More hydrogen bonds for the (structural) biologist.

Why does a given protein structure form and why is this structure stable? These fundamental biochemical questions remain fascinating and challenging problems because the physical bases of the forces that govern protein structure, stability and folding are still not well understood. Now, a general concept of hydrogen bonding in proteins is emerging. This concept involves not only N-H and O-H donor groups, but also C-H, and not only N and O as acceptor groups, but also pi-systems. We postulate that the incorporation of the entirety of these interactions leads to a more complete description of the problem, and that this could provide new perspectives and possibly new answers.

Comparative analysis of the Borrelia garinii genome.

Three members of the genus Borrelia (B.burgdorferi, B.garinii, B.afzelii) cause tick-borne borreliosis. Depending on the Borrelia species involved, the borreliosis differs in its clinical symptoms. Comparative genomics opens up a way to elucidate the underlying differences in Borrelia species. We analysed a low redundancy whole-genome shotgun (WGS) assembly of a B.garinii strain isolated from a patient with neuroborreliosis in comparison to the B.burgdorferi genome. This analysis reveals that most of the chromosome is conserved (92.7% identity on DNA as well as on amino acid level) in the two species, and no chromosomal rearrangement or larger insertions/deletions could be observed. Furthermore, two collinear plasmids (lp54 and cp26) seem to belong to the basic genome inventory of Borrelia species. These three collinear parts of the Borrelia genome encode 861 genes, which are orthologous in the two species examined. The majority of the genetic information of the other plasmids of B.burgdorferii is also present in B.garinii although orthology is not easy to define due to a high redundancy of the plasmid fraction. Yet, we did not find counterparts of the B.burgdorferi plasmids lp36 and lp38 or their respective gene repertoire in the B.garinii genome. Thus, phenotypic differences between the two species could be attributable to the presence or absence of these two plasmids as well as to the potentially positively selected genes.

Cross-link formation of phage lambda DNA in situ photochemically induced by the furocoumarin derivative angelicin.

The combined action of 365 nm ultraviolet light and xanthotoxin or angelicin inhibits the injection of phage lambda into the host. For both furocoumarin derivatives the inhibition of injection is discussed in terms of photochemically induced cross-linking of the DNA inside the phage heads; Cross-linking of DNA has previously been described for xanthotoxin (Musajo, L. and Rodighiero, G. (1972) in Photophysiology (Giese, A.C., ed.), Vol. VII, pp. 115-147, Academic Press, New York and Scott, B.R., Pathak, M.A. and Mohn, G.R. (1976) Mutation Res. 39, 30-74) but not for angelicin. The electronic structures in the first excited states calculated by means of quantum chemistry according to the Pariser-Parr-Pople method are very similar for xanthotoxin, psoralen and angelicin. Hence angelicin should be capable of acting bifunctionally like xanthotoxin, but for sterical reasons such reaction should be possible only for the folded DNA, as in phage heads, but not for diluted aqueous solution of DNA.

Computer analysis of phytochrome sequences and reevaluation of the phytochrome secondary structure by Fourier transform infrared spectroscopy.

A repertoire of various methods of computer sequence analysis was applied to phytochromes in order to gain new insights into their structure and function. A statistical analysis of 23 complete phytochrome sequences revealed regions of non-random amino acid composition, which are supposed to be of particular structural or functional importance. All phytochromes other than phyD and phyE from Arabidopsis have at least one such region at the N-terminus between residues 2 and 35. A sequence similarity search of current databases indicated striking homologies between all phytochromes and a hypothetical 84.2-kDa protein from the cyanobacterium Synechocystis. Furthermore, scanning the phytochrome sequences for the occurrence of patterns defined in the PROSITE database detected the signature of the WD repeats of the beta-transducin family within the functionally important 623-779 region (sequence numbering of phyA from Avena) in a number of phytochromes. A multiple sequence alignment performed with 23 complete phytochrome sequences is made available via the IMB Jena World-Wide Web server (http://www.imb-jena.de/PHYTO.html). It can be used as a working tool for future theoretical and experimental studies. Based on the multiple alignment striking sequence differences between phytochromes A and B were detected directly at the N-terminal end, where all phytochromes B have an additional stretch of 15-42 amino acids. There is also a variety of positions with totally conserved but different amino acids in phytochromes A and B. Most of these changes are found in the sequence segment 150-200. It is, therefore, suggested that this region might be of importance in determining the photosensory specificity of the two phytochromes. The secondary structure prediction based on the multiple alignment resulted in a small but significant beta-sheet content. This finding is confirmed by a reevaluation of the secondary structure using FTIR spectroscopy.

