Detailed analysis of the turnover of polyphosphoinositides and phosphatidic acid upon activation of phospholipases C and D in Chlamydomonas cells treated with non-permeabilizing concentrations of mastoparan

Treating Chlamydomonas moewusii cells with non-permeabilizing concentrations of mastoparan (1–5 μM) increased inositol 1,4,5-trisphosphate (InsP₃) levels up to 20-fold in a dose-dependent manner and rapidly induced deflagellation and mating-structure activation, two well-defined Ca²⁺-responses. When metabolism of the phospholipid precursors was monitored in ³²P_i-labelled cells, as much as 70% of the radioactivity in phosphatidylinositol bisphosphate (PtdInsP₂) was lost within 20 s. Thereafter, the ³²P-label in PtdInsP₂ increased to twice the control level within 10 min. A similar pattern of ³²P-labelling was also exhibited by PtdInsP. An HPLC-headgroup analysis revealed that only PtdIns4P and PtdIns(4,5)P₂ were involved and not the D3-phosphorylated isomers. Correlated with the increased polyphosphoinositide (PPI) turnover, there was a massive (5- to 10-fold) increase in ³²P-labelled phosphatidic acid (PtdOH) and, slightly later, an increase in its metabolic product, diacylglycerol pyrophosphate (DGPP), reflecting the phosphorylation of the resulting diacylglycerol (DAG) and PtdOH, respectively. Mastoparan-treatment of ³²P-labelled cells in the presence of 0.2% n-butanol increased the formation of radioactive phosphatidylbutanol (PtdBut), a specific reporter of phospholipase D (PLD) activity. This means that mastoparan activates both phospholipase C (PLC) and PLD, and thus both pathways could contribute to the increase in PtdOH. To distinguish between them, a differential labelling strategy was applied based on the fact that ³²P_i-label is slowly incorporated into structural phospholipids but rapidly incorporated into ATP. Since PLD hydrolyses a structural lipid, radioactivity only appears slowly in PtdOH_PLD (and PtdBut). In contrast, PtdOH_PLC is synthesised by phosphorylation of DAG, and therefore should rapidly incorporate radioactivity. In practice, PtdOH formed on addition of mastoparan was rapidly labelled, reflecting the specific radioactivity of the [³²P]ATP pool. Based on the production of [³²P]PtdBut, we estimate that about 5–17% of the PtdOH was generated through the PLD pathway, while the majority originated from PLC activity. Together, this is the first demonstration (i) that PLC activation is correlated with increases in Ca²⁺, InsP₃, PtdOH and DGPP, at the cost of PtdInsP and PtdInsP₂, all in one and the same cell, (ii) of the characteristics of stimulated and unstimulated PPI turnover, (iii) that stimulated turnover affects the D-4 PPI and not the 3-isomers, (iv) that PLC and PLD are activated at the same time, (v) of a simple labelling method to discriminate between the two in terms of PtdOH production. Received: 3 December 1997 / Accepted: 22 May 1998     

Improvement of P450(BM-3) whole-cell biocatalysis by integrating heterologous cofactor regeneration combining glucose facilitator and dehydrogenase in E. coli

Escherichia coli BL21, expressing a quintuple mutant of P450_BM-3, oxyfunctionalizes α-pinene in an NADPH-dependent reaction to α-pinene oxide, verbenol, and myrtenol. We optimized the whole-cell biocatalyst by integrating a recombinant intracellular NADPH regeneration system through co-expression of a glucose facilitator from Zymomonas mobilis for uptake of unphosphorylated glucose and a NADP⁺-dependent glucose dehydrogenase from Bacillus megaterium that oxidizes glucose to gluconolactone. The engineered strain showed a nine times higher initial α-pinene oxide formation rate corresponding to a sixfold higher yield of 20 mg g⁻¹ cell dry weight after 1.5 h. The initial total product formation rate was 1,000 μmol h⁻¹ μmol⁻¹ P450 leading to a total of 32 mg oxidized products per gram cell of dry weight after 1.5 h. The physiological functioning of the heterologous cofactor regeneration system was illustrated by a sevenfold increased α-pinene oxide yield in the presence of glucose compared to glucose-free conditions.     

Evidence for a light-induced H(+) conductance in the eye of the green alga Chlamydomonas reinhardtii.

