Ion channels formed by amphipathic helical peptides

Channel forming peptides (CFPs) are amphipathic peptides, of length ca. 20 residues, which adopt an -helical conformation in the presence of lipid bilayers and form ion channels with electrophysiological properties comparable to those of ion channel proteins. We have modelled CFP channels as bundles of parallel trans-bilayer helices surrounding a central ion-permeable pore. Ion-channel interactions have been explored via accessible surface area calculations, and via evaluation of changes in van der Waals and electrostatic energies as a K⁺ ion is translated along the length of the pore. Two CFPs have been modelled: (a) zervamicin-A1-16, a synthetic apolar peptaibol related to alamethicin, and (b) -toxin from Staphylococcus aureus. Both of these CFPs have previously been shown to form ion channels in planar lipid bilayers, and have been shown to have predominantly helical conformations. Zervamicin-A1-16 channels were modelled as bundles of 4 to 8 parallel helices. Two related helix bundle geometries were explored. K⁺channel interactions have been shown to involve exposed backbone carbonyl oxygen atoms. -Toxin channels were modelled as bundles of 6 parallel helices. Residues Q3, D11 and D18 generate favourable K⁺-channel interactions. Rotation of W15 about its C-C bond has been shown to be capable of occluding the central pore, and is discussed as a possible model for sidechain conformational changes in relation to ion channel gating.     

Promotion of murine antitumour activity by prothymosin α treatment: I. Induction of tumoricidal peritoneal cells producing high levels of tumour necrosis factor α

Summary The effect of prothymosin (ProT) on the survival of DBA/2 mice inoculated with syngeneic tumour cells was studied. DBA/2 mice inoculated intraperitoneally (i.p.) with 2×10⁵ syngeneic leukaemic L1210 cells developed ascites within 8–12 days and died 10–14 days later. Treatment with ProT consistently inhibited the development of ascites in 20% of the treated animals and prolonged the survival of 40%–60% of the animals up to 70 days. The most effective treatment schedule of ProT was 300 ng/mouse given i.p. at 2-day intervals for 3 weeks followed by a rest period of 7 days, prior to tumour cell inoculation. Peritoneal exudate (PE) cells collected from mice treated with the optimal dose of ProT produced, in the absence of exogenous stimulus, six- to eightfold higher levels of tumour necrosis factor (TNF) than PE cells from control mice. Furthermore these cells exhibited cytotoxic activity against several tumour cell lines including the syngeneic L1210, the TNF-insensitive P815 mastocytoma, the human MOLT-4 lymphoblastic leukaemia, as well as the murine TNF-sensitive L929 fibroblast cell line. Kinetic studies revealed that both production of TNF and tumoricidal activity peaked 7 days after the last injection of ProT and were maintained at high levels over a period of 1 month. Injections with 150 ng ProT slightly improved the survival of mice whereas higher (500 ng and 1000 ng) doses of ProT and a wide range of thymosin 1 doses remained without any effect. PE cells collected from these mice produced extremely low levels of TNF and exhibited negligible tumoricidal activity. Our data demonstrate that ProT has a protective effect in vivo against the growth of adoptively transfered tumour cells and suggest that this effect is, at least in part, mediated by ProT-activated PE cells. These cells were demonstrated to produce high levels of TNF in vitro and to exhibit activity against both TNF-sensitive and TNF-resistant cell lines.Supported by a CEC grant to Dr. M. Papamichail     

