Critical aspects on the behavior of material from the mechanical tool-workpiece interaction in single point diamond turning

Some material aspects such as grain size, purity and anisotropy exert an important influence on surface quality, especially in single point diamond turning. The aim of this paper is to present and discuss some critical factors that can limit the accuracy of ultraprecision machining of non-ferrous metals and to identify the effects of them on the cutting mechanism with single point diamond tools. This will be carried out through observations of machined surfaces and chips produced using optical and scanning electron microscopy. Solutions to reduce the influence of some of these limiting factors related with the mechanism of generation of mirror-like surfaces will be discussed.

Optimized Fast-FISH with a-satellite probes: acceleration by microwave activation

It has been shown for several DNA probes that the recently introduced Fast-FISH (fluorescence in situ hybridization) technique is well suited for quantitative microscopy. For highly repetitive DNA probes the hybridization (renaturation) time and the number of subsequent washing steps were reduced considerably by omitting denaturing chemical agents (e.g., formamide). The appropriate hybridization temperature and time allow a clear discrimination between major and minor binding sites by quantitative fluorescence microscopy. The well-defined physical conditions for hybridization permit automatization of the procedure, e.g., by a programmable thermal cycler. Here, we present optimized conditions for a commercially available X-specific a-satellite probe. Highly fluorescent major binding sites were obtained for 74oC hybridization temperature and 60 min hybridization time. They were clearly discriminated from some low fluorescent minor binding sites on metaphase chromosomes as well as in interphase cell nuclei. On average, a total of 3.43 ± 1.59 binding sites were measured in metaphase spreads, and 2.69 ± 1.00 in interphase nuclei. Microwave activation for denaturation and hybridization was tested to accelerate the procedure. The slides with the target material and the hybridization buffer were placed in a standard microwave oven. After denaturation for 20 s at 900 W, hybridization was performed for 4 min at 90 W. The suitability of a microwave oven for Fast-FISH was confirmed by the application to a chromosome 1-specific a-satellite probe. In this case, denaturation was performed at 630 W for 60 s and hybridization at 90 W for 5 min. In all cases, the results were analyzed quantitatively and compared to the results obtained by Fast-FISH. The major binding sites were clearly discriminated by their brightness

Using green fluorescent protein to understand the mechanisms of G-protein-coupled receptor regulation

G protein-coupled receptor (GPCR) activation is followed rapidly by adaptive changes that serve to diminish the responsiveness of a cell to further stimulation. This process, termed desensitization, is the consequence of receptor phosphorylation, arrestin binding, sequestration and down-regulation. GPCR phosphorylation is initiated within seconds to minutes of receptor activation and is mediated by both second messenger-dependent protein kinases and receptor-specific G protein-coupled receptor kinases (GRKs). Desensitization in response to GRK-mediated phosphorylation involves the binding of arrestin proteins that serve to sterically uncouple the receptor from its G protein. GPCR sequestration, the endocytosis of receptors to endosomes, not only contributes to the temporal desensitization of GPCRs, but plays a critical role in GPCR resensitization. GPCR down-regulation, a loss of the total cellular complement of receptors, is the consequence of both increased lysosomal degradation and decreased mRNA synthesis of GPCRs. While each of these agonist-mediated desensitization processes are initiated within a temporally dissociable time frame, recent data suggest that they are intimately related to one another. The use of green fluorescent protein from the jellyfish Aqueora victoria as an epitope tag with intrinsic fluorescence has facilitated our understanding of the relative relationship between GRK phosphorylation, arrestin binding, receptor sequestration and down-regulation.

Fluorescent ligands for studying neuropeptide receptors by confocal microscopy

This paper reviews the use of confocal microscopy as it pertains to the identification of G-protein coupled receptors and the study of their dynamic properties in cell cultures and in mammalian brain following their tagging with specific fluorescent ligands. Principles that should guide the choice of suitable ligands and fluorophores are discussed. Examples are provided from the work carried out in the authors' laboratory using custom synthetized fluoresceinylated or BODIPY-tagged bioactive peptides. The results show that confocal microscopic detection of specifically bound fluorescent ligands permits high resolution appraisal of neuropeptide receptor distribution both in cell culture and in brain sections. Within the framework of time course experiments, it also allows for a dynamic assessment of the internalization and subsequent intracellular trafficking of bound fluorescent molecules. Thus, it was found that neurotensin, somatostatin and mu- and delta-selective opioid peptides are internalized in a receptor-dependent fashion and according to receptor-specific patterns into their target cells. In the case of neurotensin, this internalization process was found to be clathrin-mediated, to proceed through classical endosomal pathways and, in neurons, to result in a mobilization of newly formed endosomes from neural processes to nerve cell bodies and from the periphery of cell bodies towards the perinuclear zone. These mechanisms are likely to play an important role for ligand inactivation, receptor regulation and perhaps also transmembrane signaling.

