A novel detection method for catecholamines using imidazole was investigated using a chemiluminescence coupled flow injection system. Imidazole catalysed decomposition of catecholamines to generate hydrogen peroxide, then the hydrogen peroxide was detected by chemiluminescence. The optimal condition for generation of hydrogen peroxide from a catecholamine was to incubate the catecholamines (53 pmol) in an imidazole solution (50 mmol/L, pH 9.0, 1.0 mL) at 60 degrees C for 30 min. Peroxide-was detected by peroxyoxalate chemiluminescence, and the rank order of the light emission intensities was as follows; dopamine (100%) >epinephrine (78%) >L-DOPA (62%) >norepinephrine (58%) >deoxyepinephrine (51%) >isoproterenol (43%) >dihydroxybenzylamine (25%). The light intensities of the reaction mixtures (corresponding to 1.06 pmol catecholamines) varied depending on the chemiluminescence (CL) detection reaction, and the rank order of the light intensity was as follows; luminol CL catalysed with horseradish peroxidase (HRP) (371%) >peroxyoxalate chemiluminescence (100%) >luminol CL catalysed with ferrycyanide (62%) >lucigenin CL (15%) >pyrogallol CL (0.8%) >purpurogallin CL (0.4%) >luminol CL (0.3%). The luminol CL reaction catalysed by HRP is recommended for the detection of peroxide in this method for catecholamines.

Catecholamines↗

PubMedDate not supplied

Total free catecholamines assay by identification of its two functional groups and micro-flow injection chemiluminescence.

We have developed a novel method of assaying total free catecholamines using sulphuric acid-derivatized beads for extracting and identifying catecholamine (CA) on the surface, and assaying the peroxide produced from CA by chemiluminescence (CL). Current assay methods for CA by electrochemical determination, fluorescence and chemiluminescence need a time-consuming separation by high-performance liquid chromatography. We eliminated this separation step by identifying the two functional groups of CA using a derivatized bead and this resulted in a highly specific CA assay. The principle is as follows: the amino group of CA was trapped by ion binding with a sulphuric acid derivative immobilized on a bead, and the diol of the CA bound to the bead was converted to peroxide with imidazole under alkaline conditions. The peroxide produced was assayed by microflow injection-horseradish peroxidase-catalysed luminol chemiluminescence. We synthesized three types of sulphuric acid-derivative immobilized beads (6.5 mm i.d.). The types of immobilized sulphuric acid derivative used were straight-chain, branched chain and benzenesulphonic, respectively. The order of the three types of beads for extracting CA was: bezenesulphonic type > branched type > straight-chain type. The optimal incubation time for generating peroxide was 30 min. The peroxide generated in the reaction solution was stable with within-run reproducibility of CV 5. 7% after incubation for 80 min. The regression equation of a standard curve for dopamine was Y = 12.8 X(2) + 476X - 373 (where Y = light intensity (RLU), X = concentration of dopamine (micromol/L)). The minimum detection limit of dopamine was 0.1 micromol/L, and the within-run reproducibility of dopamine (10.5 micromol/L) was CV 4.7% (n = 5). This method is applicable to assay of total free CA without use of HPLC.

Benzenesulfonates↗

PubMedDate not supplied

Determination of urinary free cortisol by high performance liquid chromatography with sulphuric acid-ethanol derivatization and column switching.

A rapid and highly sensitive determination method for urinary free cortisol has been developed using reversed phase high performance liquid chromatography (HPLC) with a precolumn for sulphuric acid-ethanol fluorescence derivatization and column switching. Urinary cortisol, eluted from the octadecylsilane-bonded silica (ODS) minicolumn with 90% aqueous ethanol, was derivatized with the addition of sulphuric acid only at ambient temperature. Cortisol derivatives injected directly onto the ODS precolumn were purified on-line. After switching the columns, the cortisol derivative was separated on an ODS analytical column with a retention time of 15.3 min and monitored at an emission wavelength of 520 nm (exitation wavelength of 365 nm) to decrease the detection limit to 0.26 microgram/dL (signal-to-noise ratio = 3). The automated HPLC operation resulted in good reproducibility and recovery of the stable cortisol derivative at 5 degrees C.

Chromatography, High Pressure Liquid↗

PubMedDate not supplied

A possible association of Pro12Ala polymorphism in peroxisome proliferator-activated receptor gamma2 gene with obesity in native Javanese in Indonesia.

