Crystals of urokinase type plasminogen activator complexes reveal the binding mode of peptidomimetic inhibitors

Urokinase type plasminogen activator (uPA), a trypsin-like serine proteinase, plays an important role in normal tissue re-modelling, cell adhesion, and cell motility. In addition, studies utilizing normal animals and potent, selective uPA inhibitors or genetically modified mice that lack functional uPA genes have demonstrated that uPA can significantly enhance tumor initiation, growth, progression and metastasis, strongly suggesting that this enzyme may be a promising anti-cancer target. We have investigated the structure-activity relationship (SAR) of peptidomimetic inhibitors of uPA and solved high resolution X-ray structures of key, lead small molecule inhibitors (e.g. phenethylsulfonamidino(P4)-D-seryl(P3)-L-alanyl(P2)-L-argininal(P1) and derivatives thereof) in complex with the uPA proteinase domain. These potent inhibitors are highly selective for uPA. The non-natural D-seryl residue present at the P3 position in these inhibitors contributes substantially to both potency and selectivity because, due to its D-configuration, its side-chain binds in the S4 pocket to interact with the uPA unique residues Leu97b and His99. Additional potency and selectivity can be achieved by optimizing the inhibitor P4 residue to bind a pocket, known as S1sub or S1beta, that is adjacent to the primary specificity pocket of uPA.     

Water flows in the parasitic association Rhinanthus minor/Hordeum vulgare

Using the facultative root hemiparasite Rhinanthus minor and its host Hordeum vulgare several aspects of water relations have been measured in this parasitic association. Extraction of xylem sap by the parasite from the host's roots is facilitated by con siderably higher transpiration per leaf area in the parasite than in the host and by the fact that stomata of attached Rhinanthus were open all day and night despite extremely high ABA concentrations in the leaves. By comparison, another root hemiparasite, Melampyrum arvense, parasitizing various grasses in the field, showed normal diurnal stomatal behaviour. The abnormal behaviour of Rhinanthus stomata was not due to anatomical reasons as closure could be induced by applying high external ABA concentrations. Remarkable differences have been detected between the hydraulic conductance of barley seminal roots showing relatively low values and that of Rhinanthus seminal roots showing very high values. The latter could be related to the observed high ABA concentrations in these roots. Whole plant water uptake, transpirational losses, growth-dependent deposition, and the flows of water within the plants have been measured in singly growing Rhinanthus and Hordeum plants and in the parasitic association between the two. Water uptake, deposition and transpiration in Rhinanthus were dramatically increased after attachment to the barley host; most of the water used by the parasite was extracted as xylem sap from the host, thereby scavenging 20% of the total water taken up by the host's roots. This water uptake by the parasitized host, however, due to a parasite-induced reduction in the host's growth, was decreased by 22% as compared to non-parasitized barley. The overall changes in growth-related water deposition in the host and parasite pointed to decreased shoot growth and relatively favoured root growth in the host and to strongly favoured shoot growth in the parasite. These changes in the host became more severe, when more than one Rhinanthus was parasitizing one barley plant.     

Vaccination of mice with bacteria carrying a cloned herpesvirus genome reconstituted in vivo

Bacterial delivery systems are gaining increasing interest as potential vaccination vectors to deliver either proteins or nucleic acids for gene expression in the recipient. Bacterial delivery systems for gene expression in vivo usually contain small multicopy plasmids. We have shown before that bacteria containing a herpesvirus bacterial artificial chromosome (BAC) can reconstitute the virus replication cycle after cocultivation with fibroblasts in vitro. In this study we addressed the question of whether bacteria containing a single plasmid with a complete viral genome can also reconstitute the viral replication process in vivo. We used a natural mouse pathogen, the murine cytomegalovirus (MCMV), whose genome has previously been cloned as a BAC in Escherichia coli. In this study, we tested a new application for BAC-cloned herpesvirus genomes. We show that the MCMV BAC can be stably maintained in certain strains of Salmonella enterica serovar Typhimurium as well and that both serovar Typhimurium and E. coli harboring the single-copy MCMV BAC can reconstitute a virus infection upon injection into mice. By this procedure, a productive virus infection is regenerated only in immunocompromised mice. Virus reconstitution in vivo causes elevated titers of specific anti-MCMV antibodies, protection against lethal MCMV challenge, and strong expression of additional genes introduced into the viral genome. Thus, the reconstitution of infectious virus from live attenuated bacteria presents a novel concept for multivalent virus vaccines launched from bacterial vectors.     