Use of fluorescent probes to follow membrane traffic in nerve terminals

Optical tracers in conjunction with fluorescence microscopy have become widely used to follow the movement of synaptic vesicles in nerve terminals. The present review discusses the use of these optical methods to understand the regulation of exocytosis and endocytosis of synaptic vesicles. The maintenance of neurotransmission depends on the constant recycling of synaptic vesicles and important insights have been gained by visualization of vesicles with the vital dye FM1-43. A number of questions related to the control of recycling of synaptic vesicles by prolonged stimulation and the role of calcium to control membrane internalization are now being addressed. It is expected that optical monitoring of presynaptic activity coupled to appropriate genetic models will contribute to the understanding of membrane traffic in synaptic terminals.

A continuous fluorescent assay for the determination of plasma and tissue angiotensin I-converting enzyme activity

A continuous assay using internally quenched fluorescent peptides with the general sequence Abz-peptidyl-(Dnp)P-OH (Abz = ortho-aminobenzoic acid; Dnp = 2,4-dinitrophenyl) was optimized for the measurement of angiotensin I-converting enzyme (ACE) in human plasma and rat tissues. Abz-FRK(Dnp)P-OH, which was cleaved at the Arg-Lys bond by ACE, was used for the enzyme evaluation in human plasma. Enzymatic activity was monitored by continuous recording of the fluorescence (lambdaex = 320 nm and lambdaem = 420 nm) at 37ºC, in 0.1 M Tris-HCl buffer, pH 7.0, with 50 mM NaCl and 10 µM ZnCl2. The assays can be performed directly in the cuvette of the fluorimeter and the hydrolysis followed for 5 to 10 min. ACE measurements in the plasma of 80 healthy patients with Hip-His-Leu and with Abz-FRK(Dnp)P-OH correlated closely (r = 0.90, P < 0.001). The specificity of the assay was demonstrated by the complete inhibition of hydrolysis by 0.5 µM lisinopril or captopril. Abz-FRK(Dnp)P-OH cleavage by ACE was monitored in rat lung, kidney, heart, and liver homogenates in the presence of a cocktail of inhibitors containing trans-epoxy-succinyl-L-leucylamido-(4-guanido)-butene, pepstatin, phenyl-methylsulfonyl fluoride, N-tosyl-L-phenylalanyl-chloromethyl ketone, and N-tosyl-lysyl-chloromethyl ketone to prevent undesirable hydrolysis. ACE activity in lung, heart and kidney homogenates, but not in liver homogenates, was completely abolished by 0.5 µM lisinopril or captopril. The advantages of the method are the procedural simplicity and the high sensitivity providing a rapid assay for ACE determinations.

Fluorescent quantitative PCR of Mycobacterium tuberculosis for differentiating intestinal tuberculosis from Crohn's disease

Intestinal tuberculosis (ITB) and Crohn's disease (CD) are granulomatous disorders with similar clinical manifestations and pathological features that are often difficult to differentiate. This study evaluated the value of fluorescent quantitative polymerase chain reaction (FQ-PCR) for Mycobacterium tuberculosis (MTB) in fecal samples and biopsy specimens to differentiate ITB from CD. From June 2010 to March 2013, 86 consecutive patients (38 females and 48 males, median age 31.3 years) with provisional diagnoses of ITB and CD were recruited for the study. The patients' clinical, endoscopic, and histological features were monitored until the final definite diagnoses were made. DNA was extracted from 250 mg fecal samples and biopsy tissues from each patient. The extracted DNA was amplified using FQ-PCR for the specific MTB sequence. A total of 29 ITB cases and 36 CD cases were included in the analysis. Perianal disease and longitudinal ulcers were significantly more common in the CD patients (P<0.05), whereas night sweats, ascites, and circumferential ulcers were significantly more common in the ITB patients (P<0.05). Fecal FQ-PCR for MTB was positive in 24 (82.8%) ITB patients and 3 (8.3%) CD patients. Tissue PCR was positive for MTB in 16 (55.2%) ITB patients and 2 (5.6%) CD patients. Compared with tissue FQ-PCR, fecal FQ-PCR was more sensitive (X2=5.16, P=0.02). We conclude that FQ-PCR for MTB on fecal and tissue samples is a valuable assay for differentiating ITB from CD, and fecal FQ-PCR has greater sensitivity for ITB than tissue FQ-PCR.