Proteolytic digestion of α-lactalbumin: Physiological implications

The kinetics of the partial digestion of bovine -lactalbumin (-LA) by trypsin, -chymotrypsin, and pepsin was monitored by lactose synthase activity, HPLC, and difference spectrophotometry. The relative stabilities of the various metal-bound states of -LA to trypsin and chymotrypsin at 37 and 5°C decrease in the following order: Ca(II)--LA>Zn(II), Ca(II)--LA>apo--LA. The HPLC digestion patterns of Ca(II)--LA and Zn(II), Ca(II)--LA at 5 and 37°C were similar, while the corresponding digestion patterns for apo--LA were quite different, reflecting the existence of the thermally induced denaturation states of apo--LA within this temperature region. Occupation of the first Zn(II)-binding site in Ca(II)-loaded -LA slightly alters the HPLC digestion patterns at both temperatures and accelerates the digestion at 37°C due to Zn(II)-induced shift of the thermal transition of -LA, exposing some portion of thermally denatured protein. The results suggest that the binding of Zn(II) to the first Zn(II)- (or Cu(II))-specific site does not cause any drastic changes in the overall structure of -LA. The acidic form of -LA (atpH 2.2 and 37°C) was digested by pepsin at rates similar to that for the apo- or Cu(II), Ca(II)-loaded forms by trypsin or -chymotrypsin at neutralpH. Complexation of -LA with bis-ANS affords protection against pepsin cleavage. It is suggested that the protective effects of similar small lipophilic compounds to -LA may have physiological significance (e.g., for nutritional transport).On leave from the Institute of Biological Physics, USSR Academy of Sciences, Pushchino, Moscow Region, 142292, USSR.     

Ionic channels formed byStaphylococcus aureus alpha-toxin: Voltage-dependent inhibition by divalent and trivalent cations

Summary The interaction ofStaphylococcus aureus -toxin with planar lipid membranes results in the formation of ionic channels whose conductance can be directly measured in voltage-clamp experiments. Single-channel conductance depends linearly on the solution conductivity suggesting that the pores are filled with aqueous solution; a rough diameter of 11.4±0.4 Å can be estimated for the pore. The conductance depends asymmetrically on voltage and it is slightly anion selective at pH 7.0, which implies that the channels are asymmetrically oriented into the bilayer and that ion motion is restricted at least in a region of the pore. The pores are usually open in a KCl solution but undergo a dose- and voltage-dependent inactivation in the presence of diand trivalent cations, which is mediated by open-closed fluctuations at the single-channel level. Hill plots indicate that each channel can bind two to three inactivating cations. The inhibiting efficiency follows the sequence Zn²⁺>Tb³⁺>Ca²⁺>Mg²⁺>Ba²⁺. suggesting that carboxyl groups of the protein may be involved in the binding step. A voltage-gated inactivation mechanism is proposed which involves the binding of two polyvalent cations to the channel, one in the open and one in the closed configuration, and which can explain voltage, dose and time dependence of the inactivation.     

The α-helix dipole in membranes: a new gating mechanism for ion channels

Electric dipoles placed side by side attract each other if antiparallel and repel each other if parallel. The hydrophobic -helical sections of proteins that span membranes are known to possess large electric dipole moments. The first part of the paper consists of a calculation of the interaction energies between such helices including screening effects. Interaction energies remain comparable with a typical thermal energy of KT up to separations of order 20 Å. In addition it is shown that, due solely to its dipole moment, an -helix which completely spans the membrane has an energy up to 5 KT lower than one which terminates within the membrane width. The second part of the paper describes the electrical interaction of the charge structure of a membrane channel and the protein helices that surround the pore. The gating charge transfer that is measured when a voltage sensitive ion channel switches, means that the dipole moment of the ion channel changes. This in turn results in a change in the radial forces that act between the pore and the -helices that surround it. A change in these radial forces which tend to open or to close the pore constitutes an electrically silent gating mechanism that must necessarily act subsequent to the gating charge transfer. The gating mechanism could consist of the radial translation of the neighbouring proteins or in their axial rotation under the influence of the torque that would act on a pair of approximately equidistant but oppositely directed -helices. An attempt to calculate the interaction energy of a typical pore and a single -helix spanning the membrane results in an energy of many times KT.     

