A streptomycin-resistant Escherichia coli mutant with ribosomes temperature-sensitive in the suppression of a nonsense codon.

Summary Cell free extracts from a streptomycin-resistant E. coli mutant which is also temperature-sensitive for Q phage were studied for suppression of a nonsense mutation at various temperatures. The streptomycin-resistant ribosomes of the mutant were found to be temperature-sensitive in suppression of an amber mutation in f2 phage coat protein while retaining the ability to synthesize proteins at an elevated temperature (42° C). The restriction of amber suppression at 42° C is assumed to be related to an alteration in the ribosomal protein S12 of the streptomycin-resistant mutant which also causes a change in its electrophoretic mobility.     

Outer membrane protein a and other polypeptides regulate capsular polysaccharide synthesis in E. coli K-12.

Summary capR (lon) mutants of Escherichia coli K-12 are mucoid on minimal agar because they produce large quantities of capsular polysaccharide. When such mutants are transformed to tetracycline resistance by plasmid pMC44, a hybrid plasmid that contains a 2 megadalton (Mdal) endonuclease EcoR1 fragment of E. coli K-12 DNA joined to the cloning vehicle-pSC101, capsular polysaccharide synthesis is inhibited and the transformed colonies exhibit a nonmucoid phenotype. Re-cloning of the 2 Mdal EcoR1 fragment onto plasmid pHA105, a min-colE1 plasmid, yielded plasmid pFM100 which also inhibited capsular polysaccharide synthesis in the capR mutants. A comparison of the polypeptides specified by both plasmids pFM100 and pMC44 in minicells demonstrated that seven polypeptide bands were specified by the 2 Mdal DNA, one of which was previously demonstrated to be outer membrane protein a; also known as 3b or M2 (40 kilodaltons, Kdal). Plasmid mutants no longer repressing capsular polysaccharide synthesis were either unable to specify the 40 K dal outer membrane protein a or were deficient in synthesis of 25 K dal and 14.5 K dal polypeptides specified by the 2 Mdal DNA fragment. Studies with a minicell-producing strain that also contained a capR mutation indicated that the capR gene product regulated processing of at least one normal protein, the precursor of outer membrane protein a.     

Large scale, analytical method for isolating basal lateral plasma membranes from rat duodenum.

A procedure is described for obtaining large amounts of basal lateral plasma membranes from the rat duodenal epithelium. The yield is approximately 50%, and the purification factor is 18; preparations from 25 rats routinely contain 100 mg of protein. The procedure depends on mild homogenization with a nitrogen cavitation bomb, followed by removal of brush borders by sedimentation in a weak centrifugal field. Basal lateral membranes in the resulting supernatant are partially purified by differential centrifugation in a medium which approximates their equilibrium density, and then further purified by equilibrium density gradient centrifugation in a high capacity zonal rotor. Brush border membranes may be isolated from the 450 x g pellet. Since both brush border and basal lateral membranes may be isolated from the same homogenate, this preparative procedure is suitable for such analytical purposes as determinations of distribution of enzyme activities between the two surfaces of the epithelium. The large scale of the isolation procedure makes it an appropriate starting point for purification of specific basal lateral membrane components.     

Functional disturbances in brain following injury

It was shown previously that local cerebral glucose utilization is less than 50% of normal in all cortical areas of rat brain 3 days following a focal freeze-lesion and that this effect of trauma is significantly diminished by dexamethasone (0.25 mg/Kg/day), and by indomethacin (7.5 mg/Kg single dose). To elucidate the mechanism of action of steroids and non-steroidal antiinflammatory drugs in traumatized brain, the effects of dexamethasone and indomethacin on arachidonic acid release, malondialdehyde production and prostaglandin synthesis in the lesion area were investigated. Five seconds after a freezing lesion arachidonic acid was significantly increased in the lesion area of untreated animals. Neither dexamethasone nor indomethacin had any effect on this release. The thiobarbituric acid reaction, as an estimate of malondialdehyde and non-enzymatic free radical lipoperoxide formation from unsaturated free fatty acids showed no change in the control and lesion areas of untreated and both dexamethasone and indomethacin treated groups. There was a marked increase in PGF2 alpha, PGE2, PGD2 in the lesion area of untreated animals. Indomethacin prevented the formation of prostaglandins by more than 90% while dexamethasone had no effect. These results suggest that some components of the arachidonic acid metabolism must be involved in functional disturbances resulting from trauma while steroid action is mediated in injured brain independently from the prostaglandin cascade.     

