High-density polymerase-mediated incorporation of fluorochrome-labeled nucleotides

DNA-polymerase-mediated incorporation of different fluorochrome-labeled nucleotides (FdNTPs) was investigated with the goals of optimizing the high-density labeling of probes and exploring DNA sequencing strategies that rely on the controlled, sequential addition of such compounds. By systematically evaluating variables--including polymerase type, buffer conditions, and fluorochrome chemistries--a rational strategy for the sequential addition of labeled nucleotides to a DNA template was demonstrated. A simple structural model of the polymerase-DNA template complex that considered the fluorochrome moiety of the FdNTPs and the linker length also guided this strategy. Complementary results that portend the use of simple photobleaching to enable the reliable quantitation of consecutive additions are presented.     

Impaired post-thymic development of regulatory CD4+25+ T cells contributes to diabetes pathogenesis in BB rats

One of the BB rat diabetes (diabetes mellitus (DM)) susceptibility genes is an Ian5 mutation resulting in premature apoptosis of naive T cells. Impaired differentiation of regulatory T cells has been suggested as one possible mechanism through which this mutation contributes to antipancreatic autoimmunity. Using Ian5 congenic inbred rats (wild-type (non-lyp BB) and mutated (BB)), we assessed the development of BB regulatory CD8(-)4(+)25(+)T cells and their role in the pathogenesis of DM. BB rats have normal numbers of functional CD8(-)4(+)25(+)Foxp3(+) thymocytes. The proportion of CD25(+) cells among CD8(-)4(+) recent thymic emigrants is also normal while it is increased among more mature CD8(-)4(+) T cells. However, BB CD8(-)4(+)25(+)Foxp3(+) thymocytes fail to undergo homeostatic expansion and survive upon transfer to nude BB rats while Foxp3 expression is reduced in mature CD8(-)4(+)25(+) T cells suggesting that these cells are mostly activated cells. Consistent with this interpretation, peripheral BB CD8(-)4(+)25(+) T cells do not suppress anti-TCR-mediated activation of non-lyp BB CD8(-)4(+)25(-) T cells but rather stimulate it. Furthermore, adoptive transfer of unfractionated T cells from diabetic BB donors induces DM in 71% of the recipients while no DM occurred when donor T cells are depleted of CD8(-)4(+)25(+) cells. Adoptive transfer of 10(6) regulatory non-lyp BB CD8(-)4(+)25(+) T cells to young BB rats protects the recipients from DM. Taken together, these results demonstrate that the BB rat Ian5 mutation alters the survival and function of regulatory CD8(-)4(+)25(+) T cells at the post-thymic level, resulting in clonal expansion of diabetogenic T cells among peripheral CD8(-)4(+)25(+) cells.     

Efficient high performance liquid chromatograph/ultraviolet method for determination of diclofenac and 4'-hydroxydiclofenac in rat serum

A rapid and sensitive high-performance liquid chromatographic method was developed for determination of diclofenac and its major metabolite, 4'-hydroxydiclofenac, in serum from rats treated with diclofenac. The method is simple with a one-step extraction procedure, isocratic HPLC separation, and UV detection at 280 nm. Use of N-phenylanthranilic acid as the internal standard provided good accuracy without interference by endogenous compounds or 5-hydroxydiclofenac, another metabolite of interest. Limits of detection for diclofenac and 4'-hydroxydiclofenac were 0.0225 and 0.0112 microg/ml, respectively. Average extraction efficiencies of diclofenac, 4'-hydroxydiclofenac, and the internal standard were >/=76%. The method was applied to serum collected at 3h after rats were treated with an experimentally useful dosage range of 3, 10 and 50mg/kg diclofenac. Recovery (as a percentage of dose) for the 4'-hydroxy metabolite in serum was found to consistently average from 0.10 to 0.12% following each dosage, whereas recovery of diclofenac in serum declined from 0.45 to 0.37%. Thus, the method is suitable for measurement of a major diclofenac metabolite in experimental studies.     

A novel functional module detection algorithm for protein-protein interaction networks

Background  

The sparse connectivity of protein-protein interaction data sets makes identification of functional modules challenging. The purpose of this study is to critically evaluate a novel clustering technique for clustering and detecting functional modules in protein-protein interaction networks, termed STM.     