Tnf-α and IL-1α inhibit both pyruvate dehydrogenase activity and mitochondrial function in cardiomyocytes: Evidence for primary impairment of mitochondrial function

Cytokines such as tumor necrosis factor (TNF) and Interleukin-1 (IL1) are known to influence energy metabolism and mitochondrial function in tumor and vascular smooth muscle cells. The aim of the present study was to investigate whether in cardiomyocytes mitochondrial function and PDH activity may also be impaired by TNF and IL1. Pyruvate dehydrogenase (PDH) activity and mitochondrial oxygen consumption of cultured cardiomyocytes were determined after subchronic exposure (24 h) to TNF (1, 10, 100, 1000 I.U./ml) and IL1 (0.1, 1, 10, 100 I.U./ ml).TNF- and IL1- exposure of the cardiomyocytes resulted in a concentration dependent decrease of PDH activity up to 38%. In parallel, selective oxygen consumption of the respiratory chain complexes I (NADH:ubiquinone oxidoreductase) and II (succinate:ubiquinone oxidoreductase) decreased by up to 45%. Addition of the PDH activator dichloracetate (0.01 M) resulted in complete restoration of PDH activity but not of mitochondrial function. The results suggest a primary inhibition of the mitochondrial respiratory chain by TNF and IL1 and a subsequent down regulation of PDH activity.     

Direct and indirect effects of human interferon α on renal cell carcinoma: a new in vitro assay system for evaluating cytokine-mediated antitumor effects

A highly purified natural -interferon (nIFN) was tested in vitro for direct and indirect antiproliferative activity against renal cell carcinoma (RCC), using a modified human tumor clonogenic assay and clinically achievable concentrations. In preclinical experiments, the indirect (cytokine-mediated) antiproliferative activity of nIFN was investigated using ACHN cells (established human RCC cell line). Continuous exposure to nIFN at concentrations of more than 5 IU/ml in the presence of feeder cells (a mixture of 5×10⁴ monocytes/dish and 5×10⁵ lymphocytes/dish, obtained from healthy donors) significantly inhibited colony formation of ACHN cells in comparison with growth inhibition in the absence of feeder cells (P<0.05). Various cytokines were measured in the supernatants lying over the medium on the feeder-layer agarose containing the same conditioned feeder cells. With IFN at 500 IU/ml, tumor necrosis factor (TNF) and IFN were detected at markedly high levels for 2–24 h. Neutralizing anti-TNF monoclonal antibody significantly reduced the indirect antiproliferative activity. Using our modified human tumor clonogenic assay technique, sufficient numbers of colonies for drug testing were observed in 19 of 31 surgical specimens (61.3%). In these clinical materials, nIFN at a clinically achievable concentration (50 IU/ml) significantly inhibited colony growth in the presence of feeder cells consisting of 5×10⁴ monocytes/dish and 5×10⁵ lymphocytes/dish, obtained from the patient whose tumor was examined (P<0.05). In colony-forming cases, a significant correlation between the percentage colony survival and TNF concentration in the supernatant was observed (r=–0.95,P<0.01). These results suggest that this assay system may be an appropriate technique for evaluating the antiproliferative activities of nIFN involving cytokine-mediated action, and that TNF may play an important role in this cytokine-mediated activity.This work was supported in part by a grant-in-aid for promoting research (no. 02771010) from the Ministry of Education     

Crystallographic structure of a helical lipopeptaibol antibiotic analogue

An X-ray diffraction analysis of the [Fmoc⁰,TOAC^4,8, Leu-OMe¹¹] analogue of thelipopeptaibol antibiotic trichogin A IV shows that the undecapeptide isfolded in a right-handed, mixed /3₁₀-helix. The helicalmolecules are connected in a head-to-tail arrangement along the b-axisthrough C=O···H-N intermolecular H-bonding. Thispacking mode generates a hydrophobic cavity where the FmocN-protecting groups are accommodated. The distances andangles between the nitroxide groups of the two TOAC residues, separated byone turn of the -helix, have been determined.     

