Investigation of Acidithiobacillus ferrooxidans in pure and mixed-species culture for bioleaching of Theisen sludge from former copper smelting.

AIMS: The aim of this study was to investigate the potential of bioleaching for the treatment of an environmentally hazardous waste, a blast-furnace flue dust designated Theisen sludge. METHODS AND RESULTS: Bioleaching of Theisen sludge was investigated at acidic conditions with Acidithiobacillus ferrooxidans in pure and mixed-species culture with Acidiphilium. In shaking-flask experiments, bioleaching parameters (pH, redox potential, zinc extraction from ZnS, ferrous- and ferric-iron concentration) were controlled regularly. The analysis of the dissolved metals showed that 70% zinc and 45% copper were extracted. Investigations regarding the arsenic and antimony species were performed. When iron ions were lacking, animonate (Sb(V)) and total arsenic concentration were highest in solution. The bioleaching approach was scaled up in stirred-tank bioreactors resulting in higher leaching efficiency of valuable trace elements. Concentrations of dissolved antimony were approx. 23 times, and of cobalt, germanium, and rhenium three times higher in comparison to shaking-flask experiments, when considering the difference in solid load of Theisen sludge. CONCLUSIONS: The extraction of base and trace metals from Theisen sludge, despite of its high content of heavy metals and organic compounds, was feasible with iron-oxidizing acidophilic bacteria. In stirred-tank bioreactors, the mixed-species culture performed better. SIGNIFICANCE AND IMPACT OF THE STUDY: To the best of our knowledge, this study is the first providing an appropriate biological technology for the treatment of Theisen sludge to win valuable elements.

Characterization of milk fatty acids based on genetic and herd parameters.

The objective of this study was to characterize the fatty acids (FA) in milk based on genetic and herd parameters to investigate the origin of the different FA in milk. Milk samples of 1912 Dutch Holstein-Friesian cows were analysed for 39 different FA including odd and branched-chain fatty acids. The proportion of variation caused by genetic and herd effects was calculated. In addition, genetic and herd correlations among the fatty acids were estimated and a clustering technique was used to visualise these correlations. The results indicated that in Dutch milk C12:0 is not completely synthesised de novo but also partly blood derived. It was suggested that C20:0 in milk is formed from the action of elongase enzymes on C18:0 and that the odd-chain FA C5:0-C13:0 and a part of C15:0 and C17:0 are synthesised de novo while the other part of C15:0 and C17:0 is blood derived. Furthermore, this work gives an overview of the opportunities to change the concentration of individual FA both by breeding and feeding. It is clearly shown that the extent to which the individual FA can be changed varies greatly and is dependent on the origin of the different FA in milk.

Virtual monochromatic spectral imaging versus linearly blended dual-energy and single-energy imaging during CT-guided biopsy needle positioning: Optimization of keV settings and impact on image quality.

OBJECTIVES: To compare image quality and metal artifact reduction between virtual monochromatic spectral imaging (VMSI), linearly blended dual-energy (DE) and single-energy (SE) images, each with and without dedicated iterative metal artifact reduction (iMAR) for CT-guided biopsy. MATERIALS AND METHODS: A biopsy trocar was positioned in the liver of six pigs. DE (Sn140/100kVp) and SE (120kVp/200mAs) acquisitions were performed with equivalent dose. From dual-energy datasets DE Q30-3 images and VMSI between 40-180 keV in steps of 20 keV were generated. From SE datasets I30-3 images were reconstructed. All images were reconstructed with and without iMAR. Objective image quality was analyzed applying density measurements at standardized positions (e.g. trocar tip and liver parenchyma adjacent to the trocar tip) and semi-automated threshold based segmentation. Subjective image quality was performed using semi-quantitative scores. Analyses were performed by two observers. RESULTS: At the trocar tip quantitative image analysis revealed significant difference in CT numbers between reconstructions with iMAR compared to reconstructions without iMAR for VMSI at lower keV levels (80 and 100 keV; p = 0.03) and DE (p = 0.03). For liver parenchyma CT numbers were significantly higher in VMSI at high keV compared to low keV (p≤0.01). VMSI at high keV also showed higher CT numbers compared to DE and SE images, though not the level of statistical significance. The best signal-to-noise ratio for VMSI was at 80 keV and comparable to DE and SE. Noise was lowest at 80 keV and lower than in DE and SE. Subjective image quality was best with VMSI at 80 keV regardless of the application of iMAR. iMAR significantly improved image quality at levels of 140 keV and 160 keV. Interreader-agreement was good for quantitative and qualitative analysis. CONCLUSION: iMAR improved image quality in all settings. VMSI with iMAR provided metal artifact reduction and better image quality at 80 keV and thus could improve the accurate positioning in CT-guided needle biopsy. In comparison, DE imaging did not improve image quality compared to SE.

