Quantification of the histochemical staining for carbonyles and DNA using 3-hydroxy-2-naphthoic acid hydrazide and Fast blue B

Summary Fixed cells and tissues pretreated with 4-hydroxynonenal were used as models for the histochemical demonstration of protein bound aldehydic groups. The aldehydes were stained with both a modification of the 2,4-dinitrophenylhydrazine method (2,4-DNPH) and the optimized staining using 3-hydroxy-2-naphthoic acid hydrazide and Fast blue B (NAH-FB). A correlation has been found between the specific microphotometric mean integrated maximum absorbance values of cells and tissues stained with 2,4-DNPH and with NAH-FB (cc=0.999). The maximum absorbance measured after 2,4-DNPH-staining (₃₆₇=21 000) were 1.893±0.072 (P<0.01) times that of NAH-FB-staining at 550 nm. Microphotometrically determined DNA-values of different cells stained with the NAH-FB-DNA-method correlated with those determined with methods of analytical biochemistry and published by other authors.     

Quantitation of nitric oxide-derived nitrite from activated macrophages using microdialysis sampling

An HPLC method for detecting nitrite in microdialysis samples obtained from activated RAW 264.7 macrophages in cell culture has been developed. Nitrite was quantified using a pre-column derivatization with 2,4-dinitrophenylhydrazine (2,4-DNPH) followed by HPLC-UV analysis of the azide product. For dialysates, the detection limit of nitrite was 750 nM and the quantitation limit was 2.5 microM. The microdialysis relative recovery of nitrite in the macrophage cell culture medium was determined to be 86+/-2% (n=3) at a flow rate of 0.7 microl/min. Nitrite produced from activated macrophages was measured immediately after lipopolysaccharide (LPS) stimulation using microdialysis sampling.     

改良2, 4-二硝基苯肼法测定脑内源甲醛

内源甲醛代谢失衡所造成的脑慢性损伤被认为是衰老过程中记忆丢失的危险因素之一,因此,有必要准确测定脑内不同区域甲醛的含量为相关研究提供参考.作者通过2,4-二硝基苯肼(2,4-DNPH)偶联高效液相色谱(UV-HPLC),测定了家猪脑额叶、顶叶、颞叶、枕叶、海马、小脑、脑干内源甲醛含量(75.5～83.4μmol/kg).采用10%三氯乙酸处理脑组织匀浆(pH=1.0),不但可以避免蛋白质及其他分子的干扰,还可以省略现有方法中的萃取步骤,且提高了测试的灵敏度(P<0.05).该方法的回收率约95.96%～102.04%,相对标准偏差(n=5)小于10%.结果表明,改良2,4-DNPH法用于脑内源甲醛的测定,其操作简便、可信度高.     

Cytofluorometric and cytochemical comparisons of normal and abnormal human cells from the female genital tract.

Acridine orange staining of exfoliated cells from epithelial tissues facilitates discrimination between normal and abnormal cells: abnormal cells develop highly elevated nuclear fluorescence. Comparisons of acridine orange (AO) staining with propidium iodide (PI) or Feulgen staining have shown that: (a) PI staining also provides highly elevated nuclear fluorescence from abnormal cells; (b) the distributions of nuclear fluorescence following AO or PI staining were usually not significantly different as judged by the Kolmogorov-Smirnov test; (c) fluorescence emission spectra from AO and PI stained cells are consistent with the hypothesis that both fluorochromes bind to DNA within cell nuclei; (d) DNAse treatment of AO stained normal cells eliminates the nuclear fluorescence peak from slit-scan contours; RNAse treatment has no effect on nuclear fluorescence; (e) the distribution of abnormal cell nuclear fluorescence after AO staining is usually, but not always, significantly different from the distribution of abnormal cell nuclear absorbance after Feulgen staining, with relative nuclear fluorescence being greater than relative nuclear absorbance. The hypothesis currently most consistent with these results is that elevated Feulgen DNA content can account for only part of the discrimination provided by AO staining, and that the chromatin within abnormal cells is altered so as to increase accessibility of DNA to intercalating dyes.     

