Radiation-induced chromosome aberrations: insights gained from biophysical modeling

Enzymatic misrepair of ionizing-radiation-induced DNA damage can produce large-scale rearrangements of the genome, such as translocations and dicentrics. These and other chromosome exchange aberrations can cause major phenotypic alterations, including cell death, mutation and neoplasia. Exchange formation requires that two (or more) genomic loci come together spatially. Consequently, the surprisingly rich aberration spectra uncovered by recently developed techniques, when combined with biophysically based computer modeling, help characterize large-scale chromatin architecture in the interphase nucleus. Most results are consistent with a picture whereby chromosomes are mainly confined to territories, chromatin motion is limited, and interchromosomal interactions involve mainly territory surfaces. Aberration spectra and modeling also help characterize DNA repair/misrepair mechanisms. Quantitative results for mammalian cells are best described by a breakage-and-reunion model, suggesting that the dominant recombinational mechanism during the G(0)/G(1) phase of the cell cycle is non-homologous end-joining of radiogenic DNA double strand breaks. In turn, better mechanistic and quantitative understanding of aberration formation gives new insights into health-related applications.     

The non-ionic detergent Brij 58P mimics chaperone effects

The non-ionic detergent Brij 58P is recommended as a stabilizing agent for protein storage; for example, the aggregation-prone chaperone DnaJ can be maintained in solution by low concentrations of Brij 58P. During protein folding studies with alpha-glucosidase, rhodanese and citrate synthase as model proteins, we discovered that the low concentrations of Brij 58P usually added with purified DnaJ to renaturation samples are sufficient to mimic chaperone effects with respect to prevention of protein aggregation. Furthermore, addition of Brij 58P to refolding alpha-glucosidase and citrate synthase enhanced the yield of refolded protein by a factor of two. Thus, Brij 58P can mimic chaperone effects and care should be taken when the substance is used to stabilize chaperone preparations.     

Analysis of protein-carbohydrate interaction at the lower size limit of the protein part (15-mer peptide) by NMR spectroscopy,electrospray ionization mass spectrometry,and molecular modeling

Structural analysis of minimally sized lectins will offer insights into fundamentals of intermolecular recognition and potential for biomedical applications. We thus moved significantly beyond the natural limit of lectin size to determine the structure of synthetic mini-lectins in solution, their carbohydrate selectivity and the impact of ligand binding on their conformational behavior. Using three disaccharide (Thomsen-Friedenreich antigen; Gal beta 1,3GalNAc alpha 1,R)-binding pentadecapeptides without internal disulfide bridges as role models, we successfully tested a combined strategy with different techniques of NMR spectroscopy, electrospray ionization mass spectrometry, and molecular modeling. In solution, the peptides invariably displayed flexibility with rather limited restrictions, shown by NMR experiments including nearly complete resonance assignments and molecular dynamics simulations. The occurrence of aromatic/nonpolar amino acids in the sequence did not lead to formation of a hydrophobic core known from microbial chitinase modules. Selectivity of disaccharide binding was independently observed by mass spectrometry and NMR analysis. Specific ligand interaction yielded characteristic NMR signal alterations but failed to reduce conformational flexibility significantly. We have thereby proven effectiveness of our approach to analyze even low-affinity interactions (not restricted to carbohydrates as ligands). It will be useful to evaluate the impact of rational manipulation of lead peptide sequences.     

Monocyte-expressed urokinase regulates human vascular smooth muscle cell migration in a coculture model

Interactions of vascular smooth muscle cells (VSMC) with monocytes recruited to the arterial wall at a site of injury, with resultant modulation of VSMC growth and migration, are central to the development of vascular intimal thickening. Urokinase-type plasminogen activator (uPA) expressed by monocytes is a potent chemotactic factor for VSMC and might serve for the acceleration of vascular remodeling. In this report, we demonstrate that coculture of human VSMC with freshly isolated peripheral blood-derived human monocytes results in significant VSMC migration that increases during the coculture period. Accordingly, VSMC adhesion was inhibited with similar kinetics. VSMC proliferation, however, was not affected and remained at the same basal level during the whole period of coculture. The increase of VSMC migration in coculture was equivalent to the uPA-induced migration of monocultured VSMC and was blocked by addition into coculture of soluble uPAR (suPAR). Analysis of uPA and uPAR expression in cocultured cells demonstrated that monocytes are a major source of uPA, whose expression increases in coculture five-fold, whereas VSMC display an increased expression of cell surface-associated uPAR. These findings indicate that upregulated uPA production by monocytes following vascular injury acts most likely as an endogenous activator of VSMC migration contributing to the remodeling of vessel walls.     

