High-performance liquid chromatographic determination of hippuric acid in human urine

A method is described for the determination of urinary hippuric acid by high-performance liquid chromatography. The method used ethyl acetate extraction for partial clean-up of the urine. The separation was carried out on a reversed-phase column using 20% methanol in 0.01 M aqueous potassium phosphate containing 0.5% acetic acid as a mobile phase. The column effluent was monitored with a UV detector at 254 nm. Hippuric acid was separated from other normal urine constituents in less than 10 min. Metabolites of xylene and styrene did not interfere with the assay. Analytical recoveries from urine were excellent and peak height and concentration were linearly related.     

Purification of lipase from Chromobacterium viscosum by chromatography on palmitoyl cellulose.

Palmitoyl cellulose was used to adsorb the extracellular lipase [triacylglycerol acyl-hydrolase EC 3.1.1.3] of Chromobacterium viscosum from crude enzyme solution, and the adsorbed enzyme was eluted with a suitable detergent, such as Adekatol 45-S-8 or Triton X-100. The enzyme was then purified by chromatography on a palmitoylated gauze column with an overall recovery of 71% and an increase in the specific activity of 11-fold from the supernatant fluid of bacterial cultures. Further purification procedures included fractionation with acetone, and chromatography on Sephadex G-150 and G-75 columns. Two isoenzymes were obtained, each in a homogeneous state on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis: one had a molecular weight of 120,000 and pI of 3.7 and the other a molecular weight of 30,000 with a pI of 7.3.     

Oxidative pathway of choline to betaine in the soluble fraction prepared from Arthrobacter globiformis.

One strain of bacteria which showed high H2O2-generating activity was isolated from soil and characterized as Arthrobacter globiformis based on its morphological, nutritional, and physiological characteristics. The activities of H2O2 generation, NAD reduction and oxygen consumption in the bacterial cells were examined using choline, betaine aldehyde or betaine as substrate. Choline was oxidized to betaine aldehyde under aerobic conditions in a reaction coupled with H2O2 generation and oxygen consumption. On the other hand, betaine aldehyde seemed to be oxidized to betaine through two distinct oxidative reactions, H2O2 generation (oxygen consumption) under aerobic conditions and NAD reduction under either aerobic or anaerobic conditions. These enzyme activities were found in the supernatant fraction of the sonicated cell preparation.     

Purification and characterization of choline oxidase from Arthrobacter globiformis

Choline oxidase was purified from the cells of Arthrobacter globiformis by fractionations with acetone and ammonium sulfate, and column chromatographies on DEAE-cellulose and on Sephadex G-200. The purified enzyme preparation appeared homogeneous on disc gel electrophoresis. The enzyme was a flavoprotein having a molecular weight of approx. 83,000 (gel filtration) or approx. 71,000 (sodium dodecyl sulfate--polyacrylamide disc gel electrophoresis) and an isoelectric point (pI) around pH 4.5. Identification of the reaction products showed that the enzyme catalyzed the following reactions: choline + O2 leads to betaine aldehyde + H2O2, betaine aldehyde + O2 + H2O leads to betaine + H2O2. The enzyme was highly specific for choline and betaine aldehyde (relative reaction velocities: choline, 100%; betaine aldehyde, 46%; N,N-dimethylaminoethanol, 5.2%; triethanolamine, 2.6%; diethanolamine, 0.8%; monoethanolamine, N-methylaminoethanol, methanol, ethanol, propanol, formaldehyde, acetaldehyde, and propionaldehyde, 0%). Its Km values were 1.2 mM for choline and 8.7 mM for betaine aldehyde. The optimum pH for the enzymic reaction was around pH 7.5.     

Fructose as a carbohydrate source yields stable pH and redox parameters in microcarrier cell culture

The pH and cytosolic NADH/NAD⁺ redox potential in microcarrier cultures of Madin-Darby canine kidney cells remain within physiological range when fructose is substituted for glucose in medium formulation. This difference is accounted for by the low rate of lactic acid production in cultures utilizing fructose as a primary carbohydrate source.     

Microscopic observations of skin and lymphoid organs in the hairless dog derived from the Mexican hairless

The skin and lymphoid organs of Mexican hairless dogs and their hairless offspring were examined histologically. The hairless dogs lacked most hairs except for sparse hairs on the head, tail and feet. The skin of newborn pups consisted of a thick epidermis with epidermal ingrowths forming the rudiments of hair follicles. In older dogs more than 2 months of age, however, the epidermis was thin and the ingrowths were few. Neither hair follicles nor skin glands were present. The hairy skin of the head and tail had hair follicles with sebaceous glands. Regarding the lymphoid organs, the newborn pups possessed a thymus like haired pups. But in the older dogs more than 2 months of age, the thymus was atrophied and the lymphocyte population was too sparse to demarcate the cortex and the medulla. Lymphocyte accumulation in older dogs was also poor in the spleen and mesenteric lymph nodes. The present findings indicate that the hairlessness of the Mexican hairless dogs and their descendants is accompanied by early atrophy of the thymus after birth, and is followed by poor accumulation of lymphocytes in the thymus-dependent area of the spleen and the mesenteric lymph nodes. The defect of the thymus in the hairless dog seems to be different from that in athymic nude mice and rats. Further studies are needed to elucidate the immunological response and function in hairless dogs.     

