Mechanisms for increased levels of phosphorylation of elongation factor-2 during hibernation in ground squirrels

Previously, eEF-2 phosphorylation has been identified as a reversible mechanism involved in the inhibition of the elongation phase of translation. In this study, an increased level of phosphorylation of eukaryotic elongation factor-2 (eEF-2) was observed in the brains and livers of hibernating ground squirrels. In brain and liver from hibernators, eEF-2 kinase activity was increased relative to that of active animals. The activity of protein phosphatase 2A (PP2A), a phosphatase that dephosphorylates eEF-2, was also decreased in brain and liver from hibernators. This was associated with an increase in the level of inhibitor 2 of PP2A (I(2)(PP2A)), although there was an increase in the level of the catalytic subunit of PP2A (PP2A/C) in hibernating brains and livers. These results indicate that eEF-2 phosphorylation represents a specific and previously uncharacterized mechanism for inhibition of the elongation phase of protein synthesis during hibernation. Increased levels of eEF-2 phosphorylation in hibernators appear to be a component of the regulated shutdown of cellular functions that permits hibernating animals to tolerate severe reductions in cerebral blood flow and oxygen delivery capacity.     

Characteristics of structure, composition, mass spectra, and iron release from the ferritin of shark liver (Sphyrna zygaena)

The ferritin consists of a protein shell constructed of 24 subunits and an iron core. The liver ferritin of Sphyrna zygaena (SZLF) purified by column chromatography is a protein composed of eight ferritins containing varying iron numbers ranging from 400+/-20 Fe3+/SZLF to 1890+/-20 Fe3+/SZLF within the protein shell. Nature SZLF (SZLFN) consisting of holoSZLF and SZLF with unsaturated iron (SZLFUI) to have been purified with polyacrylamide gel electrophoresis (PAGE) exhibited five ferritin bands with different pI values ranging from 4.0 to 7.0 in the gel slab of isoelectric focusing (IEF). HoloSZLF purified by PAGE (SZLFE) not only had 1890+/-20 Fe3+/SZLFE but also showed an identical size of iron core observed by transmission electron microscopy (TEM). Molecular weight of approximately 21 kDa for SZLFE subunit was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Four peaks of molecular ions at mass/charge (m/z) ratios of 10611.07, 21066.52, 41993.16, and 63555.64 that come from the SZLFE were determined by matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI-TOF MS), which were identified as molecular ions of the ferritin subunit (M+) and its polymers, namely, [M]2+, [M]+, [2M]+, and [3M]+, respectively. Both SZLFE and a crude extract from shark liver of S. zygaena showed similar kinetic characteristics of complete iron release with biphasic behavior. In addition, a combined technique of visible spectrometry and column chromatography was used for studying ratio of phosphate to Fe3+ within the SZLFE core. Interestingly, this ratio maintained invariable even after the iron release, which differed from that of other mammal ferritins.     

Direct electrical detection of hybridization at DNA-modified silicon surfaces

Electrochemical impedance spectroscopy was used to investigate the changes in interfacial electrical properties that arise when DNA-modified Si(111) surfaces are exposed to solution-phase DNA oligonucleotides with complementary and non-complementary sequences. The n- and p-type silicon(111) samples were covalently linked to DNA molecules via direct Si?C linkages without any intervening oxide layer. Exposure to solutions containing DNA oligonucleotides with the complementary sequence produced significant changes in both real and imaginary components of the electrical impedance, while exposure to DNA with non-complementary sequences generated negligible responses. These changes in electrical properties were corroborated with fluorescence measurements and were reproduced in multiple hybridization-denaturation cycles. The ability to detect DNA hybridization is strongly frequency-dependent. Modeling of the response and comparison of results on different silicon bulk doping shows that the sensitivity to DNA hybridization arises from DNA-induced changes in the resistance of the silicon substrate and the resistance of the molecular layers.     

Identification and molecular characterization of a novel y-type Glu-Dt 1 glutenin gene of Aegilops tauschii

A novel y-type high-molecular-weight glutenin subunit possessing a slightly faster mobility than that of subunit 1Dy12 in SDS-PAGE, designated 1Dy12.1^t in Aegilops tauschi, was identified by one- and two-dimensional gel and capillary electrophoresis. Its coding gene at the Glu-D ^t 1 locus was amplified with allele-specific-PCR primers, and the amplified products were cloned and sequenced. The complete nucleotide sequence of 2,807 bp containing an open reading frame of 1,950 bp and 857 bp of upstream sequence was obtained. A perfectly conserved enhancer sequence and the –300 element were present at positions of 209–246 bp and 424–447 bp upstream of the ATG start codon, respectively. The deduced mature protein of 1 Dy12.1^t subunit comprised 648 amino acid residues and had a Mr of 67,518 Da, which is slightly smaller than the 1Dy12 (68,695 Da) but larger than the 1Dy10 (67,495 Da) subunits of bread wheat, respectively, and corresponds well with their relative mobilities when separated by acid-PAGE. The deduced amino acid sequence indicated that the 1Dy12.1^t subunit displayed a greater similarity to the 1Dy10 subunit, with only seven amino acid substitutions, suggesting that this novel gene could have positive effect on bread-making quality. A phenetic tree produced by nucleotide sequences showed that the x- and y-type subunit genes were respectively clustered together and that the Glu-D ^t 1y12.1 gene of Ae. tauschii is closely related to other y-type subunit genes from the B and D genomes of hexaploid bread wheat.Communicated by H.F. Linskens     

Relationship between contents of adrenomedullin and distributions of neutral endopeptidase in blood and tissues of rats in septic shock