Ultrastructural and histochemical observations on the tegument of Gastrodiscoides hominis (Paramphistoma: Digenea).

The tegument of the paramphistome, Gastrodiscoides hominis, is basically similar to that of other digeneans. It is folded into concentrically arranged furrows and ridges bearing numerous tightly packed tubercules, and extends into the oral cavity. An area of specialized tegument is present on the ventral surface, anterior to the disc region. Mitochondria are absent from the tegumental syncytium and underlying tegumental cells, suggesting that the tegument may serve principally as a protective layer rather than in active uptake phenomena. However, extensions of the lymph and parenchyma systems are closely associated with the base of the tegumental syncytium and may provide ATP for active processes. Ciliated and non-ciliated sensory papillae are present, particularly around the oral opening. Numerous lymph channels are present in the sub-tegument and may be involved in osmoregulation.     

Studies on the ultrastructure and histochemistry of the lymph system of Gastrodiscoides hominis (Paramphistoma: Digenea).

The lymphatic system of the paramphistome, Gastrodiscoides hominis consists of numerous fluid-filled branches embedded in parenchyma and surrounded by extracellular material and is closely associated with the major organ systems of the fluke. The lymph matrix consists of a cytoplasmic syncytium within which nuclei, mitochondria and various sized granules and membranous structures occur. The granules found throughout the lymph system morphologically resemble autophagosomes and lysosomes. The lymph system provides a storage site for proteins which can be broken down to amino acids via autophagy, for subsequent mobilization and transport to tissues undergoing active protein synthesis. Many branches of the lymph system are surrounded by specialized parenchymal cells referred to as juxta-lymphatic cells. These cells are apparently associated with autophagic degradation of sequestered lymph cytoplasm, which may serve as an additional mechanism for the mobilization and transport of precursor molecules throughout the fluke via the parenchymal network.     

Ultrastructural observations on the redial tegument of Paramphistomum epiclitum from the planorbid snail, Indoplanorbis exustus.

The morphology of the tegument in the redia of Paramphistomum epiclitum (Digenea: Paramphistomidae) resembles that shown by most larval and adult digeneans; an outer surface syncytium is in continuity with the cytoplasm of in-sunken, nucleated cytons. Although tegumental cytons usually contain a single nucleus, some display up to six nuclei. The tegumental syncytium lining the pharynx of P. epiclitum rediae lack underlying cytons. The apical membrane of the tegument is elaborated by folds and microvilli, which presumably facilitate uptake of nutrients and/or exchange of ions involved in osmoregulation. A single type of secretory body, resulting from the fusion of smaller vesicles produced at Golgi complexes in the cytons, occurs throughout the tegument. Uniciliate sensory receptors occur in the surface syncytium particularly around the oral opening.     

Detection apparatus for multiple heterogeneous chemiluminescence immunoassay configurations

We describe an apparatus for measuring signals emanated from two heterogeneous chemiluminescence immunoassay (CLIA) configurations: antibody-coated polystyrene beads, in reaction tray wells, and microparticles captured by a porous matrix. An optics and fluidics design which allows the use of a common detection head for these two different assay configurations is described. The detection head moves along three Cartesian coordinates to create a localized light-tight compartment around each individual disposable reaction vessel. Reproducibility of the light seal, trigger solution delivery, and mixing is achieved for acridinium-labeled CLIA. The coated polystyrene beads configuration is tested using beta HCG, CEA, and TSH assays. The microparticle-capture configuration is tested using beta HCG and HBsAg assays. The microparticle capture CLIA has shorter incubation times and the potential for ease of automation.