Investigation of the exclusive 3He(e,e' pn)1H reaction.

Cross sections for the 3He(e,e' pn)1H reaction were measured for the first time at energy transfers of 220 and 270 MeV for several momentum transfers ranging from 300 to 450 MeV/c. Cross sections are presented as a function of the momentum of the recoil proton and the momentum transfer. Continuum Faddeev calculations using the Argonne V18 and Bonn-B nucleon-nucleon potentials overestimate the measured cross sections by a factor 5 at low recoil proton momentum with the discrepancy becoming smaller at higher recoil proton momentum.

A pure enzyme catalyzing penicillin biosynthesis.

Isopenicillin N synthetase (cyclase) has been purified to homogeneity from Cephalosporium acremonium strain C-10. The enzyme has a molecular weight of 40,000 to 42,000 and yields a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was purified in 10 percent yield by a combination of protamine sulfate and ammonium sulfate precipitations, gel filtration, and ion-exchange high-performance liquid chromatography. The purified enzyme can be stabilized with sucrose and stored at -20 degrees C for several weeks without any loss in activity.

Upper limits on perturbations of nuclear decay rates induced by reactor electron antineutrinos.

We report the results of an experiment conducted near the High Flux Isotope Reactor of Oak Ridge National Laboratory, designed to address the question of whether a flux of reactor-generated electron antineutrinos (ν¯e) can alter the rates of weak nuclear interaction induced decays of 54Mn, 22Na, and 60Co. This experiment has small statistical errors but, when systematic uncertainties are included, has null results. Perturbations greater than one part in 104 are excluded at 95% confidence level in ß± decay and electron capture processes, in the presence of an antineutrino flux of 3 × 1012 cm-2s-1. The present experimental methods are applicable to a wide range of radionuclides. Improved sensitivity in future experiments can be anticipated as we continue to better understand and reduce the dominant systematic uncertainties.

A new structural class of serine protease inhibitors revealed by the structure of the hirustasin-kallikrein complex.

BACKGROUND: Hirustasin belongs to a class of serine protease inhibitors characterized by a well conserved pattern of cysteine residues. Unlike the closely related inhibitors, antistasin/ghilanten and guamerin, which are selective for coagulation factor Xa or neutrophil elastase, hirustasin binds specifically to tissue kallikrein. The conservation of the pattern of cysteine residues and the significant sequence homology suggest that these related inhibitors possess a similar three-dimensional structure to hirustasin. RESULTS: The crystal structure of the complex between tissue kallikrein and hirustasin was analyzed at 2.4 resolution. Hirustasin folds into a brick-like structure that is dominated by five disulfide bridges and is sparse in secondary structural elements. The cysteine residues are connected in an abab cdecde pattern that causes the polypeptide chain to fold into two similar motifs. As a hydrophobic core is absent from hirustasin the disulfide bridges maintain the tertiary structure and present the primary binding loop to the active site of the protease. The general structural topography and disulfide connectivity of hirustasin has not previously been described. CONCLUSIONS: The crystal structure of the kallikrein-hirustasin complex reveals that hirustasin differs from other serine protease inhibitors in its conformation and its disulfide bond connectivity, making it the prototype for a new class of inhibitor. The disulfide pattern shows that the structure consists of two domains, but only the C-terminal domain interacts with the protease. The disulfide pattern of the N-terminal domain is related to the pattern found in other proteins. Kallikrein recognizes hirustasin by the formation of an antiparallel beta sheet between the protease and the inhibitor. The P1 arginine binds in a deep negatively charged pocket of the enzyme. An additional pocket at the periphery of the active site accommodates the sidechain of the P4 valine.

