Diameter of basalt columns derived from fracture mechanics bifurcation analysis.

The diameter of columnar joints forming in cooling basalt and drying starch increases with decreasing growth rate. This observation can be reproduced with a linear-elastic three-dimensional fracture mechanics bifurcation analysis, which has been done for a periodic array of hexagonal columnar joints by considering a bifurcation mode compatible with observations on drying starch. In order to be applicable to basalt columns, the analysis has been carried out with simplified stationary temperature fields. The critical diameter differs from the one derived with a two-dimensional model by a mere factor of 1/2. By taking into account the latent heat released at the solidification front, the results agree fairly well with observed column diameters.

Identification of (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate as a major activator for human gammadelta T cells in Escherichia coli.

The gcpE and lytB gene products control the terminal steps of isoprenoid biosynthesis via the 2-C-methyl-D-erythritol 4-phosphate pathway in Escherichia coli. In lytB-deficient mutants, a highly immunogenic compound accumulates significantly, compared to wild-type E. coli, but is apparently absent in gcpE-deficient mutants. Here, this compound was purified from E. coli DeltalytB mutants by preparative anion exchange chromatography, and identified by mass spectrometry, (1)H, (13)C and (31)P NMR spectroscopy, and NOESY analysis as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP). HMB-PP is 10(4) times more potent in activating human Vgamma9/Vdelta2 T cells than isopentenyl pyrophosphate.

Structural organization and analysis of the viral terminase gene locus of Tupaia herpesvirus.

Tupaia herpesvirus (THV) was isolated from spontaneously degenerating tissue cultures of malignant lymphoma, lung, and spleen cell cultures of tree shrews (Tupaia spp.). In order to determine the phylogenetic relatedness of THV the complete nucleotide sequence of the viral terminase (VTER) gene locus (6223 bp) of Tupaia herpesvirus strain 2 (THV-2) was elucidated and analysed. The VTER gene locus, encoding one of the most highly conserved herpes viral proteins is composed of two exons. The intron contains five potential open reading frames (ORFs). The arrangement of these ORFs is colinear with the corresponding regions in the genomes of the mammalian cytomegaloviruses. The precise primary structure of the THV-2 VTER splice junction was determined using RT-PCR and was found to be in agreement with the corresponding splice donor and acceptor sites of the mammalian cytomegaloviruses. The comparison of all six putative THV-2 proteins with the corresponding counterparts in other herpesviruses revealed that THV resides between the Human and the Murine cytomegalovirus (HCMV, MCMV). These results are in agreement with our previous statement, that THV and the known cytomegaloviruses are closely related to each other and should be classified into one taxonomic group. The genetic data presented here and in previous studies are based on the detailed comparison of highly conserved viral genes. Consequently, the classification of the Human and the cytomegaloviruses into the two genera Cyto- and Muromegalovirus, that is mainly based on overall genome structure, should be reconsidered.

Structural organization of a conserved gene cluster of Tupaia herpesvirus encoding the DNA polymerase, glycoprotein B, a probable processing and transport protein, and the major DNA binding protein.

The Tupaia herpesviruses (THVs) have been isolated from malignant lymphoma tissue cultures and from degenerating lung and spleen cell cultures of tree shrews (Tupaia spp.). Recently we succeeded in the localization of the gene locus of the THV DNA polymerase (DPOL) gene within the viral genome. Based on these results the highly conserved gene cluster of herpesviruses encoding the DPOL, the glycoprotein B (gB), a probable processing and transport protein (PRTP), and the major DNA binding protein (DNBI) was characterized in the genome of THV strain 2 (THV-2) in its entirety. The complete nucleotide sequence of the gene cluster was determined and it was discovered that the THV-2 gene products are most closely related to the corresponding proteins of mammalian cytomegaloviruses. The transcriptional activity of the four genes was confirmed by amplification of a part of the corresponding mRNAs obtained from infected cell RNA by RT-PCR. The homology values and the overall structure of the gene cluster, that shows specific colinearity with the corresponding clusters of the mammalian cytomegaloviruses, is further evidence that THV-2 is a member of the subfamily Betaherpesvirinae.

Gas-Phase cationization and protonation of neutrals generated by matrix-assisted laser desorption.

