6-Phospho-{beta}-galactosidases of Gram-positive and 6-phospho-{beta}-glucosidase B of Gram-negative bacteria: comparison of structure and function by kinetic and immunological methods and mutagenesis of the lacG gene of staphylococcus aureus

The 6-phospho-ß-galactosidase of Staphylococcus aureus,Lactococcus lactis and Lactobacillus casei and 6-phospho-ßglucosidaseB of Escherichia coli build a subfamily inside a greater enzymefamily, named the glycosal hydrolase family 1, which, hi addition,contains nine ß-glycosidases of different origins.Kinetic and immunological evidence is provided in this reportwhich strengthens the relationship of the four 6-phospho-ß-glycosidases.It is shown that the 6-phospho-ß-galactosidases and6-phospho-ß-glucosidase B are able to split aromaticß-galactoside phosphates and ß-glucosidephosphates. The turnover numbers of hydrolysis of substrateswith different epimerization at C-4 of the glycon vary up to15-fold only. Two polydonal antisera, one derived against thenative 6-phospho-ß-galactosidase from S.aureus andthe other derived against the 6-phospho-ß-glucosidaseB, cross-reacted with both enzymes. Peptides of the proteinswere separated by reverse phase HPLC. The cross-reacting peptideswere sequenced and shown to be localized at almost the sameposition in the aligned primary structures of both enzymes.An insertion of nine amino adds near these antigenic domainsis unique for the 6-phospho-ß-glycosidases and missingwithin the sequences of the ß-glycoside-specific membersof the family. The lacG gene of a 6-phospho-ß-galactosidasenegative S.aureus mutant was doned into E.coli and sequenced.In the totally inactive mutant protein only the glycine at position332 was changed to an arginine. This amino acid is part of thesequence insertion near the antigenic domain reacting with bothantisera. These data support the assumption that the regionis of great importance for the function of the enzymes and thatit is possible it determines the specificity of the phosphorylatedform of the substrates. In addition, the 6-phospho-ß-galactosidaseof S.aureus was modified by sitedirected mutagenesis of thecorresponding lacG gene hi order to replace residues Glul60and Glu375, which were suspected of being involved hi the generalacid catalysis of substrate hydrolysis, with glutamine residues.The mutant protein 160EQ retained some catalytic activity whilethe protein 375EQ was totally inactive. Glu375 is the activesite nudeophile of the 6-phospho-ß-galactosidase ofS.aureus. It is located in the sequence motif ENG where Glu358was identified as the catalytkally active nudeophile hi theß-glucosidase of Agrobacterium.     

Optimal tracing and replay for debugging message-passing parallel programs

A common debugging strategy involves reexecuting a program (on a given input) over and over, each time gaining more information about bugs. Such techniques can fail on message-passing parallel programs. Because of nondeterminacy, different runs on the given input may produce different results. This nonrepeatability is a serious debugging problem, since an execution cannot always be reproduced to track down bugs. This paper presents a technique for tracing and replaying message-passing programs. By tracing the order in which messages are delivered, a reexecution can be forced to deliver messages in their original order, reproducing the original execution. To reduce the overhead of such a scheme, we show that the delivery'order of only messages involved inraces need be traced (and not every message). Our technique makes run-time decisions to detect and trace racing messages and is usuallyoptimal in the sense that the minimal number of racing messages is traced. Experiments indicate that only 1% of the messages are often traced, gaining a reduction of two orders of magnitude over traditional techniques that trace every message. These traces allow an execution to be reproduced any number of times for debugging. Our work is novel in that we adaptively decide what to trace, and trace only those messages that introduce nondeterminacy. With our strategy, large reductions in trace size allow long-running programs to be replayed that were previously unmanageable. In addition, the reduced tracing requirements alleviate tracing bottle-necks, allowing executions to be debugged with substantially lower execution time overhead.This work was supported in part by National Science Foundation grants CCR-8815928 and CCR-9100968, Office of Naval Research grant N00014-89-J-1222, and a grant from Sequent Computer Systems, Inc.     

Using formalized temporal message-flow diagrams

Temporal message-flow diagrams (TMFDs), alternatively called sequence charts, interaction diagrams, event traces, or actor diagrams, are illustrations of a system's global message-passing activity over time, and a pictorial aid to understanding the system's behavior. They are widely used for requirements and documentation for network protocols and object-oriented applications. We present a general formalism for TMFDs, describe a suite of tools we have designed that employs this formalism, and present our experiences with these tools. The formalism and tools described serve to support and broaden the use of TMFDs in developing communicating systems.     