Comparative microarray analysis of gene expression during apoptosis-induction by growth factor deprivation or protein kinase C inhibition.

The transcriptional response of mouse pro-B cells to two different apoptotic stimuli was investigated. First, interleukin-3 (IL-3) deprivation was used to trigger programmed cell death in IL-3 dependent FL5.12 cells. Alternatively, cells were treated with the protein kinase C (PKC) inhibitor staurosporine. The temporal pattern of gene expression was followed with cDNA microarrays, covering over 8700 different mouse cDNA sequences corresponding to approximately 7900 unique genes. Messenger RNA levels of 315 genes were found to be regulated by more than twofold upon IL-3 removal, while 125 genes reacted to staurosporine treatment. Cross-comparison revealed an intersection of 34 genes similarly regulated in both pathways and thus representing candidates for common apoptosis regulators. For many expressed sequence tags (ESTs) our data suggest for the first time functions in the control of apoptosis, stress response or the cell cycle. IL-3 removal led to the repression of genes required for proliferation and to the induction of genes, linked to apoptotic and signaling pathways. Staurosporine caused predominantly activation of genes, some of which had previously been described to be involved in inflammation. Our findings indicate that cellular responses to both apoptotic stimuli influence various physiological pathways which had not previously been known to be linked.

Functional expression of a mammalian acetylcholinesterase in Pichia pastoris: comparison to acetylcholinesterase, expressed and reconstituted from Escherichia coli.

The mature rat brain acetylcholinesterase gene (T subunit, AChE) was subcloned downstream of the temperature-inducible lambda promoter PL and fused to the signal peptide of the OmpA protein. Three different expression vectors were constructed: (i) pCompmA containing the mature AChE, (ii) pComp delta TA containing a truncated AChE and (iii) pComp delta TAH containing the truncated AChE C-terminal fused to a 6xHis-tag. With all expression vectors the overexpression of AChE in Escherichia coli resulted mainly in cytoplasmic inclusion bodies (IB). However, some activity was found in the periplasmic space. The inclusion bodies were refolded in vitro, yielding up to 1.42 U/mg IB of active AChE. The refolded AChE was partially purified (approx. 300-fold) by affinity chromatography with a specific activity of approx. 250 U/mg. Removing the cysteine residue near the C-terminus (truncated AChE, delta TAChE) assuming to affect the refolding, did not increase the amount of active enzyme obtained after refolding. Purification of denatured delta TAChE-6xHis prior to refolding by Ni-NTA-chromatography increased the refolding efficiency by a factor of 1.5. Functional expression and secretion of rat brain acetylcholinesterase into the medium was achieved in Pichia pastoris. By optimizing the culture conditions, 100 mU/ml AChE in the medium was produced. In this work we are describing the functional expression of a mammalian AChE in a microbial host in good yields for the first time. The physico-chemical properties of both, the bacterial and yeast expressed AChE were compared with those of the native AChE. The properties of the yeast expressed AChE and the native AChE were similar, whereas the E. coli expressed enzyme was found to be less stable and had different inhibition properties.

Particulate ANP-sensitive guanylyl cyclase: induction in cultured human peripheral CD3+ mononuclear blood cells.

There have been conflicting reports about the occurrence and/or activity of atrial natriuretic peptide (ANP) sensitive guanylyl cyclase in the immune system. This study reports on ANP-sensitive guanylyl cyclase mRNA expression and guanylyl cyclase activity in human peripheral blood mononuclear cells (PBMC). Reverse transcription polymerase chain reaction (RT-PCR) shows that activated human PBMC of healthy blood donors express functional active ANP-sensitive guanylyl cyclase after vitro culture, whereas freshly isolated PBMC show neither specific mRNA for particulate guanylyl cyclase nor ANP-sensitive activity of this enzyme. To define the subpopulation of PBMC expressing this enzyme, cultivated PBMC were subfractioned and analyzed by RT-PCR and in situ PCR. Only CD3+ PBMC showed mRNA for ANP-sensitive guanylyl cyclase. Induction of the guanylyl cyclase required coincubation with other cells, indicating that a factor or factors secreted from cells other than CD3+ cells induces this expression. In summary, ANP-sensitive guanylyl cyclase is an inducible enzyme in human CD3+ PBMC in contrast to other cells where it is considered to be constitutive.