The hyaluronan-binding serine protease from human plasma cleaves HMW and LMW kininogen and releases bradykinin

The influence of the hyaluronan-binding protease (PHBSP), a plasma enzyme with FVII- and pro-urokinase-activating potency, on components of the contact phase (kallikrein/kinin) system was investigated. No activation or cleavage of the proenzymes involved in the contact phase system was observed. The pro-cofactor high molecular weight kininogen (HK), however, was cleaved in vitro by PHBSP in the absence of any charged surface, releasing the activated cofactor and the vasoactive nonapeptide bradykinin. Glycosoaminoglycans strongly enhanced the reaction. The cleavage was comparable to that of plasma kallikrein, but clearly different from that of coagulation factor FXIa. Upon extended incubation with PHBSP, the light chain was further processed, partially removing about 60 amino acid residues from the N-terminus of domain D5 of the light chain. These cleavage site(s) were distinct from plasma kallikrein or FXIa cleavage sites. PHBSP and, more interestingly, also plasma kallikrein could cleave low molecular weight kininogen in vitro, indicating that domains D5H and D6H are no prerequisite for kininogen cleavage. PHBSP was also able to release bradykinin from HK in plasma where the pro-cofactor circulates predominantly in complex with plasma kallikrein or FXI. In conclusion, PHBSP represents a novel kininogen-cleaving and bradykinin-releasing enzyme in plasma that shares significant catalytic similarities with plasma kallikrein. Since they are structurally unrelated in their heavy chains (propeptide), their similar in vivo catalytic activities might be directed at distinct sites where PHBSP could induce processes that are related to the kallikrein/kinin system.     

The genome of Methanosarcina mazei: evidence for lateral gene transfer between bacteria and archaea

The Archaeon Methanosarcina mazei and related species are of great ecological importance as they are the only organisms fermenting acetate, methylamines and methanol to methane, carbon dioxide and ammonia (in case of methylamines). Since acetate is the precursor of 60% of the methane produced on earth these organisms contribute significantly to the production of this greenhouse gas, e.g. in rice paddies. The 4,096,345 base pairs circular chromosome of M. mazei is more than twice as large as the genomes of the methanogenic Archaea currently completely sequenced (Bult et al., 1996; Smith et al., 1997). 3,371 open reading frames (ORFs) were identified. Based on currently available sequence data 376 of these ORFs are Methanosarcina-specific and 1,043 ORFs find their closest homologue in the bacterial domain. 544 of these ORFs reach significant similarity values only in the bacterial domain. They include 56 of the 102 transposases, and proteins involved in gluconeogenesis, proline biosynthesis, transport processes, DNA-repair, environmental sensing, gene regulation, and stress response. Striking examples are the occurrence of the bacterial GroEL/GroES chaperone system and the presence of tetrahydrofolate-dependent enzymes. These findings might indicate that lateral gene transfer has played an important evolutionary role in forging the physiology of this metabolically versatile methanogen.     

Chromosome-induced microtubule assembly mediated by TPX2 is required for spindle formation in HeLa cells

In Xenopus laevis egg extracts, TPX2 is required for the Ran-GTP-dependent assembly of microtubules around chromosomes. Here we show that interfering with the function of the human homologue of TPX2 in HeLa cells causes defects in microtubule organization during mitosis. Suppressing the expression of human TPX2 by RNA interference leads to the formation of two microtubule asters that do not interact and do not form a spindle. Our results suggest that in vivo, even in the presence of duplicated centrosomes, spindle formation requires the function of TPX2 to generate a stable bipolar spindle with overlapping antiparallel microtubule arrays. This indicates that chromosome-induced microtubule production is a general requirement for the formation of functional spindles in animal cells.     

