Iron-sulfur centers in the photosynthetic reaction center complex fromChlorobium vibrioforme. Differences from and similarities to the iron-sulfur centers in Photosystem I

The photosynthetic reaction center complex from the green sulfur bacteriumChlorobium vibrioforme has been isolated under anaerobic conditions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals polypeptides with apparent molecular masses of 80, 40, 30, 18, 15, and 9 kDa. The 80- and 18-kDa polypeptides are identified as the reaction center polypeptide and the secondary donor cytochromec ₅₅₁ encoded by thepscA andpscC genes, respectively. N-terminal amino acid sequences identify the 40-kDa polypeptide as the bacteriochlorophylla-protein of the baseplate (the Fenna-Matthews-Olson protein) and the 30-kDa polypeptide as the putative 2[4Fe-4S] protein encoded bypscB. Electron paramagnetic resonance (EPR) analysis shows the presence of an iron-sulfur cluster which is irreversibly photoreduced at 9K. Photoaccumulation at higher temperature shows the presence of an additional photoreduced cluster. The EPR spectra of the two iron-sulfur clusters resemble those of F_A and F_B of Photosystem I, but also show significantly differentg-values, lineshapes, and temperature and power dependencies. We suggest that the two centers are designated Center I (with calculatedg-values of 2.085, 1.898, 1.841), and Center II (with calculatedg-values of 2.083, 1.941, 1.878). The data suggest that Centers I and II are bound to thepscB polypeptide.     

Enhanced production of laccase in Trametes vesicolor by the addition of ethanol

In a medium containing 40 g ethanol l^–1, laccase production by Trametes versicolor was 2.6 unit per ml of the supernatant, which was over 20 times higher than that without ethanol. Laccase activity with ethanol was quite comparable to that with the well-known inducers such as veratryl alcohol, xylidine and guaiacol. With other white-rot fungi, Coriolus hirsutus and Grifola frondosa, ethanol had a similar stimulatory effect on laccase production.     

Flow cytometric determination of basophils in whole blood with n-propyl Astra Blue iodide

Within the past year, it has become apparent, in connection with its use on automatic flow cytometers, that the quality of commercially available Alcian Blue has significantly declined. A homologous series of alkylated (C1-C7) Astra Blue quaternary ammonium halides was prepared, characterized, and evaluated for the detection of basophils in whole blood. On the Technicon H6000 flow cytometer, the resolution of the basophil cluster from the main population of unstained white blood cells was found to depend on the chain length of the quaternizing alkyl group. Optimal basophil resolution was observed for the n-propyl derivative. Correlation of the new method vs Alcian Blue as the reference on the H6000 was expressed as follows: %Baso (Astra Blue) = 0.89% Baso (Alcian Blue) + 0.12% for 180 fresh whole blood samples. Within-run precision at a basophil differential count of 0.73% was characterized by SD = 0.11, identical to that obtained for Alcian Blue. Aqueous solutions of n-propyl Astra Blue iodide, in contrast to Alcian Blue, are thermally stable. Heating the reagent for 1 h at 100 degrees C did not alter solubility or cytochemical behavior. In contrast, parallel treatment of Alcian Blue yielded insoluble material by hydrolysis of the isothiouronium groups. The reagent for basophil detection comprises n-propyl Astra Blue iodide, lanthanum chloride, sodium chloride, Tween 20, and cetylpyridinium chloride. The Astra Blue derivatives were characterized by uv-vis, ir, percentage halide, paper chromatography, and 13C NMR.     

Phylogenetics of Tribe Collabieae (Orchidaceae,Epidendroideae) Based on Four Chloroplast Genes with Morphological Appraisal

Collabieae (Orchidaceae) is a long neglected tribe with confusing tribal and generic delimitation and little-understood phylogenetic relationships. Using plastid matK, psaB, rbcL, and trnH-psbA DNA sequences and morphological evidence, the phylogenetic relationships within the tribe Collabieae were assessed as a basis for revising their tribal and generic delimitation. Collabieae (including the previously misplaced mycoheterotrophic Risleya) is supported as monophyletic and nested within a superclade that also includes Epidendreae, Podochileae, Cymbidieae and Vandeae. Risleya is nested in Collabiinae and sister to Chrysoglossum, a relationship which, despite their great vegetative differences, is supported by floral characters. Ania is a distinct genus supported by both morphological and molecular evidence, while redefined Tainia includes Nephelaphyllum and Mischobulbum. Calanthe is paraphyletic and consists four clades; the genera Gastrorchis, Phaius and Cephalantheropsis should be subsumed within Calanthe. Calanthe sect. Ghiesbreghtia is nested within sect. Calanthe, to which the disputed Calanthe delavayi belongs as well. Our results indicate that, in Collabieae, habit evolved from being epiphytic to terrestrial.     