Recombinant hirustasin: production in yeast, crystallization, and interaction with serine proteases.

A synthetic gene coding for the 55-amino acid protein hirustasin, a novel tissue kallikrein inhibitor from the leech Hirudo medicinalis, was generated by polymerase chain reaction using overlapping oligonucleotides, fused to the yeast alpha-factor leader sequence and expressed in Saccharomyces cerevisiae. Recombinant hirustasin was secreted mainly as incompletely processed fusion protein, but could be processed in vitro using a soluble variant of the yeast yscF protease. The processed hirustasin was purified to better than 97% purity. N-terminal sequence analysis and electrospray ionization mass spectrometry confirmed a correctly processed N-terminus and the expected amino acid sequence and molecular mass. The biological activity of recombinant hirustasin was identical to that of the authentic leech protein. Crystallized hirustasin alone and in complex with tissue kallikrein diffracted beyond 1.4 A and 2.4 A, respectively. In order to define the reactive site of the inhibitor, the interaction of hirustasin with kallikrein, chymotrypsin, and trypsin was investigated by monitoring complex formation in solution as well as proteolytic cleavage of the inhibitor. During incubation with high, nearly equimolar concentration of tissue kallikrein, hirustasin was cleaved mainly at the peptide bond between Arg 30 and Ile 31, the putative reactive site, to yield a modified inhibitor. In the corresponding complex with chymotrypsin, mainly uncleaved hirustasin was found and cleaved hirustasin species accumulated only slowly. Incubation with trypsin led to several proteolytic cleavages in hirustasin with the primary scissile peptide bond located between Arg 30 and Ile 31. Hirustasin appears to fall into the class of protease inhibitors displaying temporary inhibition.

HLA and Graves' disease: an association with HLA-DRw3.

HLA-A, -B, and -C antigens were tested by a standard lymphocyte microcytotoxicity technique in 86 Caucasians patients from western France with Graves' disease, and the data were compared with findings in 356 healthy controls. For HLA-DR antigen typing performed by lymphocyte microcytotoxicity testing using a long incubation time, the data were compared to findings in 100 healthy controls. An increase was found in the frequency of HLA-DRw3 [51.16% of patients vs. 20% of controls, corrected P (Pc) < 0.0003; relative risk (rr), 4.19) associated with an increased frequency of HLA-B8 (44.19% of patients vs. 22.47% of controls; Pc < 0.001; rr, 2.73) and HLA-A1 (40.7% of patients vs. 28.93% of controls; Pc < 0.03; rr, 1.71). In contrast, a diminished frequency was found for HLA-B12 (12.79% vs. 31.74%; Pc < 0.01). The antigen combination B8-DRw3 was noted in 37 of the 86 Graves' disease patients compared with 13 of 100 controls (Pc < 0.00003). No association was observed between HLA antigens and the different manifestations of the disease, such as the presence of goiter and/or exophthalmos, or the severity of clinical or biochemical signs. The present findings confirm the reported increase in the frequency of HLA-B8 in patients with Graves' disease. The most striking finding was the prevalence of HLA-DRw3, which, together with recent reports on lymphocyte-defined D locus determinants pointing to an increase frequency of HLA-Dw3, suggests that the gene or genes conferring susceptibility to Graves' disease may be located close to the HLA-D (DR) region of the sixth chromosome.

Isolation and structure of the pectin lyase D-encoding gene from Aspergillus niger.

The filamentous fungus, Aspergillus niger, produces a number of extracellular pectin-degrading enzymes. We present here the isolation and the complete nucleotide sequence of the gene, pelD, coding for a pectin lyase D (PLD), which was previously described as pectin lyase I (Van Houdenhoven, Ph.D. Thesis, Wageningen, 1975). The deduced amino acid (aa) sequence corresponds to 373 aa residues including a signal peptide of 19 aa. The coding region is interrupted by four short introns (57-65 bp). The nucleotide sequence of the 5'- and 3'-flanking regions is also presented and shows no unusual features. By comparing the deduced aa sequences of the A. niger PLD and a number of bacterial pectate lyases, short regions of homology were found despite the different substrate specificities (high methoxyl-pectin versus low methoxyl-pectin or polygalacturonate) of these enzymes.