BACKGROUND: Peroxisome proliferators-activated receptor gamma (PPAR gamma) is a nuclear hormone receptor that serves as a master regulator of adipocytes-specific genes contributing to adipocytes differentiation, susceptibility to obesity, and insulin sensitivity. The substitution of proline to alanine at codon 12 of the PPAR gamma2 gene (Pro12Ala polymorphism) is most frequently identified and the associations with diabetes, obesity, and other clinical parameters have been reported and discussed in several ethnic groups. Among native Javanese ethnicity, however, there is no report about this polymorphism. AIMS AND METHODS: Aims of this study were to estimate the allele frequency of the Pro12Ala polymorphism of PPAR gamma2 gene among native Javanese in Indonesia and to investigate the relationship between this polymorphism and obesity or diabetes. This study included 540 native Javanese subjects consisting of 337 diabetic patients and 203 normal glucose tolerance subjects. Both groups included totally 160 obese subjects (body mass index > or = 25 kg/m(2)). PCR-restriction fragment length polymorphism was used for the genotype determination. RESULTS: The allele frequency of Pro12Ala polymorphism in PPAR gamma2 gene among native Javanese is lower than that in other ethnic groups. No association is seen between the Pro12Ala and diabetes (0.01 vs 0.017%, p = 0.404), a trend of the higher BMI was observed in Pro12Ala carriers in nondiabetic subjects, although this association is limited by small numbers. CONCLUSION: In this study, no association is seen between the Pro12Ala polymorphism in PPAR gamma2 gene and diabetes; a weak association with obesity is seen.

Aged↗

PubMedDate not supplied

Mating type locus-dependent stability of the Kluyveromyces linear pGKL plasmids in Saccharomyces cerevisiae.

The linear killer plasmids, pGKL1 and pGKL2, from Kluyveromyces lactis stably replicated in mitochondrial DNA-deficient (rho 0) MATa or MAT alpha haploids of Saccharomyces cerevisiae, but were unstable and frequently lost in rho 0 MATa/MAT alpha diploids, suggesting that the replication of pGKL plasmids was under the control of the MAT locus. In MATa/MAT alpha cells of S. cerevisiae, the MAT alpha gene product (alpha 2) is combined with the MATa gene product (a1) and the resultant protein, a1-alpha 2, acts to repress the expression of haploid specific genes. Experiments showed that the K. lactis linear plasmids were stably maintained in rho 0 mata1/MAT alpha diploids, indicating that the a1-alpha 2 repressor interfered with the stability of pGKL2. It was revealed by computer analysis that the consensus sequence homologous to the a1-alpha 2 repressor binding site occurred within the coding regions of pGKL2 genes which were presumed to be essential for the plasmid replication. Since the plasmids were stably maintained in diploids of K. lactis, the mating type control must not be working there.

Base Sequence↗

PubMedDate not supplied

Role of polymorphonuclear leukocytes in collagenase production in chronic otitis media.

The effect of polymorphonuclear leukocytes on collagenase production by fibroblast-like cells was studied in an in vitro cell culture system. An extract of rat intraperitoneal polymorphonuclear leukocytes induced significant collagenase production by rat skin fibroblast-like cells. Fibroblast-like cells cultured in the presence of polymorphonuclear leukocytes had many conspicuous microspikes on their surface and contained bundles of actin filaments in their cytoplasm. Polymorphonuclear leukocytes appears to be involved in bone resorption by inducing collagenase production by fibroblast-like cells, as well as by releasing their own enzymes involved in bone resorption.

Animals↗

PubMedDate not supplied

Involvement of interleukin-1 in middle ear cholesteatoma.

PURPOSE: The histopathological characteristics of middle ear cholesteatoma are hyperproliferation and differentiation of the epithelium and accompanying destruction of the bones. We directed our attention to interleukin 1 alpha (IL-1 alpha) and demonstrated localization of IL-1 alpha in the epidermis of cholesteatoma. In addition, a comparative study was made of the relationship of IL-1 alpha with the developmental stage of cholesteatoma, the degree of destruction of the auditory ossicles, presence/absence of otorrhea, and the state of subepithelium. MATERIALS AND METHODS: Middle ear cholesteatoma tissues collected during operations were used as the experimental material, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blot, and immunohistological techniques were used as methods. RESULTS: An anti-IL-1 alpha antibody-positive 17-kd band was observed in 2 of the 17 cases, whereas a 31-kd band was observed in 10 of the 17 cases. Neither the 17-kd band nor the 31-kd band was detected in normal skin tissues. The immunohistological staining showed the presence of IL-1 alpha in a region near the basal membrane of cholesteatoma epithelium. CONCLUSION: A correlation was observed between IL-1 alpha and patients with vigorous proliferation of granulation in the subepithelium. The presence of granulation in the reverse side of the eardrum plays an important role in the proliferation of cholesteatoma epithelium.