Characterization of the zinc-binding site of the histidine-proline-rich glycoprotein associated with rabbit skeletal muscle AMP deaminase

The AMP deaminase-associated variant of histidine-proline-rich glycoprotein (HPRG) is isolated from rabbit skeletal muscle by a modification of the protocol previously used for the purification of AMP deaminase. This procedure yields highly pure HPRG suitable for investigation by x-ray absorption spectroscopy of the zinc-binding behavior of the protein. X-ray absorption spectroscopy analysis of a 2:1 zinc-HPRG complex shows that zinc is bound to the protein, most probably in a dinuclear cluster where each Zn(2+) ion is coordinated, on average, by three histidine ligands and one heavier ligand, likely a sulfur from a cysteine. 11 cysteines of HPRG from different species are totally conserved, suggesting that five disulfide bridges are essential for the proper folding of the protein. At least another cysteine is present at different positions in the histidine-proline-rich domain of HPRG in all species, suggesting that this cysteine is the candidate for zinc ligation in the muscle variant of HPRG. The same conclusion is likely to be true for the six histidines used by the protein as zinc ligands. The presence in muscle HPRG of a specific zinc-binding site permits us to envisage the addition of HPRG into the family of metallochaperones. In this view, HPRG may enhance the in vivo stability of metalloenzymes such as AMP deaminase.     

Clonal expansions of mitochondrial genomes: implications for in vivo mutational spectra

It is often assumed mutant frequencies, as measured in a DNA sample, faithfully represent basic mutation rates associated with these mutations. This paradigm was extremely helpful for in vitro studies of the mechanisms of mutagenesis/repair and causes of mutations. However, in vivo, mutant fractions appear to vary dramatically and randomly from sample to sample. It's unlikely that basic mutational rates vary so much. Such variations are probably caused by clonal expansions of mutants within tissue. Whether a particular tissue sample includes an expansion or not, is a matter of chance, which explains the observed random fluctuations of mutant fractions. Well-known examples of clonal expansions involve pathological conditions such as cancer or mitochondrial disease. It is less appreciated that even in normal tissue, expansions of somatic mutants create local deviations from the "expected" mutant frequencies. The sizes of clonal expansions appear to span a wide range and thus, may affect samples of various sizes, from individual cells to individuals. In conclusion, human body appears to be a sort of a "gambling ground" for clonally expanding mutants. We speculate that expansion of early mutants rather than de novo mutation at old age may be the major source of at least some aging-specific mutants in our bodies.     

Hydrogen-induced structural changes at the nickel site of the regulatory [NiFe] hydrogenase from Ralstonia eutropha detected by X-ray absorption spectroscopy

For the first time, the nickel site of the hydrogen sensor of Ralstonia eutropha, the regulatory [NiFe] hydrogenase (RH), was investigated by X-ray absorption spectroscopy (XAS) at the nickel K-edge. The oxidation state and the atomic structure of the Ni site were investigated in the RH in the absence (air-oxidized, RH(ox)) and presence of hydrogen (RH(+H2)). Incubation with hydrogen is found to cause remarkable changes in the spectroscopic properties. The Ni-C EPR signal, indicative of Ni(III), is detectable only in the RH(+H2) state. XANES and EXAFS spectra indicate a coordination of the Ni in the RH(ox) and RH(+H2) that pronouncedly differs from the one in standard [NiFe] hydrogenases. Also, the changes induced by exposure to H(2) are unique. A drastic modification in the XANES spectra and an upshift of the K-edge energy from 8339.8 (RH(ox)) to 8341.1 eV (RH(+H2)) is observed. The EXAFS spectra indicate a change in the Ni coordination in the RH upon exposure to H(2). One likely interpretation of the data is the detachment of one sulfur ligand in RH(+H2) and the binding of additional (O,N) or H ligands. The following Ni oxidation states and coordinations are proposed: five-coordinated Ni(II)(O,N)(2)S(3) for RH(ox) and six-coordinated Ni((III))(O,N)(3)X(1)S(2) [X being either an (O,N) or H ligand] for RH(+H2). Implications of the structural features of the Ni site of the RH in relation to its function, hydrogen sensing, are discussed.     