Biodegradation and kinetics of organic compounds and heavy metals in an artificial wetland system (AWS) by using water hyacinths as a biological filter

The objective of the present study was to investigate the ability of water hyacinth (Eichhornia crassipes) to absorb organic compounds (potassium hydrogen phthalate, sodium tartrate, malathion, 2,4-dichlorophenoxy acetic acid (2,4-D), and piroxicam). For the aforementioned purpose, an artificial wetland system (AWS) was constructed and filled with water hyacinth collected from the Valsequillo Reservoir, Puebla, Mexico. Potassium hydrogen phthalate and sodium tartrate were measured in terms of chemical oxygen demand (COD) and biological oxygen demand (BOD). The present study indicated that the water hyacinths absorbed nearly 1.8–16.6 g of COD kg^?1 dm (dry mass of water hyacinth), while the absorbance efficiency of BOD was observed to be 45.8%. The results also indicated that the maximum absorbance efficiency of malathion, 2,4-D, and piroxicam was observed to be 67.6%, 58.3%, and 99.1%, respectively. The kinetics of organic compounds fitted different orders as malathion followed a zeroth-order reaction, while 2,4-D and piroxicam followed the first-order reactions. Preliminary assessment of absorption of heavy metals by the water hyacinth in the AWS was observed to be (all values in mg g^?1) 7 (Ni), 13.4 (Cd), 16.3 (Pb), and 17.5 (Zn) of dry biomass, thus proving its feasibility to depurate wastewater.     

A new test for chorismate mutase activity.

Chorismate mutase (EC 5.4.99.5) catalyzes the conversion of chorismic acid to prephenic acid. A continuous test of the enzymatic activity is based on the decrease of the absorption of the substrate chorismate at 274 nm (1). In a sensitive but discontinuous test, prephenic acid, the product of the enzymatic reaction is converted to phenylpyruvic acid. The absorbance of its enolic form is determined in alkaline solution at 320 nm (2,3). Another discontinuous test makes use of the absorption of the phenylpyruvate enol-borate complex (4,5) at 300 nm. The continuous test cannot be used when aromatic compounds are to be tested as modifiers of the enzymatic activity. Similarly, the tests based on the absorption of the enolic form of phenylpyruvic acid cannot be used when compounds which show a high absorbance in the 300 to 320 nm wavelength region are tested. This paper describes a test for chrismate mutase based on the determination of the 2,4-dinitrophenylhydrazone of the α-keto acid, phenylpyruvic acid. In alkaline solution the 2,4-dinitrophenylhydrazone of phenylpyruvic acid shows an absorption maximum at 440 nm, thus allowing one to test compounds like 3-hydroxybenzoic acid and 5-hydroxyisophthalic acid as potential inhibitors of the enzymatic reaction.     

Studies on Papanicolaou staining. III. Quantitative investigations of orangeophilia and cyanophilia

This paper provides data derived from the visible light absorbance spectra of Papanicolaou stained epithelial cells from the uterine cervix. Twenty-four types of spectra have been considered, namely, those derived from orangeophilic and cyanophilic nuclei and cytoplasms of superficial, intermediate, parabasal and dysplastic cells, and cells of carcinoma in situ and invasive carcinoma. Wavelengths of maximum absorbance and peak absorbances are tabulated. The proportions of bound orange G, eosin Y, aluminum-hematein and light green SF yellowish have been calculated. For the majority of cell types, dyebinding differences between orangeophilic and corresponding cyanophilic substrates were statistically significant. CIE coordinates were calculated from absorbance spectra; again differences between organeophilic and cyanophilic cells were statistically significant in most cases. Although the designation of cells as orangeophilic or cyanophilic is made on the basis of cytoplasmic coloration, the nucleus is also usually orangeophilic or cyanophilic. These nuclear differences are real and not due to the effects of over- and underlying cytoplasm.     

Studies on Papanicolaou Staining. III. Quantitative Investigations of Orangeophilia and Cyanophilia

This paper provides data derived from the visible light absorbance spectra of Papanicolaou stained epithelial cells from the uterine cervix. Twenty-four types of spectra have been considered, namely, those derived from orangeophilic and cyanophilic nuclei and cytoplasms of superficial, intermediate, parabasal and dysplastic cells, and cells of carcinoma in situ and invasive carcinoma. Wavelengths of maximum absorbance and peak absorbances are tabulated. The proportions of bound orange G, eosin Y, aluminum-hematein and light green SF yellowish have been calculated. For the majority of cell types, dyebinding differences between orangeophilic and corresponding cyanophilic substrates were statistically significant. CIE coordinates were calculated from absorbance spectra; again differences between orangeophilic and cyanophilic cells were statistically significant in most cases. Although the designation of cells as orangeophilic or cyanophilic is made on the basis of cytoplasmic coloration, the nucleus is also usually orangeophilic or cyanophilic. These nuclear differences are real and not due to the effects of over- and underlying cytoplasm.     

