STARNET 2: a web-based tool for accelerating discovery of gene regulatory networks using microarray co-expression data

Background  

Although expression microarrays have become a standard tool used by biologists, analysis of data produced by microarray experiments may still present challenges. Comparison of data from different platforms, organisms, and labs may involve complicated data processing, and inferring relationships between genes remains difficult.     

Diastereoselective synthesis of l-threo-3,4-dihydroxyphenylserine by low-specific l-threonine aldolase mutants

Diastereoselectivity-enhanced mutants of l-threonine aldolase (l-TA) for l-threo-3,4-dihydroxyphenylserine (l-threo-DOPS) synthesis were isolated by error-prone PCR followed by a high-throughput screening. The most improved mutant was achieved from the mutant T3-3mm2, showing a 4-fold increase over the wild-type l-TA. When aldol condensation activity was examined using whole cells of T3-3mm2, its de was constantly maintained at 55% during the batch reactions for 80 h, yielding 3.8 mg l-threo-DOPS/ml.     

Direct calibration of PICKY-designed microarrays

Background  

Few microarrays have been quantitatively calibrated to identify optimal hybridization conditions because it is difficult to precisely determine the hybridization characteristics of a microarray using biologically variable cDNA samples.     

Structural studies of neuropeptides composed ofl -Asp andd -Glu by13C-NMR spectroscopy

¹³C-NMR spectral data are presented for four neuropeptides composed ofl-Asp, Ac-l-Asp, andd-Glu, which contain and peptide linkages. The data for the various compounds are compared to related dipeptides, one of which is a known neuropeptide, in order to gain structural information about these compounds. Electron-nuclear relaxation rates were used to elucidate the metal-ion binding sites of these species.     

Comparative studies on S-adenosyl-l -methionine binding sites of protein N-methyltransferases, using 8-azido-S-adenosyl-l -methionine as photoaffinity probe

Employing a photoaffinity labeling procedure with 8-azido-S-adenosyl-l-[methyl-³H]methionine (8-N₃-Ado[methyl-³H]Met), the binding sites for S-adenosyl-l-methionine (AdoMet) of three protein N-methyltransferases [AdoMet:myelin basic protein-arginine N-methyltransferase (EC2.1.1.23); AdoMet:histone-arginin N-methyltransferase (EC2.1.1.23); and AdoMet:cytochromec-lysine N-methyltransferase (EC2.1.1.59)] have been investigated. The incorporation of the photoaffinity label into the enzymes upon UV irradiation was highly specific. In order to define the AdoMet binding sites, the photolabeled enzymes were sequentially digested with trypsin, chymotrypsin, and endoproteinase Glu-C. After each proteolytic digestion, radiolabeled peptide from each enzyme was resolved on HPLC first by gradient elution and further purified by an isocratic elution. Retention times of the purified radiolabeled peptides from the three enzymes from the corresponding proteolysis were significantly different, indicating that their sizes and compositions were different. Amino acid composition analysis of these peptides confirmed further that the AdoMet binding sites of these protein N-methyltransferases are quite different.     

n-carbamoyl-d-p-hydroxyphenylglycine production using immobilized d-hydantoinase from recombinant E. coli

An immobilized d-hydantoinase was characterized and employed to produce n-carbamoyl-d-p-hydroxyphenylglycine (CpHPG) in a repeated batch process. The V_max and K_m of the immobilized d-hydantoinase at 50°C were 6.28 mm min⁻¹ g⁻¹ biocatalyst and 71.6 mm, respectively. The product CpHPG did not inhibit the activity of d-hydantoinase. Optimal reaction temperature was 60°C. A decrease in activity of immobilized d-hydantoinase due to thermal inactivation could be described as first-order decay; the deactivation energy was 23.97Kcal mol⁻¹. Under process conditions (50°C, 10% w/v substrate, and pH 8.5), the half-life of the immobilized d-hydantoinase was eight batches. The attrition of immobilized d-hydantoinase particles with a large amount of insoluble substrate particles during stirring resulted in fine biocatalyst particles. In addition to the thermal inactivation, the loss of fine biocatalyst particles during the recovery step contributed to the low operational stability.     

Determination of two sites of automethylation in bovine erythrocyte protein (d -aspartyl/l -isoaspartyl) carboxyl methyltransferase

Protein (d-aspartyl/l-isoaspartyl) carboxyl methyltransferase (PCM, E.C. 2.1.1.77) was previously shown to be enzymatically methyl esterified in an autocatalytic manner at altered aspartyl residues; methyl esters are observed in a subpopulation of the enzyme termed thePCM fraction [Lindquist and McFadden (1994),J. Protein Chem. 13, 23–30]. The altered aspartyl sites serving as methyl acceptors inPCM have now been localized by using proteolytic enzymes and chemical cleavage techniques in combination with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry to identify fragments of the [³H]automethylated enzyme that contain a [³H]methyl ester. Methylation was positively identified at positions Asn₁₈₈ and Asp₂₁₇ in the enzyme sequence, a consequence of the spontaneous alteration of these sites tol-isoaspartyl ord-aspartyl sites and their methylation by active PCM molecules. The identification of more than one site of automethylation shows thatPCM is not a homogeneous population of damaged PCM molecules, but rather a complex population of molecules with a variety of age-altered damage sites.Abbreviations PCM protein (d-aspartyl/l-isoaspartyl) carboxyl methyltransferase - EDTA disodium ethylenediaminetetraacetate - PMSF phenylmethylsulfonyl fluoride - TEA trifluoroacetic acid - HPLC high-pressure liquid chromatography     

