The Late Endosomal ClC-6 Mediates Proton/Chloride Countertransport in Heterologous Plasma Membrane Expression

Members of the CLC protein family of Cl⁻ channels and transporters display the remarkable ability to function as either chloride channels or Cl⁻/H⁺ antiporters. Due to the intracellular localization of ClC-6 and ClC-7, it has not yet been possible to study the biophysical properties of these members of the late endosomal/lysosomal CLC branch in heterologous expression. Whereas recent data suggest that ClC-7 functions as an antiporter, transport characteristics of ClC-6 have remained entirely unknown. Here, we report that fusing the green fluorescent protein (GFP) to the N terminus of ClC-6 increased its cell surface expression, allowing us to functionally characterize ClC-6. Compatible with ClC-6 mediating Cl⁻/H⁺ exchange, Xenopus oocytes expressing GFP-tagged ClC-6 alkalinized upon depolarization. This alkalinization was dependent on the presence of extracellular anions and could occur against an electrochemical proton gradient. As observed in other CLC exchangers, ClC-6-mediated H⁺ transport was abolished by mutations in either the “gating” or “proton” glutamate. Overexpression of GFP-tagged ClC-6 in CHO cells elicited small, outwardly rectifying currents with a Cl⁻ > I⁻ conductance sequence. Mutating the gating glutamate of ClC-6 yielded an ohmic anion conductance that was increased by additionally mutating the “anion-coordinating” tyrosine. Additionally changing the chloride-coordinating serine 157 to proline increased the NO₃⁻ conductance of this mutant. Taken together, these data demonstrate for the first time that ClC-6 is a Cl⁻/H⁺ antiporter.     

Assessment of Threat Status and Management Effectiveness in Kakamega Forest, Kenya

To counteract an increasing biodiversity decline, parks and protected areas have been established worldwide. However, many parks lack adequate management to address environmental degradation. To improve management strategies simple tools are needed for an assessment of human impact and management effectiveness of protected areas. This study quantifies the current threats in the heavily fragmented and degraded tropical rainforest of Kakamega, western Kenya. We recorded seven disturbance parameters at 22 sites in differently managed and protected areas of Kakamega Forest. Our data indicate a high level of human impact throughout the forest with illegal logging being most widespread. Furthermore, logging levels appear to reflect management history and effectiveness. From 1933 to 1986, Kakamega Forest was under management by the Forest Department and the number of trees logged more than 20 years ago was equally high at all sites. Since 1986, management of Kakamega Forest has been under two different organizations, i.e. Forest Department and Kenya Wildlife Service. The number of trees logged illegally in the last 20 years was significantly lower at sites managed by the Kenya Wildlife Service. Finally, logging was lower within highly protected National and Nature Reserves as compared to high logging within the less protected Forest Reserves. Reflecting management effectiveness as well as protection status in Kakamega Forest, logging might therefore provide a valuable quantitative indicator for human disturbance and thus an important tool for conservation managers. Logging might be a valuable indicator for other protected areas, too, however, other human impact such as e.g. hunting might also prove to be a potential indicator.     

Tissue plasminogen activator: peptide analyses confirm an indirectly derived amino acid sequence, identify the active site serine residue, establish glycosylation sites, and localize variant differences

Tissue plasminogen activator, separated into variants I and II (differing in Mr by 2000-3000), was reduced and [14C]carboxymethylated. Fragments from cleavages with enzymes and cyanogen bromide (CNBr) were separated by reverse-phase high-performance liquid chromatography and subjected to sequence degradations. All seven CNBr fragments were purified and found to be compatible with the cDNA-derived amino acid sequence [Pennica, D., Holmes, W. E., Kohr, W. J., Harkins, R. N., Vehar, G. A., Ward, C. A., Bennett, W. F., Ylverton, E., Seeburg, P. H., Heynecker, H. L., Goeddel, D. V., & Collen, D. (1983) Nature (London) 301, 214-221]. Chemical characterization of 93% of the 527 residues recovered in 50 peptides confirmed the indirectly deduced primary structure of the protein. The tryptic peptide patterns from the two variants were found to differ for one peptide (T15). Since carbohydrate was present in this peptide for variant I and since a marked difference in chromatographic behavior for T15 was observed in variant II, we conclude that carbohydrate differences in this peptide (i.e., Asn-184 in the numbering system of the cDNA-derived amino acid sequence) are the explanation for the size differences between variants I and II. Carbohydrate was also found at two other positions in the protein, corresponding to Asn-117 and Asn-448. However, a fourth potential glycosylation site, Asn-218, is apparently not utilized for carbohydrate attachment. The enzyme is inactivated by diisopropyl phosphorofluoridate, which covalently modifies the serine residue corresponding to position 478, identifying this as the active site serine residue.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and identification of two structural variants of porcine tissue plasminogen activator by affinity adsorption on fibrin

Porcine tissue plasminogen activator has been purified from delipidized heart tissue by affinity adsorption to fibrin. A crude fraction is prepared from an acid tissue extract by precipitation with ammonium sulphate. The tissue activator of this fraction is isolated by adsorption on fibrin and elution with KSCN. The procedure also includes chromatography on arginine-Sepharose and two gel-filtration steps. The final product has a specific activity of 250 000 IU/mg (±16 000) as compared to an international urokinase reference preparation. The yield calculated from the active ammonium sulphate precipitate is about 28%. An approx. 7 000-fold increase of specific activity is obtained, most of which is achieved in the fibrin step. The native tissue plasminogen activator consists of a single chain molecule with a molecular weight of 64 000 as measured by SDS-polyacrylamide gel electrophoresis. In a previous report, it was claimed that the activator is composed of two disulphide-connected polypeptide chains. These results were due to a preparation artefact, caused by proteolytic activity present in the tissue extracts. The introduction of the protease inhibitor aprotinin and 6-amino-hexanoic acid in the purification procedure has abolished the effect of the protease contaminant, leading to the production of a one-chain activator. Treatment with plasmin transforms the native, one-chain tissue activator into a variant composed of two chains of about equal size (M_r 32 000) connected by disulphide bonding. This modified activator is indistinguishable from the one obtained at insufficient protection against proteolytic enzymes. The cleavage by plasmin causes about an 8-fold increase of amidolytic activity as measured on $\text{H}\text{-}\text{D}\text{-}\text{Val}\text{-}\text{Gly}\text{-}\text{Arg}\text{-p-}\text{nitroanalide}$. The fibrinolytic activity as measured by clot lysis is only slightly increased. The physiological significance of the cleavage is discussed.     

Biological synthesis of platinum nanoparticles using Diopyros kaki leaf extract

The leaf extract of Diopyros kaki was used as a reducing agent in the ecofriendly extracellular synthesis of platinum nanoparticles from an aqueous H₂PtCl₆·6H₂O solution. A greater than 90% conversion of platinum ions to nanoparticles was achieved with a reaction temperature of 95°C and a leaf broth concentration of >10%. A variety of methods was used to characterize the platinum nanoparticles synthesized: inductively coupled plasma spectrometry, transmission electron microscopy, energy-dispersive X-ray spectroscopy, X-ray photoelectron spectroscopy, and Fourier-transform infrared spectroscopy (FTIR). The average particle size ranged from 2 to 12 nm depending on the reaction temperature and concentrations of the leaf broth and PtCl₆ ²⁻. FTIR analysis suggests that platinum nanoparticle synthesis using Diopyros kaki is not an enzyme-mediated process. This is the first report of platinum nanoparticle synthesis using a plant extract.