Environmental stress and bacterial infection in channel catfish, Ictalurus punctatus Rafinesque

Channel catfish, Ictalurus punctatus , were injected intraperitoneally with a sublethal dose of Aerornonas hydrophila and then stressed for 144 h by being maintained either in a dissolved oxygen concentration of 1·5 mg/1, 1·2 mg/1 total ammonia, and/or 6·5 mg/1 free CO₂ with a continuous inflow of water. A significant difference in percentage of mortality was noted between treatments ( P < 0·05). The trunk kidneys of surviving stressed fish had significantly higher total bacterial counts than non-stressed controls. A. hydrophila was isolated from 67% of the stressed fish and 9% of the control fish. Edwardsiella tarda , apparently endemic in the population, was isolated from 43% of the stressed fish and 7% of the control fish. Histopathological lesions were in the gills, liver, spleen, trunk kidney, and head kidney of stressed fish, but not control fish.     

Use of generic fast gradient liquid chromatography–tandem mass spectroscopy in quantitative bioanalysis

Short narrow analytical HPLC columns have been used successfully with high linear flow-rates and combined with mass spectrometric detection to produce a generic approach to quantitative bioanalysis. The approach has been used to validate several assays in the low ng/ml region and an example is given in this paper. When combined with a simple solid-phase extraction process the need for complicated, time consuming method development has been removed for the majority of pharmaceutical compounds. The approach takes advantage of not only the extra selectivity of the MS–MS detector but the excellent resolution and peak shape produced by gradient elution.     

Antioxidant Properties of the Major Polyphenolic Compounds in Broccoli

We have examined the antioxidant activity of the major phenolic compounds in Broccoli: two flavonol glycosides (quercetin 3-O-sophoroside and kaemp-ferol 3-O-sophoroside) and four hydroxycinnamic acid esters (1,2'-disinapoyl-2-feruloyl gentiobiose, 1-sinapoyl-2-feruloyl gentiobiose, 1,2,2'-trisinapoyl gentiobiose and 1,2-disinapoyl gentiobiose). The Trolox C equivalent antioxidant capacity (TEAC) and inhibition of iron/ascorbate-induced lipid per-oxidation of phosphatidyl choline vesicles were measured. In the aqueous phase TEAC assay, the two flavonol glycosides were less active than their respective aglycones. TEAC values for the hydroxycinnamic acid esters were less than the sum of their constituent hydroxycinnamic acids on a molar basis. Quercetin 3-O-sophoroside was a potent inhibitor of lipid peroxidation, in contrast to kaempferol 3-O-sophoroside. The hydroxycinnamic acid esters were highly effective at preventing lipid damage with the exception of 1,2,2'-trisinapoyl gentiobiose. The six compounds analysed herein demonstrate the antioxidant activity of the major phenolics in broccoli and indicate the effect on antioxidant activity of sugar substitutions in the phenolic B ring.     

Resolution of pea legumin subunits by high-performance liquid chromatography

Pea legumin was dissociated into its component subunits by 6 M urea: these were subsequently fractionated by FPLC using a combination of Mono P, Mono Q, and Mono S columns. The resolution and speed of separation were greatly improved in comparison with previous fractionations. Twelve discrete fractions were obtained and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Six "normal" legumin subunits (Mr 60,000) were identified as well as some "large" (Mr 66,000) and "small" (Mr 44,000) subunits. A few polypeptides of unknown origin were also observed. Four subunits were purified to homogeneity as adjudged by electrophoresis and HPLC and in sufficient yields to permit further studies. Anomalous electrophoretic behavior of the legumin subunits was also observed.     

High resolution "ultra performance" liquid chromatography coupled to oa-TOF mass spectrometry as a tool for differential metabolic pathway profiling in functional genomic studies

The combination of a new 1.7 mum reversed-phase packing material, and a chromatographic system, operating at ca. 12,000 psi, (so-called ultra performance liquid chromatography, UPLC) has enabled dramatic increases in chromatographic performance to be obtained for complex mixture separation. This increase in performance is manifested in improved peak resolution, together with increased speed and sensitivity. Here, we show that UPLC offers significant advantages over conventional reversed-phase HPLC amounting to a more than doubling of peak capacity, an almost 10-fold increase in speed and a 3- to 5-fold increase in sensitivity compared to that generated with a conventional 3.5 microm stationary phase. The first functional genomic application of UPLC-MS technology is illustrated here with respect to multivariate metabolic profiling of urines from males and females of two groups of phenotypically normal mouse strains (C57BL19J and Alpk:ApfCD) and a "nude mouse" strain. We have also compared this technology to conventional HPLC-MS under similar analytical conditions and show improved phenotypic classification capability of UPLC-MS analysis together with increased ability to probe differential pathway activities between strains as a result of improved analytical sensitivity and resolution.     

