Physical microhabitat requirements of freshwater pearl mussels,Margaritifera margaritifera (L.)

Surface sediment diatoms from the east coast of Lake Tanganyika were analysed using ordination and classification techniques, and compared with assemblages previously described from the northern part of the lake. Grain-size analyses were performed on subsamples. Four groups of diatom assemblages were recognised. The first group clusters samples taken in the north, far from the Rusizi river mouth. The second group comprises samples taken on silty sediment along the Tanzanian coast, including one sample taken near the mouth of the Malagarazi river and those from the northernmost part of the lake. The third group comprises surface sediments along the Burundian coast (near Ramba and Magara), and the fourth is characterised by epipsammic taxa. A sample taken near the central arm of the Malagarazi river is included in the latter group. The impact of small rivers on the diatom assemblages in the surface sediments is restricted to the mouth area.     

Transport of amino acids and ammonium in mycelium of Agaricus bisporus.

Mycelium of Agaricus bisporus took up methylamine (MA), glutamate, glutamine and arginine by high-affinity transport systems following Michaelis-Menten kinetics. The activities of these systems were influenced by the nitrogen source used for mycelial growth. Moreover, MA, glutamate and glutamine uptakes were derepressed by nitrogen starvation, whereas arginine uptake was repressed. The two ammonium-specific transport systems with different affinities and capacities were inhibited by NH(+)(4), with a K(i) of 3.7 microM for the high-velocity system. The K(m) values for glutamate, glutamine and arginine transport were 124, 151 and 32 microM, respectively. Inhibition of arginine uptake by lysine and histidine showed that they are competitive inhibitors. MA, glutamate and glutamine uptake was inversely proportional to the intracellular NH(+)(4) concentration. Moreover, increase of the intracellular NH(+)(4) level caused by PPT (DL-phosphinotricin) resulted in an immediate cessation of MA, glutamine and glutamate uptake. It seems that the intracellular NH(+)(4) concentration regulates its own influx by feedback-inhibition of the uptake system and probably also its efflux which becomes apparent when mycelium is grown on protein. Addition of extracellular NH(+)(4) did not inhibit glutamine uptake, suggesting that NH(+)(4) and glutamine are equally preferred nitrogen sources. The physiological importance of these uptake systems for the utilization of nitrogen compounds by A. bisporus is discussed.     

Chlamydia isopodii sp. n., an obligate intracellular parasite of Porcellio scaber

In an ultrastructural study of the hepatopancreas of Porcellio scaber, an obligate intracellular parasite, Chlamydia, was noted in the epithelial cells. Although the infection was found to extend the entire length of the hepatopancreas, it was most extensive in the glandular region. Indirect immunofluorescence testing revealed no cross-reactivity with either lymphogranuloma venereum or psittacosis antisera.     

Redox study of electron donation to P-680 in Photosystem II

Flash-induced absorption changes at 820 nm were studied as a function of redox potential in Tris-extracted Photosystem II oxygen-evolving particles and Triton subchloroplast fraction II particles. The rereduction kinetics of P-680+ in both preparations showed biphasic recovery phases with half-times of 42 and 625 microseconds at pH 4.5. The magnitude of the 42 microseconds phase of P-680+ rereduction was strongly dependent on the redox potential of the medium. This absorption transient, attributed to electron donation from D1 (the secondary electron donor in oxygen-inhibited chloroplasts), titrated as a single redox component with a midpoint potential of +240 +/- 35 mV. The experimentally determined midpoint potential was found to be independent of pH over the tested range 4.5-6.0. In contrast, the magnitude of the 625 microseconds phase of P-680+ rereduction was independent of redox potential between +350 and +100 mV. These results are interpreted in terms of a model in which an alternate electron donor with Em approximately equal to 240 mV, termed D0, serves as a rapid donor (t 1/2 less than or equal to 2 microseconds) to P-680+ in Tris-extracted and Triton-treated Photosystem-II preparations. According to this model, the slower electron donor, D1, is functional only when D0 becomes oxidized.     

Rapid quantitative analysis of binary mixtures of Escherichia coli strains using pyrolysis mass spectrometry with multivariate calibration and artificial neural networks

Pyrolysis mass spectrometry (PyMS) and multivariate calibration were used to show the high degree of relatedness between Escherichia coli HB101 and E. coli UB5201. Next, binary mixtures of these two phenotypically closely related E. coli strains were prepared and subjected to PyMS. Fully interconnected feedforward artificial neural networks (ANNs) were used to analyse the pyrolysis mass spectra to obtain quantitative information representative of the level of E. coli UB5201 in E. coli HB101. The ANNs exploited were trained using the standard back propagation algorithm, and the nodes used sigmoidal squashing functions. Accurate quantitative information was obtained for mixtures with >3% E. coli UB5201 in E. coli HB101. To remove noise from the pyrolysis mass spectra and so lower the limit of detection, the spectra were reduced using principal components analysis (PCA) and the first 13 principal components used to train ANNs. These PCA-ANNs allowed accurate estimates at levels as low as 1% E. coli UB5201 in E. coli HB101 to be predicted. In terms of bacterial numbers, it was shown that the limit of detection for PyMS in conjunction with ANNs was 3 × 10⁴ E. coli UB5201 cells in 1·6 × 10⁷ E. coli HB101 cells. It may be concluded that PyMS with ANNs provides a powerful and rapid method for the quantification of mixtures of closely related bacterial strains.     