Selective staining of human platelet glycoproteins using nitrocellulose transfer of electrophoresed proteins and peroxidase-conjugated lectins

Platelet proteins (0.5-5 micrograms) were electrophoresed in a one-dimensional or an unreduced-reduced, two-dimensional sodium dodecyl sulfate gel system. The separated proteins were then transferred electrophoretically to nitrocellulose and reacted with peroxidase-conjugated lectins. Visualization of specific glycoproteins which bound the lectins was made by the chromogenic reaction catalyzed by peroxidase utilizing 3,3'-diaminobenzidine as the substrate. Wheat germ agglutinin specifically reacted with and allowed the visualization of glycoprotein Ib. Peanut agglutinin also specifically stained glycoprotein Ib after treatment of the nitrocellulose transferred proteins with neuraminidase. Ricinus communis agglutinin I stained thrombospondin, a 260 kDa protein, and factor VIII. Concanavalin A stained mainly glycoproteins IIb, III, IV, and V. Glycoproteins Ia, Ic, IIa, and other minor glycoproteins could be separated by unreduced-reduced, two-dimensional gel electrophoresis and were stained weakly with wheat germ agglutinin conjugates. These techniques were found to be reproducible as well as easily applied to the analysis and identification of platelet glycoproteins, particularly when dealing with a limited amount of platelets.     

Colorectal Cancers Mimic Structural Organization of Normal Colonic Crypts

Colonic crypts are stereotypical structures with distinct stem cell, proliferating, and differentiating compartments. Colorectal cancers derive from colonic crypt epithelia but, in contrast, form morphologically disarrayed glands. In this study, we investigated to which extent colorectal cancers phenocopy colonic crypt architecture and thus preserve structural organization of the normal intestinal epithelium. A subset of colon cancers showed crypt-like compartments with high WNT activity and nuclear β-Catenin at the leading tumor edge, adjacent proliferation, and enhanced Cytokeratin 20 expression in most differentiated tumor epithelia of the tumor center. This architecture strongly depended on growth conditions, and was fully reproducible in mouse xenografts of cultured and primary colon cancer cells. Full crypt-like organization was associated with low tumor grade and was an independent prognostic marker of better survival in a collection of 221 colorectal cancers. Our findings suggest that full activation of preserved intestinal morphogenetic programs in colon cancer requires in vivo growth environments. Furthermore, crypt-like architecture was linked with less aggressive tumor biology, and may be useful to improve current colon cancer grading schemes.     

Calcium binding mode of gamma-carboxyglutamic acids in conantokins.

Conantokin-T (con-T) and conantokin-G (con-G) are two highly homologous peptide toxins found in Conus venom. The former is a 21-residue peptide with four gamma-carboxyglutamic acid (Gla) residues (at positions 3, 4, 10 and 14), while the latter is a 17-residue peptide with five gamma-carboxyglutamic acid residues (at positions 3, 4, 7, 10 and 14). Despite the apparent similarity in number and relative positions of the gamma-carboxyglutamic acid residues, (113)Cd-NMR studies indicated a distinct metal binding behavior for con-G and con-T. There appears to be four binding sites in con-G in contrast to one metal binding site in con-T. To elucidate the mode of calcium binding by the gamma-carboxyglutamic acid residues in these conantokins, we designed various analogous peptides with their gamma-carboxyglutamic acid replaced by other amino acid residues. (113)Cd-NMR experiments on conantokin analogues reveal that the major difference in the number of metal binding sites between con-G and con-T is due to the residue at position 7. We also performed molecular simulations to calculate the relative binding free energies of several potential binding sites. Based on our theoretical and experimental results, we propose a 'four-site' binding model for conantokin-G and a 'single-site' binding model for conantokin-T.     

<Emphasis Type="Italic">In vitro</Emphasis> Enhancement of Lactate Esters on the Percutaneous Penetration of Drugs with Different Lipophilicity

Lactate esters are widely used as food additives, perfume materials, medicine additives, and personal care products. The objective of this work was to investigate the effect of a series of lactate esters as penetration enhancers on the in vitro skin permeation of four drugs with different physicochemical properties, including ibuprofen, salicylic acid, dexamethasone and 5-fluorouracil. The saturated donor solutions of the evaluated drugs in propylene glycol were used in order to keep a constant driving force with maximum thermodynamic activity. The permeability coefficient (K _p), skin concentration of drugs (SC), and lag time (T), as well as the enhancement ratios for K _p and SC were recorded. All results indicated that lactate esters can exert a significant influence on the transdermal delivery of the model drugs and there is a structure-activity relationship between the tested lactate esters and their enhancement effects. The results also suggested that the lactate esters with the chain length of fatty alcohol moieties of 10–12 are more effective enhancers. Furthermore, the enhancement effect of lactate esters increases with a decrease of the drug lipophilicity, which suggests that they may be more efficient at enhancing the penetration of hydrophilic drugs than lipophilic drugs. The influence of the concentration of lactate esters was evaluated and the optimal concentration is in the range of 5∼10 wt.%. In sum, lactate esters as a penetration enhancer for some drugs are of interest for transdermal administration when the safety of penetration enhancers is a prime consideration.