Fast sampling,rapid filtration apparatus: Principal characteristics and validation from studies ofd-glucose transport in human jejunal brush-border membrane vesicles

Summary Kinetic data in (brush-border) membrane vesicles which rely on the validity of the initial rate assumption for their interpretation and depend on tracer flux studies using the rapid filtration technique for their experimental measurement have been limited to some extent by the absence of techniques that would allow for real-time data analysis. In this paper, we report on our successful design of a fast sampling, rapid filtration apparatus (FSRFA) which seems to fill up this technical gap since showing the following characteristics: (i) rapid injection (5 msec) and mixing (less than 100 msec) of small amounts of vesicles (10–40 l) with an incubation medium (0.2–1.0 ml); (ii) fast (20 to 80 msec depending on the sample volume) and multiple (up to 18 samples at a maximal rate of 4/sec) sampling of the uptake mixture followed by rapid quenching in the stop solution (approximately 5 msec) according to a predetermined time schedule (any time combination from 0.25 to 9999 sec); and (iii) fast, automated, and sampling-synchronized filtration and washings of the quenched uptake medium (only 15–20 sec are necessary for the first filtration followed by two washings and extra filtrations). As demonstrated using adult human jejunal brush-border membrane vesicles and Na⁺-d-glucose cotransport as models, the FSRFA accurately reproduces the manual aspects of the rapid filtration technique while allowing for very precise initial rate determinations. Moreover, the FSRFA has also been designed to provide as much versatility as possible and, in its present version, allows for a very precise control of the incubation temperature and also permits a few efflux protocols to be performed. Finally, its modular design, which separates the fast sampling unit from the rapid filtration device, should help in extending its use to fields other than transport measurement.     

The R1 gene conferring race-specific resistance to Phytophthora infestans in potato is located on potato chromosome V.

Summary Late blight in potato is caused by the fungusPhytophthora infestans and can inflict severe damage on the potato crop. Resistance toP. infestans is either based on major dominantR genes conferring vertical, race-specific resistance or on minor genes inducing horizontal, unspecific resistance. A dihaploid potato line was identified which carried theR1 gene, conferring vertical resistance to allP. infestans races, with the exception of those homozygous for the recessive virulence allele of the locusV1. The F₁ progeny of a cross between this resistant parent P(R1) and P(r), a line susceptible to all races, was analysed for segregation ofR1 and of restriction fragment length polymorphism (RFLP) markers distributed on the potato RFLP map comprising more than 300 loci. TheR1 locus was mapped to chromosome V in the interval between RFLP markers GP21 and GP179. The map position ofR1 was found to be very similar to the one ofRx2, a dominant locus inducing extreme resistance to potato virus X.     

Sequence of a novel HLA-DQB1 allele

The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence data base and have been assigned the accesion number M74842. The name DQB1*0304 has been officially assigned by the WHO Nomenclature Committee in November 1991. This follows the agreed policy that, subject to the conditions stated in the most recent Nomenclature Report (WHO Nomenclature Committee for factors of the HLA system, 1991), names will be assigned to new sequences as they are identified. List of such new names will be published in the following WHO Nomenclature Report.     

Production and properties of xylanases from thermophilic actinomycetes

30 strains of xylanolytic thermophilic actinomycetes were isolated from composted grass and cattle manure and identified as members of the generaThermomonospora, Saccharomonospora, Microbispora, Streptomyces andActinomadura. Screening of these strains for extracellular xylanase indicated that strains ofSaccharomonospora andMicrobispora generally were poor xylanase producers (0.5–1.5 U/ml) whereas relatively high activities were observed in cultures ofStreptomyces andActionomadura (4–12 U/ml).A preliminary characterization of the enzymes of strains of the latter genera suggested that xylanases of all the strains ofActinomadura exhibited higher thermostabilities than those ofStreptomyces. To evaluate the potential of thermophilicActinomadura for industrial applications, xylanases of three strains were studied in more detail. The highest activity levels for xylanases were observed in cultures grown on xylan and wheat bran. The optimal pH and temperature for xylanase activities ranged from 6.0 to 7.0 and 70 to 80°C. The enzymes exhibited considerable thermostability at their optimum temperature. The half-lives at 75°C were in the range from 6.5 to 17h. Hydrolysis of xylan by extracellular xylanases yielded xylobiose, xylose and arabinose as principal products. Estimated by the amount of reducing sugars liberated the degree of hydrolysis was 55 to 65%. Complete utilization of xylan is presumably achieved by -xylosidase activities which could be shown to be largely cell-associated in the 3Actinomadura strains.     