Structural determinants of plant lignans for the formation of enterolactone in vivo

The quantity of mammalian lignans enterolactone (ENL) and enterodiol (END) and of plant lignans secoisolariciresinol (SECO) and 7-hydroxymatairesinol (HMR) excreted in a 24-h rat urine sample was measured after a single p.o. dose of an equivalent quantity of secoisolariciresinol diglycoside (SDG), secoisolariciresinol (SECO), matairesinol (MR), 7-hydroxymatairesinol (HMR) and ENL. Plant lignans (SECO and HMR) were partially absorbed as such. The aglycone form of SECO was more efficiently converted into mammalian lignans END and ENL than the glycosylated form, SDG. Of plant lignans, MR produced the highest quantities of ENL: the quantity was over twofold compared with HMR or SDG. The majority of the animals, which had been given SECO, excreted higher quantities of END than ENL into urine, but ENL was the main lignan metabolite after SDG. The highest quantities of ENL in urine were measured after the administration of ENL as such. The (-)SECO isolated from Araucaria angustifolia was converted into (-)ENL only. The administration of (-)SDG, which was shown to produce (+)SECO, resulted in excretion of (+)ENL only and (-)HMR was converted into (-)ENL only. This confirmed that the absolute configurations at C8 and C8' are not changed during the microbial metabolism. Whether the biological effects are enantiomer-specific, remains to be resolved.     

Isolation and Characterization of Two Forms of an Acidic Bromelain Stem Proteinase

Two forms of an acidic bromelain proteinase isolated from crude bromelain, an extract from pineapple stem, were found by a two-step FPLC purification procedure. The basic main components were removed by cation exchange chromatography and the breakthrough fraction was further resolved by anion exchange chromatography into 15 protein fractions, only two of which, called SBA/a and SBA/b, were proteolytically active. These components were characterized by electrospray mass spectroscopy (ESMS), isoelectric focusing, N-terminal amino acid sequence analysis, monosaccharide analysis, and enzymatic parameters. The molecular masses of SBA/a and SBA/b were determined by ESMS to be 23,550 and 23,560, respectively. The isoelectric points (pI) of the two bands of SBA/a were 4.8 and 4.9; SBA/b focused as a single band at pI = 4.8. Partial N-terminal amino acid sequences (11 residues) were identical to SBA/a and SBA/b and identical with those of stem bromelain, the basic main proteinase of the pineapple stem, and fruit bromelain, the acidic main proteinase of the pineapple fruit. Both components are highly glycosylated; hydrolysis of SBA/a yielded about twofold more monosaccharide per protein than SBA/b. The comparison of the catalytic properties of SBA/a with those of SBA/b revealed no relevant differences in the hydrolysis of three peptidyl-NH-Mec substrates and in the inhibition profiles using chicken cystatin and E-64, indicating that these components can be considered as two forms of a single enzyme. Both forms are scarcely inhibited by chicken cystatin and slowly inactivated by E-64, hence are nontypical cysteine proteinases of the papain superfamily.     

Structure and dynamics of human interphase chromosome territories in vivo

A new approach is presented which allows the in vivo visualization of individual chromosome territories in the nuclei of living human cells. The fluorescent thymidine analog Cy3-AP3-dUTP was microinjected into the nuclei of cultured human cells, such as human diploid fibroblasts, HeLa cells and neuroblastoma cells. The fluorescent analog was incorporated during S-phase into the replicating genomic DNA. Labelled cells were further cultivated for several cell cycles in normal medium. This well-known scheme yielded sister chromatid labelling. Random segregation of labelled and unlabelled chromatids into daughter nuclei resulted in nuclei exhibiting individual in vivo detectable chromatid territories. The territories were composed of subcompartments with diameters ranging between approximately 400 and 800 nm which we refer to as subchromosomal foci. Time-resolved in vivo studies demonstrated changes of positioning and shape of territories and subchromosomal foci. The hypothesis that subchromosomal foci persist as functionally distinct entities was supported by double labelling of chromatin with CldU and IdU, respectively, at early and late S-phase and subsequent cultivation of corresponding cells for 5–10 cell cycles before fixation and immunocytochemical detection. This scheme yielded segregated chromatid territories with distinctly separated subchromosomal foci composed of either early- or late-replicating chromatin. The size range of subchromosomal foci was similar after shorter (2 h) and longer (16 h) labelling periods and was observed in nuclei of both living and fixed cells, suggesting their structural identity. A possible functional relevance of chromosome territory compartmentalization into subchromosomal foci is discussed in the context of present models of interphase chromosome and nuclear architecture. Received: 10 November 1997 / Accepted: 24 November 1997