A novel approach to the study of RBCs based on the collection of three-dimensional high-resolution AFM images and on the measure of the surface roughness of their plasma membrane is presented. The dependence of the roughness from several parameters of the imaging was investigated and a general rule for a trustful analysis and comparison has been suggested. The roughness of RBCs is a morphology-related parameter which has been shown to be characteristic of the single cells composing a sample, but independent of the overall geometric shape (discocyte or spherocyte) of the erythrocytes, thus providing extra-information with respect to a conventional morphology study. The use of the average roughness value as a label of a whole sample was tested on different kinds of samples. Analyzed data revealed that the quantitative roughness value does not change after treatment of RBCs with various commonly used fixation and staining methods while a drastic decrease occurs when studying cells with membrane-skeletal alteration both naturally occurring or artificially induced by chemical treatments. The present method provides a quantitative and powerful tool for a novel approach to the study of erythrocytes structure through an ultrastructural morphological analysis with the potential to give information, in a non-invasive way, on the RBCs function.
From skin secretions of the European frog Bombina bombina, a new peptide has been isolated that contains 60 amino acids, including 10 cysteine residues. Its sequence was determined by automated Edman degradation and confirmed by analysis of the cDNA encoding the precursor. A search in the databanks demonstrated that the pattern of cysteine residues in this skin peptide is similar to the ones found in protease inhibitors from Ascaris and in a segment of human von Willebrand factor. The 3D structure of the trypsin inhibitor from Ascarissuum could be used as a template to build a model of the amphibian peptide. In addition, we have demonstrated that this constituent of skin secretion is indeed an inhibitor of trypsin and thrombin, with Ki values in the range of 0.1 to 1 pM. The new peptide was thus named BSTI for Bombina skin trypsin/thrombin inhibitor. With few exceptions, these were rather small molecules containing less than 30 amino acids. As for their biological function, two main groups can be discerned. One comprises a multitude of peptides interacting with specific receptors present in the nervous system, the gastrointestinal tract and other organs of mammals. Many of these peptides are in fact similar or even identical to hormones and neurotransmitters. The second group encompasses antimicrobial peptides, at least some of which exert their function through direct binding to the phospholipid bilayer of cell membranes.Recently, a new family of peptides, the xenoxins, was isolated and characterized from skin secretions of Xenopus laevis (Kolbe et al., 1993). These 66-amino-acid peptides are related to neurotoxins and cytotoxins from snake venoms, but they are apparently devoid of toxic activity. In the course of our analysis of the skin secretion of Bombina bombina, a main fraction containing peptides with an apparent molecular mass of 8-10 kDa was isolated. In view of the earlier findings on xenoxins, we Reprint requests to: G. Kreil, Institute of Molecular Biology, Billrothstrasse 11, A-5020 Salzburg, Austria; e-mail: gkreil@oeaw.ac.at.wanted to test whether similar constituents were also present in skin secretion of this species. Here we present our analysis of one of the two main components of this fraction. As we have shown, this is a trypsin inhibitor related to the protease inhibitors from Ascaris (Grasberger et al., 1994; Huang et al., 1994).The peptide has been termed Bombina skin trypsin inhibitor, abbreviated BSTI. Results Isolation and amino acid sequence of BSTISkin secretions from B. bombina were collected and extracted with n-butanol. The aqueous phase was chromatographed over ConA-Sepharose to remove glycoproteins, proteoglycans, mucins etc. The eluate was further fractionated by molecular sieve chromatography (see the Materials and methods). One of the main peaks contained polypeptides with an apparent molecular weight of 8-10 kDa, as shown by SDS-PAGE. Separation of this fraction by reverse-phase HPLC yielded three peaks (see Fig. 1). Of these, the main constituent was investigated f...
The S-conjugation rates of the free-reacting thiols present on each component of rat hemoglobin with 5,5-dithio-bis(2,2-nitrobenzoic acid) (DTNB) have been studied under a variety of conditions. On the basis of their reactivity with DTNB (0.5 mM), three classes of thiols have been defined as follows: fast reacting (fHbSH), with t1 ⁄2 <100 ms; slow reacting (sHbSH), with t1 ⁄2 30 -50 s; and very slow reacting (vsHbSH), with t1 ⁄2 180 -270 s. Under paraphysiological conditions, fHbSH (identified with Cys-125(H3)) conjugates with DTNB 100 times faster than glutathione and ϳ4000 times more rapidly than (v)sHbSH (Cys-13␣(A11) and Cys-93(F9)). Such characteristics of fHbSH reactivity that are independent of the quaternary state of hemoglobin are mainly due to the following: (i) its low pK (ϳ6.9, the cysteinyl anion being stabilized by a hydrogen bond with Ser-123(H1)) and (ii) the large exposure to the solvent (as measured by analysis of a model of the molecular surface) and make these thiols the kinetically preferred groups in rat erythrocytes for S-conjugation. In addition, because of the high cellular concentration (8 mM, i.e. four times that of glutathione), fHbSHs are expected to intercept damaging species in erythrocytes more efficiently than glutathione, thus adding a new physiopathological role (direct involvement in cellular strategies of antioxidant defense) to cysteinyl residues in proteins.Human but not rat erythrocytes are reported (1) to be able to restore the cellular pool of GSH, which is strongly decreased after treatment with diazenedicarboxylic acid bis(N,N-dimethylamide), a thiol-oxidizing agent known by the trivial name diamide (2). Such a difference in redox behavior was attributed to a lower enzymatic capacity in reducing disulfides of rat red cells relative to the human ones (3). However, recent work in this laboratory demonstrated that the reversibility of such process can be observed also in rat erythrocytes, depending on diamide dose (4). Additional evidence (3) may suggest that these differences in behavior between rat and human erythrocytes could be related to diversities in reactivity of sulfhydryl groups of the corresponding hemoglobins.
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