Probing 2-dimensional protein-protein interactions on model membranes

This protocol describes an in vitro approach for measuring the kinetics and affinities of interactions between membrane-anchored proteins. This method is particularly established for dissecting the interaction dynamics of cytokines with their receptor subunits. For this purpose, the receptor subunits are tethered in an orientated manner onto solid-supported lipid bilayers by using multivalent chelator lipids. Interaction between the ligand with the receptor subunits was probed by a combination of surface-sensitive spectroscopic detection techniques. Label-free detection by reflectance interferometry is used for following assembly of the membrane and tethering of the receptor subunits in quantitative terms. Total internal reflection spectroscopy is used for monitoring ligand binding to the membrane-anchored receptor, for monitoring ligand-receptor interactions by FRET and for monitoring ligand-exchange kinetics. These assays can be used for determining the affinities and stabilities of ligand-receptor complexes in plane of the membrane. The techniques described in this protocol can be established in 2-3 months.     

Synthesis of a multivalent chelator lipid for stably tethering histidine-tagged proteins onto membranes

This protocol describes the synthesis of a lipid-like molecule carrying a head group containing two nitrilotriacetic acid moieties. This multivalent chelator lipid can be incorporated into lipid membranes, to which histidine-tagged protein can then be tethered in an oriented fashion. Possible applications of this lipid are protein tethering to solid-supported membranes, to lipid vesicles or to live cells. As compared to conventional monovalent chelator lipids, this lipid can achieve highly stable tethering of proteins by the multivalent chelator head. The eight-step synthesis described in this protocol can be completed within 4-5 weeks.     

Flap Endonuclease 1 Mechanism Analysis Indicates Flap Base Binding Prior to Threading

FEN1 cleaves 5′ flaps at their base to create a nicked product for ligation. FEN1 has been reported to enter the flap from the 5′-end and track to the base. Current binding analyses support a very different mechanism of interaction with the flap substrate. Measurements of FEN1 binding to a flap substrate show that the nuclease binds with similar high affinity to the base of a long flap even when the 5′-end is blocked with biotin/streptavidin. However, FEN1 bound to a blocked flap is more sensitive to sequestration by a competing substrate. These results are consistent with a substrate interaction mechanism in which FEN1 first binds the flap base and then threads the flap through an opening in the protein from the 5′-end to the base for cleavage. Significantly, when the unblocked flap length is reduced from five to two nucleotides, FEN1 can be sequestered from the substrate to a similar extent as a blocked, long flap substrate. Apparently, interactions related to threading occur only when the flap is greater than two to four nucleotides long, implying that short flaps are cleaved without a threading requirement.     

Biochemical and molecular characterization of a rhizobitoxine-producing <Emphasis Type="Italic">Bradyrhizobium</Emphasis> from pigeon pea plants

Out of a total of 8 bacterial strains isolated from the root nodules of pigeon pea plants grown in arid region, five were identified as rhizobia based on biochemical test and confirmed by 16S rDNA sequencing. PCR based screening for the rtxA gene (involved in biosynthesis of rhizobitoxine) revealed that the gene was present in one strain identified biochemically and genetically as belonging to species Bradyrhizobium (BS KT-24). The strain was resistant to phosphomycin, nalidixic acid, kanamycin, gentamicin and neomycin but sensitive towards streptomycin and spectinomycin. Bioinformatic-tool-guided phylogenetic analysis of rtxA gene revealed its distinctiveness from other known rtxA genes (present in B. japonicum, B. elkanii and Xanthomonas oryzae). The rhizobitoxine producing strain BS KT-24 is considered to exhibit better survival and nodulation protection besides competitiveness for pigeon pea and other legumes grown under abiotic stress and, thus, be a candidate in practical aspect of rhizobitoxine production by rhizobium and its application as rhizobial inoculants.     

Mineralization of 4-aminobenzenesulfonate (4-ABS) by Agrobacterium sp. strain PNS-1

A bacterial strain, PNS-1, isolated from activated sludge, could utilize sulphanilic acid (4-ABS) as the sole organic carbon and energy source under aerobic conditions. Determination and comparison of 16S r DNA sequences showed that the strain PNS-1 is closely related to the species of Agrobacterium genus. Growth on 4-ABS was accompanied with ammonia and sulfate release. TOC results showed complete mineralization of sulphanilic acid. This strain was highly specific for 4-ABS as none of the sulphonated aromatics used in the present study including other ABS isomers were utilized. Strain PNS-1 could, however, utilize all the tested monocyclic aromatic compounds devoid of a sulfonate group. No intermediates could be detected either in the growth phase or with dense cell suspensions. Presence of chloramphenicol completely inhibited 4-ABS degradation by cells pregrown on succinate, indicating that degradation enzymes are inducible. No plasmid could be detected in the Agrobacterium sp. Strain PNS-1 suggesting that 4-ABS degradative genes may be chromosomal encoded.