Voltammetric studies on the electrochemical behaviour of membrane-entrapped hemes

Summary The electrochemical behaviour of Fe(III)-protoporphyrin IX entrapped into a cellulose triacetate membrane has been investigated by cyclic voltammetry. The physical entrapment into a solid matrix does not modify the redox properties of the entrapped berries, which also act as efficient promoters in the electrochemistry of cytochromec. Such a system represents a promising example of a simple solid-state promoter, and stimulates further investigations in order to obtain more complex systems that may be of significance for basic and applied bioelectrochemistry.     

Cloning, characterization, and modeling of a monoclonal anti-human transferrin antibody that competes with the transferrin receptor.

In this report we describe the isolation and characterization of a monoclonal antibody against human serum transferrin (Tf) and the cloning and sequencing of its cDNA. The antibody competes with the transferrin receptor (TR) for binding to human Tf and is therefore expected to bind at or very close to a region of interaction between Tf and its receptor. From the deduced amino acid sequence, we constructed a 3-dimensional model of the variable domains of the antibody based on the canonical structure model for the hypervariable loops. The proposed structure of the antibody is a first step toward a more detailed characterization of the antibody-Tf complex and possibly toward a better understanding of the Tf interaction with its receptor. The model might prove useful in guiding site-directed mutagenesis studies, simplifying the experimental elucidation of the antibody structure, and in the use of automatic procedures to dock the interacting molecules as soon as structural information about the structure of the human Tf molecule will be available.     

Cooperative effect of inositol hexakisphosphate, bezafibrate, and clofibric acid on the spectroscopic properties of the nitric oxide derivative of ferrous human hemoglobin

The cooperative effect of inositol hexakisphosphate (IHP), bezafibrate (BZF), and clofibric acid (CFA) on the spectroscopic (EPR and absorbance) properties of the nitric oxide derivative of ferrous human hemoglobin (HbNO) has been investigated quantitatively. In the presence of IHP, BZF, and CFA, the X-band EPR spectra and the absorption spectra in the Soret region of HbNO display the same basic characteristics described in the presence of 2,3-diphosphoglycerate (2,3-DPG), which have been attributed to a low affinity conformation of the tetramer. Addition to HbNO of two allosteric effectors together (such as IHP and BZF, or IHP and CFA) further stabilizes the low affinity conformation of the ligated hemoprotein (i.e., HbNO). Moreover, in the presence of saturating amounts of IHP, the affinity of BZF and CFA for HbNO increases by about fifteenfold. Likewise, in the presence of both IHP and BZF, as well as in IHP and CFA, the oxygen affinity for ferrous human hemoglobin (Hb) is reduced with respect to that observed in the presence of IHP, BZF, or CFA alone, which in turn is lower than that reported in the absence of any allosteric effector. All the data were obtained at pH 7.0 (in 1.0 × 10⁻¹ M N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid]/NaOH buffer system plus 1.0 × 10⁻¹ M NaCl), as well as at 100 K and/or 20°C. The results here reported represent clearcut evidence for the cooperative and specific (i.e., functionally relevant) binding of IHP, BZF, and CFA to Hb.     

Three high-lysine mutations control the level of ATP-binding HSP70-like proteins in the maize endosperm.

The synthesis and deposition of seed storage proteins in maize are affected by several dominant and recessive mutants. The effect of three independent mutations, floury-2 (fl2), Defective endosperm-B30 (De-B30), and Mucronate (Mc), that reduce zein level in the endosperm were investigated. These mutations also control the level of b-70, a polypeptide bound to protein bodies, which is separable into the two isoforms b-70I and b-70II by two-dimensional gel electrophoresis. Both isoforms are overexpressed 10-fold in fl2; however, only b-70I is present in De-B30 and Mc, which contain an amount of total b-70 isoforms fivefold higher than in the wild type. Both b-70I and b-70II resemble heat shock protein (HSP70) in that they bind ATP, cross-react with anti-HSP antibodies, and have N-terminal sequence homology to HSP70. All maize protein body-located b-70 characteristics are typical of those of chaperone-like HSPs. A third protein, b-70III, similar in size to but slightly more acidic than b-70I and b-70II, also binds ATP and reacts with the same antibody, providing evidence for the presence in endosperm extracts of a cytosolic chaperone-like protein. The level of b-70III was not altered by the mutations studied. The results suggested that the repression effect of the three mutations on zein accumulation may be mediated by the alteration of a zein transport or zein assembly process involving b-70I and b-70II.     

The v-src oncogene may not be responsible for the increased radioresistance of hematopoietic progenitor cells expressing v-src.

