To determine the origin of non-vagal afferent fibers innervating the heart of guinea pigs, capsaicin was injected into the ventricular myocardium to induce depletion of substance P (SP). The lower cervical, upper thoracic and lumbar spinal ganglia, as well as the left atrium and base of ventricles, were assayed for SP depletion by using the enzyme-linked immunosorbent assay (ELISA) and immunohistochemical procedures. Capsaicin affected spinal ganglia from the 3 regions differently. The substance P level in lumbar spinal ganglia remained fairly constant, while the level of SP from cervical and thoracic regions declined significantly. At the maximal depletion dosage (173 μg of capsaicin/kg), SP concentration decreased 72.3% in cervical spinal ganglia, 45.5% in thoracic ganglia and 56.1% in the atrium. The lack of SP depletion in lumbar ganglia indicates that capsaicin acted locally on cardiac afferents rather than systemically. Data from this study suggest that capsaicin-sensitive neurons of the heart have cell bodies in the lower cervical spinal ganglia as well as in the upper thoracic spinal ganglia.

Lipid transfer inhibitor protein (LTIP) is an important regulator of cholesteryl ester transfer protein function. We report the development of an immunoassay for LTIP and its use to quantify LTIP in plasma of varying lipid contents. A rabbit antibody against bacterially produced recombinant LTIP detected two LTIP isoforms in plasma differing in carbohydrate content. This antibody was used in a competitive, enzyme-linked immunoassay that uses partially purified LTIP bound to microtiter plates. To optimize LTIP immunoreactivity, plasma samples required preincubation in 1% Tween-20 and 0.5% Nonidet P-40. In normolipidemic plasma, LTIP averaged 83.5 mg/ml. LTIP was 31% higher in males than in females. LTIP was positively associated with HDL cholesterol in normolipidemic males but not in females. In hypertriglyceridemic males, LTIP was only 56% of control values, whereas in hypertriglyceridemic females, LTIP tended to increase. Additionally, in males with normal cholesterol and triglyceride (TG) ≤ 200 mg/dl, LTIP varied inversely with plasma TG. Overall, we have confirmed the negative association between plasma TG levels and LTIP previously suggested by a small data set, but now we demonstrate that this effect is seen only in males. The mechanisms underlying this gender-specific response to TG, and why LTIP and HDL levels correlate in males but not in females, remain to be determined.

A new enzyme activity assay has been developed for the target N8-acetylspermidine deacetylase, a not-well-studied but essential enzyme in the polyamine interconversion and reutilization pathway. The enzyme assay, based on mass spectrometric detection of a specific reaction product following sample introduction by flow injection, was shown to have a sensitivity of smaller than 1 micromolar and typical RSD of 3-10%. The linear range for analyte was from 1 μM to 100 μM, with R2 > 0.992. The new assay avoids the use of radio labels. Sample preparation is straightforward, and high specificity is provided by the selected reaction monitoring, SRM, using a triple quadrupole mass spectrometer. Acetylputrescine was used for the first time as the substrate for the assay of N8-acetylspermidine deacetylase in lieu of N8 -acetylspermidine. The crude enzyme extracted from rat liver had an apparent Km value of 80.6 μM for acetylputrescine and a Vmax of 1.1 nmol mg-1 min-1. Enzyme extracted from frozen rat liver was compared with that from fresh rat liver. Frozen rat liver extraction had similar kinetics parameters with the fresh preparation and had a specific activity of 0.8 nmol mg-1 min-1. N8-acetylspermidine deacetylase was partially purified by protein precipitation and gell filtration chromatography. Affinity chromatography was tentatively applied for further isolation of the enzyme, but was not yet successful.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第19770号 / 農博第2166号 / 新制||農||1040(附属図書館) / 学位論文||H28||N4986(農学部図書室) / 32806 / 京都大学大学院農学研究科食品生物科学専攻 / (主査)教授 河田 照雄, 教授 保川 清, 教授 橋本 渉 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