Toxicology of simple and complex mixtures.

Humans are exposed to mixtures of chemicals, rather than to individual chemicals. From a public health point of view, it is most relevant to answer the question of whether or not the components in a mixture interact in a way that results in an increase in their overall effect compared with the sum of the effects of the individual components. In this article, options for the hazard identification and risk assessment of simple and complex chemical mixtures will be discussed. In addition, key research needed to continue the development of hazard characterization of chemical mixtures will be described. Clearly, more collaboration among toxicologists, model developers and pharmacologists will be necessary.

Molecular dynamics simulation reveals conformational switching of water-mediated uracil-cytosine base-pairs in an RNA duplex.

A 4 ns molecular dynamics simulation of an RNA duplex (r-GGACUUCGGUCC)(2 )in solution with Na+ and Cl- as counterions was performed. The X-ray structure of this duplex includes two water-mediated uracil-cytosine pairs. In contrast to the other base-pairs in the duplex the water-mediated pairs switch between different conformations. One conformation corresponds to the geometry of the water-mediated UC pairs in the duplex X-ray structure with water acting both as hydrogen-bond donor and acceptor. Another conformation is close to that of a water-mediated UC base-pair found in the X-ray structure of the 23 S rRNA sarcin/ricin domain. In this case the oxygen of the water molecule is linked to two-base donor sites. For a very short time also a direct UC base-pair and a further conformation that is similar to the one found in the RNA duplex structure but exhibits an increased H3(U)...N3(C) distance is observed. Water molecules with unusually long residence times are involved in the water-mediated conformations. These results indicate that the dynamic behaviour of the water-mediated UC base-pairs differs from that of the duplex Watson-Crick and non-canonical guanine-uracil pairs with two or three direct hydrogen bonds. The conformational variability and increased flexibility has to be taken into account when considering these base-pairs as RNA building blocks and as recognition motifs.

C-H...pi-interactions in proteins.

A non-redundant set of 1154 protein structures from the Protein Data Bank was examined with respect to close interactions between C-H-donor and pi-acceptor groups. A total of 31,087 interactions were found to satisfy our selection criteria. Their geometric parameters suggest that these interactions can be classified as weak hydrogen bonds.A set of 12 interaction classes were defined based on the division of the donors into three groups and the acceptors into four groups. These classes were examined separately, and the respective interactions described in detail in each class. Most prominent were interactions between aliphatic C-H donors and aromatic pi-acceptors and interactions between aromatic C-H donors and aromatic pi-acceptors. About three-quarters of the Trp-rings, half of all Phe and Tyr-rings and a quarter of all His-rings were found to be involved as acceptors in C-H...pi-interactions. On the donor side, a preference for aromatic C-H groups was observed, but also for the aliphatic side-chains of the long, extended amino acid residues Lys, Arg and Met, and the Pro ring. The average distance between the C-donor and the center-of-mass of the pi-acceptor was observed to be significantly longer in the 174 protein structures determined at >2.5 A resolution. Also, the distribution is significantly wider. This resolution dependence suggests that the force fields commonly used for the refinement of protein structures may not be adequate. C-H...pi-interactions involving aromatic groups either as donor or as acceptor groups are found mostly in the interior of the protein. The more hydrophilic the participating groups are, the closer to the surface are the interactions located. About 40 % of all C-H...pi-interactions occur between amino acid residue side-chains that are separated by nine or less residues in sequence. Dependent on the interaction class, different preferences for secondary structure, residue type and side-chain conformation were observed. It is likely that the C-H...pi-interactions contribute significantly to the overall stability of a protein.

Evaluation of synergism or antagonism for the combined action of antiviral agents.

The criteria used for the evaluation of synergism or antagonism for the combined action of antiviral agents are critically reviewed. The isobole method is the only generally applicable approach which does not require any assumptions about the shape of dose-response curves. A microcomputer-based isobole method is proposed which constructs response surfaces and a complete set of isoboles in the dose range under study from a relatively small number of experiments. The objective of this work is to take a step towards standardization of definitions, terminology, and methods for the evaluation of synergism or antagonism in antiviral drug combination experiments.

