Mechanism of Proteinase Release from Lactococcus lactis subsp. cremoris Wg2

The procedure generally used for the isolation of extracellular, cell-associated proteinases of Lactococcus lactis species is based on the release of the proteinases by repeated incubation and washing of the cells in a Ca²⁺-free buffer. For L. lactis subsp. cremoris Wg2, as many as five incubations for 30 min at 29°C are needed in order to liberate 95% of the proteinase. Proteinase release was not affected by chloramphenicol, which indicates that release is not the result of protein synthesis during the incubations. Ca²⁺ inhibited, while ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) stimulated, proteinase release from the cells. The pH optimum for proteinase release ranged between 6.5 and 7.5, which was higher than the optimum pH of the proteinase measured for casein hydrolysis (i.e., 6.4). Treatment of cells with the serine proteinase inhibitor phenylmethylsulfonyl fluoride prior to the incubations in Ca²⁺-free buffer reduced the release of the proteinase by 70 to 80%. The residual proteinase remained cell associated but could be removed by the addition of active L. lactis subsp. cremoris Wg2 proteinase. This suggests that proteinase release from cells of L. lactis subsp. cremoris Wg2 is the result of autoproteolytic activity. From a comparison of the N-terminal amino acid sequence of the released proteinase with the complete amino acid sequence determined from the nucleotide sequence of the proteinase gene, a protein of 180 kilodaltons would be expected. However, a proteinase with a molecular weight of 165,000 was found, which indicated that further hydrolysis had occurred at the C terminus.     

Classification and ordination of macroinvertebrate assemblages to predict stream acidity in upland Wales

Macroinvertebrates were collected from riffles at 104 sites in upland Wales during April and July 1984. Species assemblages were ordinated by DECORANA, classified by TWINSPAN and related to stream chemistry and other environmental factors using correlation and multiple discriminant analysis. DECORANA axis 1 was most strongly correlated with pH and aluminium concentration whilst axis 2 correlated with stream gradient and flow. Four TWINSPAN site groups established in each season were also principally related to pH and aluminium concentration, and reflected overall taxon-richness; differences between groups were most apparent during spring, when catchment forest cover and taxon-richness were also related. A dichotomous key based on indicator species was established for each season with the coleopteran Hydraena gracilis Germar and the Ephemeroptera, including Baetis rhodani Pictet, important indicators at Level 1. We propose that these indicator systems may be used for the rapid detection and assessment of acid waters throughout Wales, and that the methodology is applicable generally.     

Pseudomonas aeruginosa outer membrane protein F produced inEscherichia coli retains vaccine efficacy

Pseudomonas aeruginosa outer membrane protein F was purified by extraction from polyacrylamide gels of cell envelope proteins of anEscherichia coli strain expressing the cloned gene for protein F. Antisera directed against protein F purified fromP. aeruginosa PAO1 reacted with thisE. coli strain by immunofluorescence assay and immunoblotting, whereas these antisera were nonreactive withE. coli strains lacking thePseudomonas protein F gene. The protein F purified from thisE. coli strain was used to immunize mice by intramuscular injection of 10 µg of protein F preparation on days 1 and 14, followed by burn and challenge of the mice on day 28. As compared with control mice immunized withE. coli K-12 lipopolysaccharide, immunization with theE. coli-derived protein F afforded significant protection against subsequent challenge with heterologous Fisher-Devlin immunotype 5 and 6 strains ofP. aeruginosa. Antisera from mice immunized with theE. coli-derived protein F reacted at bands corresponding to protein F and 2-mercaptoethanol-modified protein F upon immunoblotting against cell envelope proteins of the PAO1, immunotype 5, and immunotype 6 strains ofP. aeruginosa and theE. coli strain containing the cloned F gene, but failed to react at these sites in anE. coli strain lacking the F gene. These data demonstrate thatP. aeruginosa protein F produced inE. coli through genetic engineering techniques retains its vaccine efficacy in the complete absence of anyP. aeruginosa lipopolysaccharide.     

Computer-assisted grading of adenocarcinoma in prostatic aspirates

Conventional cytologic grading of fine needle aspirates of prostatic adenocarcinoma has been shown neither to be reproducible nor to correlate well with histologic grading. This study developed a tumor grade classification based on computerized cytomorphometric features and compared the results to conventional grading of companion tissue sections. The image analysis system evaluated architectural features of the aspirates (mainly cell cluster features and interrelationships) as well as nuclear features. Thirty-five prostatic adenocarcinomas (8 well, 19 moderately and 8 poorly differentiated) were evaluated. Discriminant functions based on data collected at medium and high resolution distinguished between aspirates from low-grade (well-differentiated) and high-grade (poorly differentiated) adenocarcinomas with 81% accuracy. Moderately differentiated cancers could not be classified as a distinct group. This study suggests that accurate grading of prostatic adenocarcinoma in fine needle aspirate smears requires the evaluation of medium-resolution features related to specimen cellularity and uniformity or crowding of cell clusters as well as of high-resolution features of nuclear area, perimeter and coarseness of chromatin texture. These findings are compared to those of other schemes for the cytologic grading of prostatic aspirates.     

