Selectivity of lipases and esterases towards phenol esters

The selectivity of 28 lipases and esterases in the hydrolysis of butanoates of o-, m- or p-substituted phenols was investigated in a microtiterplate format. The phenols released during enzyme-catalyzed hydrolysis were converted in situ with Gibbs’ reagent to form a blue indophenol complex, which was quantified spectrophotometrically at 600 nm. Substantial differences in rates were found, which exhibits that the type and position of the substituent at the alkyl group has a strong influence on the selectivity of the enzymes. For various enzymes, the p-nitro derivative was the best substrate, whereas for other enzymes the m-Cl-derivative was preferentially hydrolyzed. Analysis of the data using the Hammett equation showed that sometimes the observed changes followed a predictable trend, but in several cases the result is very unexpected.     

Generation of baculovirus vectors for the high-throughput production of proteins in insect cells

The baculovirus expression system is one of the most popular methods used for the production of recombinant proteins but has several complex steps which have proved inherently difficult to adapt to a multi-parallel process. We have developed a bacmid vector that does not require any form of selection pressure to separate recombinant virus from non-recombinant parental virus. The method relies on homologous recombination in insect cells between a transfer vector containing a gene to be expressed and a replication-deficient bacmid. The target gene replaces a bacterial replicon at the polyhedrin loci, simultaneously restoring a virus gene essential for replication. Therefore, only recombinant virus can replicate facilitating the rapid production of multiple recombinant viruses on automated platforms in a one-step procedure. Using this vector allowed us to automate the generation of multiple recombinant viruses with a robotic liquid handler and then rapidly screen infected insect cell supernatant for the presence of secreted proteins.     

Cloning and gene map assignment of the Xiphophorus DNA ligase 1 gene

Fishes represent the stem vertebrate condition and have maintained several gene arrangements common to mammalian genomes throughout the 450 Myr of divergence from a common ancestor. One such syntenic arrangement includes the GPI-PEPD enzyme association on Xiphophorus linkage group IV and human chromosome 19. Previously we assigned the Xiphophorus homologue of the human ERCC2 gene to linkage group U5 in tight association with the CKM locus. CKM is also tightly linked to the ERCC2 locus on human chromosome 19, leading to speculation that human chromosome 19 may have arisen by fusion of two ancestral linkage groups which have been maintained in fishes. To investigate this hypothesis further, we isolated and sequenced Xiphophorus fish genomic regions exhibiting considerable sequence similarity to the human DNA ligase 1 amino acid sequence. Comparison of the fish DNA ligase sequence with those of other species suggests several modes of amino acid conservation in this gene. A 2.2-kb restriction fragment containing part of an X. maculatus DNA ligase 1 exon was used in backcross hybrid mapping with 12 enzyme or RFLP loci. Significant linkage was observed between the nucleoside phosphorylase (NP2) and the DNA ligase (LIG1) loci on Xiphophorus linkage group VI. This assignment suggests that the association of four DNA repair-related genes on human chromosome 19 may be the result of chance chromosomal rearrangements.      

Electro-optical behaviour of CuFe2O4@rGO nanocomposite for nonenzymatic detection of uric acid via the electrochemical method

Uric acid (UA) is a blood and urine component obtained as a metabolic by-product of purine nucleotides. Abnormalities in UA metabolism cause crystal deposition as monosodium urate and lead to various diseases such as gout, hyperuricemia, Lesch–Nyhan syndrome, etc. Monitoring these diseases requires a rapid, sensitive, selective, and portable detection approach. Therefore, this study demonstrates the hydrothermal synthesis of CuFe₂O₄/reduced graphene oxide (rGO) nanocomposite for selective detection of UA. After the nanocomposite synthesis, characterization was performed by X-ray diffraction spectroscopy, Fourier transform infrared spectroscopy, Raman spectroscopy, X-ray photoelectron spectroscopy, UV–visible spectrometry, atomic force spectroscopy, scanning electron microscopy, and electrochemical analysis. Furthermore, from the electrochemical analysis using cyclic voltammetry (CV), kinetic studies were carried out by varying the scan rate to obtain the diffusion coefficient, surface concentration, and rate of charge transfer to achieve a calibration curve that indicates the quasi reversible nature of the fabricated electrode with a linear regression coefficient of oxidation (R²: 0.9992) and reduction (R²: 0.9971) peaks. Moreover, the fabricated nonenzymatic amperometric sensor to detect UA with a linearity (R²: 0.9989) of 1–400 μM was highly sensitive (2.75 × 10⁻⁴ mAμM⁻¹ cm⁻²) and had a lower limit of detection (0.01231 μM) at pH 7.5 in phosphate-buffered saline solution. Therefore, the CuFe₂O₄/rGO/ITO-based nonenzymatic sensor could detect interfering agents and spiked real bovine serum samples with higher sensitivity and selectivity for UA detection.     

