Rapid determination of 5-fluorouracil in plasma using capillary electrophoresis

A rapid, simple and sensitive capillary electrophoresis (CE) method used for the determination of 5-fluorouracil in rabbit plasma is described in the present paper. In this method, samples were simply pretreated by a solvent extraction procedure prior to injection. With a running buffer composed of 30 mM Tris-H(3)PO(4) (pH 7.0) and 5% isopropanol, 5-fluorouracil was easily separated from the external standard alpha-phenethylol as well as other substances existed in the plasma. A linearity of 5-fluorouracil was determined in the range from 0.17 to 42.50 microg/ml with a correlation coefficient of 0.999. A limit of quantitation (LOQ) corresponding to signal-to-noise ratio of 10 was obtained (LOQ=0.08 microg/ml). The method was successfully used for determining the 5-fluorouracil in real plasma samples from rabbits.     

The severe acute respiratory syndrome (SARS) coronavirus NTPase/helicase belongs to a distinct class of 5' to 3' viral helicases

The putative NTPase/helicase protein from severe acute respiratory syndrome coronavirus (SARS-CoV) is postulated to play a number of crucial roles in the viral life cycle, making it an attractive target for anti-SARS therapy. We have cloned, expressed, and purified this protein as an N-terminal hexahistidine fusion in Escherichia coli and have characterized its helicase and NTPase activities. The enzyme unwinds double-stranded DNA, dependent on the presence of a 5' single-stranded overhang, indicating a 5'o 3' polarity of activity, a distinct characteristic of coronaviridae helicases. We provide the first quantitative analysis of the polynucleic acid binding and NTPase activities of a Nidovirus helicase, using a high throughput phosphate release assay that will be readily adaptable to the future testing of helicase inhibitors. All eight common NTPs and dNTPs were hydrolyzed by the SARS helicase in a magnesium-dependent reaction, stimulated by the presence of either single-stranded DNA or RNA. The enzyme exhibited a preference for ATP, dATP, and dCTP over the other NTP/dNTP substrates. Homopolynucleotides significantly stimulated the ATPase activity (15-25-fold) with the notable exception of poly(G) and poly(dG), which were non-stimulatory. We found a large variation in the apparent strength of binding of different homopolynucleotides, with dT24 binding over 10 times more strongly than dA24 as observed by the apparent Km.     

OsSET1, a novel SET-domain-containing gene from rice

A novel SET-domain-containing gene OsSET1 was isolated from rice (Oryza sativa L.). Its deduced protein consists of 895 amino acids. OsSET1 has a high degree of structure similarity to other SET-domain-containing genes such as CLF in higher plants and E(z) in animals. RT-PCR showed that the gene expresses throughout the entire plant. A transient expression assay in onion epidermis revealed that the OsSET1 protein is localized in nuclei. Over-expression of the SET domain of OsSET1 in Arabidopsis resulted in altered shoot development at seedling stages.     

EST analysis of gene expression in the tentacle of Cyanea capillata

Jellyfish, Cyanea capillata, has an important position in head patterning and ion channel evolution, in addition to containing a rich source of toxins. In the present study, 2153 expressed sequence tags (ESTs) from the tentacle cDNA library of C. capillata were analyzed. The initial ESTs consisted of 198 clusters and 818 singletons, which revealed approximately 1016 unique genes in the data set. Among these sequences, we identified several genes related to head and foot patterning, voltage-dependent anion channel gene and genes related to biological activities of venom. Five kinds of proteinase inhibitor genes were found in jellyfish for the first time, and some of them were highly expressed with unknown functions.     

5-Azido neuraminic acid thioglycoside as sialylation donor

5-Azido neuraminic acid thioglycoside with O-benzyl protecting groups was synthesized. The sialylations of this new donor type showed good alpha-selectivities for certain primary hydroxyls.     

The effects of lysosomotropic agents on normal and INCL cells provide further evidence for the lysosomal nature of palmitoyl-protein thioesterase function

Fatty acylation of proteins on cysteine residues is a common post-translational modification that plays roles in protein-membrane and protein-protein interactions. Recently, we described a lysosomal palmitoyl-protein thioesterase that removes long-chain fatty acids from lipid-modified cysteine residues in proteins. Deficiency in palmitoyl-protein thioesterase results in a human lysosomal storage disorder, infantile neuronal ceroid lipofuscinosis (INCL), which primarily affects the central nervous system. The pathological hallmark of the disorder is the accumulation of granular osmiophilic deposits (GROD) that resemble lipofuscin, or aging pigment. In previous work, we have shown that [35S]cysteine-labeled lipid thioesters derived from fatty acylated proteins accumulate in cultured cells derived from palmitoyl-protein thioesterase-deficient patients. In the present work, we show that the lipid cysteine thioesters accumulate in the lysosomal fraction, and we further show that the appearance of these compounds in the organic phase is blocked by inhibitors of lysosomal proteolysis, demonstrating through biochemical means the lysosomal nature of the site of palmitoyl-protein thioesterase action. Furthermore, substrates for palmitoyl-protein thioesterase accumulate even in normal cells after leupeptin or chloroquine treatment. This was demonstrated by subjecting extracts of treated cells to exhaustive proteolysis to release protein-bound cysteine lipid for analysis. Cysteamine, a lysosomotropic drug recently proposed for the treatment of INCL, was found to have effects similar to leupeptin and chloroquine, suggesting that its mechanism of action may be more complex than previously understood.     

Axonal protein synthesis provides a mechanism for localized regulation at an intermediate target

As axons grow past intermediate targets, they change their responsiveness to guidance cues. Local upregulation of receptor expression is involved, but the mechanisms for this are not clear. Here protein synthesis is traced within individual axons by introducing RNAs encoding visualizable reporters. Individual severed axons and growth cones can translate proteins and also export them to the cell surface. As axons reach the spinal cord midline, EphA2 is among the receptors upregulated on at least some distal axon segments. Midline reporter upregulation is recapitulated by part of the EphA2 mRNA 3' untranslated region, which is highly conserved and includes known translational control sequences. These results show axons contain all the machinery for protein translation and cell surface expression, and they reveal a potentially general and flexible RNA-based mechanism for regulation localized within a subregion of the axon.