Estudo das características de adsorção de água e da estabilidade das microcápsulas de óleo essencial de laranja na seleção de material de parede

Este trabalho consistiu no estudo e comparação das características de adsorção de água de três amostras de microcápsulas de óleo essencial de laranja, obtidas pela secagem por atomização de três diferentes emulsões preparadas pela adição de óleo essencial de laranja (oel), a uma solução aquosa de material de parede (mp) constituída de capsul (5,0, 0,0 e 10,0%), goma arábica (5,0, 10,0 e 0,0%) sendo constante para as três emulsões a maltodextrina (36,0%), água (44,0%) e óleo essencial (10,0%). A microencapsulação foi realizada a 220 e 110° C de ar de entrada e saída do secador usando um atomizador rotativo a 20.000rpm. Com base à determinação das isotermas de adsorção de água a 30, 40 e 50° C e usando o modelo de GAB para ajustar os pontos experimentais foram avaliadas as características das isotermas, a estabilidade e área superficial de adsorção de água das diferentes amostras de microcápsulas obtidas. Os resultados indicaram ser importante o estudo das características de adsorção de água para estimar a estabilidade das microcápsulas de oel e a comparação destas mostrou que as microcápsulas obtidas pela secagem por atomização da emulsão preparada com 5,0% de capsul e 5,0% de goma arábica apresentaram o melhor resultado.

Microencapsulação de óleo essencial de laranja: seleção de material de parede

No presente trabalho se procedeu à comparação de agentes microencapsulantes, material de parede (mp), na microencapsulação de óleo essencial de laranja (material ativo) através da secagem por atomização. Foram preparadas três amostras de emulsões pela adição de óleo essencial de laranja a uma solução aquosa do mp composta de capsul (5,0, 0,0 e 10,0%), goma arábica (5,0, 10,0 e 0,0%) sendo constante para as três amostras maltodextrina (36,0%), água (44,0%) e óleo essencial (10,0%). Foram avaliadas as curvas de secagem das emulsões frente à retenção do óleo essencial de laranja e a tendência de formação de dobras superficiais das partículas e verificou-se a hipótese "menor a tendência de formação de dobras na superfície das microcápsulas maior a retenção de material ativo". A microencapsulação foi obtida pela secagem por atomização, com temperaturas de 220ºC e 110ºC do ar de entrada e de saída da câmara de secagem, respectivamente, e com atomizador rotativo (20.000rpm). A comparação das microcápsulas obtidas a partir das três amostras de emulsões mostrou que aquela preparada com 10,0% de capsul e 0,0% de goma arábica apresentou o maior resultado. O período de taxa constante de secagem desta mistura é curto e com maior retenção de umidade após a secagem. As microcápsulas obtidas apresentaram maior retenção de óleo essencial e dobras superficiais menos pronunciadas decorrentes do menor entumescimento das gotículas durante a secagem.

Preparação de um material de referência certificado para controle de agrotóxicos em hortifrutigranjeiros: estudo da homogeneidade

Neste trabalho são apresentados os esforços para garantir a homogeneidade de um material de referência. Níveis residuais (mg.kg-1) de quatro agrotóxicos (γ-HCH, fenitrotiona, clorpirifós e procimidona) foram adicionados à polpa de tomate com o objetivo de se preparar um material de referência certificado. As propriedades mais importantes desses materiais são a homogeneidade e a estabilidade. Antes de serem enviados a outros laboratórios, os materiais de referência precisam ter sua homogeneidade verificada. Nas etapas prévias, amostras foram avaliadas de modo a prover dados sobre o tratamento mais adequado para minimizar a variabilidade analítica do lote preparado e garantir a qualidade da amostra candidata a material de referência certificado, tão bem como a estimativa da incerteza associada à homogeneidade. A análise de variância fornece informações sobre a variabilidade do lote preparado e o grau de invariabilidade da amostra fortificada. Depois da preparação, as amostras foram expostas à radiação gama na dose de 2 kGy e submetidas a um estudo interlaboratorial para certificação. As concentrações certificadas dos agrotóxicos após caracterização foram 0,191 ± 0,047 mg.kg-1; 0,192 ± 0,068 mg.kg-1; 0,225 ± 0,076 mg.kg-1e 0,177 ± 0,051 mg.kg-1 para o γ-HCH, a fenitrotiona, clorpirifós e procimidona respectivamente.