Quantum-chemical ab initio study on the adenine-difluorotoluene complex--a mimic for the adenine-thymine base pair.

Recent experiments have shown that difluorotoluene (F), a nonpolar isostere for thymine (T), codes efficiently and specifically for adenine (A) in DNA replication. F has almost the same shape as thymine but it is unable to form conventional hydrogen bonds with adenine. Therefore, it has been claimed that not hydrogen bonding but shape complementary may be important for the selection of the correct bases by DNA-replicating enzymes. In order to gain deeper insight into structure, charge distribution and energetics of the A-F and A-T base pairs we have performed quantum-chemical ab initio and density functional calculations at the HF, MP2 and B3LYP levels. The interaction energy of the A-F complex amounts to -3.8 kcal/mol (MP2) and is thus substantially smaller than typical ab initio interaction energies for Watson-Crick or non-canonical base pairs. The A-T and A-F complexes are planar and their overall geometries are similar (root-mean-square deviation: 0.4 A). The calculated donor acceptor atom distances in A-T are in good agreement with the experimental mean values obtained from an analysis of 21 high resolution DNA structures. However, A-F shows a base pair opening as compared to A-T. Even though the interaction energy in the A-F base pair is small, the distances for the N6-H...F and N1...H-C3 contacts are still below the sum of the van-der-Waals radii, which means that the interaction is not governed by van-der-Waals forces alone. If the experimental findings can be confirmed, then our results indicate that DNA polymerase is able to retain high fidelity with base pairs of much smaller interaction energies than found for the conventional Watson-Crick and non-canonical base pairs.

A molecular dynamics simulation study of coaxial stacking in RNA.

We report on unrestrained molecular dynamics simulations of an RNA tetramer binding to a tetra-nucleotide overhang at the 5'-end of an RNA hairpin (nicked structure) and of the corresponding continuous hairpin with Na+ as counterions. The simulations lead to stable structures and in this way a structural model for the coaxially stacked RNA hairpin is generated. The stacking interface in the coaxially stacked nicked hairpin structure is characterized by a reduced twist and shift and a slightly increased propeller twist as compared to the continuous system. This leads to an increased overlap between C22 and G23 in the stacking interface of the nicked structure. In the simulations the continuous RNA hairpin has an almost straight helical axis. On the other hand, the corresponding axis for the nicked structure exhibits a marked kink of 39 degrees. The stacking interface exhibits no increased flexibility as compared to the corresponding base pair step in the continuous structure.

Quantum-chemical analysis of C-H...O and C-H...N interactions in RNA base pairs--H-bond versus anti-H-bond pattern.

Geometries and interaction energies of unusual UU and AA base pairs with one standard hydrogen bond (H-bond) and additional C-H...O or C-H...N contacts have been determined by quantum-chemical methods taking into account electron correlation. Whereas the C-H bond length in the UU C-H...O contact increases upon complex formation (H-bond pattern), the C-H bond of the AA C-H....N interaction is shortened (anti-H-bond pattern). The same properties are found for model complexes between U or A and formaldehyde that have intermolecular C-H...acceptor contacts but no standard H-bonds. Both the C-H...acceptor H-bond and anti-H-bond interactions are attractive. A possible influence of the donor CH group charge distribution on the interaction pattern is discussed.

Zero interaction response surfaces for combined-action assessment.

Combined-action assessment requires first the calculation of combination effects expected for the case of no interaction. In a second step the experimental combination effects have to be compared to the expected ones in order to classify the combination as synergistic or antagonistic. Adopting the response surface methodology, a new procedure for the calculation of zero interaction response surfaces which can be applied both to various criteria for defining zero interaction and to various types of dose-response relations is proposed. The approach requires only information from single-agent dose-response relations, but provides nevertheless a comprehensive overview over the complete dose range under study. It can replace the tedious isobolographic analysis if the dose-additivity criterion is adopted.

Influence of leukemia P388 on the pharmacokinetics of mitoguazone in B6D2F1 mice.

