Tetsuo Yamane

Zinc Protoporphyrin in the Erythrocytes of Patients with Lead Intoxication and Iron Deficiency Anemia

The fluorescent porphyrin in the erythrocytes of patients with lead intoxication or with iron deficiency anemia is zinc protoporphyrin that is bound to globin moieties, probably at heme binding sites.

Cholesterol Hydroperoxide Formation in Red Cell Membranes and Photohemolysis in Erythropoietic Protoporphyria

3β-Hydroxy-5α-hydroperoxy-Δ6-cholestene is produced in protoporphyrin-containing red blood cell ghosts irradiated with approximately 400-nanometer light in the presence of oxygen. Incorporation of this cholesterol photooxidation product into normal red blood cells leads to increased osmotic fragility and eventual hemolysis. These results may be relevant to photohemolysis associated with erythropoietic protoporphyria.

Nuclear magnetic resonance determination of ligand-induced conformational changes in myoglobin.

Abstract We report the results of a high-resolution proton nuclear magnetic resonance at 220 MHz of deoxymyoglobin and three diamagnetic complexes of myoglobin with oxygen, carbon monoxide and ethylisocyanide. We emphasize the resonances which are shifted from their normal positions by ring currents from aromatic rings, particularly the porphyrin ring, because these ring current shifts are very sensitive to changes of the relative co-ordinates of the observed protons and the ring. A specific resonance at −6.1 p.p.m., with intensity of about three protons, has been attributed to the protons of an aromatic residue, shifted upfield by the ring currents of the heme group. A possible origin of this resonance is Phe CD1. The observed change of this resonance of 0.2 p.p.m. upon oxygenation corresponds to a movement of a few tenths of an angstrom unit. Slightly larger shifts upon oxygenation of resonances attributed to methyl groups of aliphatic residues are interpreted in terms of movements of about 0.5 A. A comparison of the complete spectra of Mb ‡ and MbO2 shows that there are many small differences of 0.1 to 0.2 p.p.m. which indicate widespread small structural changes upon oxygenation. Differences in the ring-current-shifted resonances amongst various ligated forms including cyanoferrimyoglobin, indicate the existence of structural differences in the protein when the ligand is changed.

Crotamine is a novel cell-penetrating protein from the venom of rattlesnake Crotalus durissus terrificus

Herein we report that crotamine, a small lysine‐ and cysteine‐rich protein from the venom of the South American rattlesnake, can rapidly penetrate into different cell types and mouse blastocysts in vitro. In vivo, crotamine strongly labels cells from mouse bone marrow and spleen and from peritoneal liquid, as shown by fluorescent confocal laser‐scanning microscopy. Nuclear localization of crotamine was observed in both fixed and unfixed cells. In the cytoplasm, crotamine specifically associates with centrosomes and thus allows us to follow the process of centriole duplication and separation. In the nucleus, it binds to the chromosomes at S/G2 phase, when centrioles start dividing. Moreover, crotamine appears as a marker of actively proliferating cells, as shown by 5‐BrdU cell‐proliferation assay. Crotamine in the micromolar range proved nontoxic to any of the cell cultures tested and did not affect the pluripotency of ES cells or the development of mouse embryos.

Nuclear magnetic resonance study of cyanoferrimyoglobin; Identification of pseudocontact shifts

Abstract The high-resolution proton magnetic resonance spectrum of sperm whale cyanoferrimyoglobin in solution in water was measured at 220 MHz, and it was compared with the spectrum in deuterium oxide. Exchangeable protons gave three peaks with strongly temperature-dependent shifts in the region −13 to −24 p.p.m., measured downfield from 2:2-dimethyl-2-silapentane-5-sulphonate. They were shifted by interactions with the unpaired electrons. Four other exchangeable peaks, which had shifts with much smaller temperature dependences, were observed in the region −10 to −14 p.p.m. In the upfield region, four non-exchangeable peaks which had been thought to have areas corresponding to two protons each, and which had been assigned to methylene protons of the heme propionate sidechains, have been found to have areas corresponding to three protons, and they must be assigned to methyl groups. Since they are paramagnetically shifted, as are other methyl peaks at low field, there are too many paramagnetically shifted methyl peaks in the spectrum to be accounted for by contact interactions alone. This means that pseudocontact interactions, which do not depend on spin delocalization through covalent bonds, must be appreciable. Estimates of pseudocontact shifts allow assignments to be made for the three paramagnetically shifted exchangeable peaks, namely, the protons N 3 H of His E7, N 1 H of His F8 and the peptide NH of His F8. Since pseudocontact shifts have been found to be appreciable, some of the peak assignments made earlier for non-exchangeable peaks are now uncertain.