Localization of androgen receptor in the follicle and corpus luteum of the primate ovary during the menstrual cycle

Ovarian androgens may act locally to modulate follicular and luteal function in various species. This study examined the distribution of androgen receptors within the primate ovary throughout the menstrual cycle. Ovaries were collected from rhesus and cynomolgus monkeys during the early, mid-, and late (n = 3-5 per stage) follicular and luteal phases of the cycle. The tissues were processed for indirect immunocytochemical localization of androgen receptors with a specific monoclonal antibody against human androgen receptor (AN1-15). In addition, ovaries (n = 3) were collected from rhesus monkeys for biochemical detection of androgen receptor using 3H-androgen and AN1-15. Specific immunocytochemical staining, as determined by comparing adjacent tissue sections incubated with either AN1-15 or a nonspecific control antibody, was exclusively nuclear. Androgen receptor was detected in the germinal epithelium and ovarian stroma at all stages of the cycle. The thecal and granulosa cells of growing follicles, and of many but not all atretic follicles, contained androgen receptors. Luteinizing granulosa cells of the periovulatory follicle and luteal cells from the early and midluteal phase stained intensely for androgen receptor. Regressing corpora lutea of the late luteal phase also stained for androgen receptor; however, fully regressed corpora lutea in the early follicular phase of the next cycle did not exhibit receptor staining. Luteal cells that were androgen receptor-positive also stained histochemically for the presence of 3 beta-hydroxysteroid dehydrogenase. Sucrose gradient analysis with radiolabeled androgen demonstrated a shift in the androgen receptor peak in monkey ovarian tissue upon addition of AN1-15, confirming the presence of androgen receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increased accuracy and speed of absorption cytometric DNA measurements by automatic corrections for nuclear darkness

We have developed a method of calculating the average local absorbance (ALA) of a nucleus from the integrated nuclear absorbance and area. One can use the ALA, along with nuclear areas measured at different point absorbance thresholds, to determine whether a nucleus is stained too lightly or too darkly for accurate absorption measurements; this allows selection of an optimal light wavelength for the performance of these measurements. The ALA can also be used for automatic and instantaneous correction of integrated absorbance values from darkly stained cells. This allows the rapid measurement of the integrated absorbances of a large number of nuclei that are heterogeneous in stain intensity. Coefficients of variation of approximately 3% are obtained for the integrated absorbances of nuclei of nontransformed G0/G1 cells. This correction method can be applied with any image densitometer that generates both integrated absorbance and area values.     

Muscle protein changes following eccentric exercise in humans

This study characterized changes in the protein composition of human muscle tissue after eccentric exercise. Four subjects performed 70 maximum eccentric, isokinetic actions of the forearm flexors with one arm. The other arm served as control. A biopsy of the biceps muscle of each arm was taken 2 days after exercise when muscles were very sore (mean = 8.0; 1 = normal; 10 = very, very sore), and muscle damage was documented by a mean decrease of 0.2 radians in the relaxed elbow angle. Proteins from the biopsy tissue were solubilized in a high ionic strength buffer containing several proteolytic inhibitors. Protein concentrations of the extracts were determined and identical amounts loaded onto sodium dodecyl sulfate (SDS) polyacrylamide gels (7.5, 12.5, and 17.5%). Densitometric analysis of the Coomassie brilliant blue stained gels revealed alterations in the amounts of three protein bands in the exercised tissue relative to the control. These changes were in the linear portion of the graph of absorbance versus protein amount. Wilcoxon's signed rank test showed the first two of the following bands to increase significantly in amount (P less than 0.062). The average percentage changes [mean (SEM)] for these bands were 63 (21), 39 (5), and 82 (35). The corresponding molecular weights determined from known standards were 76300 (860), 33200 (310), and 12000 (80) daltons, respectively. These changes imply that the increased synthesis, decreased degradation, or some combination thereof, of these three proteins may be necessary for the repair or regeneration response to exercise-induced muscle damage.     

High-throughput colorimetric assays optimized for detection of ketones and aldehydes produced by microbial cell factories

Randomized strain and pathway engineering are critical to improving microbial cell factory performance, calling for the development of high-throughput screening and selection systems. To facilitate this effort, we have developed two 96-well plate format colorimetric assays for reliable quantification of various ketones and aldehydes from culture supernatants, based on either a vanillin-acetone reaction or the 2,4-dinitrophenylhydrazine (2,4-DNPH) reagent. The vanillin-acetone assay enabled accurate and selective measurement of acetone titers up to 2 g l⁻¹ in a minimal culture medium. The 2,4-DNPH-based assay can be used for a wide range of aldehydes and ketones, shown here through the optimization of conditions for 15 different compounds. Both assays were implemented to improve acetone production from different substrates by an engineered Escherichia coli strain. The fast and user-friendly colorimetric assays proposed here open the potential for iterative rounds of (automated) strain and pathway engineering and screening, facilitating the efforts towards further boosting production titers of industrially relevant ketones and aldehydes.     