The ionization mechanisms involved in matrix-assisted ultraviolet laser desorption/ionization (MALDI) were studied with a time-of-flight mass spectrometer. When protonated or cationized quasimolecular ions generated by MALDI are not extracted promptly, their abundance is a function of the delay time between laser irradiation and ion extraction, maximizing at an optimum delay time (DTM) of a few hundred nanoseconds. The ion abundance at DTM exceeds that of prompt extraction by a factor of 2 or more. Increasing the cation density near the sample surface reduces the DTM, whereas increasing the desorption laser irradiance has the opposite effect. The enhancement suggests extensive gas-phase ion-molecule reactions after irradiation by the desorption laser has ceased.

Delayed extraction time-of-flight MALDI mass spectrometry of proteins above 25,000 Da.

Matrix-assisted laser desorption/ionization (MALDI) with delayed extraction (DE) has been optimized for mass analysis of high-mass proteins and glycoproteins with masses above 25,000 Da. Under optimized experimental conditions, i.e. using a weak extraction field strength and a long delay time, a steep drop in mass resolution above 30,000 Da is no longer observed and an improvement of more than a factor of 10 is obtained compared with the non-DE case, at least up to 66 kDa in a 1.2 m time-of-flight mass analyzer. On this level of resolution the factors limiting further improvements become apparent, i.e. adduct ion formation between matrix and analyte, but also cationization and further non-matrix-related adducts, as well as prompt fragmentation. Moreover, heterogeneity of the sample is often the reason for the detection of broad signals for larger proteins. Within these limitations, DHBs (a 9:1 mixture of 2,5-dihydroxybenzoic acid and 2-hydroxy-5-methoxybenzoic acid) gave by far the best results.

Cyclophosphamide levels in serum and spinal fluid of multiple sclerosis patients treated with immunosuppression.

The cyclophosphamide content of urine, serum and spinal fluid of immuno-suppressed multiple sclerosis patients was determined using a newly developed off-line combination of high pressure liquid chromatography and field desorption mass spectrometry. Although urinary excretion of the drug varied considerably between patients, the levels in serum and spinal fluid were in the same range, a relationship that may facilitate control of the dosage.

Determination of cyclophosphamide in urine, serum and cerebrospinal fluid of multiple sclerosis patients by field desorption mass spectrometry.

The levels of cyclophosphamide in samples of urine, serum and cerebrospinal fluid of multiple sclerosis patients, who had received the drug orally (4 x 100 mg/day) have been determined by field desorption mass spectrometry using the principle of stable isotope dilution. After thorough preparation of the samples, concentrations of cyclophosphamide of 10 to 60 micrograms/ml in urine and 200 to 400 ng/ml in serum and cerebrospinal fluid have been determined from 0.6-3 ml samples. The first quantitative data for cyclophosphamide in cerebrospinal fluid of multiple sclerosis patients could be established without the use of radioactive material. The relatively high level of the parent drug found in the fluid at the end of a 3-weeks treatment may shed some light on the mechanism of action of this drug in the treatment of the disease.

Laser desorption ionization mass spectrometry of bioorganic molecules.

Matrix-assisted laser desorption/ionization (LDI) mass spectrometry (MS) is a very young method that has overcome the mass limitations for the mass spectrometry of biopolymers (1-4). Four years ago, a UV-absorbing matrix was used to extend the accessible mass range of UV-LDI of peptides, and a strong dependence on the UV absorption properties of the matrix was demonstrated. The mol-wt determination capability is now ca. 300,000, and since the method is still evolving, its potential is far from fully exploited. Two other mass spectrometric methods are currently applicable to high-molecular mass determination; plasma desorption mass spectrometry ( Chapter 10 in this vol.) enables the production and detection of molecular ions of proteins up to about 30 kDa. Another recent technique, electrospray MS (5), works by spraying a solution of sample into an electric field, producing highly charged droplets, from which molecular ions are desorbed. The characteristic feature of electrospray mass spectra is a distribution of multiple-charged molecular ions, allowing mass measurement of proteins up to 70 kDa (see Chapter 8 ).

Analysis and characterization of the complete genome of tupaia (tree shrew) herpesvirus.