Purification and characterization of GDP L -Fuc: N-acetyl β D -glucosaminide α1→6fucosyltransferase from human blood platelets

-6 L -Fucosyltransferase (1,6FucT; EC 2.4.1.68) from human platelets, the enzyme that is released into serum during coagulation of blood, was purified 100,000-fold. The purification required three sequential chromatographic steps: chromatofocusing, affinity column chromatography on GnGn-Gp(asialo-aglacto-transferrin glycopeptide)-CH-Sepharose, and gel filtration of Sephadex G-200. The final preparation contained a protein that migrated as a single discrete band Mr of 58,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditions, and as a single enzymatically active peak Mr of 58,000 in gel filtration. Although the purified enzyme utilized the biantennary GnGn-Gp as substrate, it was twice as active with the triantennary oligosaccharide when the Man 1,3 antenna was substituted with GlcNac1,4. On the other hand the tetraantennary oligosaccharide was not a preferred substrate. The Km values for the substrate asialo-agalactotransferrin-glycopeptide, and GDP L -fucose were 29 and 28 M, respectively. The optimum pH of the enzyme was 6.0. The activity of 1,6FucT was abolished in the presence of -mercaptoethanol. Divalent cations such as Mg2+ and Ca2+ activated, but Cu2+, Zn2+ and Ni2+ strongly inhibited the activity.     

Structure of α-α-Hairpins with Short Connections in Globular Proteins

The analysis of conformations of more than 100 --hairpins with closely packed helical segments and connections up to four amino acid residues in length was carried out. Five types of the connections were revealed, and their and values on the Ramachandran map were found. Each type of --hairpins was shown to have a unique sequence pattern for hydrophobic and hydrophilic residues.     

Endogenous interferon α/β produced by Kupffer cells inhibits interleukin-1, tumor necrosis factor α production and interleukin-2-induced activation of nonparenchymal liver cells

Summary We have previously reported liver-specific interferon (IFN) / production by murine Kupffer cells that was not observed with other tissue macrophages incubated in the absence of stimulators such as IFN or lipopolysaccharide (LPS). Consequently, while interleukin-2 (IL-2) alone induced pronounced lymphokine-activated killer (LAK) activity from splenocytes, combination of anti-IFN/ antibody with IL-2 was required to generate significant LAK activity from nonparenchymal liver cells. This endogenous IFN/ production by Kupffer cells was not induced by LPS because (a) addition of polymyxin B did not abolish the positive effects of anti-IFN/ antibody on nonparenchymal liver cells, and (b) similar results were obtained when comparing the responses of LPS-responsive C3HeB/FeJ and LPS-hyporesponsive C3H/HeJ mice. The possibility of hepatotropic infection was also ruled out in that anti-IFN/ antibody enhanced hepatic but not splenic LAK cell induction in vitro in both conventional and germfree C3H/HeN mice. IFN/ played an autoregulatory role by down-regulating the production of IL-1 and tumor necrosis factor by Kupffer cells. However, the augmenting effect of anti-IFN/ antibody on LAK induction from non-parenchymal liver cells was not mediated through an increase in the level of either IL-1 or TNF, as specific antisera against either cytokine did not abrogate this positive effect. Finally, flow-cytometry analysis showed that IFN/ significantly diminished the expression of IL-2 receptor chain, indicating an inhibition of LAK cell generation at a relatively early stage of induction.This work is supported by NIH grant RO1-28 835 and by Medical Research Funds from the Veterans Administration     

Characterization of active barley alpha-amylase 1 expressed and secreted by Saccharomyces cerevisiae

Recombinant barley -amylase 1 isozyme was constitutively secreted by Saccharomyces cerevisiae. The enzyme was purified to homogeneity by ultrafiltration and affinity chromatography. The protein had a correct N-terminal sequence of His-Gln-Val-Leu-Phe-Gln-Gly-Phe-Asn-Trp, indicating that the signal peptide was efficiently processed. The purified -amylase had an enzyme activity of 1.9 mmol maltose/mg protein/min, equivalent to that observed for the native seed enzyme. The k _cat/K _m was 2.7 × 10² mM^–1.s^–1, consistent with those of -amylases from plants and other sources.     