Preparação de um material de referência certificado para controle de agrotóxicos em hortifrutigranjeiros: estudo da estabilidade

Neste trabalho são apresentados os resultados dos estudos de estabilidade referentes à produção de um material de referência certificado. Foram avaliados níveis residuais de concentração dos agrotóxicos γ-HCH, fenitrotiona, clorpirifós e procimidona em polpa de tomate. A pasteurização e a irradiação gama foram empregadas à polpa de tomate, visando manter a integridade da amostra candidata a material de referência. A polpa foi preparada e dividida em duas partes. Cada parte foi fortificada com os referidos agrotóxicos na faixa de concentração de 0,1 a 0,2 mg.kg-1. Uma das partes foi submetida à pasteurização a 90 °C por 4 minutos e a outra parte foi irradiada com dose de 2,0 kGy depois de homogeneizada. A estabilidade e a incerteza da amostra, correspondente ao período de tempo avaliado, foram determinadas através da análise de regressão em conjunto com a ANOVA. Os resultados indicaram que ambos os procedimentos de preparo da amostra são adequados para a conservação da polpa de tomate fortificada com os quatro agrotóxicos, com vantagem para o tratamento por irradiação com a dose de 2,0 kGy.

Microparticles obtained by complex coacervation: influence of the type of reticulation and the drying process on the release of the core material

Microparticles obtained by complex coacervation were crosslinked with glutaraldehyde or with transglutaminase and dried using freeze drying or spray drying. Moist samples presented Encapsulation Efficiency (%EE) higher than 96%. The mean diameters ranged from 43.7 ± 3.4 to 96.4 ± 10.3 µm for moist samples, from 38.1 ± 5.36 to 65.2 ± 16.1 µm for dried samples, and from 62.5 ± 7.5 to 106.9 ± 26.1 µm for rehydrated microparticles. The integrity of the particles without crosslinking was maintained when freeze drying was used. After spray drying, only crosslinked samples were able to maintain the wall integrity. Microparticles had a round shape and in the case of dried samples rugged walls apparently without cracks were observed. Core distribution inside the particles was multinuclear and homogeneous and core release was evaluated using anhydrous ethanol. Moist particles crosslinked with glutaraldehyde at the concentration of 1.0 mM.g-1 protein (ptn), were more efficient with respect to the core retention compared to 0.1 mM.g-1 ptn or those crosslinked with transglutaminase (10 U.g-1 ptn). The drying processes had a strong influence on the core release profile reducing the amount released to all dry samples

Presence of central nervous system tissues as bovine spongiform encephalopathy specified risk material in Turkish raw meat ball (cig kofte)

Abstract Bovine Spongiform Encephalopathy (BSE) is a virulent disease which may infect by affecting the central nervous system (CNS) tissues in cattle and causes degeneration in nerves. Central nervous system tissues such as brain and spinal cord which are classified as specified risk materials (SRMs) are regarded to be main source of infection. The contamination of the meat with the specific risk materials (SRMs) can occur in phases of slaughter, fragmentation of carcass and processing. This study was conducted in order to investigate the existence of CNS tissues in raw meat ball (cig kofte) which is commonly consumed in the Southeastern Region of Turkey, particularly in Şanlıurfa. For this purpose, 145 samples of raw meat ball were tested. The enzyme-linked immunosorbent assay (ELISA) kits (Ridascreen risk material 10/5, R-biofarm GmbH) which determine glial fibrillary acidic protein (GFAP) as determinant were used. As a result of the analyses, positivity was detected in 21 of totally 145 samples of raw meat ball (14.48%). 6 (4.14%) of the samples gave low level of positivity (≥ 0.1 standard absorbance), 10 (6.90%) gave medium level of positivity (>0.2 standard absorbance) and 5 (3.45%) gave high level of positivity (≥0.5 standard absorbance). As a consequence, meats are contaminated in any phase of both slaughter and meat production even if accidentally. Regarding this matter, necessary measures should be taken and hygiene rules should be applied.