for TXA2.

Autoimmunity and selected environmental factors of disease induction.

Autoimmune diseases may be induced by physical and/or chemical environmental factors. A review of the available literature on mercuric chloride, iodine, silicone, anilides, L-tryptophan, vinyl chloride, and canavanine suggests three general mechanisms by which they may induce disease. First, oxidative damage probably is a frequent process involved in disease induction and pathogenesis. Second, certain compounds also may generate antigen-specific immune responses that could then cross-react with self-tissues. Other xenobiotics might bind to self-tissues and increase self-tissue immunogenicity. Third, physical and chemical agents may also modulate the immune system. Finally, in response to controversies surrounding the influence of human activities on global climate changes, the immunosuppressive effects of ozone and ultraviolet radiation are discussed.

Left main coronary trunk compression by dilated main pulmonary artery in atrial septal defect. Report of three cases.

Narrowing of the left main coronary trunk, which was compressed by the dilated pulmonary artery, was associated with atrial septal defect in three adults. One of them had severe pulmonary hypertension. Coronary angiograms revealed localized narrowing of the left main coronary trunk, and the left main coronary trunk had a concave shape. No stenosis of other coronary arteries was observed. In all patients the atrial septal defect was closed with a polytetrafluoroethylene patch. In the patient with 75% narrowing of the left main coronary trunk, aorta-coronary bypass was performed; it was not performed in the two with 50% narrowing. In two survivors postoperative coronary angiograms showed that the narrowing of the left main coronary trunk improved or disappeared. These results suggest that markedly dilated pulmonary arteries easily compress the left main coronary trunk and cause narrowing, which improves after atrial septal defect closure.

Stimulatory effect of reconstituted basement membrane components (matrigel) on the colony formation of a panel of human lung cancer cell lines in soft agar.

Lung cancers have been distinguished into small-cell lung cancer (SCLC) and non-small cell-lung cancer (NSCLC) types on the basis of their clinical behaviors and their responses to treatment. Moreover, growth of most SCLC cell lines in liquid culture medium is nonadherent, while that of most NSCLC cell lines is adherent. In this study, we examined the effect of matrigel (reconstituted basement membrane components), which is known to have growth-stimulatory activity on various human tumor cell lines in immunodeficient mice, on soft-agar colony formation of a panel of SCLC and NSCLC cell lines to clarify its mechanism of growth stimulation of cancer cells. Matrigel enhanced colony formation of all 9 NSCLC cell lines and 4 of 9 SCLC cell lines. There was a statistically significant difference (P < 0.01) between colony formations with and without matrigel of NSCLC cell lines, but not for SCLC cell lines. In liquid culture medium, all 9 NSCLC lines and 3 of 9 SCLC lines adhered to plastic dishes, whereas the other SCLC lines did not. Matrigel enhanced colony formation of all 3 adherent-type SCLC lines and 1 of 6 nonadherent-type NSCLC lines. Matrigel enhanced colony formation of both of 2 adherent-type non-lung cancer cell lines and 1 of 2 nonadherent-type leukemia cell lines. Neither transforming growth factor beta, collagen type IV, fibronectin, nor laminin, which are components of matrigel, enhanced colony formation of an NSCLC cell line in soft agar. The increase in the colony number of the NSCLC cell line by matrigel was abrogated by the protein kinase inhibitors staurosporine and UCN-01.