The tupaia herpesvirus (THV) was isolated from spontaneously degenerating tissue cultures of malignant lymphoma, lung, and spleen cell cultures of tree shrews (Tupaia spp.). The determination of the complete nucleotide sequence of the THV strain 2 genome resulted in a 195,857-bp-long, linear DNA molecule with a G+C content of 66.5%. The terminal regions of the THV genome and the loci of conserved viral genes were found to be G+C richer. Furthermore, no large repetitive DNA sequences could be identified. This is in agreement with the previous classification of THV as the prototype species of herpesvirus genome group F. The search for potential coding regions resulted in the identification of 158 open reading frames (ORFs) regularly distributed on both DNA strands. Seventy-six out of the 158 ORFs code for proteins that are significantly homologous to known herpesvirus proteins. The highest homologies found were to primate and rodent cytomegaloviruses. Biological properties, protein homologies, the arrangement of conserved viral genes, and phylogenetic analysis revealed that THV is a member of the subfamily Betaherpesvirinae. The evolutionary lineages of THV and the cytomegaloviruses seem to have branched off from a common ancestor. In addition, it was found that the arrangements of conserved genes of THV and murine cytomegalovirus strain Smith, both of which are not able to form genomic isomers, are colinear with two different human cytomegalovirus (HCMV) strain AD169 genomic isomers that differ from each other in the orientation of the long unique region. The biological properties and the high degree of relatedness of THV to the mammalian cytomegaloviruses allow the consideration of THV as a model system for investigation of HCMV pathogenicity.

Differentiation of 'isobaric' peptides and human milk oligosaccharides by exact mass measurements using electrospray ionization orthogonal time-of-flight analysis.

Exact mass determination is performed by electrospray ionization orthogonal time-of-flight mass analysis. For peptides in the mass range of 1200-1500 Da a mass error of < 5 ppm is achieved with internal calibration within a single mass measurement provided peak intensities are high. Peptides containing isobaric amino acids like glutamine and lysine can thus be easily differentiated by their mass. In cases where more than one of these isobaric amino acids are present, the position of the amino acid can be revealed by exactly determining the mass differences between adjacent Yi" fragment ions in the collision-induced dissociation spectrum. Mass determination accuracy can be enhanced to 0.5-2 ppm by averaging over 8-10 mass measurements. Thereby compositional analysis of human milk oligosaccharides in a mixture can be performed in the mass range up to 3000 Da, even for low-intensity molecule-ion signals and for isobaric compounds with a mass difference of only 0.025 Da.

Influence of pressure in the first pumping stage on analyte desolvation and fragmentation in nano-ESI MS.

In ESI MS, some classes of biomolecules are detected only with low signal intensities due to difficulties in achieving efficient analyte desolvation, either because an analyte tends to fragment already at gentle desolvation conditions (i.e., noncovalent protein complexes or nucleotides) or because an analyte requires very strong activation in order to remove solvent molecules (i.e., carbohydrates). Even though the pressure in the first pumping stage of the ESI instrument is known to have an influence on the desolvation conditions, it has never been the focus of a detailed investigation. The role of the pressure in the first pumping stage is systematically interrogated in this study for several model substances. Ion signal intensities and signal-to-noise ratios are significiantly enhanced if the pressure in the first pumping stage is increased and adjusted, and analyte fragmentation can be substantially reduced. Thus, besides thermal heating and the acceleration in the nozzle-skimmer region, which are usually optimized, the pressure in the first pumping stage is an additional important desolvation parameter.

Nano-electrospray ionization mass spectrometry: addressing analytical problems beyond routine.

The advent of nano-electrospray ionization (nano-ESI) has considerably extended the usability of ESI in the analytical mass spectrometric laboratory. One of the remarkable features of nano-ESI is its extremely low sample consumption. Only a few microliters of analyte solution (10(-5)-10(-8) M) are sufficient for molecular weight determination and structural investigations by MS/MS. But nano-ESI is more than just a minimized-flow ESI; the low solvent flow rate also affects the mechanism of ion formation. As a consequence, the area of ESI-MS applications is significantly enhanced. Oligosaccharides, glycosides as well as glycoproteins can be analyzed more easily than with normal ion spray. The same holds for the analysis of non-covalent complexes sprayed directly from aqueous solutions.

Isolation, identification and determination of cyclophosphamide and two of its metabolites in urine of a multiple sclerosis patient by high pressure liquid chromatography and field desorption mass spectrometry.

The off-line combination of high pressure liquid chromatography and field desorption mass spectrometry has been used for the simultaneous isolation, identification and determination of cyclophosphamide and two of its metabolites, 4-ketocyclophosphamide and carboxyphosphamide in urine from a patient suffering from multiple sclerosis. Cyclophosphamide and its metabolites were separated using reverse phase liquid chromatography. Field desorption mass spectrometry was employed for identification and quantification. The technique applied needs no derivatization for analysis. The limits of detection by field desorption mass spectrometry for 1, 2 and 3 are factor of about 4 X 10(3)-10(5) lower than those of a common variable ultraviolet detector. Quantitative determination was carried out using the method of stable isotope dilution with deuterated analogues of 1, 2 and 3. In a pilot study, the ratio of 1:2:3 was determined to 1:0.02:0.6. One ml of urine is sufficient for simultaneous analysis of the three compounds. The typical analysis time, including separation by liquid chromatography and field desorption measurement, is about 30 minutes.