A "two-objective, one-area" procedure in absorption microphotometry and its application using an inverted microscope

A 'two-objective, one-area' method and related equations are suggested to measure absorbance of microscopic stained objects. In such work, the measuring field invariably includes an image of the object and some clear area surrounding the image. The total intensity in the two areas is measured photometrically, using two different objectives, and substituted in the equation for absorbance. The equation is independent of the term representing intensity from the clear area and hence the error in the measurement of absorbance is reduced. The limitations of the 'two-objective, one-area' method are discussed and its pragmatic operation described with an experimental setup involving an inverted microscope. The method permits measurement of intensity in a part of a stained cell while the rest of the cell remains in the field of view. The method is applied to measure absorbance in Giemsa stained ascites cells and Feulgen stained liver and Human Amnion cells.     

An antibody raised against in vitro-derived human mast cells identifies mature mast cells and a population of cells that are Fc epsilon RI(+), tryptase(-), and chymase(-) in a variety of human tissues.

Selective markers for human mast cells are of paramount importance for understanding their role in physiological and pathological processes. A mouse monoclonal antibody (MAb) designated 2C7, raised against in vitro-derived human mast cells, was used in immunoenzymatic analysis of sections from a variety of human organs. Double immunolabeling with 2C7 and tryptase, chymase, Fc epsilon RIalpha, and c-kit was performed on cryostat tissue sections from skin, colon, uterus, breast, stomach, bladder, and lung. MAb 2C7 stained greater than 93% of the tryptase(+) or chymase(+) mast cells in all tissues examined. In addition, the majority of cells stained with the tryptase or chymase also stained for Fc epsilon RIalpha. However, there were a significant number of Fc epsilon RIalpha(1) cells in all tissues studied that were tryptase(-) and/or chymase(-). In contrast, MAb 2C7 in double immunoenzymatic staining co-localized with 93-96% of the Fc epsilon RIalpha(1) cells in all tissues. Analysis for c-kit expression on the different tissues revealed that the majority of tryptase(+) or chymase(+) cells in skin, uterus, bladder, and lung stained with c-kit. However, only approximately 70-78% of tryptase(+) cells in colon and stomach were c-kit(+). These data suggest that MAb 2C7 appears to identify mature mast cells and a population of Fc epsilon RIalpha(1), chymase(-), and tryptase(-) cells in a variety of human tissues.     

Kinetic and structural insight into the mechanism of BphD, a C-C bond hydrolase from the biphenyl degradation pathway

Kinetic and structural analyses of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) hydrolase from Burkholderia xenovorans LB400 (BphD(LB400)) provide insight into the catalytic mechanism of this unusual serine hydrolase. Single turnover stopped-flow analysis at 25 degrees C showed that the enzyme rapidly (1/tau(1) approximately 500 s(-1)) transforms HOPDA (lambda(max) = 434 nm) into a species with electronic absorption maxima at 473 and 492 nm. The absorbance of this enzyme-bound species (E:S) decayed in a biphasic manner (1/tau(2) = 54 s(-1), 1/tau(3) = 6 s(-1) approximately k(cat)) with simultaneous biphasic appearance (48 and 8 s(-1)) of an absorbance band at 270 nm characteristic of one of the products, 2-hydroxypenta-2,4-dienoic acid (HPD). Increasing solution viscosity with glycerol slowed 1/tau(1) and 1/tau(2) but affected neither 1/tau(3) nor k(cat), suggesting that 1/tau(2) may reflect diffusive HPD dissociation, and 1/tau(3) represents an intramolecular event. Product inhibition studies suggested that the other product, benzoate, is released after HPD. Contrary to studies in a related hydrolase, we found no evidence that ketonized HOPDA is partially released prior to hydrolysis, and, therefore, postulate that the biphasic kinetics reflect one of two mechanisms, pending assignment of E:S (lambda(max) = 492 nm). The crystal structures of the wild type, the S112C variant, and S112C incubated with HOPDA were each determined to 1.6 A resolution. The latter reveals interactions between conserved active site residues and the dienoate moiety of the substrate. Most notably, the catalytic residue His265 is hydrogen-bonded to the 2-hydroxy/oxo substituent of HOPDA, consistent with a role in catalyzing ketonization. The data are more consistent with an acyl-enzyme mechanism than with the formation of a gem-diol intermediate.     

