Disruption of energy metabolism and smoltification during exposure of juvenile atlantic salmon (Salmo salar) to low pH

Juvenile Atlantic salmon were held for 76 days at pH 4.7 during the period when the final stages of smoltification normally occur. Control salmon (pH 6.5) had significant increases in weight, length and liver somatic index which were not observed in those held at low pH. Condition factor decreased in both groups but to a significantly greater extent in the low pH group. After 15 days levels of ADP and glucose were higher and AEC, CP and glycogen were lower in muscles of salmon exposed to low pH. These qualitative differences were maintained until the end of the experiment. ATP and total adenylate concentrations in muscle were lower after 62 days of exposure to low pH compared to controls. The levels of ATP, total adenylates, AEC and glucose were consistently higher in livers of salmon exposed to low pH than those of controls. There were no differences in liver concentrations of AMP, CP and glycogen between the two treatments or with time of exposure within each treatment. The results suggest that exposure of juvenile Atlantic salmon to low pH increased gluconeogenesis and decreased food intake to the detriment of growth.     

Identification and purification of overlapping cosmid clones of the region Xq24-qter of human X chromosome using HPLC.

The assembly of a large physical map of genomes requires simultaneous analysis of many cosmid clones for overlapping regions. The search for overlapping regions may be achieved by various means. High-performance liquid chromatography (HPLC) provides an alternative to gel electrophoresis since microgram amounts of each DNA fragment may be collected into individual test tubes for further analysis. HPLC has been used to identify overlapping cosmid clones from a pool of cosmid DNA containing the terminal portion of the long arm of the human X chromosome (Xq24-qter). Among 400 cosmids analyzed, 3 were shown to overlap.     

Reconstitution of RecBC DNase activity from purified Escherichia coli RecB and RecC proteins

The Escherichia coli RecB protein, normally synthesized in low amounts, has been amplified by linkage of the recB gene to the phage lambda leftward promoter in an expression plasmid. From strains harboring this plasmid, RecB protein has been purified to homogeneity by a simple procedure which includes affinity chromatography on a column of RecC protein bound to agarose. The purified RecB protein has DNA-dependent ATPase activity but no exonuclease activity. RecC protein alone has neither ATPase nor exonuclease activity. However, when combined together, the RecB and RecC proteins show the ATP-dependent double-stranded exonuclease properties characteristic of the RecBC DNase.     

Xyloside transport by XylP, a member of the galactoside-pentoside-hexuronide family.

This paper describes the functional characterization of the xyloside transporter, XylP, of Lactobacillus pentosus with the aid of a spectroscopy-based assay system. In order to monitor the transport reaction, the natural xyloside isoprimeverose, a building block of hemicellulose, and the analogue methyl-isoprimeverose were chemically synthesized by a new and efficient procedure. The XylP protein was purified by metal affinity chromatography, following high level expression in Lactococcus lactis from the nisin-inducible promoter. The purified XylP protein was incorporated into liposomes, in which the glucose dehydrogenase from Acinetobacter calcoaceticus (sGDH) was entrapped. sGDH can oxidize aldose sugars in the presence of dichlorophenol-indophenol as electron acceptor. The coupled assay thus involves XylP-mediated isoprimeverose uptake followed by internal oxidation of the sugar by sGDH, which can be monitored from the reduction of 2,6-dichlorophenol-indophenol at 600 nm. The uptake of isoprimeverose was stimulated by the presence of the non-oxidizable methyl-isoprimeverose on the trans-side of the membrane, indicating that exchange transport is faster than unidirectional downhill uptake. Unlike other members of the galactoside-pentoside-hexuronide family, XylP does not transport monosaccharides (xylose) but requires a glycosidic linkage at the anomeric carbon position. Consistent with a proton motive force-driven mechanism, the uptake was stimulated by a membrane potential (inside negative relative to outside) and inhibited by a pH gradient (inside acidic relative to outside). The advantages of the here-described transport assay for studies of carbohydrate transport are discussed.     

Trypanosoma rhodesiense blood forms express all antigen specificities relevant to protection against metacyclic (insect form) challenge

Monoclonal antibodies were used to demonstrate the expression of four distinct metacyclic (infective insect form) trypanosome antigens on blood forms of T. rhodesiense. Metacyclic antigens were consistently expressed on the blood forms on days 4 and 5 of the first parasitemia after metacyclic infection of C57BL/6 mice. In different mice examined, the percent of blood forms expressing metacyclic antigens ranged from 46 to 85%. Immunization with irradiated day-5 blood form trypanosomes was protective against metacyclic challenge, indicating that all antigen specificities relevant to protective immunization against metacyclic challenge are expressed on blood form trypanosomes. Blood forms, in contrast to metacyclic forms, can be isolated in quantities sufficient for purification of antigens and genetic cloning.     

An assay for the detection of specific binding of 3-methylcholanthrene to rat liver cytosolic proteins using DEAE-cellulose

A method for the detection of the specific binding of 3-methylcholanthrene to rat liver cytosolic proteins is described. The separation of the protein-bound 3-methylcholanthrene from the free 3-methylcholanthrene was achieved using a batch DEAE-cellulose technique. Extraction of the DEAE-cellulose with 0.3 M KCl allowed the selective release and measurement of the amount of protein-bound 3-methylcholanthrene. The assay was optimized for the following parameters: time of incubation with DEAE-cellulose, time required for salt extraction, protein concentration, the concentration of KCl required to elute the specific binding proteins, the amount of DEAE-cellulose required to bind the specific binding proteins, and ligand specificity. The sedimentation properties of those 3-methylcholanthrene-binding proteins which were extracted with salt from DEAE-cellulose were examined on 5 to 20% sucrose gradients; the major binding species sedimented as a broad peak at 4.5 S.