The dephosphorylation of inositol 1,4-bisphosphate to inositol in liver and brain involves two distinct Li+-sensitive enzymes and proceeds via inositol 4-phosphate.

1. Hydrolysis of both enantiomers of inositol 1-phosphate and both enantiomers of inositol 4-phosphate to inositol is inhibited by LiCl in liver and brain. 2. The phosphatase activity is predominantly soluble. 3. Inositol 1,4-bisphosphate is also hydrolysed by the soluble fraction of liver and brain. 4. Bisphosphatase activity is inhibited by LiCl, but is less sensitive than monophosphatase activity. 5. The product of bisphosphatase in liver and brain is inositol 4-phosphate.     

N-acetylglucosaminyltransferase substrates prepared from glycoproteins by hydrazinolysis of the asparagine-N-acetylglucosamine linkage. Purification and structural determination of oligosaccharides with mannose andN-acetylglucosamine at the non-reducing termini

Sixteen asparagine-linked oligosaccharides ranging in size from (Man)₂(GlcNAc)₂ (Fuc)₁ to (GlcNAc)₆(Man)₃(GlcNAc)₂ were obtained from human ₁-acid glycoprotein and fibrinogen, hen ovomucoid and ovalbumin, and bovine fetuin, fibrin and thyroglobulin by hydrazinolysis, mild acid hydrolysis and glycosidase treatment. The oligosaccharides hadN-acetylglucosamine at the reducing termini and mannose andN-acetylglucosamine residues at the non-reducing termini and were prepared for use asN-acetylglucosaminyltransferase substrates. Purification of the oligosaccharides involved gel filtration and high performance liquid chromatography on reverse phase and amine-bonded silica columns. Structures were determined by 360 MHz and 500 MHz proton nuclear magnetic resonance spectroscopy, fast atom bombardment-mass spectrometry and methylation analysis. Several of these oligosaccharides have not previously been well characterized.Abbreviations bis bisecting GlcNAc - DMSO dimethylsulfoxide - FAB fast atom bombardment - Fuc l-fucose - Gal d-galactose - GLC gas-liquid chromatography - GlcNAc or Gn N-acetyl-d-glucosamine - HPLC high performance liquid chromatography - Man or M d-mannose - MES 2-(N-morpholino)ethanesulfonate - MS mass spectrometry - NMR nuclear magnetic resonance - PIPES piperazine-N,N-bis(2-ethane sulfonic acid) the nomenclature of the oligosaccharides is shown in Table 1.     

Omega-hydroxylation of prostaglandin E1 in the isolated perfused lungs of pregnant rabbits

Cytochrome P450PG omega is induced in the rabbit lung in a gestational age-dependent manner and hydroxylates certain eicosanoids at their terminal, or omega (omega), carbon. This enzyme has been isolated from microsomal fractions and its activity has been characterized (Williams, D.E., et al., J. Biol. Chem. 259; 14600-14608, 1984). The experiments presented here examine the omega-hydroxylation activity of the intact lung during presentation of an eicosanoid substrate, prostaglandin E1 (PGE1), to the lung vasculature. Isolated, perfused lungs from three pregnant and four nonpregnant rabbits were injected with [3H]-PGE1. One-second fractions were collected from the perfusion effluent and were analyzed for metabolism of PGE1. Lungs isolated from pregnant rabbits metabolized PGE1 mainly to two polar derivatives, 20-hydroxy-PGE1 and 13,14-dihydro-15-keto-20-hydroxy-PGE1, whereas lungs from nonpregnant rabbits yielded mainly a relatively nonpolar metabolite, 13,14-dihydro-15-keto-PGE1. These metabolites were identified by coelution with standards that were generated enzymatically in vitro and whose structures were confirmed by gas chromatography/mass spectrometry (GC/MS).     