Adrenomedullin (ADM), a multifunction peptide with important roles in regulating cardiovascular homeostasis, has the vasodilatory properties and is of particular interest in the pathophysiology of sepsis. ADM levels in plasma and tissues are regulated by the proteolysis of neutral endopeptidase (NEP), the major enzyme of ADM degradation. We observed the NEP activity in the plasma, the activity and distribution of NEP and its mRNA expression in the tissues of rats in septic shock to study the possible role of NEP in elevating tissue ADM concentration during sepsis. ADM level increases progressively during sepsis except in the jejunum. Rats in early phase of shock (ES) showed diverse changes in tissue NEP activity. Plasma NEP activity, tissue NEP activity and its protein and mRNA expression in the left ventricle, aorta, jejunum and lung in the late phase of shock (LS) rats were lower than those in ES and the control, but no statistical change of NEP activity in the kidney was observed. The level of ADM was inversely correlated with NEP activity in the plasma, ventricle and aorta and positively correlated with NEP activity in the jejunum. Thus, in sepsis, the local concentration and action of ADM in tissues may be differentially regulated by NEP.     

Synthesis and biological evaluation of bis and monocarbonate prodrugs of 10-hydroxycamptothecins

In an effort to improve the stability of labile lactone ring of camptothecins, the bis and mono-alkyl carbonate prodrugs of 10-hydroxycamptothecins were synthesized and their chemical and enzymatical stability as well as antitumor activity were studied. The in vitro evaluation of the stability of these carbonates indicates that the 10,20-biscarbonates are firstly hydrolyzed to afford the stable 20-monocarbonates. And the 10-carbonates are not stable in human plasma, mouse plasma and pH7.4 phosphate buffer, while the 20-carbonates are relatively stable in the three media and can be readily cleaved by porcine liver esterase. The overall toxicity of the tested carbonate against mice bearing S180 sarcoma is much lower when compared with the parent compound, and the antitumor activity is maintained.     

Remodeling of the adventitia during coronary arteriogenesis

We studied the role of the adventitia in adaptive arteriogenesis during the phase of active growth of coronary collateral vessels (CV) induced by chronic occlusion of the left circumflex coronary artery in canine hearts. We used electron microscopy and immunoconfocal (IF) labeling for bFGF, matrix metalloproteinase (MMP)-2, MMP-9, tissue-type plasminogen activator (tPA), its inhibitor (PAI-1), fibronectin (FN), and Ki-67. Proliferation of smooth muscle cells and adventitial fibroblasts was evident. Quantitative IF showed that adventitial MMP-2, MMP-9, and FN were 9.2-, 7.5-, and 8.6-fold, bFGF was 5.1-fold, and PAI-1 was 3.4-fold higher in CV than in normal vessels (NV). The number of fibroblasts was 5-fold elevated in CV, but the elastic fiber content was 25-fold greater in NV than in CV. Perivascular myocyte damage and induction of endothelial nitric oxide synthase in peri-CV capillaries indicate expansion of CV. It was concluded that adventitial activation is associated with the development of CV through cell proliferation, production of growth factors, and induction of extracellular proteolysis thereby contributing to remodeling during adaptive arteriogenesis.     

Structure determination,apoptosis induction,and telomerase inhibition of CFP-2, a novel lichenin from Cladonia furcata

A great deal of experimental evidence has accumulated in the past several decades, suggesting that polysaccharides have wide bioactivities. Cladonia furcata polysaccharide, CFP-2, a water-soluble lichenin with a mean Mr 7.6 x 10(4), was first obtained by 0.25 M NaOH solution extraction, ethanol precipitation, DEAE-cellulose, and Sephadex G-200 column chromatography. Gas chromatography of acid hydrolyzate of CFP-2 suggested that it was composed of D-glucose, D-galactose, and D-mannose in the molar ratios of 8:1:1. Periodate oxidation, Smith degradation, IR, and NMR spectroscopy analysis revealed that CFP-2 had a backbone consisting of alpha-(1-->3) and alpha-(1-->4)-linked D-glucopyranosyl residues substituted at O-6 with beta-(1-->6)-linked D-galactopyranosyl residue and alpha-(1-->6)-linked D-mannopyranosyl residue. CFP-2 was able to reduce viability of cultured HL-60 and K562 cells. The antiproliferative properties of CFP-2 appeared to be attributable to its induction of apoptotic cell death as determined by ultrastructural change, internucleosomal DNA fragmentation, and increased proportion of the subdiploid cell population. To elucidate molecular events in the apoptosis, protein expressions of Bcl-2, Bax, Fas, and FasL were measured by Western blotting using specific antibodies in HL-60 cells. The level of Bcl-2 remained largely unchanged, but the Bax, Fas, and FasL expression showed up-regulation. Moreover, the telomerase activity analyzed by TRAP-ELISA assay in HL-60 cells treated with CFP-2 decreased as compared with the untreated control cells. These results suggest that CFP-2 could have a possible cancer therapeutic potential.     

The binding potential between the cholera toxin B-oligomer and its receptor

Employing atomic force microscopy as an in situ molecular force probe, we have measured the binding strength between cholera toxin B-pentamer (ctB) and its membrane receptor, ganglioside G(M1). By application of the basic principle of the reaction rate theory, key parameters of the ligand-receptor binding potential can be obtained from our data. The potential has a well with a spatial span of about 2.5 A and a depth of at least six times the thermal energy at room temperature. The very short range nature of the binding potential leads to the specificity of the ctB-G(M1) coupling.