Rhodopsin-mediated photoreceptor currents, I(P), of the unicellular alga Chlamydomonas reinhardtii were studied under neutral and acidic conditions. We characterized the kinetically overlapping components of the first, flash-induced inward current recorded from the eye, I(P1), as a low- and high-intensity component, I(P1a) and I(P1b), respectively. They peak between 1 and 10 ms after the light-flash and are both likely to be carried by Ca(2+). I(P1a) and I(P1b) exhibit half-maximal photon flux densities, Q(1/2), of approximately 0.14 and 58 microE m(-2), and maximal amplitudes of approximately 4.9 and 38 pA, respectively. At acidic extracellular pH values (pH 3-5), both I(P1) currents are followed by distinct H(+) currents, I(P2a) and I(P2b), with maxima after approximately 5 and 100 ms, respectively. Because the Q(1/2) values of I(P1b) and I(P2b) virtually coincide with Q(1/2) of rhodopsin bleaching, we suggest that the respective conductances G(1b) and G(2b) are closely coupled to the rhodopsin, whereas the low light-saturating conductances G(1a) and G(2a) reflect transducer-activated states of a second rhodopsin photoreceptor system.     

Excitation energy transfer in phycobiliproteins of the cyanobacterium <Emphasis Type="Italic">Acaryochloris marina</Emphasis> investigated by spectral hole burning

The cyanobacterium Acaryochloris marina developed two types of antenna complexes, which contain chlorophyll-d (Chl d) and phycocyanobilin (PCB) as light-harvesting pigment molecules, respectively. The latter membrane-extrinsic complexes are denoted as phycobiliproteins (PBPs). Spectral hole burning was employed to study excitation energy transfer and electron–phonon coupling in PBPs. The data reveal a rich spectral substructure with a total of four low-energy electronic states whose absorption bands peak at 633, 644, 654, and at about 673 nm. The electronic states at ~633 and 644 nm can be tentatively attributed to phycocyanin (PC) and allophycocyanin (APC), respectively. The remaining low-energy electronic states including the terminal emitter at 673 nm may be associated with different isoforms of PC, APC, or the linker protein. Furthermore, the hole burning data reveal a large number of excited state vibrational frequencies, which are characteristic for the chromophore PCB. In summary, the results are in good agreement with the low-energy level structure of PBPs and electron–phonon coupling parameters reported by Gryliuk et al. (BBA 1837:1490–1499, 2014) based on difference fluorescence line-narrowing experiments.     

Effect of replacing maize grain and soybean meal with a xylose-treated wheat grain on feed intake and performance of dairy cows

This study evaluated wheat grain which was treated with xylose in aqueous Ca–Mg lignosulphonate solution at elevated temperatures (WeiPass®) in order to reduce ruminal degradation of starch and crude protein. The two tested isoenergetic and isonitrogenous diets contained on dry matter (DM) basis either 16% maize grain and 6.4% soybean meal (Diet CON) or 17.8% xylose-treated wheat and 4.6% soybean meal (Diet Wheat). Thirty-six German Holstein dairy cows were assigned to one of the two groups according to parity, body weight after calving, and milk yield during the previous lactation. Data collection started at 21 d before the expected calving date until 120 d in milk. The average of DM intake, energy-corrected milk (ECM) yield, and milk fat and protein yields (all given as kg/d) were 18.9, 28.7, 1.25, and 1.02 for Diet CON and 19.3, 32.5, 1.36, and 1.11 for Diet Wheat, respectively. Only ECM and milk protein yields were greater (p < 0.05) for cows receiving Diet Wheat. In conclusion, the xylose-treated wheat grain can replace maize grain and part of soybean meal in diets for lactating dairy cows and may be an alternative feedstuff depending on overall ration composition and availability and costs of grain sources.     

Phospho-eNOS Ser-114 in human mesenchymal stem cells: constitutive phosphorylation, nuclear localization and upregulation during mitosis

Activity of endothelial nitric oxide synthase (eNOS) is modulated by protein-protein interaction and phosphorylation at specific serine or threonine residues. Using immunofluorescence analysis we show here that proliferating mesenchymal stem cells (MSCs) derived from human bone marrow exhibit cytosolic and pronounced nuclear localization of eNOS. Examination of phosphorylated eNOS subspecies revealed that eNOS phosphorylated at Ser-114 is heavily enriched in the nucleus, whereas eNOS phosphorylated at Ser-1177 is localized at filamentous structures in the cytosol that are abundant in the perinuclear region. Phosphorylation of eNOS at Ser-114 but not at Ser-1177 was strongly increased in cells shortly before mitosis and decreased to normal level after completed cell division. Double immunofluorescence analysis revealed that subcellular localization of 8-hydroxyguanosine immunoreactivity was overlapping with eNOS phosphorylated at Ser-114 in human MSCs providing evidence that phosphorylation at this residue is linked to the generation of superoxide anions. As expected there was only a weak colocalization between eNOS phosphorylated at Ser-1177 and caveolin-1. Different from many other cell systems, human MSCs accumulate eNOS in the nucleus without an acute stimulus. eNOS constitutively phosphorylated at distinct amino acid residues is targeted to different subcellular compartments pointing to an important role of specific phosphorylation events in the life cycle of proliferating human MSCs.     

