Ultrastructural injury to human spermatozoa after freezing and thawing

The ultrastructure of human spermatozoa at various stages of the freezing and thawing process was studied. In addition to conventional fixations, a freeze-substitution method was used to examine spermatozoa before they were thawed. Dilution in a glycerol-egg yolk-citrate medium caused slight swelling of the acrosome. During slow freezing, when large ice crystals grow in the diluent, the sperm plasmalemma became tighter, the mitochondria had more angular profiles and there was a reduction in electron density of the acrosomal contents. After thawing, the apical segment of the acrosome usually became swollen and the mitochondria appeared rounded. We deduce that these ultrastructural changes occur either during or after the thawing procedure.     

Purification and properties of the soluble cytochromes c-550 and c-556 from the bacterium Aquaspirillum itersonii

Two c-type cytochromes were isolated from cells of the gram-negative bacterium Aquaspirillum itersonii grown under low aeration in the presence of nitrate. The major component, cytochrome c-550, was equated with the (single) c-type cytochrome previously reported to be present in this organism [Clark-Walker, G. D. & Lascelles, J. (1970) Arch. Biochem. Biophys. 136, 153-159], although a significantly higher molecular mass was apparent in the present work. The complete amino acid sequence of this cytochrome is reported in the accompanying paper. A second soluble c-type cytochrome, designated c-556, was also isolated. The molecular mass, isoelectric point, spectrum, midpoint oxidation reduction potential and amino acid composition of this monoheam cytochrome are reported. The possible relationship of this cytochrome to other cytochromes c-556 is discussed.     

Histamine H2 receptors on chondrocytes derived from human, canine and bovine articular cartilage.

Histamine (1-100 microM) induced a concentration-dependent increase in intracellular cyclic AMP in monolayer cultures of human, canine and foetal-bovine articular chondrocytes. The dose-response curve for histamine in each culture was progressively displaced to the right with increasing concentrations of cimetidine, an H2-receptor antagonist. The histamine-induced cyclic AMP elevation in human articular chondrocytes was also significantly decreased by ranitidine, another H2 antagonist, but not by the H1 antagonists mepyramine and chlorpheniramine. These findings indicate that histamine activates chondrocyte adenylate cyclase through an H2 receptor. The cyclic AMP response of human chondrocytes to histamine was many times greater than that measured for synovial fibroblasts under similar conditions. Such findings suggest that mast-cell-chondrocyte interactions in vivo may contribute to changed chondrocyte metabolism in joint disease.     

Reflectance and transmittance of light by leaves

Spectrophotometric transmittance and reflectance curves were recorded for wavelengths from 0.45 (in some cases 0.34) to 2.7 micrometers for faces and backs of leaves and for stacked leaves of several plant species. Measurements were made at different angles of illumination. Leaf spectrophotometric curves were compared with curves for leaf extracts, potato tuber tissue, glass beads in water, and frozen leaves to demonstrate the physical bases for the leaf curves. Leaves were infiltrated with liquids of different refractive indices for further comparison of spectrophotometric curves. Goniophotometric reflectance curves were recorded, giving visible reflectance and degree of polarization as functions of viewing angle for two different angles of illumination.     

Purification of rheumatoid synovial collagenase and its action on soluble and insoluble collagen.

1. The neutral collagenase released into the culture medium by explants of ehrumatoid synovial tissue has been purified by ultrafiltration and column chromatography, utilising Sephadex G-200, Sephadex QAE A-50 and Sephadex G-100 superfine. 2. The final collagenase preparation had a specific activity against thermally reconstituted collagen fibrils of 312 mug collagen degraded min-1 mg enzyme protein-1, representing more than a 1000-fold increase over that of the active culture medium. 3. Electrophoresis in polyacrylamide disc-gels with and without sodium dodecyl sulphate showed the enzyme to migrate as a single protein band. Elution experiments from polyacrylamide gels and chromatography columns have provided no evidence for the existence of more than one collagenase. 4. The molecular weight of the enzyme, as determined by dodecylsulphate-polyacrylamide gel electrophoresis, was 33000. 5. Data obtained from sutdies with the ion-exchange resin and from gel electrophoresis in acid and alkaline buffer systems suggested a basically charged enzyme. 6. It did not hydrolyse the synthetic collagen peptide Pz-Pro-Leu-Gly-Pro-D-Arg and non-specific protease activity was absent. 7. The collagenase attacked undenatured collagen in solution at 25 degrees C resulting in a 58% loss of viscosity and producing the two characteristic products TCA(3/4) and TCB(1/4). 8. At 37 degrees C and pH 8.0 both reconstituted collagen fibrils and gelatin were degraded to peptides of less than 10000 molecular weight. 9. As judged by the release of soluble hydroxyproline peptides and electron microscopic appearances the enzyme degraded human insoluble collagens derived from tendon and soft juxta-articular tissues although rates of attack were less than with reconstituted fibrils. 10. The data suggests that pure rheumatoid synovial collagenase at 37 degrees C and neutral pH can degrade gelatin, reconstituted fibrils and insoluble collagens without the intervention of non-specific proteases. 11. The different susceptibilities of various collagenous substrates to collagenase attack are discussed.     