Adult↗

PubMedDate not supplied

Treatment of advanced primary lung cancer associated with malignant pleural effusion by the combination of immunotherapy and chemotherapy.

Twelve patients with advanced primary lung cancers associated with malignant pleural effusion were treated with intrathoracic instillation of recombinant interleukin-2 with or without in-vitro-sensitized cells. Two cases achieved complete response, and 7 partial response. The adverse effects seen in the protocol were marginal, and the protocol was well-tolerated and feasible. Furthermore, 4 cases were treated with the combination of systemic chemotherapy and adoptive immunotherapy. Of these, 3 cases responded well to the therapy and have shown a complete response for more than 20 months, indicating that adoptive immunotherapy together with chemotherapy might be a beneficial treatment for advanced lung cancer patients.

Adenocarcinoma↗

PubMedDate not supplied

Effects of 10 min tilt and visual directional information on ocular counterrolling.

The effects of 10 min of static tilt on ocular counterrolling (OCR) were investigated. Seven healthy volunteers were tilted 45 degrees around the naso-occipital axis, fixed at that position for 10 min, and the pre- and post-tilt OCRs were recorded. OCR was video-recorded using an infrared CCD camera and subjected to computer image analysis. The subjects were directed to fixate on an imagined target in the dark (experiment 1), or viewed an earth vertical, striped pattern placed in front of his/her eyes to enhance the recognition of extent of body tilt (experiment 2). In experiment 1, the average OCRs recorded before and after 10 min of tilt showed no significant changes. However, in experiment 2, OCR decreased significantly after 10 min of tilt. These results suggest that visual perception of spatial verticality can produce time-dependent adaptation of OCR.

Adaptation, Ocular↗

PubMedDate not supplied

Histology, pathogenesis, and treatment of cholesteatoma.

Factors present in cholesteatoma connective tissue induce bone resorption in chronic otitis media. The presence of skin and its products appears to exacerbate the destructive influence of connective tissue. One exacerbating factor is pressure, shown in an animal model to increase bone resorption by influencing the subepithelial connective tissue. Consideration of pathogenic factors provides a rationale for modifications in the treatment of middle ear cholesteatoma.

Animals↗

PubMedDate not supplied

Effects of pressure on bone resorption in the middle ear of rats.

Laminaria, a hygroscopic seaweed, was used to induce pressure within the rat tympanic cavity. Resorption of the bulla and cochlear walls occurred in all animals within 2 weeks. Cochlear fistulas were observed in four of nine animals after 2 weeks and in nine of 11 animals after 3 weeks. Preswelled laminaria inserted in the same area induced minimal bone resorption after 2 and 3 weeks. In animals given indomethacin following insertion of laminaria, both granulation-tissue formation and bone resorption were inhibited. Collagenase appeared localized in the inflammatory granulation tissue in the bone-resorption area, especially in fibroblasts, mononuclear cells, and osteoclasts. Results of this study suggested that pressure created by laminaria caused bone resorption by stimulating osteoclasts and granulation tissue.

Animals↗

PubMedDate not supplied

Pathogenic factors in bone resorption in cholesteatoma.

Factors present in skin appear to enhance bone resorption in chronic otitis media. These skin factors were replicated in a series of experiments using an animal model. The presence of activated granulation tissue is a universal finding in bone resorption in otitis media. Skin promotes bone resorbing connective tissue by the action of keratin as a foreign body, by the enhancement of middle ear sepsis, by stimulation and activation of inflammatory cells and most importantly through the creation of pressure.

Animals↗

PubMedDate not supplied

Endoscopic transethmosphenoidal approach for pituitary tumors with image guidance.

The advantages of endoscopic transethmosphenoidal surgery for pituitary tumors using a navigation system were reported. The surgical technique was as follows. First, sphenoidal sinuses were opened via the bilateral ethmoidal sinuses and the olfactory clefts. Then the septum of the sphenoidal sinuses was resected. Next, an endoscope was inserted via the left nasal cavity and fixed in place. The tumor was then removed via the right nasal cavity. Our approach for pituitary tumors provided sufficient working space and permitted the surgeon to carry out the procedure using both hands. In addition, use of the InstaTrak System made it possible to recognize the orientation of the surgical field in the sella turcica. Thus, the tumor could be resected more easily and safely. It is concluded that this approach will be particularly useful for patients with narrow nasal cavities or poor development of the paranasal sinuses.

Adenoma↗