Identification of a tomatinase in the tomato-pathogenic actinomycete Clavibacter michiganensis subsp. michiganensis NCPPB382

The insertion site of a transposon mutant of Clavibacter michiganensis subsp. michiganensis NCPPB382 was cloned and found to be located in the gene tomA encoding a member of the glycosyl hydrolase family 10. The intact gene was obtained from a cosmid library of C. michiganensis subsp. michiganensis. The deduced protein TomA (543 amino acids, 58 kDa) contains a predicted signal peptide and two domains, the N-terminal catalytic domain and a C-terminal fibronectin III-like domain. The closest well-characterized relatives of TomA were tomatinases from fungi involved in the detoxification of the tomato saponin alpha-tomatine which acts as a growth inhibitor. Growth inhibition of C. michiganensis subsp. michiganensis by alpha-tomatine was stronger in the tomA mutants than in the wild type. Tomatinase activity assayed by deglycosylation of alpha-tomatine to tomatidine was demonstrated in concentrated culture supernatants of C. michiganensis subsp. michiganensis. No activity was found with the tomA mutants. However, neither the transposon mutant nor a second mutant constructed by gene disruption was affected in virulence on the tomato cv. Moneymaker.     

Processing of the human transferrin receptor at distinct positions within the stalk region by neutrophil elastase and cathepsin G

The ectodomain of the human transferrin receptor (TfR) is released as soluble TfR into the blood by cleavage within a stalk. The major cleavage site is located C-terminally of Arg-100; alternative cleavage sites are also present. Since the cleavage process is still unclear, we looked for proteases involved in TfR ectodomain release. In the supernatant of U937 histiocytic cells we detected alternatively cleaved TfR (at Glu-110). In membrane fractions of these cells we identified two distinct proteolytic activities responsible for TfR cleavage within the stalk at either Val-108 or Lys-95. Both activities could be inhibited by serine protease inhibitors, but not by inhibitors of any other class of proteases. Protein purification yielded a 28 kDa protein that generated the Val-108 terminus. The protease activity could be ascribed to neutrophil elastase according to the substrate specificity determined by amino acid substitution analysis of synthetic peptides, an inhibitor profile, the size of the protease and the use of specific antibodies. The results of analogous experiments suggest that the second activity is represented by another serine protease, cathepsin G. Thus, membrane-associated forms of neutrophil elastase and cathepsin G may be involved in alternative TfR shedding in U937 cells.     

Antimicrobial resistance among urinary tract infection (UTI) isolates in Europe: results from the SENTRY Antimicrobial Surveillance Program 1997

The SENTRY Antimicrobial Surveillance Program was established to monitor the occurrence and antimicrobial susceptibility of bacterial pathogens via an international network of sentinel hospitals. Twenty European hospitals referred a total of 887 urinary tract infection (UTI) isolates to the European SENTRY reference laboratory during the period October–December 1997. Ninety percent of the referred species were represented by Escherichia coli (52%), Enterococcus spp. (12%), Klebsiella spp. (7%), Proteus spp. (7%), Pseudomonas aeruginosa (7%), and Enterobacter spp. (5%). The susceptibility of E. coli isolates to penicillins was less than 60%, while almost all of the isolates were susceptible to piperacillin/tazobactam (98% susceptibility), cephalosporins (98%), and carbapenems (100%). Amikacin was the best aminoglycoside (99.8% susceptibility). The susceptibility to quinolones was only 88–89%, with highest levels of resistance observed for isolates from Portugal, Italy, England, The Netherlands, and some centers in France, Spain, and Poland. The susceptibility of Klebsiella spp. to the newer generations of cephalosporins was 82–95% and to the carbapenems 100%. Amikacin was again the best aminoglycoside (94% susceptibility). The susceptibility of Enterobacter spp. to any ß-lactam antibiotic was poor, except for the carbapenems (100% susceptibility) and cefepime (90% susceptibility), while the susceptibility to aminoglycosides was 80–89%. Proteus spp. showed complete susceptibility to cefepime, ceftriaxone, the carbapenems, and piperacillin/tazobactam, while the susceptibility of P. aeruginosa isolates was poor, with best results for the carbapenems (susceptibility 89%), piperacillin/tazobactam (susceptibility 84%), and amikacin and ticarcillin (susceptibility to both 80%). Enterococcus spp. showed the highest susceptibility to vancomycin (98%), teicoplanin (98%), and ampicillin (94%).