Calcium and phytochrome control of leaf unrolling in dark-grown barley seedlings

The red light-stimulated component of unrolling in sections from 7-d-old dark-grown barley (Hordeum vulgare L.) leaves is inhibited by ethyleneglycol-bis-(-aminoethyl ether)-N,N,N,N-tetracetic acid (EGTA). A free-Ca²⁺ activity of less than 40 M restores the ability to respond to red light, but only if supplied within 1 h of red light. Magnesium ions are an ineffective substitute. At least two processes in unrolling appear to be Ca²⁺-sensitive.Fluence-response measurements indicate that the levels of the far-red-absorbing from of phytochrome (Pfr) still present 4 h after red-light treatment should be above saturation for the unrolling response; consequently, loss of Pfr does not explain the loss in effectiveness of Ca²⁺ during prolonged EGTA treatment. However, if a further red-light treatment is given simultaneously with Ca²⁺ addition 4 h after the initial light stimulus, then full unrolling occurs in EGTA-treated sections. These data indicate that, under normal circumstances, a functional change in the properties of Pfr must occur, uncoupling it from the transduction chain.Abbreviations EGTA ethyleneglycol-bis-(-aminoethylether)-N,N,N,N,-tetracetic acid - FR far-red light - Mes 2-(N-morpholino)ethanesulphonic, acid - Pfr far-red absorbing form of phytochrome - Pr red-absorbing form of phytochrome - R red light     

The loci of perception for phytochrome control of internode growth in light-grown mustard: Promotion by low phytochrome photoequilibria in the internode is enhanced by blue light perceived by the leaves

Under continuous white light (WL), extension growth of the first internode in Sinapis alba L. was promoted by low red (R): far-red (FR) ratios reaching the stem and-or the leaves. Conversely, the growth promotion by end-of-day light treatments was only triggered by FR perceived by the leaves and cotyledons, while FR given to the growning internode alone was tatally ineffective. Continuous WL+FR given to the internode was also in-effective if the rest of the shoot remained in darkness. Both the background stem growth, and the growth promotion caused by either an end-of-day FR pulse or continuous WL+FR given to the internode, increased with increasing fluence rates of WL given to the rest of the shoot. The increase by WL of the growth-stimulatory effect of low phytochrome photoequilibria in the internode appears to be mediated by a specific blue-light-absorbing photoreceptor, as blue-deficient light from sodium-discharge lamps, or from filtered fluorescent tubes, promoted background stem growth similarly to WL but did not amplify the response to the R:FR ratio in the internode. Supplementing the blue-deficient light (94 mol·m^-2·s^-1) with low fluence rates of blue (<9 mol·m^-2·s^-1) restored the promotive effect of low R:FR reaching the internode.Abbreviations BL blue light - FR far-red light - PAR photosynthetically active radiation - Pfr/P ratio between the FR-absorbing form and total phytochrome - R red light - SOX low-pressure sodium lamp - WL white light Supported by the Consejo Nacional de Investigaciones Cientificas y Técnicas (República Argentina) and the ORS scheme (UK)     

Biochemical analysis of the activation of adherent neutrophils in vitro

The role played by neutrophil oxidative responses in host defense and injury is an area of active investigation. In order to study neutrophil responses in vitro, methods are required for cell purification, enumeration, and quantification of activation responses, which mimic the in vivo situation as closely as possible. In this communication (and its companion paper, Albertine et al., 1988) improved methods for all of these tasks are described and applied to investigate neutrophil structure-function relationships in vitro and in vivo. Human neutrophils were purified by using a series of platelet-poor plasma-Percoll gradients (51, 62, 76 and 80% in Percoll). This modification of previously published procedures results in consistently successful neutrophil purification and has allowed us to purify neutrophils from bronchoalveolar lavage fluid as well as blood. Activation of human and sheep neutrophils (superoxide anion production) was quantitated by the reduction of ferricytochrome c using a microtiter plate reader to measure the increase in absorbance at 550 nm from adherent neutrophils. Adherence of neutrophils was quantitated by measurement of LDH in cells lysed with Triton X-100 using a new method which uses readily available commercial reagents and can quantitate the LDH content of as few as 5000 neutrophils (or the LDH released from 5% of 100,000 neutrophils). Assay conditions for superoxide anion were optimized, limitations both in assay design and instruments used to measure OD were explored and enumerated, and these methods were used to quantitate sheep and human neutrophil activation responses. Using methods described in Albertine et al. (1988) for fixing neutrophils in microtiter wells after assay of their functional capacity, we have studied the same cells functionally and morphologically. We have used these techniques to study blood and alveolar neutrophils from a patient with acute respiratory failure. His alveolar neutrophils displayed 67% of the activation response as peripheral neutrophils (4.31 +/- 0.12 nmol superoxide released per 250,000 neutrophils at 60 min vs. 6.38 +/- 0.18 in blood, P less than 0.01) and structural changes which suggested previous activation in vivo. These studies demonstrate that similar morphological changes are observed in neutrophils activated with phorbol myristate acetate in vitro, as are observed in cells which have been activated by pathophysiologic processes in vivo.