Free and polymerized tubulin in cultured bone cells and Chinese hamster ovary cells: the influence of cold and hormones

A low pH method of liposome-membrane fusion (Schneider et al., 1980, Proc. Natl. Acad. Sci. U. S. A. 77:442) was used to enrich the mitochondrial inner membrane lipid bilayer 30-700% with exogenous phospholipid and cholesterol. By varying the phospholipid-to- cholesterol ratio of the liposomes it was possible to incorporate specific amounts of cholesterol (up to 44 mol %) into the inner membrane bilayer in a controlled fashion. The membrane surface area increased proportionally to the increase in total membrane bilayer lipid. Inner membrane enriched with phospholipid only, or with phospholipid plus cholesterol up to 20 mol %, showed randomly distributed intramembrane particles (integral proteins) in the membrane plane, and the average distance between intramembrane particles increased proportionally to the amount of newly incorporated lipid. Membranes containing between 20 and 27 mol % cholesterol exhibited small clusters of intramembrane particles while cholesterol contents above 27 mol % resulted in larger aggregations of intramembrane particles. In phospholipid-enriched membranes with randomly dispersed intramembrane particles, electron transfer activities from NADH- and succinate-dehydrogenase to cytochrome c decreased proportionally to the increase in distance between the particles. In contrast, these electron- transfer activities increased with decreasing distances between intramembrane particles brought about by cholesterol incorporation. These results indicate that (a) catalytically interacting redox components in the mitochondrial inner membrane such as the dehydrogenase complexes, ubiquinone, and heme proteins are independent, laterally diffusible components; (b) the average distance between these redox components is effected by the available surface area of the membrane lipid bilayer; and (c) the distance over which redox components diffuse before collision and electron transfer mediates the rate of such transfer.     

A study of functionally active amino acids involved in the interaction of HLA-A2 or HLA-A3 molecules with cytolytic T lymphocytes

A large series of HLA-A2/HLA-A3 recombinant genes were generated by using the in vivo recombination technique. These genes have each been modified in the last two-thirds of the third exon such that one or several HLA-A2-specific substitutions have been made in the HLA-A3 gene and vice versa. The recombinant genes were transfected into the murine cell line P815 and the transfectants were used as targets for a series of 20 human CTL lines or clones specific for HLA-A2 or HLA-A3, or restricted by HLA-A2 and specific for influenza A. Several patterns of anti-HLA-A2, anti-HLA-A3, and HLA-A2-restricted anti-influenza CTL activity were observed and when uncloned cell lines were studied, a progressive selection of some clones with a similar pattern of activity was regularly found. From the comparison of these different patterns the following conclusions can be drawn: 1) In most but not all cases both domains of the class I molecule were essential for CTL recognition, but residue 152 was critically important for the majority of CTL tested; 2) amino acids 114/116 were also critical in most cases, and their position close to amino acid 152 in the tertiary structure of the molecule may have some functional significance; and 3) amino acid 161, although highly conserved, plays an unexpected but very important role in CTL function.     

Molecular Analysis of the P-M Gonadal Dysgenesis Cline in Eastern Australian Drosophila Melanogaster

The latitudinal cline in P-M gonadal dysgenesis potential in eastern Australia has been shown to comprise three regions which are, from north to south respectively, P, Q, and M, with the P-to-Q and Q-to-M transitions occurring over relatively short distances. The P element complements of 30 lines from different regions of the cline were determined by molecular techniques. The total amount of P element-hybridizing DNA was high in all lines, and it did not correlate in any obvious way with the P-M phenotypes of individual lines. The number of potentially full-sized P elements per genome was high in lines from the P regions, but variable or low among lines from the Q and M regions, and thus declined overall from north to south. A particular P element deletion-derivative, the KP element, occurred in all the tested lines. The number of KP elements was low in lines from the P region, much higher in lines from the Q region, and highest among lines from the M region, thus forming a cline reciprocal to that of the full-sized P elements. Another transposable element, hobo, which has been described as causing dysgenic traits similar to those of P-M hybrid dysgenesis, was shown to be present in all lines and to vary among them in number, but not in any latitudinal pattern. The P-M cline in gonadal dysgenesis potential can be inferred to be based on underlying clinal patterns of genomic P element complements. P activity of a line was positively correlated with the number of full-sized P elements in the line, and negatively correlated with the number of KP elements. Among Q and M lines, regulatory ability was not correlated with numbers of KP elements.     

Oligochaeta in Spartina stems: the microdistribution of Enchytraeidae and Tubificidae in a salt marsh,Sapelo Island,USA

The distribution and abundance of Enchytraeidae and Tubificidae in and around Spartina alterniflora plants in a tidal salt marsh on Sapelo Island, Georgia, USA were studied using two different sampling techniques: wet funnel extraction and stem dissection. At least 80% of all worms inhabited leaf sheaths at the bases of S. alterniflora plants, and densities were low in sediment, root and surface debris samples. Oligochaete densities were dependent on the position within the marsh, the height on stems and the stage of sheath decay. Six predominant species were identified and included Marionina appendiculata, Marionina spartinae, Marionina waltersi, Marionina paludis, and Monopylephorus parvus. Individual species were distributed differently on stems and enchytraeids were more common than tubificids on standing-dead and further up S. alterniflora stems. Estimates of oligochaete densities in salt marsh habitats are increased dramatically when the numbers of worms on stems are considered. Possible advantages of the stem microhabitat are discussed in relation to the biology and ecology of oligochaetes.     

The importance of fixation procedures on DNA template and its suitability for solution-phase polymerase chain reaction and PCR in situ hybridization

Summary Conventional solution-phase polymerase chain reaction (PCR) and in situ PCR/PCR in situ hybridization are powerful tools for retrospective analysis of fixed paraffin wax-embedded material. Amplification failure using these techniques is now encountered in some centres using archival fixed tissues. Such ailures may not only be due to absent target DNA sequences in the tissues, but may be a direct effect of the type of fixative, fixation time and/or fixation temperature used. The type of nucleic acid extraction procedure applied will also influence amplification results. This is particularly true with in situ PCR/PCR in situ hybridization.To examine these effects in solution-phase PCR, -globin gene was amplified in 100 mg pieces of tonsillar tissue fixed in Formal saline, 10% formalin, neutral buffered formaldehyde, Carnoy's, Bouin's, buffered formaldehyde sublimate, Zenker's, Helly's and glutaraldehyde at 0 to 4°C, room temperature and 37°C fixation temperatures and for fixation periods of 6, 24, 48 and 72 hours and 1 week. DNA extraction procedures used were simple boiling and 5 days' proteinase K digestion at 37°C. Amplified product was visible primarily yet variably from tissue fixed in neutral buffered formaldehyde and Carnoy's, whereas fixation in mercuric chloride-based fixatives produced consistently negative results. Room temperature and 37°C fixation temperature appeared most conducive to yielding amplifiable DNA template. Fixation times of 24 and 48 hours in neutral buffered formaldehyde and Carnoy's again favoured amplification.Fixed SiHa cells (containing 1–2 copies of HPV 16) were examined using PCR in situ hybridization for the amplification of HPV 16. Discrete and diffuse amplification signals were obtained. Neutral buffered formaldehyde fixation for 12–24 hours yielded amplifiable material suitable for use with PCR in situ hybridization. Overall amplification success within cellular preparations was 40%, with non-specific background staining also seen. Possible technical problems encountered with PCR in situ hybridization are discussed.