This thesis describes the development of a way to study the N8 - acetylspermidine deacetylase enzyme activity. The method created in this thesis emphasizes sensitivity, accuracy and safety. In this study, HeLa cells were cultured and extracted to yield a crude N8 - acetylspermidine deacetylase enzyme mixture. By measuring the decrease of N8 - acetylspermidine and the increase of spetmidine, N8 -acetylspermidine deacetylase enzyme activity can be determined using either a Varian 1200L LC/MS/MS or an API 3000 LC-ES (+)/MS/MS. An acetylation-derivatization method was developed because N8 -acetylspermidine and spermidine are hard to purify from a biological sample since they are not retained on a CIS solid phase extraction column or on a RP HPLC (high performance liquid chromatography) reverse phase column due to their small molecular weight and high polarity. The quantitation of N8 -acetylspem1indine over the range 2ng/ul to 5pg/ul was fit by linear regression as y = 1.064x + 0.218 with an R-squared value of 0.9996, where y is the peak area of the fragment-ion SRM (selected reaction monitoring: m/z: 188/114) chromatograms from N8 -acetylspermindine and x is the concentration of N8 - acetylspermindine. Acetylation of spermidine (SPD) and N8-acetylspermidine (N8AcSPD) with d6-acetic anhydride produces the d9 labeled triacetylated derivative of SPD and d6 labled triacetylated spermidine derivative of N8AcSPD. These triacetylated forms are retained on a C18 column. MS/MS gives characteristic m/z fragment ions for the derivatized species: N8AcSPD (278 to 215), NlAcSPD (278 to 218) and SPD (281 to 218). The fragment-ion SRM (selected reaction monitoring) chromatograms are used for the quantitation. A plot of peak area ratios for known mixtures of N8AcSPD and total SPD versus the molar ratios of N8AcSPD and total SPD was found to fit a linear regression line withy= 0.705x + 0.035 with an R-squared value of 0.9919. Quantitation of d6- and dg-tri-acetylspermidine by LC/MS/MS is possible at the low levels of materials found in cell extracts since the separation method results in a lower limit of quantitation. This approach enables the study of N8 - acetylspermidine deacetylase enzyme activity.

Platinum-Group Metal (PGM) nanostructures as advantageous alternatives to natural peroxidases have drawn great attention because of their superior catalytic activities, which can effectively enhance performance of enzyme-based in vitro diagnostics. The catalytic activity of metal nanoparticle peroxidase mimics can depend on their size, shape, elemental composition, and surface ligand of PGM nanostructures. Therefore, to develop optimal peroxidase mimics for a few bioanalytical and diagnostic applications, such as enzyme-linked immunosorbent assay (ELISA), it is important to investigate how structural aspects of PGM nanoparticles correlate with the ability of the nanoparticles to serve as functional mimics of protein peroxidase enzymes. In summary, this dissertation has studied: 1) iridium (Ir), platinum (Pt) and Ir/Pt bimetallic nanowire structures as peroxidase mimics, and the effect of different wires' length on their peroxidase-like activities and certain application of sandwich ELISA for the detection of carcinoembryonic antigen (CEA, a cancer biomarker); 2) ultra-small Ir nanoparticles, with an average size of 1.1 nm, supported by WO2.72 nanowire with high catalytic activity. Those Ir nanoparticles were applied to sandwich ELISA and competitive ELISA for sensitive detection of CEA and aflatoxin B1 (AFB1, a carcinogenic toxin), respectively; 3) the size effect of peroxidase mimics on their catalytic activities and performance in biosensing application, where Pd-Ir core-shell nanoparticles were used as a type of model peroxidase mimics. These studies may significantly stimulate further investigations of PGM nanostructures as peroxidase mimics and other potential applications in in vitro diagnostics.