Novel BNIP1 variants and their interaction with BCL2 family members.

By PCR and EST database searches we have identified three novel BNIP1 splice variants, and found that one of them, BNIP1-b, contains a highly conserved BH3 domain. The BNIP1 gene has been assigned to chromosome 5q33-34. Using in vitro protein-protein interaction assays, all BNIP1 variants were shown to interact with BCL2 and also with BCL2L1 (previously Bcl-xL). These interactions are BH3-independent. Furthermore, the BNIP1 variants cannot interact with BAX. The results suggest that the BNIP1 variants are novel members of the BCL2 family but function through a mechanism different from other BH3-only members.

The circulating bioactive form of human guanylin is a high molecular weight peptide (10.3 kDa).

Guanylin is a peptide isolated from rat intestine that stimulates intestinal guanylate cyclase. We describe here the purification of circulating guanylin from human hemofiltrate. By N-terminal protein sequence analysis 47 amino acids were determined. This sequence corresponds to the positions 22 to 68 of the prohormone deduced from the cDNA sequence of human proguanylin. Mass spectral analysis of the circulating peptide showed the molecular weight to be 10,336 Da, which corresponds to the mass calculated from position 22 to the C-terminus of the peptide predicted from the cDNA sequence. Circulating guanylin markedly increased the cyclic GMP content of T84 cells. Our data show that the hormonal form of guanylin is circulating as a 10.3-kDa peptide in human blood.

Thrombolytic and haemorrhagic effects of bolus doses of tissue-type plasminogen activator and a hybrid plasminogen activator with prolonged plasma half-life (K2tu-PA: CGP 42935).

K2tu-PA is a hybrid plasminogen activator linking the kringle 2 domain of tissue-type plasminogen activator (t-PA) to the catalytic protease domain of single-chain urokinase-type plasminogen activator (scu-PA). K2tu-PA, as t-PA has high affinity for fibrin and is activated by fibrin but has a longer plasma half-life (over 30 min). The aim of this study was to compare the effects of bolus doses of recombinant t-PA (rt-PA) and K2tu-PA, on: 1) lysis of performed thrombi (fibrinolysis), 2) accretion of new fibrin on pre-existing thrombi during fibrinolysis (thrombus growth), 3) thrombolysis as assessed by reduction of thrombus weight and 4) systemic plasma proteolysis and blood loss from a standard wound. A jugular vein thrombosis model and an ear bleeding model were adopted in rabbits. Saline produced 11 +/- 2% fibrinolysis. rt-PA, 0.2 mg/kg, 0.4 mg and 0.8 mg/kg produced 35 +/- 4%, 54 +/- 4% and 78 +/- 6% fibrinolysis, respectively. K2tu-PA, at the same doses, produced 39 +/- 5%, 57 +/- 6% and 83 +/- 6% fibrinolysis, respectively. Thus, no differences in the fibrinolytic activity of rt-PA and K2tu-PA were observed. Injection of saline was followed by an accretion of 56.4 +/- 5.9 micrograms of radioactive new fibrin on the thrombi. The injection of the three increasing doses of rt-PA was followed by an accretion of 54.9 +/- 5.3 micrograms, 49.1 +/- 6.1 micrograms and 47.2 +/- 4.8 micrograms.(ABSTRACT TRUNCATED AT 250 WORDS)

Properties of chimeric (tissue-type/urokinase-type) plasminogen activators obtained by fusion at the plasmin cleavage site.