The aim of the present study was to investigate the influence of different stages of leukemia P388 on the pharmacokinetics of the antineoplastic agent mitoguazone in mice. It could be shown that, independent of the tumor stage investigated, the total clearance of mitoguazone is slightly reduced reflecting a moderate increase of AUC in the serum of leukemia-bearing animals. Furthermore, in an advanced tumor stage the drug levels in kidneys, liver, spleen and serum were found to be elevated to some extent in comparison to tumor-free controls in contrast to an earlier stage of leukemia. In conclusion, the tumor stage has to be considered as an important factor to which extent a neoplasia may alter the pharmacokinetics of drugs used for anticancer chemotherapy.

Influence of melanoma B16 on the pharmacokinetics of mitoguazone in mice.

In this study, the influence of different stages and transplantation routes of the experimentally widely used solid tumor melanoma B16 on the pharmacokinetics of the antineoplastic agent mitoguazone was investigated in B6D2F1 mice. It could be shown that changes of the pharmacokinetic parameters as well as the distribution pattern of this drug were clearly influenced and dependent on the tumor stage but not by the tumor inoculation route. Advanced melanoma (d16) led to a sharp decrease in the terminal elimination half-life as well as to decreased spleen levels and increased initial liver concentrations of the drug. With respect to the results obtained in leukemia P388-bearing mice it can be concluded that the tumor stage as well as the tumor model are to be considered as important factors in which way and to which extent a tumor may alter the pharmacokinetics of antineoplastic agents.

Model-based analysis of survival experiments with zero and non-zero final survivors.

Modified logistic, Weibull and Gompertz survival models are described which are suitable for the model-based analysis of survival experiments with both zero and non-zero final survivors. They are applied to 3 examples: the influence of food restriction and body fat on the life span of mice and the effect of an immunosuppressant and of an antiviral agent on experimentally virus-infected mice. It is shown that besides the median survival time and the fraction of animals finally surviving the hazard rate at the median survival time, the ratio of maximum to minimum survival time and the area under the survival curve are useful parameters for a more detailed analysis of survival experiments. Hazard plots display differences between experiments more clearly than survival curves. The models described provide a powerful tool which is appropriate for a great variety of different survival experiments. The model-based analysis of survival data is also a prerequisite for the development of computerized data bases on the survival behaviour of laboratory animals. The approach presented can be easily generalized to cases for which spontaneous mortality and mortality due to an experimental challenge interfere.

Image library of biological macromolecules.

An Image Library of Biological Macromolecules is described, which contains image and text files related to structures of biological macromolecules. Currently, the Library has approximately 3000 image files of approximately 300 structures of biological macromolecules whose coordinates are available in the Protein Data Bank and in the Nucleic Acid Database. The entries include all RNA structures, approximately 70 DNA structures, 150 proteins and a few carbohydrates. The Library contains further images of amino acids, of standard and modified nucleotides and of nucleic acid model structures. Each entry consists of an annotation file with bibliographic and sequence information and possibly comments, of a color-coded distance plot and of structure images. Almost all of the images are available both in a mono and in a stereo representation. Standard procedures for generating these images were strictly avoided. Therefore, mixed rendering, coloring and labeling techniques were used extensively. Since May 1995 the Library has a growing division of images in the new Virtual Reality Modeling Language (VRML) format. The Image Library of Biological Macromolecules can be accessed via the World-Wide Web (http://www.imb-jena.de/IMAGE.html). There is a large number of structures determined by experimental and/or modeling techniques which are not intended to be included into the Protein Data Bank or Nucleic Acid Database for some reason. The Image Library could be a repository of these structures and of images of these and other structures of biological macromolecules including structures which are not known at atomic detail. Authors who are willing to make available images or coordinates to the scientific community via the Image Library of Biological Macromolecules are requested to contact the author.

Zero interaction response surfaces, interaction functions and difference response surfaces for combinations of biologically active agents.

In the field of combination experiments there is wide-spread confusion over definitions, terminology and methods for the evaluation of interaction between biologically active agents. According to our view the widely used isobole approach is the method of choice. In this contribution it is shown how the combination of the classical isobole approach with response surface modeling and computer graphics leads to powerful new methods for the assessment of interaction of biologically active agents. In particular, zero interaction response surfaces, difference response surfaces and interaction functions are proposed. Zero interaction response surfaces represent surfaces which display zero interaction in the whole dose range. Difference response surfaces display the difference between an actual response surface and the corresponding zero interaction response surface. Interaction functions are a generalization of the index of interaction, which describe the dose dependence of this quantity.