500-MHz 1H-NMR studies of ribosomal proteins isolated from 70-S ribosomes of Escherichia coli

A method for the large-scale isolation of ribosomal proteins is described avoiding pre-separation of 30-S and 50-S subunits. Five proteins isolated in this way were studied with high-resolution 1H NMR at 500 MHz. These are S21, L18, L25, L30 and L33. The results show that L18, L25 and L30 exhibit tertiary structure in solution and indications for secondary structure in S21 are found. Protein L33 appears to be a random coil. Several resonances in the 1H NMR spectra are assigned to particular protons of amino acid residues, e.g. the aromatic ring protons of tyrosines and histidines, and epsilon-protons of lysines.     

Regulation of adenylate cyclase in electropermeabilized Dictyostelium discoideum cells.

In Dictyostelium discoideum cells the enzyme adenylate cyclase is functionally coupled to cell surface receptors for cAMP. Coupling is known to involve one or more G-proteins. Receptor-mediated activation of adenylate cyclase is subject to adaptation. In this study we employ an electropermeabilized cell system to investigate regulation of D. discoideum adenylate cyclase. Conditions for selective permeabilization of the plasma membrane have been described by C.D. Schoen, J. C. Arents, T. Bruin, and R. Van Driel (1989, Exp. Cell Res. 181, 51-62). Only small pores are created in the membrane, allowing exchange of exclusively low molecular weight substances like nucleotides, and preventing the loss of macromolecules. Under these conditions functional protein-protein interactions are likely to remain intact. Adenylate cyclase in permeabilized cells was activated by the cAMP receptor agonist 2'-deoxy cAMP and by the nonhydrolyzable GTP-analogue GTP gamma S, which activates G-proteins. The time course of the adenylate cyclase reaction in permeabilized cells was similar to that of intact cells. Maximal adenylate cyclase activity was observed if cAMP receptor agonist or GTP-analogue was added just before cell permeabilization. If these activators were added after permeabilization adenylate cyclase was stimulated in a suboptimal way. The sensitivity of adenylate cyclase activity for receptor occupation was found to decay more rapidly than that for G-protein activation. Importantly, the adenylate cyclase reaction in permeabilized cells was subject to an adaptation-like process that was characterized by a time course similar to adaptation in vivo. In vitro adaptation was not affected by cAMP receptor agonists or by G-protein activation. Evidently electropermeabilized cells constitute an excellent system for investigating the positive and negative regulation of D. discoideum adenylate cyclase.     

A missense mutation Pro157 Arg in lipoprotein lipase (LPLNijmegen) resulting in loss of catalytic activity.

Here we report on the molecular defect that leads to a deficiency of lipoprotein lipase (LPL) activity in a proband of Dutch descent. Southern-blot analysis of the LPL gene from the patient did not reveal any major DNA rearrangements. Sequencing of polymerase-chain-reaction-amplified DNA revealed that the proband is a homozygote for G725C, resulting in a substitution of Pro157 for Arg. This substitution alters a restriction site for PvuII, which allowed rapid identification of the mutant allele in family members. Site-directed mutagenesis and transient expression of the mutant LPL in COS cells produced an enzymatically inactive protein, establishing the functional significance of this mutation. This naturally occurring mutation which alters the Pro157 adjacent to Asp156 of the proposed catalytic triad, indicates that this region of the protein is indeed crucial for LPL catalytic activity.     

Biodegradation of alpha- and beta-hexachlorocyclohexane in a soil slurry under different redox conditions

Aerobic conditions proved to be best for the microbiol conversion of alpha-hexachlorocyclohexane (alpha-HCH) in a soil slurry. The dry soil contained 400 mg of alpha-HCH per kg. This xenobiotic compound was mineralized within about 18 days at an initial rate of 23 mg/kg of soil per day by the mixed native microbial population of the soil. The only intermediate that was detected during breakdown was pentachlorocyclohexene, which was detected at very small concentrations. Alpha-HCH was also bioconverted under methanogenic conditions. However, a rather long acclimation period (about 30 days) was necessary before degradation started, at a rate of 13 mg/kg of soil per day. Mass balance calculations showed that about 85% of the initial alpha-HCH that was present was converted to monochlorobenzene, 3,5-dichlorophenol, and a trichlorophenol isomer, possibly 2,4,5-trichlorophenol. Under both denitrifying and sulfate-reducing conditions, no significant bioconversion of alpha-HCH was observed. The beta isomer of HCH was recalcitrant at all of the four redox conditions studied. We propose that the specific spatial chloride arrangement of the beta isomer is responsible for its stability. The results reported here with complex soil slurry systems showed that alpha-HCH is, in contrast to the existing data in the literature, best degraded biologically in the presence of oxygen.     

