Invasion status of Florida bass Micropterus floridanus (Lesueur, 1822) in South Africa

Largemouth bass Micropterus salmoides are a popular North American angling species that was introduced into South Africa in 1928. To enhance the largemouth bass fisheries, Florida bass Micropterus floridanus were introduced into KwaZulu Natal, South Africa, in 1980. Knowledge on the status of M. floridanus in South Africa is required, because it lives longer and reaches larger sizes than M. salmoides, which may result in heightened impacts on native biota. Because M. floridanus are morphologically similar, but genetically distinct from M. salmoides, the distribution of this species was assessed by genetically screening 185 Micropterus sp. individuals sampled from 20 localities across South Africa using the mitochondrial ND2 gene. Individuals with mitochondrial DNA matching M. salmoides were recovered from 16 localities, whereas M. floridanus mitochondrial DNA was recovered from 13 localities. At nine localities (45%), the mitochondrial DNA of both species was detected. These results demonstrate M. floridanus dispersal to multiple sites across South Africa.     

Subcellular distribution of superoxide dismutases in human neutrophils. Influence of myeloperoxidase on the measurement of superoxide dismutase activity

We have identified two distinct pools of superoxide dismutase in fractions of human peripheral neutrophils obtained by the isopycnic fractionation of homogenates of the latter with linear sucrose gradients. Superoxide dismutase activity, observed with polyacrylamide gels impregnated with Nitro Blue Tetrazolium, was present in: (1) the mitochondrial fraction [density (rho) 1.169g/ml], containing the high-molecular-weight KCN-resistant enzyme, and (2) the cytoplasm fraction, containing the low-molecular-weight KCN-sensitive enzyme. Superoxide dismutase activity, observed with a quantitative assay involving cytochrome c, was present in: (1) the mitochondria, (2) the cytoplasm, and (3) the azurophil-granule fractions (rho=1.206 and 1.222g/ml). No substantial enzyme activity was observed in specific-granule fractions (rho=1.187g/ml) or in the membranous fraction (rho=1.136g/ml) in either assay. The apparent superoxide dismutase activity observed in the azurophil granules with the cytochrome c assay was attributable not to true superoxide dismutase but to myeloperoxidase, an enzyme found solely in the azurophil granules. In the presence of H(2)O(2), human neutrophil myeloperoxidase oxidized ferrocytochrome c. Thus, in the cytochrome c assay for superoxide dismutase, the oxidation of ferrocytochrome c by myeloperoxidase mimicked the inhibition of reduction of ferricytochrome c by superoxide dismutase. When myeloperoxidase was removed from azurophilgranule fractions by specific immuno-affinity chromatography, both myeloperoxidase and apparent superoxide dismutase activities were removed. It is concluded that there is no detectable superoxide dismutase in either the azurophil or specific granules of human neutrophils. Mitochondrial superoxide dismutase, 15% of the total dismutase activity of the cells, occurred only in fractions of density 1.160g/ml, where isocitrate dehydrogenase and cytochrome oxidase were also observed.     

Schmid's metaphyseal chondrodysplasia mutations interfere with folding of the C-terminal domain of human collagen X expressed in Escherichia coli.

Human collagen X contains a highly conserved 161-amino acid C-terminal non-triple helical domain that is homologous to the C-terminal domain of collagen VIII and to the C1q module of the human C1 enzyme. We have expressed this domain (residues 545-680) in Escherichia coli as a glutathione S-transferase fusion protein. The purified fusion protein trimerizes spontaneously in vitro, and after thrombin cleavage, the purified C-terminal domain trimer (46.2 kDa) is extremely stable and trypsin-resistant. Mutations within the C-terminal domain have been observed in patients with Schmid's metaphyseal chondrodysplasia (SMCD). Some of these mutations (Y598D, G618V, W651X, or H669X; X is the stop codon) were constructed by site-directed mutagenesis. Each mutation had identical consequences regarding the fusion protein: 1) absence of trimeric formation, 2) copurification of the approximately 60-kDa GroEL chaperone protein, and 3) sensitivity of the monomeric fusion protein to trypsin digestion. These results show that the C-terminal domain of collagen X is sufficient to produce a very stable and compact trimer in the absence of collagen Gly-X-Y repeats. Moreover, mutations causing SMCD interfere in this system with the correct folding of the C-terminal domain. The existence of a similar mechanism in chondrocytes might explain the relative homogeneity of phenotypes in SMCD despite the diversity of mutations.     

Down-regulation of cinnamoyl-CoA reductase in tomato (Solanum lycopersicum L.) induces dramatic changes in soluble phenolic pools

Health-beneficial properties of many secondary plant metabolites have created much interest into the control of their biosynthesis in crop species. Phenolic compounds, including flavonoids, hydroxycinnamates, and tannins, make up an important group of such phytonutrients. They are formed via the phenylpropanoid pathway and share common precursors with lignin, an insoluble cell wall-associated polymer. In this study, the aim was to reduce lignin biosynthesis so as to enhance the availability of these precursors and, thereby, stimulate the production of soluble, potentially health-promoting, phenolic compounds in tomato (Solanum lycopersicum L.). First two tomato genes encoding cinnamoyl-CoA reductase (CCR), a key enzyme in the formation of lignin monomers, were identified and characterized. Transgenic plants exhibiting a reduced lignin content were subsequently obtained through an RNAi strategy targeting one of these genes. As anticipated, the total level of soluble phenolics was higher in stems and leaves of the transformants as compared with control plants. This was correlated with an increased antioxidant capacity of the corresponding plant extracts. Analysis of the soluble phenolic fraction by HPLC-MS revealed that vegetative organs of CCR down-regulated plants contained higher amounts of chlorogenic acid and rutin, and accumulated new metabolites undetectable in the wild type, such as N-caffeoyl putrescine and kaempferol rutinoside. In fruits, CCR down-regulation triggered the moderate accumulation of two new compounds in the flesh, but the total phenolic content was not affected. Although the prospects of exploiting such a strategy for crop improvement are limited, the results provide further insight into the control of the phenylpropanoid pathway in the Solanaceae.     