An enzyme-linked immunosorbent assay (ELISA) using polyclonal antibodies has been developed for the detection of the fluorescing group of Pseudomonas. The assay was used as a rapid test (6h) for predicting the shelf-life of pasteurized fluid milk. Milk samples were held at 7°C and tested weekly until determined to be unacceptable by daily sensory evaluation. Sterile milk samples were inoculated with target concentrations of 0 (control), 100, and 1000 cells/ml of Pseudomonas fluorescens on day 0. Samples were tested before and after preliminary incubations. Preliminary incubations conducted include milk alone and milk with broth (1:1) for 18h at 21°C. ELISA and plate counts were performed before and after preliminary incubation to determine the number of pseudomonads present and the relationship between ELISA and plate counts. These numbers were correlated to the shelf-life of each sample, as determined by sensory evaluation. Samples undergoing a preliminary incubation with only milk gave the best correlation to shelf-life (R=0.86). / Master of Science

Bovine mastitis is the most important economic disease to the dairy industry with losses estimated at 2 billion dollars per year in the United States. Staphylococcus aureus (.§.. aureus) is the primary cause of contagious mastitis. Conventional culture methods (National Mastitis Council) were used as a basis for comparing the ability of the enzyme linked immunosorbent assay. ProStaph Iâ ¢, to identify s. aureus. The test had an accuracy of 96%, with a sensitivity of 90% and a specificity of 97%. Results indicated that rinsing teat-cup liners with a 25 ppm iodophor or 100 ppm chlorine solution reduced the presence of S. aureus on the liners by 97%. ProStaph I was used to rapidly screen DHIA preserved milk samples in 10 Virginia cooperator herds. Herds were classified as high (>10% infected) or low prevalence (<10% infected). There were six high prevalence herds after the first test. Average prevalence of cows scoring Ab +2 and +3 was 11.9% ± 7.9. Over the seven month study, prevalence of positive cows declined significantly (P<.OI), but somatic cell count remained relatively unchanged (P>.lO). Four herds continued to have >10% of the animals infected. Incidence of new infection averaged 3.6% ± 2.8 from the first to the last test. Chronic cows averaged 6.9% ± 4.8 over the seven month study. Analysis of variance showed significant (P<.Ol) effects of herd on ProStaph I score J milk yield, and see. Elevated ProStaph I scores were highly correlated (P <.01) with increases in lactation number. ProStaph I changed quadratically (P<.Ol) with increasing SCC. Somatic cell count increased (P<.OI) as ProStaph I score increased. / Master of Science

<p>Försämrat immunförsvar och ökad risk att drabbas av bakterie- och virusinfektioner förekommer hos patienter med blodsjukdomarna multipelt myelom, Waldenströms makroglobulinemi samt hos vissa patienter med blodsjukdomen monoklonal gammopati av oklar signifikans. Infektionerna kräver ofta antibiotikabehandling och behandling med antivirala medel. I dagsläget är det svårt att förutsäga vilka av patienterna som kommer att drabbas av svåra och ibland livshotande infektioner. Därför ges många av patienterna förebyggande antibiotikabehandling.</p><p>I studiens början sattes en enzyme-linked immunosorbent assay (ELISA) för detektion av antikroppar mot <em>Moraxella catarrhalis </em>upp. I studien undersöktes om antikroppstitrar i serum mot bakterien <em>Moraxella catarrhalis</em> var lägre hos patientgrupperna än hos friska kontrollpersoner i samma ålder och om variationer förekom mellan patientgrupperna samt hur kontrollgrupper i olika åldrar skiljde sig från varandra. Kontrollgrupperna som undersöktes var mellan 20-40 år, 40-60 år samt 60 år och äldre.</p><p>Resultatet var att patienterna med multipelt myelom hade lägst antikroppstitrar, patienter med monoklonal gammopati av oklar signifikans hade något högre och patienter med Waldenströms makroglobulinemi hade ännu högre antikroppstitrar. Kontrollgruppen äldre än 60 år hade högre antikroppstitrar än både kontrollgruppen 20-40 år och 40-60 år. Lägst antikroppstitrar hade kontrollgrupp 40-60 år men ingen signifikant skillnad påvisades mellan kontrollgrupp 20-40 år och 40-60 år.</p>