Disappearance of factor VIII antibodies upon frequent administration of factor VIII.

20 haemophilia patients known to have antibodies against F VIII for at least more than three years were treated on a regular base with 25 U/kg b.w. F VIII every other day. All 5 patients with previous maximal anti F VIII antibody levels between 5 and 60 BU/ml showed a decrease of antibody level and normal F VIII recovery within 1-2 months. From 12 patients with previous antibody levels above 60 BU/ml, 8 showed a disappearance of antibodies within 2-26 months. In 3 patients in whom no previous highest inhibitor level was known, one was treated successfully. Another group of 6 young patients in whom an inhibitor against F VIII had just (less than 3 months) developed, was treated with F VIII as soon as an inhibitor was detected. The dose infused was 25 U/kg b.w. F VIII twice weekly. In 5 patients this regimen was successful within 1-7 months. In the 6th patient the dosage was increased to every other day. One year after the beginning of therapy no inhibitor was detectable. So our results show that regular administration of F VIII in intermediate or low dose can lead to rapid disappearance of anti F VIII antibodies especially in patients with moderate inhibitor levels.     

Forced prey-predator oscillations

In this paper existence and stability of subharmonic solutions of the Volterra-Verhulst equations with a periodic coefficient are analyzed by the method of Urabe. The study supports the view that the observed 4- and 10-yr cycles of prey-predator systems are due to seasonal fluctuations.AMS(MOS) classification: 92A15, 34C25, 34A50     

En‐face optical coherence tomography for the detection of cancer in prostatectomy specimens: Quantitative analysis in 20 patients

The increase histopathological evaluation of prostatectomy specimens rises the workload on pathologists. Automated histopathology systems, preferably directly on unstained specimens, would accelerate the pathology workflow. In this study, we investigate the potential of quantitative analysis of optical coherence tomography (OCT) to separate benign from malignant prostate tissue automatically. Twenty fixated prostates were cut, from which 54 slices were scanned by OCT. Quantitative OCT metrics (attenuation coefficient, residue, goodness‐of‐fit) were compared for different tissue types, annotated on the histology slides. To avoid misclassification, the poor‐quality slides, and edges of annotations were excluded. Accurate registration of OCT data with histology was achieved in 31 slices. After removing outliers, 56% of the OCT data was compared with histopathology. The quantitative data could not separate malignant from benign tissue. Logistic regression resulted in malignant detection with a sensitivity of 0.80 and a specificity of 0.34. Quantitative OCT analysis should be improved before clinical use.     

Association of plasma lipids and apolipoproteins with the insulin response element in the apoC-III promoter region in familial combined hyperlipidemia.

The apoAI-CIII-AIV gene cluster, located on chromosome 11, contributes to the phenotype of familial combined hyperlipidemia (FCH), but this contribution is genetically complex. Combinations of haplotypes, based on three restriction enzyme polymorphisms: XmnI and MspI sites, 5' of the start site of the apoA-I gene and SstI polymorphism in the 3' untranslated region of exon 4 of the apoC-III gene, were analyzed to characterize their effect on the expression of severe hyperlipidemia. An epistatic interaction was demonstrated: the S2 allele on one haplotype was synergistic in its hyperlipidemic effect to the X2M2 allele on the other haplotype (Dallinga-Thie, G. M. et al. J. Clin. Invest. 1997. 99: 953-961). In the present study two additional polymorphic sites in the insulin response element (IRE) of the apoC-III gene promoter, T-455C: FokI restriction site, C-482T: MspI restriction site, were studied in 34 FCH pedigrees including 34 probands, 220 hyperlipidemic relatives, 300 normolipidemic relatives, and 236 spouses. In contrast to the earlier data for the other polymorphisms in this gene cluster (XmnI, MspI/AI, and SstI), there were no differences in frequency distributions of the T-455C and the C-482T variants between probands, hyperlipidemic and normolipidemic relatives and spouses. No significant associations between plasma lipid traits and DNA variants in the IRE were observed. Analysis of combinations of haplotypes based on the five polymorphisms in the gene cluster provided further evidence for a dominant role of the SstI polymorphism as a major susceptibility locus in FCH. The inclusion of the IRE markers did not improve genetic informativeness, nor our understanding of the observed synergistic relationship associated with the high risk combination of haplotypes in FCH families.     

Book Review Essay

Book reviewed in this article: Louis Dumont. German Ideology: From France to Germany and Back.