Chondrocalcin is identical with the C-propeptide of type II procollagen.

The primary structure of the cartilage matrix molecule chondrocalcin has been found to be identical with that of the C-propeptide of type II procollagen by comparing sequence analyses of the N-terminal regions and of tryptic peptides derived from chondrocalcin. This implies that in type II collagen the C-propeptide of type II collagen is employed not only in the assembly of the triple helix of type II collagen, as demonstrated previously, but in calcifying cartilage it may also be involved in those events leading to cartilage calcification, as earlier indicated.     

Heterogeneous binding of high mobility group chromosomal proteins to nuclei

A dramatic difference is observed in the intracellular distribution of the high mobility group (HMG) proteins when chicken embryo fibroblasts are fractionated into nucleus and cytoplasm by either mass enucleation of cytochalasin-B-treated cells or by differential centrifugation of mechanically disrupted cells. Nuclei (karyoplasts) obtained by cytochalasin B treatment of cells contain more than 90 percent of the HMG 1, while enucleated cytoplasts contain the remainder. A similar distribution between karyoplasts and cytoplasts is observed for the H1 histones and the nucleosomal core histones as anticipated. The presence of these proteins, in low amounts, in the cytoplast preparation can be accounted for by the small percentage of unenucleated cells present. In contrast, the nuclei isolated from mechanically disrupted cells contain only 30-40 percent of the total HMGs 1 and 2, the remainder being recovered in the cytosol fraction. No histone is observed in the cytosol fraction. Unike the higher molecular weight HMGs, most of the HMGs 14 and 17 sediment with the nuclei after cell lysis by mechanical disruption. The distribution of HMGs is unaffected by incubating cells with cytochalasin B and mechanically fractionating rather than enucleating them. Therefore, the dramatic difference in HMG 1 distribution observed using the two fractionation techniques cannot be explained by a cytochalasin-B-induced redistribution. On reextraction and sedimentation of isolated nuclei obtained by mechanical cell disruption, only 8 percent of the HMG 1 is released to the supernate. Thus, the majority of the HMG 1 originally isolated with these nuclei, representing 35 percent of the total HMG 1, is stably bound, as is all the HMGs 14 and 17. The remaining 65 percent of the HMGs 1 and 2 is unstably bound and leaks to the cytosol fraction under the conditions of mechanical disruption. It is suggested that the unstably bound HMGs form a protein pool capable of equilibrating between cytoplasm and stably bound HMGs.     

Human collagen ''fingerprints'' produced by clostridopeptidase A digestion and high-pressure liquid chromatography.

Samples (1-2mg) of purified human type I, II and III collagens and alpha1(I) and alpha2 chains were digested with clostridiopeptidase A and the released peptides analysed by ion-exchange high-pressure liquid chromatography. Specific 'fingerprints' were produced for each type of collagen. The reproducible nature of these 'fingerprints' and the reconstitution of the type I 'fingerprint' from the 'fingerprints' of the component alpha1(I) and alpha2 chains showed that the specificity of these 'fingerprints' was related to the primary structure of each type of collagen. In addition, some of the differences observed between the 'fingerprints' of the alpha1(I) and alpha2 chains of type I collagen were shown to be suitable for the quantitative analysis of these chains.     

Glycerophosphocholine metabolism in higher plant cells. Evidence of a new glyceryl-phosphodiester phosphodiesterase

Glycerophosphocholine (GroPCho) is a diester that accumulates in different physiological processes leading to phospholipid remodeling. However, very little is known about its metabolism in higher plant cells. (31)P-Nuclear magnetic resonance spectroscopy and biochemical analyses performed on carrot (Daucus carota) cells fed with GroPCho revealed the existence of an extracellular GroPCho phosphodiesterase. This enzymatic activity splits GroPCho into sn-glycerol-3-phosphate and free choline. In vivo, sn-glycerol-3-phosphate is further hydrolyzed into glycerol and inorganic phosphate by acid phosphatase. We visualized the incorporation and the compartmentation of choline and observed that the major choline pool was phosphorylated and accumulated in the cytosol, whereas a minor fraction was incorporated in the vacuole as free choline. Isolation of plasma membranes, culture medium, and cell wall proteins enabled us to localize this phosphodiesterase activity on the cell wall. We also report the existence of an intracellular glycerophosphodiesterase. This second activity is localized in the vacuole and hydrolyzes GroPCho in a similar fashion to the cell wall phosphodiesterase. Both extra- and intracellular phosphodiesterases are widespread among different plant species and are often enhanced during phosphate deprivation. Finally, competition experiments on the extracellular phosphodiesterase suggested a specificity for glycerophosphodiesters (apparent K(m) of 50 microM), which distinguishes it from other phosphodiesterases previously described in the literature.