Checkpoint arrest signaling in response to UV damage is independent of nucleotide excision repair in Saccharomyces cerevisiae

The recognition of DNA double-stranded breaks or single-stranded DNA gaps as a precondition for cell cycle checkpoint arrest has been well established. However, how bulky base damage such as UV-induced pyrimidine dimers elicits a checkpoint response has remained elusive. Nucleotide excision repair represents the main pathway for UV dimer removal that results in strand interruptions. However, we demonstrate here that Rad53p hyperphosphorylation, an early event of checkpoint signaling in Saccharomyces cerevisiae, is independent of nucleotide excision repair (NER), even if replication as a source of secondary DNA damage is excluded. Thus, our data hint at primary base damage or at UV damage (primary or secondary) that does not need to be processed by NER as the relevant substrate of damage-sensing checkpoint proteins.     

Regular ingestion of opuntia robusta lowers oxidation injury

The influence of opuntia robusta (prickly pear), a traditionally used dietary nutrient against diabetes mellitus among the American Indian population, was examined in 15 young patients suffering from familial heterozygous isolated hypercholesterolemia. Oxidation injury was determined via 8-epi-PGF(2 alpha)in plasma, serum and urine. Daily consumption of 250 g broiled edible pulp of prickly pear had no influence on body weight and body fat composition. Total cholesterol was lowered (P<0.01) as was LDL-cholesterol (P<0.04). No significant changes were observed either in triglycerides or in HDL. Prickly pear induced a significant decrease in plasma (27.9+/-3.3-->25.6+/-3.2;P<0.03), serum (302.0+/-11.4-->283.2+/-14.5;P<0.0003) and urinary (355.9+/-18.4-->323.9+/-16;P<0.00002) 8-epi-PGF(2alpha)values. The findings on a decrease of 8-epi-PGF(2alpha)were more pronounced in females than in males, the highest significance being found in urine, while, in contrast, the effects on total- and LDL-cholesterol were more pronounced in males. A prerunning 4 weeks period of dietary counseling had no significant effect on either of the parameters examined. These findings indicate that the regular ingestion of opuntia robusta is able to significantly reduce in-vivo oxidation injury in a group of patients suffering from familial hypercholesterolemia. This traditional food of the American Indians thus may have a significant cardiovascular benefit.     

Overcoming the problems associated with poor spectra quality of the protein kinase Byr2 using residual dipolar couplings

For the Ras-binding domain of the protein kinase Byr2, only a limited number of NOE contacts could be initially assigned unambiguously, as the quality of the NOESY spectra was too poor. However, the use of residual (1)H-(15)N dipolar couplings in the beginning of the structure determination process allows to overcome this problem. We used a three-step recipe for this procedure. A previously unknown structure could be calculated reasonably well with only a limited number of unambiguously assigned NOE contacts.     

The SNAREs vti1a and vti1b have distinct localization and SNARE complex partners

Two mammalian proteins, vtila and vtilb, are homologous to the yeast Q-SNARE Vtilp which is part of several SNARE complexes in different transport steps. In vitro experiments suggest distinct functions for vtila and vtilb. Here we compared the subcellular localization of endogenous vtila and vtilb by immunofluorescence and immuno-electron microscopy. Both proteins had a distinct but overlapping localization. vtila was found predominantly on the Golgi and the TGN, vtilb mostly on tubules and vesicles in the TGN area and on endosomes. vti1a coimmunoprecipitated with VAMP-4, syntaxin 6, and syntaxin 16. These four SNAREs could assemble into a SNARE complex of conserved structure because one SNARE motif of each subgroup is present. vtila-beta, VAMP-4, syntaxin 6, and syntaxin 16 are coenriched with small synaptic vesicles and with clathrin-coated vesicles isolated from rat brain synaptosomes. Therefore, this SNARE complex may have a role in synaptic vesicle biogenesis or recycling.