Differentiation of lysine/glutamine in peptide sequence analysis by electrospray ionization sequential mass spectrometry coupled with a quadrupole ion trap.

Electrospray ionization coupled to a quadrupole ion trap mass spectrometer is used to differentiate between the isobaric amino acids lysine and glutamine in sequence analysis of peptides. Collision-induced dissociation is used for fragmentation. Several isobaric peptides with one or more lysines or glutamines at different positions were investigated. The ambiguous amino acid either in the peptide chain or at the C- or N-terminus can be clearly identified based on specific side chain fragment ions resulting from MS3 or MS4 of B- and Y"-fragment ions.

Analysis of the first complete DNA sequence of an invertebrate iridovirus: coding strategy of the genome of Chilo iridescent virus.

Chilo iridescent virus (CIV), the type species of the genus Iridovirus, a member of the Iridoviridae family, is highly pathogenic for a variety of insect larvae. The virions contain a single linear ds DNA molecule that is circularly permuted and terminally redundant. The coding capacity and strategy of the CIV genome was elucidated by the analysis of the complete DNA nucleotide sequence of the viral genome (212,482 bp) using cycle sequencing by primer walking technology. Both DNA strands were sequenced independently and the average redundancy for each nucleotide was found to be 1.85. The base composition of the viral genomic DNA sequence was found to be 71.37% A+T and 28.63% G+C. The CIV genome contains 468 open reading frames (ORFs). The size of the individual viral gene products ranges between 40 and 2432 amino acids. The analysis of the coding capacity of the CIV genome revealed that 50% (234 ORFs) of all identified ORFs were nonoverlapping. The comparison of the deduced amino acid sequences to entries in protein data banks led to the identification of several genes with significant homologies, such as the two major subunits of the DNA-dependent RNA polymerase, DNA polymerase, protein kinase, thymidine and thymidylate kinase, thymidylate synthase, ribonucleoside-diphosphate reductase, major capsid protein, and others. The highest homologies were detected between putative viral gene products of CIV and lymphocystis disease virus of fish (LCDV). Although many CIV putative gene products showed significant homologies to the corresponding viral proteins of LCDV, no colinearity was detected when the coding strategies of the CIV and LCDV-1 were compared to each other. An intriguing result was the detection of a viral peptide of 53 amino acid residues (ORF 160L) showing high homology (identity/similarity: 60.0%/30.0%) to sillucin, an antibiotic peptide encoded by Rhizomucor pusillus. Iridovirus homologs of cellular genes possess particular implications for the molecular evolution of large DNA viruses.

Quantitative investigations on the diaplacental transfer of thallium by field desorption mass spectrometry.

The placental transfer of thallium cations in pregnant mice was investigated by determining the thallium concentrations in fetal and maternal tissue 0.5 to 24 h after application of thallium. The maternal dose of thallium was 8 mg/kg body weight throughout. Uterus and fetus were found not to differ from other organs like heart and liver in time course and magnitude of thallium uptake with an initial surge during the first few hours of exposure to thallium and a rapid decrease to steady 12 and 24 h values somewhat lower than those found in the kidney. Diaplacental transfer is therefore assumed comparatively rapid and a specific placental barrier for thallium does not seem to exist. For the determination of thallium concentrations Field Desorption Mass Spectrometry was utilized as a reliable, fast and sensitive method for the analysis of metal cations in biological material. This method does not require extensive pretreatment of the tissue and total sample amounts in the range of milligrams and less are sufficient for quantitative analysis.

Determination of lithium and rubidium in physiological fluids and tissues of rabbits during the reproductive phase.

1. The natural concentrations of lithium and rubidium were determined during the reproductive phase of rabbits by field desorption mass spectrometry. 2. Samples of serum, milk, amniotic fluid and placenta tissue were analysed. 3. The concentration changes in serum during the reproductive phase were between 2.61 and 5.02 micrograms/l for lithium and between 107.78 and 136.28 micrograms/l for rubidium. 4. Correlations between the concentration in maternal serum and bone growth of the fetus as well as the formation of milk were found. 5. Concentrations of 9.30 micrograms/l lithium and 1780.00 micrograms/l rubidium in the milk lead to the assumption that these trace metals are essential for the metabolism of the young rabbit.