Dynamic sweating response of man to infrared irradiation in various spectral regions

In an attempt to detect differences in the thermal effect of infrared irradiation of different wavelengths, transient sweating response to infrared irradiation in various spectral regions was examined. In Series 1, the ventral or dorsal surface of the nude subject was irradiated repetitively for a period of 4 min (2 min on, 2 min off) by each of three kinds of infrared heaters with main emissivity in near-infrared (NIR; 0.7–2.8 m), intermediate-infrared (MIR; 1.5–5.8 m), and far-infrared (FIR; 2.8–25 m) regions. The sweating response on a non-irradiated area tended to be the greatest with MIR, while the magnitude of the sweating response on the irradiated area showed no consistent differences among various wavelengths. The results infer that MIR stimulated cutaneous thomoreceptors most effectively, while its direct effect on local sweat gland activity was minimal. In Series 2, the effects of 9–12 min irradiations in more restricted ranges of wavelength were compared by the combination of the three kinds of heaters with filters (translucent to wavelength ranges of 1.3–2.7, 2.7–3.5, 3.6–8.0 m, respectively). The sweating response on a remote area was predominantly greater with the range of 2.7–3.5 m than with the other wavelength ranges, while the local effect on sweating was minimal with this range. The results of Series 2 reinforce those of Series 1, indicating that the degree of stimulation of cutaneous thermoreceptors and of direct thermal effect on sweat gland activity differ with spectral regions incident on the skin, thus affecting local and remote effects on the sweating response.     

Alteration of mitochondrial genomes containing atpA genes in the sexual progeny of cybrids between Raphanus sativus cms line and Brassica napus cv. Westar

Summary We have investigated the fate of the mitochondrial genomes of cybrids derived from donor-recipient protoplast fusion between X-irradiated Raphanus sativus (cms line) and iodoacetamide-treated Brassica napus cv. Westar. Two out of ten fusion products were male-sterile with the diploid chromosome number of B. napus. The mitochondrial (mt) genomes of the cybrids and their progeny were further analyzed by DNA-DNA hybridizaion using the pea mitochondrial ATPase subunit gene (atpA) as a probe. One cybrid, 18-3, had a 3.0 kb fragment characteristic of B. napus and a 2.0 kb non-parental fragment when the BamHI-digested DNA was hybridized with the probe. In the first-backcrossed progeny of this cybrid, the hybridization pattern was not stably inherited. A 4.0 kb radish fragment, not detectable in the cybrid, appeared in one of the BC₁ generation siblings, and the 2.0 kb non-parental fragment was lost in another. The hybridization patterns in BC₁ progeny siblings of cybrid 12-9 were also varied. The alteration of mtDNA in the cybrid progeny continued to the BC₂ generation. There was no clear evidence of a heteroplasmic state or of sub-stoichiometric molecules in the mt genome of cybrid 18-3. A possible cause of the observed alteration in the mt genome is discussed.     

Agrobacterium-mediated transformation and regeneration of kiwi fruit

Summary Genetically transformed kiwi fruit (Actinidia deliciosa) plants were obtained from hypocotyl and stem segments co-cultured with Agrobacterium tumefaciens strain EHA101 harboring a binary vector, pLAN411 or pLAN421, which contained the neomycin phosphotransferase II (nptII) gene and the -glucuronidase (GUS) gene. After co-culturing with the A. tumefaciens, the hypocotyl or stem segments were cultured on a selection medium containing 25g/ml kanamycin and 500g/ml Claforan. After one month in culture, shoots had regenerated from the cuttings. Green shoots were analyzed for NPTII activity and GUS activity. Eighty-five percent of the green shoots examined expressed the nptII and GUS genes. GUS histochemical assays revealed strong GUS expression in guard cells, mesophyll cells, and trichomes.     

Immuno-cross-reactivity of histidine and dopa decarboxylases

Immuno-cross-reactivity between histidine decarboxylase (HDC) and dopa decarboxylase (DDC) was investigated. By comparing the cDNA sequences of rat HDC with rat and guinea-pig DDCs, we found a region that may possibly be related to the cross-reactivity of anti-rat HDC antibody with guinea-pig DDC. The peptide encoded by this region was synthesized and anti-peptide antibody was prepared. We also purified HDC and DDC homogeniously from fetal rat liver and guinea-pig liver, respectively. On immunoblotting, anti-peptide antibody recognized both rat HDC and guinea-pig DDC. Anti-HDC polyclonal antibody which also recognizes both enzymes detected only rat HDC when it was absorbed by the peptide. This result indicates that this region is responsible for the immuno-cross-reactivity of anti-rat HDC antibody with guinea-pig DDC.