An RNA folding method capable of identifying pseudoknots and base triples

MOTIVATION: Recently, we described a Maximum Weighted Matching (MWM) method for RNA structure prediction. The MWM method is capable of detecting pseudoknots and other tertiary base-pairing interactions in a computationally efficient manner (Cary and Stormo, Proceedings of the Third International Conference on Intelligent Systems for Molecular Biology, pp. 75-80, 1995). Here we report on the results of our efforts to improve the MWM method's predictive accuracy, and show how the method can be extended to detect base interactions formerly inaccessible to automated RNA modeling techniques. RESULTS: Improved performance in MWM structure prediction was achieved in two ways. First, new ways of calculating base pair likelihoods have been developed. These allow experimental data and combined statistical and thermodynamic information to be used by the program. Second, accuracy was improved by developing techniques for filtering out spurious base pairs predicted by the MWM program. We also demonstrate here a means by which the MWM folding method may be used to detect the presence of base triples in RNAs. AVAILABILITY: http://www.cshl.org/mzhanglab/tabaska/j axpage. html CONTACT: tabaska@cshl.org      

Anti-SLA seropositive autoimmune hepatitis sera recognize distinct subunits of glutathione S-transferase: high prevalence of the Ya autoantigen.

We have recently reported that anti-SLA seropositive autoimmune hepatitis (AIH) patients develop autoantibodies against glutathione S-transferase (GST). GSTs are multifunctional enzymes mediating hepatic detoxification of cytotoxic and genotoxic compounds and are also involved in biliary secretion. We have observed varying reactivity of individual AIH sera towards several GST isoenzymes. Since the GST subunits have very similar molar masses and therefore are not satisfactorily resolved by one-dimensional gel electrophoresis, we have performed their fractionation by reverse-phase high performance liquid chromatography (HPLC) to better separate the individual GST isoenzymes. 4 individual GST subunits were isolated as judged by electrophoretic analysis of the 4 distinct peaks. The identity of isolated proteins was unequivocally determined by protein sequencing. Isolated subtypes were loaded on 15% SDS gels and blotted. Immunoblotting was performed with eleven anti-SLA positive sera that displayed differential reactivity with total GSTs. Fractionation of the GSTs by HPLC did not impair their ability to react with specific autoantibodies. Interestingly, the majority of GST-positive AIH sera reacted with one or two GST subtypes, only two sera recognized 3 subunits. Ya was most prevalent autoantigen. Autoantibodies against Yb2 were detected solely in one serum. This pattern of reactivity indicates that individual patients' sera discriminate between GST subunits despite their sequence homology. It is well known that the GST variants differ within their amino-terminal part while the residual moiety is highly conserved. It would suggest that autoantibodies recognize distinct epitopes located within amino-terminus of individual GST variants.     

Genbank accession #: AF139098

Plant Molecular Biology -     

Geographical and temporal variation in the diet of Bank Cormorants Phalacrocorax neglectus in South Africa

The Bank Cormorant Phalacrocorax neglectus is endemic to the Benguela upwelling ecosystem off southwest Africa and is classified as Endangered owing to a recent large reduction in its number. It is thought that food scarcity, including a decreased abundance of West Coast rock lobster Jasus lalandii, has been a major driver of the decrease, yet its diet in South Africa is poorly known. We collected 941 pellets regurgitated by Bank Cormorants, at 18 South African breeding colonies during 1975–1985, and 1 523 pellets at 17 colonies during 1995–2002. The species composition of the diet (% numbers) was significantly different between the two periods, with widespread decreases in proportions of rock lobster in the west and of octopus and cuttlefish Sepia spp. at most localities. These taxa were replaced in the diet by fish, including Gobiidae and Clinidae. The pelagic goby Sufflogobius bibarbatus, an important prey of Bank Cormorants in Namibia, was absent from pellets collected in 1975–1985 but common at northern localities from 1995–2002. Composition of the diet by frequency of occurrence was only determined for 1995–2002, when rock lobster was present in 67% of all samples collected, cuttlefish in 39%, and Clinidae in 32%. Data for 1975–1985 and 1995–2002 showed that carapace lengths of rock lobsters eaten by Bank Cormorants averaged 56 mm (range 22–82 mm) and 50 mm (range 22–75 mm), respectively, which compares to the minimum legal size of 75 mm for fisheries in South Africa. This energy- rich prey item was an important constituent of the diet in the winter breeding period.