Crotamine Mediates Gene Delivery into Cells through the Binding to Heparan Sulfate Proteoglycans

Recently we have shown that crotamine, a toxin from the South American rattlesnake Crotalus durissus terrificus venom, belongs to the family of cell-penetrating peptides. Moreover, crotamine was demonstrated to be a marker of centrioles, of cell cycle, and of actively proliferating cells. Herein we show that this toxin at non-toxic concentrations is also capable of binding electrostatically to plasmid DNA forming DNA-peptide complexes whose stabilities overcome the need for chemical conjugation for carrying nucleic acids into cells. Interestingly, crotamine demonstrates cell specificity and targeted delivery of plasmid DNA into actively proliferating cells both in vitro and in vivo, which distinguishes crotamine from other known natural cell-penetrating peptides. The mechanism of crotamine penetration and cargo delivery into cells was also investigated, showing the involvement of heparan sulfate proteoglycans in the uptake phase, which is followed by endocytosis and peptide accumulation within the acidic endosomal vesicles. Finally, the permeabilization of endosomal membranes induced by crotamine results in the leakage of the vesicles contents to the cell cytosol.

Properties of cell penetrating peptides (CPPs)

Different approaches have been developed for the introduction of macromolecules, proteins and DNA into target cells. Viral (retroviruses, lentiviruses, etc.) and nonviral (liposomes, bioballistics etc.) vectors as well as lipid particles have been tested as DNA delivery systems. However, all of them share several undesirable effects that are difficult to overcome, such as unwanted immunoresponse and limited cell targeting. The discovery of the cell penetrating peptides (CPPs) showing properties of macromolecules carriers and enhancers of viral vectors, opened new opportunities for the delivery of biologically active cargos, including therapeutically relevant genes into various cells and tissues. This review summarizes recent data about the best characterized CPPs as well as those sharing cell‐penetrating and cargo delivery properties despite differing in the primary sequence. The putative mechanisms of CPPs penetration into cells and interaction with intracellular structures such as chromosomes, cytoskeleton and centrioles are addressed. We further discuss recent developments in overcoming the lack of cells specificity, one of the main obstacles for CPPs application in gene therapy. In particular, we review a newly discovered affinity of CPPs to actively proliferating cells. IUBMB Life, 58: 7 ‐ 13, 2006

Assignments of the paramagnetically shifted heme methyl nuclear magnetic resonance peaks of cyanometmyoglobin by selective deuteration

Abstract Three of the four paramagnetically shifted heme methyl nuclear magnetic resonance peaks of cyanometmyoglobin could be assigned by comparing the proton nuclear magnetic resonance spectra of myoglobins reconstituted from selectively deuterated hemes. These spectra indicate that the fourth methyl nuclear magnetic resonance peak has to be looked for outside the region −9 to −43 parts per million.

High resolution nuclear magnetic resonance study of base pairing in four purified transfer RNA molecules

Abstract The high resolution nuclear magnetic resonance spectra of hydrogen bonded protons in four purified tRNA molecules are reported. From the temperature and concentration dependence it is shown that these resonances arise from intramolecular hydrogen bonds.

Nuclear magnetic resonance study of the binding of fluoride ions to carboxypeptidase A

Abstract The single zinc atom of carboxypeptidase A has been replaced by manganese to form CPA(Mn) ‡ following the method of Coleman & Vallee (1960). Fluoride ions have been shown not to inhibit the enzymic activity of CPA(Mn) and we have studied the broadening of the 19 F nuclear magnetic resonance caused by interactions with the paramagnetic manganese. From the strength of the interaction we conclude that fluoride can bind directly to the manganese in the enzyme without affecting either the enzymic activity or the interactions of the manganese with the water protons. The inhibitor β-phenylpropionate removed the 19 F broadening by CPA(Mn). The same inhibition constant described this reduction of broadening in the fluoride solutions and the reduction of enzymic activity in the chloride and fluoride solutions of CPA(Mn). These results, in conjunction with our previous nuclear magnetic resonance studies of the water protons, which showed there was one (or more) water ligand of the metal, mean that if the manganese in CPA(Mn) has three protein ligands, as the X-ray crystal structure shows for zinc in CPA(Zn), then the total number of metal ligands is five or more.