Integration of data systems and technology improves research and collaboration for a superfund research center

Large collaborative centers are a common model for accomplishing integrated environmental health research. These centers often include various types of scientific domains (e.g., chemistry, biology, bioinformatics) that are integrated to solve some of the nation's key economic or public health concerns. The Superfund Research Center (SRP) at Oregon State University (OSU) is one such center established in 2008 to study the emerging health risks of polycyclic aromatic hydrocarbons while using new technologies both in the field and laboratory. With outside collaboration at remote institutions, success for the center as a whole depends on the ability to effectively integrate data across all research projects and support cores. Therefore, the OSU SRP center developed a system that integrates environmental monitoring data with analytical chemistry data and downstream bioinformatics and statistics to enable complete "source-to-outcome" data modeling and information management. This article describes the development of this integrated information management system that includes commercial software for operational laboratory management and sample management in addition to open-source custom-built software for bioinformatics and experimental data management.     

Hesperidin Promotes Osteogenesis and Modulates Collagen Matrix Organization and Mineralization In Vitro and In Vivo

This study evaluated the direct effect of a phytochemical, hesperidin, on pre-osteoblast cell function as well as osteogenesis and collagen matrix quality, as there is little known about hesperidin’s influence in mineralized tissue formation and regeneration. Hesperidin was added to a culture of MC3T3-E1 cells at various concentrations. Cell proliferation, viability, osteogenic gene expression and deposited collagen matrix analyses were performed. Treatment with hesperidin showed significant upregulation of osteogenic markers, particularly with lower doses. Mature and compact collagen fibrils in hesperidin-treated cultures were observed by picrosirius red staining (PSR), although a thinner matrix layer was present for the higher dose of hesperidin compared to osteogenic media alone. Fourier-transform infrared spectroscopy indicated a better mineral-to-matrix ratio and matrix distribution in cultures exposed to hesperidin and confirmed less collagen deposited with the 100-µM dose of hesperidin. In vivo, hesperidin combined with a suboptimal dose of bone morphogenetic protein 2 (BMP2) (dose unable to promote healing of a rat mandible critical-sized bone defect) in a collagenous scaffold promoted a well-controlled (not ectopic) pattern of bone formation as compared to a large dose of BMP2 (previously defined as optimal in healing the critical-sized defect, although of ectopic nature). PSR staining of newly formed bone demonstrated that hesperidin can promote maturation of bone organic matrix. Our findings show, for the first time, that hesperidin has a modulatory role in mineralized tissue formation via not only osteoblast cell differentiation but also matrix organization and matrix-to-mineral ratio and could be a potential adjunct in regenerative bone therapies.     

Diffusion soldering for stable high-temperature thin-film bonds

Diffusion soldering is a special bonding technique to produce joints at a moderate temperature that are subsequently stable at higher temperatures. The search for material systems extending the upper-temperature limit of stability requires information from the pertinent phase diagrams and the reaction kinetics. Combining experimental studies on phase equilibration in powder samples with bulk and thin-film diffusion couples is a useful approach for a systematic search. Promising candidates for dsoldering are Pt-In or Pd-In, and molybdenum is also an effective diffusion barrier against the attack of liquid tin.     

Regional mass flux balancing for controlling gentle soil remediation operations

Extraction of heavy metals by accumulating plants is a method which is currently in development for the "gentle" remediation of contaminated agricultural soils (phytoremediation, see [2]). Areal contaminant mass flux balances are basic criteria for the design of such remediations and their control. A framework for the integration of contaminant balances relating the field scale of remediation with the regional scale of soil monitoring networks is provided by the recently developed method PROTERRA [3]. The objective of the study presented in this paper was to test the suitability of PROTERRA for planning and monitoring gentle soil remediations. For this purpose we applied the PROTERRA method to the contaminated agricultural land in and around Dornach, Switzerland, to assess copper flux balances.The calculations showed that atmospheric deposition and the application of pesticides and manure are important pathways for the inputs of copper. The copper export with a special maize cultivar accumulating heavy metals would be about three times higher than the average metal export with crops. A moderate increase of sewage sludge application would lead to a substantial increase of the copper input. Decisions to remediate the soil should take the uncertainty of mass flux balances both on field scale as well as on regional scale into account. Therefore, an important need of further development of the PROTERRA method is the integration of uncertainty analysis on both scales.