Infection of the IL-3-dependent, myeloid progenitor cell line 32D cl 3 with murine retroviruses that contain either the wild-type or a temperature-sensitive mutant v-src can render these cells growth-factor independent. These cells also became resistant to gamma irradiation administered at the low-dose rate of 0.05 Gy/min, which is used clinically. The v-src-dependent nature of resistance to gamma irradiation was examined by studying four clones of 32D cl 3 cells that had been infected with a retrovirus carrying the tsLA31A mutant of v-src. The tyrosine-specific kinase activity of this mutant is dramatically reduced at the nonpermissive temperature of 39 degrees C. Cells transformed by v-src and grown at either 34 or 39 degrees C, in the presence or absence of IL-3, demonstrated a significantly higher D0 compared to parental cells examined under identical conditions. In addition, expression of v-src abrogated the synergistic killing effect of heat and gamma irradiation. The D0 of parental 32D cl 3 cells kept at 39 degrees C after gamma irradiation was reduced significantly compared to the D0 of these cells kept at 34 degrees C. This contrasts with data from 32D cl 3 cells infected with either the wild-type v-src or the temperature-sensitive mutant, neither exhibited a synergistic effect in the D0 at either 34 or 39 degrees C. Therefore, while continuous expression of a v-src gene product is required for maintenance of the growth-factor-independent state, v-src does not appear to be responsible for the increased gamma-radiation resistance of these cells at low dose rate.     

Microvesicles as promising biological tools for diagnosis and therapy

ABSTRACT

Introduction: Shed by most cells, in response to a myriad of stimuli, extracellular vesicles (EVs) carry proteins, lipids, and various nucleic acids. EVs encompass diverse subpopulations differing for biogenesis and content. Among these, microvesicles (MVs) derived from plasma membrane, are key regulators of physiopathological cellular processes including cancer, inflammation and infection. This review is unique in that it focuses specifically on the MVs as a mediator of information transfer. In fact, few proteomic studies have rigorously distinguished MVs from exosomes.

Areas covered: Aim of this review is to discuss the proteomic analyses of the MVs. Many studies have examined mixed populations containing both exosomes and MVs. We discuss MVs’ role in cell-specific interactions. We also show their emerging roles in therapy and diagnosis.

Expert commentary: We see MVs as therapeutic tools for potential use in precision medicine. They may also have potential for allowing the identification of new biomarkers. MVs represent an invaluable tool for studying the cell of origin, which they closely represent, but it is critical to build a repository with data from MVs to deepen our understanding of their molecular repertoire and biological functions.     

Oxidized albumin. The long way of a protein of uncertain function

Background

Proteins are extremely reactive to oxidants and should represent a potential target of instable reactive oxygen. This may represent a problem for plasma proteins since they may be directly modified in vivo in a compartment where antioxidant enzymatic systems are scarcely represented. On the other hand, it is possible that some plasma components have evolved over time to guarantee protection, in which case they can be considered as anti-oxidants.

Scope of review

To present and discuss main studies which addressed the role of albumin in plasma antioxidant activity mainly utilizing in vitro models of oxidation. To present some advances on structural features of oxidized albumin deriving from studies carried out on in vitro models as well as albumin purified in vivo from patients affected by clinical conditions characterized by oxidative stress.

Major conclusions

There are different interaction with HOCl and chloramines. In the former case, HOCl produces an extensive alteration of ²³⁸Trp and ¹⁶²Tyr, ⁴²⁵Tyr, ⁴⁷Tyr, while thiol groups are only partially involved. Chloramines are extremely reactive with the unique free SH group of albumin (³⁴Cys) with the formation of sulfenic and sulfinic acid as intermediates and sulfonic acid as end-product. Oxidized albumin has a modified electrical charge for the addition of an acidic residue and presents α-helix and random coil reorganization with subtle changes in domain orientation.

General significance

Albumin, is the major antioxidants in plasma with a concentration (0.8 mM) higher than other antioxidants by an exponential factor. Functional and protective roles in the presence of oxidative stress must be defined. This article is part of a Special Issue entitled Serum Albumin.     

Plasma factors controlling atrial natriuretic peptide (ANP) aggregation: role of lipoproteins

We have previously shown that human plasma atrial alpha-natriuretic peptide (alpha-hANP) sequestering is a protective phenomenon against amyloid aggregation. In the present work, the possible role of lipoproteins as alpha-hANP binding factors has been investigated in vitro using an experimental model, developed in our laboratory, that allows to work at physiological concentrations. This approach consists of gel filtration on Sephacryl S-300 HR of big alpha-[(125)I]hANP generated in phosphate buffered saline or in human normal plasma supplemented or not with lipoproteins. The results of these experiments indicate that high density lipoproteins (HDL) are responsible for the ANP binding phenomenon observed in vitro, while low density lipoproteins and very low density lipoproteins do not directly interact with ANP. Moreover, the HDL remodeling process occurring in vitro has been analyzed during plasma incubation by monitoring the redistribution of lipids and apolipoproteins among the HDL subclasses. The changes in HDL size and composition observed in incubated plasma were compared with the redistribution of endogenous and labeled big ANP. The obtained results revealed that both tend to follow the molecular rearrangement in plasma of apolipoprotein A-I containing particles and suggested that, among HDL species, the small particles are mainly involved in the ANP binding phenomenon. This hypothesis was further demonstrated by ligand blotting experiments that confirmed the existence of differences in the ability of HDL particles to bind alpha-[(125)I]hANP.