A importância em conhecer o padrão alimentar em mosquitos da Família Culicidae permite esclarecer alguns aspectos relacionados à transmissão de zoonoses e estimar o grau de contato humano-vetor que é fator relevante em estudos epidemiológicos. Com o objetivo de explorar o comportamento alimentar dessa Família, em área epizoótica de febre amarela silvestre, foram coletados exemplares nos municípios de Santo Antônio das Missões e Garruchos, Estado do Rio Grande do Sul. Fêmeas ingurgitadas foram obtidas por aspiração em ambiente de mata, no período de setembro de 2005 a abril de 2007 e identificadas segundo fonte de sangue ingerido através da técnica imunoenzimática ELISA de captura no sistema avidinabiotina. Foram testadas seis fontes de alimento: ave, bovino, eqüino, humano, macaco e rato. Os resultados obtidos mediante a padronização de anticorpos monoclonais possibilitaram demonstrar pela primeira vez o reconhecimento de sangue humano ingerido nesses mosquitos pelo emprego da subclasse IgG1 e comprovar a sensibilidade e especificidade da técnica ELISA de captura. No município de Santo Antônio das Missões, de um total de 190 amostras, 60,9% reagiram para sangue de boi, 23,6% para humano, 9,9% para ave, 1,9% para macaco e 3,7% para combinações de dois hospedeiros. Quanto às amostras referentes ao município de Garruchos, das 158 fêmeas capturadas na área Cachoeirinha pode-se observar reatividade para ave (16%), boi (29,6%), humano (36,8%), cavalo (4%), macaco (0,8%) e combinações de hospedeiros (12,8%), enquanto que para as 149 fêmeas pertencentes à área de São José, detectou-se sangue ingerido de boi em (51,5%), ave e humano (11,5%), macaco (6,2%), cavalo (0,8%) e mistos (18,5%). Aedes scapularis, Aedes crinifer, Culex (Culex) spp., Haemagogus leucocelaenus apresentaram maior número de fêmeas ingurgitadas nos dois municípios. Os resultados obtidos com Aedes scapularis sugerem ecletismo, conforme combinações detectadas em amostras de sangue de diferentes fontes. Haemagogus leucocelaenus apresentou a maior proporção de amostras contendo sangue humano em relação às demais fontes e essa característica traz implicações, por ser espécie incriminada na transmissão e por se tratar de área de ocorrência de epizootias de febre amarela. / The knowledge of mosquitoes Culicidae host feeding patterns permits to clarify some aspects related to zoonosis transmission and to estimate the degree of human-vector contact which is relevant in epidemiological studies. Aiming to explore the feeding behavior of these mosquitoes, specimens were collected in the municipalities of Santo Antônio das Missões and Garruchos, Rio Grande do Sul, an epizootic area of sylvatic yellow fever. Engorged females were collected by aspiration from forested areas from September 2005-April 2007 and their blood meals were identified using the avidin-biotin system of immunoenzymatic ELISA capture. Six blood meal sources were tested: bird, cattle, horse, human, monkey and rat. The result achieved with the species-specific IgG1 mAb was unprecedented for mosquito blood meal identification and reinforced the sensibility and specificity of the immunoenzymatic ELISA capture. Of the 190 samples from Santo Antônio das Missões, 60.9% reacted to cattle, 23.6% to human, 9.9% to bird, 1.9% to monkey and 3.7% to mixed blood meals. In Garruchos, of the 158 females collected in Cachoeirinha, 16.0% reacted to bird, 29.6% to cattle, 36.8% to human, 4.0% to horse, 0.8% to monkey and 12.8% to mixed blood, while of the 149 engorged females from São José, blood from cattle accounted for 51.5%, of blood identified, bird and human 11.5%, monkey 6.2%, horse 0.8% and mixed blood 18.5%. Blood engorged females of Aedes scapularis, Aedes crinifer, Culex (Culex) spp., Haemagogus leucocelaenus predominated in the two municipalities. The results obtained with Aedes scapularis suggests its eclecticism, according to the combinations of blood which were detected from different sources. Haemagogus leucocelaenus was found to have the highest proportion of samples containing human blood in comparison with other sources, which has implications, on account of being incriminated in the transmission and also for taking into consideration the outbreaks reported that underline the risk of yellow fever.