Assessment of histogenesis and proliferative potential in cytologic specimens of human brain tumors. Value of immunocytochemistry and nucleolar organizer regions.

The value of immunocytochemistry and nucleolar organizer regions (NORs) for the histogenetic identification and the estimation of the proliferative potential of brain tumors was assessed by the investigation of imprint smears of 51 neurosurgical tumor specimens. A panel of five monoclonal antibodies was used to cover a broad range of immunohistochemical markers. For the assessment of NORs, a silver staining technique (AgNOR) was used. NORs were enumerated and measured by means of an interactive image analysis system. The immunocytochemical results were similar for the smears and paraffin-embedded sections for 95.6% of the investigations performed and for 76.2% of the cases. Glial fibrillary acidic protein (GFAP) was positive in 9 of 17 tumors of glial origin, but was negative in 9 metastatic tumors. Vimentin was positive in 10 of 10 and fibronectin in 9 of 10 meningiomas investigated. The number of NORs increased steadily with the increasing grade of malignancy. Especially in glioblastomas, the number of NORs per cell exhibited a wide range, which might reflect the heterogeneity of these neoplasms. Metastases revealed a higher number of NORs per cell than did glioblastomas. In the cytologic differential diagnosis of these tumors, an absence of GFAP expression combined with a high NOR count is suggestive of a metastatic tumor.     

Evolution of a B2 tagged sequence from a long-range repeat family in the genus Mus

A long-range repeat family of more than 50 kb repeat size is clustered in Chromosomes (Chr) 1 of Mus musculus and M. spretus. In M. musculus this long-range repeat family shows considerable variation of copy-number frequency and contains coding regions for at least two genes. In an intron of a gene, which is part of the repeat, a B2 small interspersed repetitive element (SINE) is inserted at identical positions. The B2 element is present in all copies of the long-range repeat family; it was presumably a component of the ancestral single-copy precursor sequence that gave rise by amplification to the repeat family. Copies of the long-range repeat family vary with respect to the number of TAAA tandem repeats in the A-rich 3 end region of the B2 element. As inferred from polymerase chain reaction (PCR) data, presence and frequency of repeat number variants in the (TAAA)_n block are strain and species specific. The B2 element and its flanking regions were sequenced from two copies of the long-range repeat family. Sequence divergence between the two copies (only non-CG base substitutions and deletions/insertions) was determined to be 2.6%. Based on the drift rate in human Alu elements and a correction for the higher drift rates in rodents, and estimate for the divergence time of 1.7 million years was calculated. Since the long-range repeat family is present in M. musculus and M. spretus, it must have evolved by amplification before the separation of the two species about 1–4 million years ago.     

Mitogen Stimulation of Lymphocytes in Pigs with Hereditary Vitamin C Deficiency

Recently an inherited vitamin G deficiency in the pigs presumably based on an autosomal recessive gene was decribed* Homozygotes are in contrast to heterozygotes and normal pigs unable to synthesize ascorbic acid. In an experiment comprising 3 littermate pigs, 2 homozygous and 1 heterozygous for the vitamin C deficiency gene, the influence of ascorbic acid depletion, and repletion on mitogen stimulation of peripheral blood lymphocytes was studied. Ascorbic acid depletion of the vitamin C dependent pigs resulted in a rapid decline in plasma ascorbic acid. Response of lymphocytes to stimular tion with Concanavalin A (Con A) and phytohemagglutinin M (PHA) decreased more slowly reaching a minimum, which coincidedi with the occurrence of the first clinical symptoms of scurvy. Following resupplementation with vitamin C the plasma content of ascorbic acid rapidly returned to normal, while the lymphocyte response to Con A and PHA stimulation only gradually approached the initial values. The repletion with ascorbic acid caused a transitory increase in the response to pokeweed mitogen (PWM) stimulation. The significance of these findings in relation to the cellular immune system in normal pigs is discussed.     

Isolation and characterization of a 3-chlorobenzoate degrading pseudomonad

A pseudomonad has been isolated from sewage, which can utilize 3-chlorobenzoic acid as a sole carbon source. In cells grown on benzoate the enzymes of the -ketoadipic acid pathway are present. Considerable enzymic activities for chlorinated substrates were found in benzoate grown cells only for the oxygenation of 3-chlorobenzoate and the dehydrogenation of 3- and 5-chloro-3,5-cyclohexadiene-1,2-diol-1-carboxylic acid. 3-Chlorobenzoate grown cells show additional high activities for the turnover of 3- and 4-chlorocatechols and chloromuconic acids.Abbreviations Used DHB (-)-3,5-cyclohexadiene-1,2-diol-1-carboxylic acid (derived from the trivial name, dihydrodihydroxybenzoate) - 3- and 5-Cl-DHB correspondingly 3- and 5-chloro-3,5-cyclohexadiene-1,2-diol-1-carboxylic acid     

