Use of191Pt radiotracer for the development of enrichment procedures to detect natural levels of platinum in biological and environmental materials

Biological Trace Element Research - The191Pt-radiotracer is a powerful tool to develop separation and preconcentration methods for Pt. The radiotracer was produced either through190Pt...     

The crystal structure of dihydrofolate reductase from Thermotoga maritima: molecular features of thermostability

Two high-resolution structures have been obtained for dihydrofolate reductase from the hyperthermophilic bacterium Thermotoga maritima in its unliganded state, and in its ternary complex with the cofactor NADPH and the inhibitor, methotrexate. While the overall fold of the hyperthermophilic enzyme is closely similar to monomeric mesophilic dihydrofolate reductase molecules, its quaternary structure is exceptional, in that T. maritima dihydrofolate reductase forms a highly stable homodimer. Here, the molecular reasons for the high intrinsic stability of the enzyme are elaborated and put in context with the available data on the physical parameters governing the folding reaction. The molecule is extremely rigid, even with respect to structural changes during substrate binding and turnover. Subunit cooperativity can be excluded from structural and biochemical data. Major contributions to the high intrinsic stability of the enzyme result from the formation of the dimer. Within the monomer, only subtle stabilizing interactions are detectable, without clear evidence for any of the typical increments of thermal stabilization commonly reported for hyperthermophilic proteins. The docking of the subunits is optimized with respect to high packing density in the dimer interface, additional salt-bridges and beta-sheets. The enzyme does not show significant structural changes upon binding its coenzyme, NADPH, and the inhibitor, methotrexate. The active-site loop, which is known to play an important role in catalysis in mesophilic dihydrofolate reductase molecules, is rearranged, participating in the association of the subunits; it no longer participates in catalysis.     

Non-linear viscoelastic behavior of abdominal aortic aneurysm thrombus

The objective of this work was to determine the linear and non-linear viscoelastic behavior of abdominal aortic aneurysm thrombus and to study the changes in mechanical properties throughout the thickness of the thrombus. Samples are gathered from thrombi of seven patients. Linear viscoelastic data from oscillatory shear experiments show that the change of properties throughout the thrombus is different for each thrombus. Furthermore the variations found within one thrombus are of the same order of magnitude as the variation between patients. To study the non-linear regime, stress relaxation experiments are performed. To describe the phenomena observed experimentally, a non-linear multimode model is presented. The parameters for this model are obtained by fitting this model successfully to the experiments. The model cannot only describe the average stress response for all thrombus samples but also the highest and lowest stress responses. To determine the influence on the wall stress of the behavior observed the model proposed needs to implemented in the finite element wall stress analysis.     

Nucleotide sequence analysis of the CT operon of the Vibrio cholerae classical strain 569B

The complete nucleotide sequence of the Vibrio cholerae classical strain 569B was determined. The results prove the exactness of the amino acid CT B sequence published by Takao et al. (1985, Eur. J. Biochem. 146, 503-508). A comparison is made with already reported CT genes.     

The primer specificity of cytoplasmic poly(A) polymerase from cryptobiotic gastrulae of Artemia salina

Poly(A) polymerase has been purified to near homogeneity from the cytoplasm of Artemia salina as described previously (Roggen, E and Slegers, H. (1985) Eur. J. Biochem. 147, 225–232). Affinity chromatography on poly(A)-Sepharose 4B separates the enzyme preparation into two fractions. In standard assay conditions poly(A) polymerase fraction I (poly(A)-Sepharose 4B unbound) and fraction II (poly(A)-Sepharose 4B bound) have specific activities of 2.4 and 8.0 μmol AMP/h per mg enzyme, respectively. Poly(A) polymerase fraction II shows a high primer specificity towards the 17 S poly(A)-containing mRNP. Depending on the reaction conditions used, poly(A) sequences of 140 ± 15 AMP residues/μg enzyme are synthesized on the latter primer. In contrast, poly(A) polymerase fraction I only elongates oligo(A) primers efficiently. An endogenous RNA is detected in poly(A) polymerase II preparations. This RNA has a length of 83 ± 2 nucleotides and is a component of a 60 kDa particle. After removal of the latter the specificity of poly(A) polymerase fraction II for the 17 S poly(A)-containing mRNP is abolished and the characteristics of the enzyme resemble those of poly(A) polymerase I.     