Evidence for a non-antioxidant,dose-dependent role of α -lipoic acid in caspase-3 and ERK2 activation in endothelial cells

Endothelial cell apoptosis contributes to atherosclerosis and may be exacerbated by oxidative stress. Results from clinical trials using antioxidant supplementation are equivocal and could be enhanced by antioxidants with additional non-antioxidant properties such as -lipoic acid and -tocopherol. The aim of this study was to investigate the effects of these antioxidants on cytoprotective pathways and endothelial apoptosis. Endothelial cells were incubated with -lipoic acid and -tocopherol, alone or in combination, prior to incubation with H₂O₂ or staurosporine. -lipoic acid pre-treatment alone increased caspase-3 activity in a dose-dependent manner. Both H₂O₂ and staurosporine increased DNA fragmentation and caspase-3 activity and pre-treatment of cells with -lipoic acid and/or -tocopherol failed to prevent stress-induced apoptosis. Neither antioxidant treatments nor apoptotic inducers alone altered expressions of Bcl-2, Bax, HSP70 or pERK1/2 or pJNK. -lipoic decreased pERK2 in staurosporine-treated cells in a dose-dependent manner. These findings indicate that pre-incubation with -lipoic acid and -tocopherol, alone or in combination, does not protect against oxidative- or non-oxidative-induced apoptosis in endothelial cells. Moreover, we have demonstrated a non-antioxidant, dose-dependent role of -lipoic acid in caspase-3 and ERK2 activation. These data provide an insight and indicate caution in the use of high doses of -lipoic acid as an antioxidant.     

Effects of natural interferon α, natural tumor necrosis factor α and their combination on human mesothelioma xenografts in nude mice

Effects of human natural interferon (nIFN) alone, human natural tumor necrosis factor (nTNF) alone and their combination (OH-1) were tested on three human mesothelioma lines implanted in nude mice. Tumors were transplanted subcutaneously by trocar on treatment day –12. nIFN was given intraperitoneally (i.p.) at a dose of 2 × 10⁷ or 2 × 10⁸ IU kg^–1 day^–1, 5 days a week for 3 weeks. nTNF was given i.p. at a dose of 2 × 10⁷ or 2 × 10⁸ U kg^–1 day^–1 in the same schedule as that of nIFN. Tumor diameters were serially measured and tumor volumes were calculated. Antitumor effects were assessed by two methods: comparison of final tumor volumes in treated and control groups (T/C), and changes in median average total tumor volume. The treatment produced no clinically discernible toxicities. nIFN had strong inhibitory activity against all three human mesothelioma lines. nTNF alone had modest activity only at the high dose used. The combination of the two produced activity essentially similar to that produced by nIFN alone. High-dose nIFN may have a role as an active agent in the treatment of patients with mesothelioma.     

Protein dynamics studied by NMR

The results of NMR studies using several nuclei indicate that proteins have considerable internal mobility. The most obvious is the mobility of side-chains. This mobility is general on the exterior surfaces but extends internally in a differential way. The functional value of surface mobility concerns both on and off rates of ligand binding (e.g. metal ions and parts of substrates) and protein/protein interactions. The mobility, which indicates that recognition is more in the hand-in-glove class than in the lock-in-key class, makes for a modified view of the specificity of protein interactions. Thus, fast on/off systems cannot be as selective as slower systems. Segmental mobility of proteins is considered in the context of protein secondary structure. The least mobile segments are the -sheet and the tight -turn. Mobility is always possible for, but not within, rod-like helices and in loose turns. Many examples are given and the importance of mobility in molecular machines is described. Finally, examples are given of virtually random-coil proteins, segments, and linker regions between domains and the functional value of such extremely dynamic regions of proteins is discussed.This work is based on a lecture at the EBSA Symposium, organised by the Italian Biophysical Society (S.I.B.P.A.), Tabiano Terme, September 1992     

Biochemical identification and immunological localization of two non-keratin polypeptides associated with the terminal differentiation of avian scale epidermis