Two hybrid plasminogen activators (K2tu-PA and FK2tu-PA), linking the kringle 2 domain or the finger plus the kringle 2 domains of tissue-type plasminogen activator (t-PA) to the catalytic domain of single-chain urokinase-type plasminogen activator (scu-PA) were studied. At variance with similar constructs previously reported, they were obtained by fusion of the t-PA and scu-PA derived portions at their plasmin cleavage site (between Arg275 of t-PA and Ile159 of scu-PA), thus eliminating from scu-PA the two peptide bonds (Glu143-Leu144 and Arg156-Phe157) that lead to low molecular weight scu-PA and to thrombin-inactivated tcu-PA. The specific activities of K2tu-PA and FK2tu-PA, as measured by fibrin plate were 2.5 x 10(6) and 1.0 x 10(6) t-PA equivalent units/mg, respectively. Activation of plasminogen by hybrid PAs was stimulated by both CNBr-digested fibrinogen (40- and 80-fold) and Des-A-fibrin monomers (6- and 12-fold). The relatively weak stimulation of chimeric PAs by minimally degraded fibrin monomers was consistent with their reduced fibrin binding capacity. Like scu-PA, the chimeric PAs, in the single-chain form, were insensitive to inhibition, as they retained full activity after prolonged incubation in plasma and did not interact with SDS-reactivated recombinant PAI-1. The concentration producing 50% lysis of blood clots in 3 h was 0.5 microgram/ml for K2tu-PA and 1 microgram/ml for FK2tu-PA, as compared to 0.5 microgram/ml and > 2 micrograms/ml for t-PA and scu-PA, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

A hybrid plasminogen activator binds to the u-PA receptor and has a reduced thrombolytic potency in vivo.

Fibrinolytic properties of four hybrids of u-PA and t-PA, all containing the u-PA growth factor domain and binding to recombinant human u-PA receptor expressed in CHO cells, were compared. Highest fibrin stimulation was observed with uK2tPA which when compared to t-PA in the rabbit system, had a considerably prolonged circulatory half-life in vivo. Compared to an equimolar dose of t-PA, 0.4 mg/kg uK2tPA caused a similar consumption of alpha 2-antiplasmin and fibrinogen and a considerably greater prolongation of the ex-vivo blood clotting time. Nevertheless, this dose of uK2tPA was inactive in the jugular vein thrombosis assay. This lack of thrombolytic activity is presumably due to the presence of a functional u-PA growth factor domain, which in binding uK2tPA to cellular blood elements possibly retards its penetration into the blood clot and in this manner could neutralize the potential thrombolytic activity of the t-PA kringle 2 and protease domains in uK2tPA.

Thrombolytic activity of two chimeric recombinant plasminogen activators (FK2tu-PA and K2tu-PA) in rabbits.

The aim of this study was to evaluate the thrombolytic activity of two hybrid plasminogen activators (HPAs) in a rabbit jugular vein thrombosis model. In the two HPAs the kringle-2 domain (K2tu-PA) or the finger and the kringle-2 domains (FK2tu-PA) of tissue-type plasminogen activator (t-PA) are linked to the catalytic protease domain of single chain urokinase type plasminogen activator (scu-PA). The two HPAs were compared with rt-PA and scu-PA on a weight/weight basis. K2tu-PA, FK2tu-PA, rt-PA and scu-PA were infused at doses of 0.4, 0.8 and 1.2 mg/kg over 3 h. Saline served as control. Saline produced 11 +/- 2% thrombolysis. The three doses of K2tu-PA led to 38 +/- 4%, 66 +/- 5% and 89 +/- 7% thrombolysis, respectively; the three doses of FK2tu-PA: 18 +/- 3%, 29 +/- 5% and 33 +/- 6%, respectively; the three doses of rt-PA 32 +/- 2%, 49 +/- 3% and 68 +/- 6%, respectively; the three doses of scu-PA 16 +/- 2%, 24 +/- 3% and 32 +/- 4%, respectively. K2tu-PA and rt-PA showed a statistically significant higher thrombolytic activity than FK2tu-PA and scu-PA at the three tested doses (p less than 0.01). The thrombolytic activity of K2tu-PA was significantly higher than rt-PA at the two higher doses (p less than 0.01). Both K2tu-PA and rt-PA produced a statistically significant reduction of fibrinogen, alpha 2-antiplasmin and plasminogen 3 h after the start of the infusions of the two higher doses.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of aging on the lipid order and composition of rat adipocyte ghosts.

An analysis of the cholesterol/phospholipid ratio of adipocyte ghosts from rat epididymal fat pads shows a significant increase with age (P less than 0.005). An attempt to correlate these changes with the order of the lipid matrix was made using the stearic acid spin label 2-(3-carboxypropyl)-4, 4-dimethyl-2-tridecyl-3-oxazolidinyloxyl [I(12,3)]. Although order was negatively correlated with temperature in preparations from both 6- and 24-month-old rats, no effect of age could be detected.