Automethylation of protein (d -aspartyl/l -isoaspartyl) carboxyl methyltransferase, a response to enzyme aging

A question that is central to understanding the mechanisms of aging and cellular deterioration is whether enzymes involved in recognition and metabolism of spontaneously damaged proteins are themselves damaged, either becoming substrates for their own activity; or being unable to act upon themselves, initiating cascades of cellular damage. We show here byin vitro experiments that protein (d-aspartyl/l-isoaspartyl) carboxyl methyltransferase (PCM) from bovine erythrocytes does methylate age-dependent amino acid damage in its own sequence. The subpopulation that is methylated, termed thePCM fraction, appears to be formed through age-dependent deamidation of an asparaginyl site to either anl-isoaspartyl ord-aspartyl site because (a) the stoichiometry of automethylation of purified PCM is less than 1%, a value typical of the substoichiometric methylation of many other aged protein substrates, (b)PCM is slightly more acidic than the bulk of PCM, and (c) the methyl esterified site inPCM has the characteristic base-lability of this type of methyl ester. Also, the methyl group is not incorporated into the enzyme as an active site intermediate because the incorporated methyl group is not chased onto substrate protein. The effect of enzyme dilution on the rate of the automethylation reaction is consistent with methylation occurring between protein molecules, showing that the pool of PCM is autocatalytic even though individual molecules may not be. The automethylation and possible self-repair of the PCM pool has implications for maintaining thein vivo efficiency of methylation-dependent protein repair.     

A facile synthesis of 5-thio-l-fucose and 5-thio-d-arabinose from d-arabinose

5-Thio-l-fucopyranose tetraacetate was synthesized in 11 steps from or d-arabinose diethyl dithioacetal by one-carbon elongation at C-5. Highly diastereo-selective addition of MeLi in ether to a derivative was achieved to give the corresponding 6-deoxy-β-d-altrofuranose isomer in good yield. A sulfur atom was introduced at C-5 of 6-deoxy-d-altrofuranose derivatives via substitution of a 5-tosylate with KSAc in HMPA with inversion of configuration, giving 5-thio-l-fucopyranose. A derivative was also prepared from 6-deoxy-β-d-altrofuranose derivatives. 5-Thio-d-arabinopyranose tetraacetate, the 5-demethyl analog of 5-thio-l-fucose, was also synthesized from in 5 steps. 5-Thio-d-arabinose showed weak inhibitory activity against α-l-fucosidase from bovine kidney (K_{i = 0.77 mM}).     

A microplate assay for d-xylose/ d-glucose isomerase

A modification of the resorcinol method of Kulka¹ for the determination of ketoses is described. Though being a stopped enzyme test, it is much more sensitive than the carbazol method and by applying microtiter plates and measuring with an ELISA reader, a large number of tests can be performed within a short time, thereby facilitating initial velocity studies.

The test is linear up to a concentration of 2.5 m -xylulose even in the presence of 10 m -xylose and 2 m -fructose in the presence of 10 m -glucose. The sensitivity is 25 μ for xylulose and 38 μ for fructose. The test method is insensitive to perturbations of substances frequently used in isolation procedures such as ammonium sulfate, Triton X-100, PEG 6000, sodium dodecyl sulfate, and ethanol in moderate concentrations.     

Physiologie der Schnecke und desCorti schen Organs

Ohne ZusammenfassungMit 37 Abbildungen.     

Isolation and characterization of sets of anti-l -fucose and anti-bovine serum albumin antibodies directed against a glycoconjugate ofl -fucose and bovine serum albumin

A set of anti-carbohydrate antibodies and a set of anti-protein antibodies were isolated from the serum of rabbits immunized with a glycoconjugate ofl-fucose and bovine serum albumin. The sets were separated by affinity chromatography by a two-column method on adsorbents withl-fucose or bovine serum albumin ligands. Isoelectrofocusing results showed that the anti-carbohydrate antibodies consisted of 11 molecular species and the anti-bovine serum albumin antibodies consisted of seven molecular species. The anti-carbohydrate antibodies are all of the IgG type while the anti-protein antibodies contain three types of globulin molecules, IgA, IgG, and IgM. The former antibodies should be useful as markers for unique glycoproteins of diseased cells and the latter antibodies may be useful for investigating the mechanism of simultaneous synthesis of three types of immunoglobulins.