Summary The expression of two previously uncharacterized polypeptides produced in epidermal cells of chick reticulate and scutate scales during late embryonic scale histogenesis and in hatchling birds has been studied biochemically and immunologically. These polypeptides have been identified by two-dimensional pH gradient gel electrophoresis as basic in charge, with apparent molecular weights of 20 and 23 kD, and they have been characterized immunologically and by amino acid analysis as non-keratin in nature. Monoclonal antibodies which react with both polypeptides have been used for immunohistochemical and immunogold electron-microscopic localization. Immunoreactivity was observed in suprabasal cells of reticulate scale epidermis, where it codistributed with bundles of -type cytokeratins in the -keratin-rich layers of epidermis known as the alpha stratum and in suprabasal cells of the outer epidermal surface of scutate scales, where it codistributed with -and -type keratin filament bundles in the -keratin-rich layers of epidermis known as the beta stratum.     

Rapid and Efficient Synthesis of Peptides Containing α,α-dialkylamino acids employing KOBt

Several Fmoc-,-dialkylamino acids and their acid chlorides have been prepared, isolated and characterised. The synthesis of peptides containing sterically hindered dialkylamino acids has been accomplished using acid chloride/KOBt in dichloromethane. The yields as well as the purity of the peptides were satisfactory.     

Effect of murine interferon α/β on tumor-induced suppressor function

T-lymphocyte-mediated immunosuppression has been described in several animal models and in man. In animal models, T-cell-mediated immunosuppression can hasten the development of cancers, permit the growth of tumors in immunocompetent hosts, and inhibit otherwise effective antitumor immunotherapy. Cyclophosphamide can abrogate the T-cell-mediated immunosuppression. However, inappropriately administered cyclophosphamide can adversely affect antitumor immunity. On the basis of data showing that interferon / (IFN/) and IFN selectively abrogate the T-cell-mediated dinitrofluorobenzene-specific suppressor function, we investigated the efficacy of purified murine IFN/ in manipulating tumorinduced T-cell-mediated immunosuppression in the wellcharacterized P815 mastocytoma model. In this model, generation of cytotoxicity in vitro and its inhibition by T cells correlates with antitumor immunity in vivo. We report that IFN/ selectively diminishes the generation of tumor-induced suppressor activity.     

New syntheses of α-methyl- and α,α′-dimethylspermine

-Methylspermine and ,-dimethylspermine were synthesized in high overall yields starting from N-(benzyloxycarbonyl)-3-aminobutanol in order to study polyamine biochemistry in vitro and in vivo.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 2, 2005, pp. 200–205.Original Russian Text Copyright © 2005 by Grigorenko, Vepsalainen, Jarvinen, Keinanen, Alhonen, Janne, Khomutov.     

Tumor necrosis factor α modifies resistance to interferon α in vivo: First clinical data

Summary Patients with Philadelphia-positive chronic-phase chronic myelogenous leukemia (CML) resistant to interferon (IFN) were treated in a phase I/II study with recombinant human tumor necrosis factor to overcome IFN resistance. Doses of 40, 80, 120 or 160 µg/m² TNF were given as 2-h infusions on 5 consecutive days every 3 weeks. IFN (4 × 10⁶ IU/m² s.c., daily) treatment was continued. Six patients were treated, completing 1–24 (median, 12) treatment cycles. Five of the six patients achieved partial hematological remission, while the remaining patient had to stop treatment because of WHO grade 4 thrombocytopenia following the first TNF cycle. No complete hematologic remission or cytogenetic improvement was seen. Side-effects were similar to those described for both substances alone. Maximum tolerable TNF doses usually varied between 80 µg/m² and 160 µg/m². To examine possible pathways of TNF activity in these patients, interferon receptor status and (2–5)-oligoadenylate synthetase levels were examined in peripheral blood mononuclear cells. Both parameters remained unchanged during TNF treatment. These preliminary data point to significant clinical efficacy of additionally applyed TNF in IFN-resistant CML patients.