Somatic Embryogenesis and Regeneration of in Plantlets in Pomegranate

Plantlets were regenerated from somatic embryos originatingfrom cotyledonary tissues of pomegranate (Punica granatum) throughmultiple somatic embryogenesis. Embryogenic cell clusters proliferatedvigorously with regular sub-culturing after 20 d on RBM-II mediumcontaining 1 µM kinetin (KN), 2 µM benzylamino purine(BAP) and 5 µM 2,4-dichlorophenoxyacetic acid (2, 4-D).Developmental stages of somatic embryos were expressed on sub-culturingwith a low level of 2, 4-D (2.5 µM). Embryogenic initialscells were small, round to oval, thick-walled, contained densecytoplasm which stained with acetocarmine and were usually attachedto non-embryogenic cells. Embryo maturation was obtained onRBM-III and IV media to produce young seedlings on the initiationof the first long tap root. Punica granatum L., pomegranate, multiple somatic embryogenesis, callus culture, plant regeneration     

Analysis of a range of crown gall and normal plant tissues for Ti plasmid-determined compounds

Summary Twenty-five plant tissues from several species, including thirteen crown gall tissues, were analysed for the full range of unusual compounds (the opines) whose synthesis in crown gall cells and utilization by Agrobacterium tumefaciens are genetically determined by the Ti plasmids found in this bacterial species. A technique for the analysis of the non-guanidino opines by GC and GC/MS is described. None of the opines were detected in any of the various normal tissues analysed. In the crown gall tissues, on the other hand, these compounds were often present at very high levels. The type of opines found in the crown gall tissues was dependent on the strain of initiating bacterium.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - GC gas chromatography - GC/MS coupled gas chromatography-mass spectrometry - HFB heptafluorobutyryl - SIM selected ion monitoring - TLC thin layer chromatography     

Microspectrophotometric Studies of Romanowsky Stained Blood Cells. IV. Maturation of Myeloid and Erythroid Cell Lines in Bone Marrow

A quantitative characterization has been made of azure B/eosin stained cells from bone marrow. Two cell lines were followed: the myeloid line (white cell blast, promyelocyte, neutrophilic myelocyte, neutrophilic metamyelocyte, neutrophilic hand, neutrophilic segmented) and the erythroid line (rubriblast, prorubricyte, rubricyte, metarubricyte, diffusely basophilic erythrocyte, erythrocyte). A consensus scheme was used to obtain the “true” classification of the cells. Cell types were characterized by three methods: absorbance spectra, dye binding, and chromaticities. Within both cell lines nuclear maturation is accompanied by an overall increase in peak absorbance with little shift in the position of the maximum. Generally, binding of azure B and eosin increases; azure B dimer/monomer ratios show a slight downward trend during maturation. Changes in chromaticities are to bluish purples of increasing saturation. Cytoplasmic changes accompanying maturation are much more striking than nuclear changes, and again the two cell lines show similarities. Generally, there is decreased binding of azure B during maturation. In the erythroid line, the Soret band of hemoglobin becomes increasingly prominent. Chromaticities change from bluish purples to purplish pinks, particularly in the erythroid line.     

Microspectrophotometric studies of Romanowsky stained blood cells. IV. Maturation of myeloid and erythroid cell lines in bone marrow

A quantitative characterization has been made of azure B/eosin stained cells from bone marrow. Two cell lines were followed: the myeloid line (white cell blast, promyelocyte, neutrophilic myelocyte, neutrophilic metamyelocyte, neutrophilic band, neutrophilic segmented) and the erythroid line (rubriblast, prorubricyte, rubricyte, metarubricyte, diffusely basophilic erythrocyte, erythrocyte). A consensus scheme was used to obtain the "true" classification of the cells. Cell types were characterized by three methods: absorbance spectra, dye binding, and chromaticities. Within both cell lines nuclear maturation is accompanied by an overall increase in peak absorbance with little shift in the position of the maximum. Generally, binding of azure B and eosin increases; azure B dimer/monomer ratios show a slight downward trend during maturation. Changes in chromaticities are to bluish purples of increasing saturation. Cytoplasmic changes accompanying maturation are much more striking than nuclear changes, and again the two cell lines show similarities. Generally, there is decreased binding of azure B during maturation. In the erythroid line, the Soret band of hemoglobin becomes increasingly prominent. Chromaticities change from bluish purples to purplish pinks, particularly in the erythroid line.