The enzyme that cleaves apolipoprotein A-II upon in vitro incubation of human plasma high-density lipoprotein-3 with blood polymorphonuclear cells is an elastase

The proteolytic activity directed against apolipoprotein A-II (apo-A-II) which is released from human blood polymorphonuclear cells (PMN) when they are incubated with human plasma high-density lipoprotein-3 (HDL3) was studied to assess the properties and site specificity of the enzyme. When 125I-apo-A-II-labeled HDL3 was incubated with the PMN protease at 37 degrees C, a complete cleavage of apo-A-II was observed which paralleled the formation of bands of approximately 11,000 and 7,000 daltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 7,000-dalton component had the following N-terminal sequence: NH2-Thr-Asp-Tyr-Gly-Lys-Asp-Leu-Met-Glu-Lys. This corresponds to residues 19 through 28 of the intact apo-A-II monomer. Methoxysuccinyl (MeO-Suc)-Ala-Ala-Pro-Val-chloromethylketone-(CH2Cl) caused a 90% inhibition of apo-A-II hydrolysis at the highest concentration tested (6 X 10(-4)M). Besides apo-A-II, the PMN enzyme also hydrolyzed a synthetic substrate, MeO-Suc-Ala-Ala-Pro-Val-4-nitroanilide and its 4-methylcoumaryl-7-amide analogue. The protease appeared to have a mass of 28,000 daltons as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the [3H]diisopropylfluorophosphate-labeled PMN enzyme. That the PMN enzyme which cleaves apo-A-II is an elastase was derived from the following criteria: 1) cleavage at the Val-X bond in apo-A-II and in the two synthetic substrates studied; 2) prevention of the cleavage by MeO-Suc-Ala-Ala-Pro-Val-CH2Cl, a known specific elastase inhibitor; and 3) a mass comparable to that reported for a pure PMN elastase. These studies establish that apolipoproteins can be suitable substrates for enzymes of the elastase family.     

Partial purification of the maturation-promoting factor MPF from unfertilized eggs of Xenopus laevis

A 200-fold purification of the maturation-promoting factor or MPF from unfertilized eggs of Xenopus laevis is reported for the first time. Purification was achieved by three successive column chromatographies on hydroxyapatite, trisacryl blue and L-arginine-agarose. The presence of MPF was assessed by the usual maturation criteria after injections of test material into immature stage VI unstimulated X. laevis oocytes: the precocious appearance of the maturation spot (within 45-120 min), the germinal vesicle breakdown, the presence of the first polar body and the second metaphase spindle. Purification was monitored by the decrease of the minimal amount of protein injected in a constant volume (50 nl) required to induce 50% frequency of germinal vesicle breakdown. This amount decreased from 500 ng in the crude extract to 2.5 ng in the 200-fold purified material. Analysis by SDS-PAGE of the crude extract showed about 40 Coomassie-blue-stained polypeptides with molecular masses ranging from 300 kDa to 20 kDa, whereas in the 200-fold purified MPF only 5 stained polypeptides were revealed, with molecular masses of 62, 53, 49, 39 and 37 kDa. In vitro phosphorylations for the detection of kinase activities for endogenous and exogenous substrates were monitored by analysis of autoradiograms of SDS-PAGE, after treatment of fractions with [gamma-32P]ATP. Only inactive fractions eluted from columns ahead of MPF, and fractions containing MPF activity were tested. Phosphorylation of numerous stained polypeptides was demonstrated in the crude MPF extract and exogenous substrates such as phosvitin, casein and histone type II-AS were also strongly phosphorylated. In the MPF fraction, purified on hydroxyapatite, a polypeptide of 53 kDa was more highly and specifically phosphorylated and the presence of kinase activities was observed for the above three exogenous substrates. In the 100-fold and 200-fold purified MPF, phosphorylation of endogenous substrates could not be shown and kinase activities for the above three substrates were drastically decreased as compared with the crude and purified MPF obtained after hydroxyapatite column chromatography. However, neither endogenous phosphorylations nor kinase activities with the above exogenous substrates could be shown in inactive fractions eluted ahead of MPF at the different purification steps. Some characteristics of the purified material are also described in this paper.