The purification of an erythroid protein which binds to enhancer and promoter elements of haemoglobin genes.

An erythroid nuclear protein (EF1), originally detected as a protein binding within the nuclease hypersensitive site upstream of the chicken beta H-globin gene, has been purified. This protein of 37,000-39,000 molecular weight binds to three sites within the hypersensitive region: one between the CCAAT and TATA boxes, the second (further upstream) next to a NF1 binding site, and the third adjacent to a regulatory element found in a number of beta-globin genes. The EF1 protein also binds to an erythroid-specific promoter element of the mouse alpha-globin gene and to two sites within the chicken beta A-globin enhancer. These six EF1-binding sites are related by the consensus sequence A/TGATAA/GG/C. A minor protein of molecular weight 72,000 which co-purifies with EF1 also binds to the same sequences.     

Binding of human complement C8 to C9: role of the N-terminal modules in the C8 alpha subunit

Human C8 is one of five components of the membrane attack complex of complement (MAC). It is composed of a disulfide-linked C8alpha-gamma heterodimer and a noncovalently associated C8beta chain. The C8alpha and C8beta subunits contain a pair of N-terminal modules [thrombospondin type 1 (TSP1) + low-density lipoprotein receptor class A (LDLRA)] and a pair of C-terminal modules [epidermal growth factor (EGF) + TSP1]. The middle segment of each protein is referred to as the membrane attack complex/perforin domain (MACPF). During MAC formation, C8alpha mediates binding and self-polymerization of C9 to form a pore-like structure on the membrane of target cells. In this study, the portion of C8alpha involved in binding C9 was identified using recombinant C8alpha constructs in which the N- and/or C-terminal modules were either exchanged with those from C8beta or deleted. Those constructs containing the C8alpha N-terminal TSP1 or LDLRA module together with the C8alpha MACPF domain retained the ability to bind C9 and express C8 hemolytic activity. By contrast, those containing the C8alpha MACPF domain alone or the C8alpha MACPF domain and C8alpha C-terminal modules lost this ability. These results indicate that both N-terminal modules in C8alpha have a role in forming the principal binding site for C9 and that binding may be dependent on a cooperative interaction between these modules and the C8alpha MACPF domain.     

A novel and highly conserved collagen (pro(alpha)1(XXVII)) with a unique expression pattern and unusual molecular characteristics establishes a new clade within the vertebrate fibrillar collagen family

The type XXVII collagen gene codes for a novel vertebrate fibrillar collagen that is highly conserved in man, mouse, and fish (Fugu rubripes). The pro(alpha)1(XXVII) chain has a domain structure similar to that of the type B clade chains (alpha1(V), alpha3(V), alpha1(XI), and alpha2(XI)). However, compared with other vertebrate fibrillar collagens (types I, II, III, V, and XI), type XXVII collagen has unusual molecular features such as no minor helical domain, a major helical domain that is short and interrupted, and a short chain selection sequence within the NC1 domain. Pro(alpha)1(XXVII) mRNA is 9 kb and expressed by chondrocytes but also by a variety of epithelial cell layers in developing tissues including stomach, lung, gonad, skin, cochlear, and tooth. By Western blotting, type XXVII antisera recognized multiple bands of 240-110 kDa in tissue extracts and collagenous bands of 150-140 kDa in the conditioned medium of the differentiating chondrogenic ATDC5 cell line. Phylogenetic analyses revealed that type XXVII, together with the closely related type XXIV collagen gene, form a new, third clade (type C) within the vertebrate fibrillar collagen family. Furthermore, the exon structure of the type XXVII collagen gene is similar to, but distinct from, those of the genes coding for the type A or B clade pro(alpha) chains.