The initial characterization of the iron environment in lipoxygenase by M?ssbauer spectroscopy

The incorporation of 57Fe into two lipoxygenase isoenzymes from soybeans has been achieved making possible the first observations of the iron environment in these proteins using M?ssbauer spectroscopy. Immature soybean seeds were grown in tissue culture medium supplied with 57Fe. The iron in the active lipoxygenases that were isolated from the cultured seeds was readily detected in M?ssbauer measurements. It was unequivocally demonstrated that the native enzyme contains high-spin Fe(II). Based on the sign of the electric field gradient, the most likely ligand sphere for the iron in native lipoxygenase consists of oxygen and nitrogen ligands in a roughly octahedral field of symmetry. It was possible to detect M?ssbauer signals in highly concentrated samples of native lipoxygenases containing 57Fe at natural abundance. The spectra obtained for enriched and natural abundance native enzyme had the same high-spin Fe(II) M?ssbauer parameters. This confirmed that the environment of the iron in enzymes isolated from cultured seeds and dry soybeans were the same. The M?ssbauer spectra (4.2-250 K) for samples of both isoenzymes after oxidation of the iron in native enzyme by the product of lipoxygenase catalysis were extremely broad (20 mm/s) with no obvious narrow resonance lines. This was the result of the existence of paramagnetically broadened spectra for such samples even at relatively high temperature as evidenced by the appropriate EPR signal. A small molecule containing an iron site sharing many of these M?ssbauer and electron paramagnetic resonance properties with lipoxygenase was identified: Fe(II)/(III).diethylenetriaminepentaacetic acid.     

Mutagenic, electrochemical, and crystallographic investigation of the cytochrome b5 oxidation-reduction equilibrium: involvement of asparagine-57, serine-64, and heme propionate-7

A gene coding for lipase-solubilized bovine liver microsomal cytochrome b5 has been synthesized, expressed in Escherichia coli, and mutated at functionally critical residues. Characterization of the recombinant protein revealed that it has a reduction potential that is approximately 17 mV lower than that of authentic wild-type protein at pH 7 (25 degrees C). Structural studies determined that the recombinant protein differed in sequence from authentic wild-type cytochrome b5 owing to three errors in amidation status in the published sequence for the protein on which the gene synthesis was based. The structural origin of the lower reduction potential exhibited by the triple mutant has been investigated through X-ray crystallographic determination of the three-dimensional structure of this protein and is attributed to the presence of Asp-57 within 3.3 A of heme vinyl-4 in the mutant. In addition, the model developed by Argos and Mathews [Argos, P., & Mathews, F.S. (1975) J. Biol. Chem. 250, 747] for the change in cytochrome b5 oxidation state has been studied through mutation of Ser-64 to Ala. In this model, Ser-64 is postulated to stabilize the oxidized protein through H-bonding interactions with heme propionate-7 that orients this propionate group 6.2 A from the heme iron. Spectroelectrochemical studies of a mutant in which Ser-64 has been changed to an alanyl residue demonstrate that this protein has a reduction potential that is 7 mV lower than that of the wild-type protein; moreover, conversion of the heme propionate groups to the corresponding methyl esters increases the potential by 67 mV.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human fibroblasts show expression of the leukotriene-A4-hydrolase gene, which is increased after simian-virus-40 transformation

Human fibroblasts in cell culture converted the epoxide intermediate leukotriene A4 into the potent chemotaxin leukotriene B4. The identity of leukotriene B4 was ascertained by its mobility in reverse-phase high performance liquid chromatography, ultraviolet spectroscopy and gas chromatography/mass spectrometry. The presence of the enzyme responsible for the conversion (i.e. leukotriene A4 hydrolase), as well as the corresponding mRNA, were demonstrated by Western and Northern blot analyses. Leukotriene-A4-hydrolase enzyme activity, protein and mRNA were all enhanced (approximately threefold) in human fibroblasts that had been transformed by simian virus 40.     

Nucleotide sequence of Rattus norvegiens mitochondrial DNA that includes the genes for tRNAile, tRNAgln and tRNAf-met

The nucleotide sequence of a segment of mtDNA from Rattus norvegiens (rat) which contains the genes for tRNA^ile, tRNA^gl and tRNA^f-met has been determined. A detailed comparison has been made between this sequence and the corresponding sequences of mouse, human and bovine mtDNAs with regard to the primary and secondary structure of the tRNA genes, the regions connecting the tRNA genes, and the regions flanking the tRNA genes which code for the carboxyl terminus of URF-1 and the amino terminus of URF-2. No differences were found in the nucleotide sequences of the genes for tRNA^ile, tRNA^gln and tRNA^f-met in mtDNAs from three different female lines of rats (SASCO-1, SASCO-2 and Wild-UT) that differ by substitutions of 0.8% to 1.8% of their total nucleotides.     

Two different species of murein transglycosylase in Escherichia coli.

We demonstrated that Escherichia coli murein transglycosylase exists in two forms. After mechanical disruption of the cells, one form was found in the soluble fraction and the other, in the cell envelope. The two enzymes differed with respect to molecular weight, isoelectric point, solubility in aqueous buffers, and to some extent in their requirements for maximal catalytic activity. The molecular weight of the membrane-bound transglycosylase (35,000) was half that of the soluble enzyme. Whether the high-molecular-weight soluble protein is a precursor of the membrane-bound enzyme species remains to be elucidated.