PocketOptimizer 2.0: A modular framework for computer-aided ligand-binding design

The ability to design customized proteins to perform specific tasks is of great interest. We are particularly interested in the design of sensitive and specific small molecule ligand-binding proteins for biotechnological or biomedical applications. Computational methods can narrow down the immense combinatorial space to find the best solution and thus provide starting points for experimental procedures. However, success rates strongly depend on accurate modeling and energetic evaluation. Not only intra- but also intermolecular interactions have to be considered. To address this problem, we developed PocketOptimizer, a modular computational protein design pipeline, that predicts mutations in the binding pockets of proteins to increase affinity for a specific ligand. Its modularity enables users to compare different combinations of force fields, rotamer libraries, and scoring functions. Here, we present a much-improved version––PocketOptimizer 2.0. We implemented a cleaner user interface, an extended architecture with more supported tools, such as force fields and scoring functions, a backbone-dependent rotamer library, as well as different improvements in the underlying algorithms. Version 2.0 was tested against a benchmark of design cases and assessed in comparison to the first version. Our results show how newly implemented features such as the new rotamer library can lead to improved prediction accuracy. Therefore, we believe that PocketOptimizer 2.0, with its many new and improved functionalities, provides a robust and versatile environment for the design of small molecule-binding pockets in proteins. It is widely applicable and extendible due to its modular framework. PocketOptimizer 2.0 can be downloaded at https://github.com/Hoecker-Lab/pocketoptimizer .     

Domain Evolution in the α-Amylase Family

The available amino acid sequences of the α-amylase family (glycosyl hydrolase family 13) were searched to identify their domain B, a distinct domain that protrudes from the regular catalytic (β/α)₈-barrel between the strand β3 and the helix α3. The isolated domain B sequences were inspected visually and also analyzed by Hydrophobic Cluster Analysis (HCA) to find common features. Sequence analyses and inspection of the few available three-dimensional structures suggest that the secondary structure of domain B varies with the enzyme specificity. Domain B in these different forms, however, may still have evolved from a common ancestor. The largest number of different specificities was found in the group with structural similarity to domain B from Bacillus cereus oligo-1,6-glucosidase that contains an α-helix succeeded by a three-stranded antiparallel β-sheet. These enzymes are α-glucosidase, cyclomaltodextrinase, dextran glucosidase, trehalose-6-phosphate hydrolase, neopullulanase, and a few α-amylases. Domain B of this type was observed also in some mammalian proteins involved in the transport of amino acids. These proteins show remarkable similarity with (β/α)₈-barrel elements throughout the entire sequence of enzymes from the oligo-1,6-glucosidase group. The transport proteins, in turn, resemble the animal 4F2 heavy-chain cell surface antigens, for which the sequences either lack domain B or contain only parts thereof. The similarities are compiled to indicate a possible route of domain evolution in the α-amylase family. Received: 4 December 1996 / Accepted: 13 March 1997     

Degradation of chlorophenols by a defined mixed microbial community

Synthetic sewage containing phenol, acetone, and alkanols plus 4-chlorophenol or a mixture of isomeric chlorophenols is completely degraded by a defined mixed culture with Pseudomonas sp. strain B13 as a chlorocatechol-dissimilating member of the community. Total degradation of the organic carbon was indicated by release of stoichiometric amounts of chloride and low content of dissolved organic carbon in the cell-free effluents. During adaptation to high loads of chlorophenols the initial meta-cleavage activity was completely replaced by ortho-cleavage activity of type I and II. In the fully acclimated culture, hybrid strains such as Alcaligenes sp. strain A7-2 were detected, which are more competitive than Pseudomonas sp. strain B13 with respect to chlorophenol degradation.     

Critical Reactions in Fluorobenzoic Acid Degradation by Pseudomonas sp. B13

3-Chlorobenzoate-grown cells of Pseudomonas sp. B13 readily cometabolized monofluorobenzoates. A catabolic pathway for the isomeric fluorobenzoates is proposed on the basis of key metabolites isolated. Only 4-fluorobenzoate was utilized and totally degraded after a short period of adaptation. The isoenzymes for total degradation of chlorocatechols, being found during growth with 3-chlorobenzoate or 4-chlorophenol, were not induced in the presence of fluorobenzoates. Correspondingly, only the ordinary enzymes of the benzoate pathway were detected in 4-fluorobenzoate-grown cells. Ring cleavage of 3-fluorocatechol was recognized as a critical step in 3-fluorobenzoate degradation. 2-Fluoro-cis,cis-muconic acid was identified as a dead-end metabolite from 2- and 3-fluorobenzoate catabolism. During 2-fluorobenzoate cometabolism, fluoride is eliminated by the initial dioxygenation.