Inheritance of the S113L mutation within an inbred family with carnitine palmitoyltransferase enzyme deficiency

Deficiency of carnitine palmitoyltransferase type II (CPT II) is a clinically heterogeneous autosomal recessive disorder of lipid metabolism. The most common mutation in the CPT 11 gene is the S113L mutation, which substitutes leucine for serine at amino acid position 113. We studied an inbred family with three affected cousins with CPT II deficiency and found the S113L mutation to be present in a homozygous state in all three patients. Pedigree analysis traced the S 113L mutation back to one common ancestor. Although the patients in this family have an identical genotype at the CPT II locus, their clinical picture ranges from asymptomatic to lethal.     

Sequences of the 5S rRNAs of the thermo-acidophilic archaebacterium Sulfolobus solfataricus (Caldariella acidophila) and the thermophilic eubacteria Bacillus acidocaldarius and Thermus aquaticus.

We have determined the nucleotide sequences of the 5 S rRNAs of three thermophilic bacteria: the archaebacterium Sulfolobus solfataricus, also named Caldariella acidophila, and the eubacteria Bacillus acidocaldarius and Thermus aquaticus. A 5 S RNA sequence for the latter species had already been published, but it looked suspect on the basis of its alignment with other 5 S RNA sequences and its base-pairing pattern. The corrected sequence aligns much better and fits in the universal five helix secondary structure model, as do the sequences for the two other examined species. The sequence found for Sulfolobus solfataricus is identical to that determined by others for Sulfolobus acidocaldarius. The secondary structure of its 5 S RNA shows a number of exceptional features which distinguish it not only from eubacterial and eukaryotic 5 S RNAs, but also from the limited number of archaebacterial 5 S RNA structures hitherto published. The free energy change of secondary structure formation is large in the three examined 5 S RNAs.     

The response of <Emphasis Type="Italic">Cupriavidus metallidurans</Emphasis> CH34 to spaceflight in the international space station

The survival and behavior of Cupriavidus metallidurans strain CH34 were tested in space. In three spaceflight experiments, during three separate visits to the ‘International Space Station’ (ISS), strain CH34 was grown for 10–12 days at ambient temperature on mineral agar medium. Space- and earth-grown cells were compared post-flight by flow cytometry and using 2D-gel protein analysis. Pre-, in- and post-flight incubation conditions and experiment design had a significant impact on the survival and growth of CH34 in space. In the CH34 cells returning from spaceflight, 16 proteins were identified which were present in higher concentration in cells developed in spaceflight conditions. These proteins were involved in a specific response of CH34 to carbon limitation and oxidative stress, and included an acetone carboxylase subunit, fructose biphosphate aldolase, a DNA protection during starvation protein, chaperone protein, universal stress protein, and alkyl hydroperoxide reductase. The reproducible observation of the over-expression of these same proteins in multiple flight experiments, indicated that the CH34 cells could experience a substrate limitation and oxidative stress in spaceflight where cells and substrates are exposed to lower levels of gravity and higher doses of ionizing radiation. Bacterium C. metallidurans CH34 was able to grow normally under spaceflight conditions with very minor to no effects on cell physiology, but nevertheless specifically altered the expression of a few proteins in response to the environmental changes.     

Fractionation of vanadium complexes in serum, packed cells and tissues of Wistar rats by means of gel filtration and anion-exchange chromatography

Male Wistar rats were intraperitoneally injected with [(48)V]vanadium tracer to (1) investigate the distribution of vanadium over different tissues and (2) study the distribution of vanadium over the proteins and peptides in serum, packed cells and homogenates of tissues by means of liquid chromatography experiments (size exclusion, ion exchange). Target organs were primarily kidney, bone, spleen and liver. In serum we found that vanadium was mainly bound to transferrin; however, a small amount was also bound to albumin. Besides these two complexes, a significant part of vanadium occurred as readily exchangeable ("free") vanadium. In packed cells, vanadium is mainly bound to hemoglobin and to two abundant low molecular mass complexes. The chromatograms of tissues (kidney, liver, testes, spleen and lung) show similar high molecular mass complexes (vanadium co-elutes with ferritin, transferrin and hemoglobin). Between the low molecular mass complexes there are similar peaks for spleen, testes and kidneys on the one hand, and liver and lung on the other hand, albeit the differences are small. In the case of lung, there is an additional low molecular mass peak.     

RNAA as compared to ICP-MS for the analysis of normal human serum

The merits of radiochemical neutron activation analysis (RNAA) and inductively coupled plasma mass spectrometry (ICP-MS) are critically discussed for the determination of trace and ultratrace elements in normal human serum. For RNAA, two semiautomated procedures, allowing the determination of up to 18 elements, are briefly described. ICP-MS has a series of interesting features for the determination of trace elements. Matrix and spectral interferences can mostly be avoided or corrected for. After a simple 5- or 10-fold dilution and addition of an internal standard, more than 20 elements can be measured precisely and accurately.