Molecular Evidence for a Deep Clade of Dunnarts (Marsupialia: Dasyuridae: Sminthopsis)

Sminthopsis is the most speciose genus of living dasyurid marsupials and, along with its close relatives Antechinomys and Ningaui, constitutes the clade Sminthopsini. Phylogenetic relationships among the 23 species in this clade have been the subject of much morphological and molecular investigation, including a recent integration of penis morphology (in Sminthopsis) with molecular systematics. Several phylogenetic issues remain open, however, including the monophyly of Sminthopsis and branching order among early sminthopsin lineages. In this study, we revisit sminthopsin systematics with an expanded molecular data set, including new DNA sequences from mitochondrial (valine transfer-RNA and 16S ribosomal RNA) and nuclear (interphotoreceptor retinoid binding protein and beta-fibrinogen) loci, along with previously published sequences of cytochrome b, 12S ribosomal RNA, control region, and protamine P1. Our results again fail to establish the monophyly of Sminthopsis, but do provide a clearer resolution of early sminthopsin branching. Specifically, our phylogeny suggests three major groups of Sminthopsis species: S. longicaudata (perhaps the sister of Antechinomys); the Macroura species group of previous authors (S. crassicaudata, S. macroura, S. virginiae, S. douglasi, and S. bindi); and the remaining 13 species allied with the Murina species group. Our results depart from previous molecular findings by reuniting S. ooldea with the Murina group, while resolving S. psammophila as sister to the hairy-footed dunnarts (S. hirtipes and S. youngsoni). We suggest that this conflict traces to anomalous phylogenetic signal in previously published cytochrome b sequences. Penis morphology maps reasonably well onto our phylogeny, requiring parallel origination of only one of the ten morphotypes described for Sminthopsis.     

Copper uptake and translocation in chicory (Cichorium intybus L. cv Grasslands Puna) and tomato (Lycopersicon esculentum Mill. cv Rondy) plants grown in NFT system. II. The role of nicotianamine and histidine in xylem sap copper transport

The effect of rooting media Cu concentration (0.05–20 mg Cu L^-1) on amino acid concentrations and copper speciation in the xylem sap of chicory and tomato plants was measured using 6 week old plants grown in a nutrient film technique system (NFT). Irrespective of the Cu concentration in the nutrient solutions, more than 99.68% and 99.74% of total Cu in tomato and chicory xylem sap was in a bound form. When exposed to high Cu concentrations in the rooting media, amino acid concentrations in the sap increased. Relative to other amino acids, the concentrations of glutamine (Gln), histidine (His), asparagine (Asn), valine (Val), nicotianamine (NA) and proline (Pro) in tomato xylem saps, and His, γ-aminobutyric acid (Gaba), glutamic acid (Glu), leucine (Leu), NA and phenylalanine (Phe) in chicory xylem saps showed the greatest increases. The data indicate that induced synthesis of some free amino acids as a specific and proportional response to Cu treatment. For a single complexation amino acid, the solution Cu²⁺concentration vs pH titration curve for NA at 0.06–0.07 mM was most similar, closely followed by His at 0.5–0.6 mM, to the solution Cu²⁺concentration behaviour in both tomato and chicory xylem sap. It is concluded that increased Cu concentrations in the rooting media induced selective synthesis of certain amino acid which include NA, His, Asn and Gln which have high stability constants with Cu. NA and His have the highest binding constants for Cu and the concentrations of NA and His in chicory and tomato xylem saps can account for all the bound Cu carried in the sap. This revised version was published online in June 2006 with corrections to the Cover Date.