Optimal left ventricular diameter measurement in subjects with sigmoid septum: comparison with three-dimensional left ventricular volume.

BACKGROUND: The purpose of this study was to determine the most appropriate measurement of left ventricular (LV) end-diastolic diameter for subjects with the sigmoid septum (SS) by measuring the LV end-diastolic diameter at the base and mid-ventricle and by examining the relationship between these measurements and the three-dimensional (3D) echocardiographic LV end-diastolic volume. METHODS: In 91 patients who underwent echocardiography for screening cardiovascular abnormalities, the aorto-septal angle (ASA) was measured as an index of the sigmoid septum. LV end-diastolic diameter was measured at the base and mid-ventricular level (DDbase and DDmid, respectively), and their average value was calculated (DDavg). By using 3D echocardiography, LV end-diastolic volume (EDV3D) was measured. RESULTS: Among 91 patients, 48 patients had narrow ASA (< 120 degrees) and were divided into the sigmoid septum (SS) group, and the remaining 43 patients were divided into the non-SS group. In the SS group, all DDbase, DDmid, and DDavg were significantly correlated with EDV3D (r = 0.59, 0.80, and 0.76, respectively), and the correlation coefficient between DDbase and EDV3D was significantly lower than that between DDmid and EDV3D (p < 0.01). On the other hand, in the non-SS group, all DDbase, DDmid, and DDavg were significantly correlated with EDV3D (r = 0.77, 0.85, and 0.84, respectively), and the correlation coefficient between DDbase and EDV3D was statistically comparable to that between DDmid and EDV3D (p = 0.12). ASA was significantly correlated with the difference of DDmid minus DDbase (r = - 0.71, p < 0.001). CONCLUSIONS: In patients with SS, DDmid and DDavg were well reflected the 3D echocardiographic LV end-diastolic volume.

Identification of deleterious recessive haplotypes and candidate deleterious recessive mutations in Japanese Black cattle.

Intensive use of a few elite sires has increased the risk of the manifestation of deleterious recessive traits in cattle. Substantial genotyping data gathered using single-nucleotide polymorphism (SNP) arrays have identified the haplotypes with homozygous deficiency, which may compromise survival. We developed Japanese Black cattle haplotypes (JBHs) using SNP array data (4843 individuals) and identified deleterious recessive haplotypes using exome sequencing of 517 sires. We identified seven JBHs with homozygous deficiency. JBH_10 and JBH_17 were associated with the resuming of estrus after artificial insemination, indicating that these haplotypes carried deleterious mutations affecting embryonic survival. The exome data of 517 Japanese Black sires revealed that AC_000165.1:g.85341291C>G of IARS in JBH_8_2, AC_000174.1:g.74743512G>T of CDC45 in JBH_17, and a copy variation region (CNVR_27) of CLDN16 in JBH_1_1 and JBH_1_2 were the candidate mutations. A novel variant AC_000174.1:g.74743512G>T of CDC45 in JBH_17 was located in a splicing donor site at a distance of 5 bp, affecting pre-mRNA splicing. Mating between heterozygotes of JBH_17 indicated that homozygotes carrying the risk allele died around the blastocyst stage. Analysis of frequency of the CDC45 risk allele revealed that its carriers were widespread throughout the tested Japanese Black cattle population. Our approach can effectively manage the inheritance of recessive risk alleles in a breeding population.

Non-Alcoholic Fatty Liver Disease Markers Associated with Fasting Serum Insulin and Urinary Albumin Excretion Independent of Fasting Plasma Glucose.

OBJECTIVE: We examined the association between non-alcoholic fatty liver disease (NAFLD) markers and fasting serum immunoreactive insulin (FIRI) and urinary albumin excretion (UAE). SUBJECTS AND METHODS: This study comprised Periods I and II from January 2007 to May 2009, and from June 2009 to December 2011, respectively. After excluding people with ethanol intake ≥210 g/week in men and ≥140 g/week in women, 961 people (613 men, 348 women; mean age: 44 years) were included. We evaluated the fatty liver using ultrasonography score (FLUS) and measured liver enzymes. RESULTS: The mean observation period was 25 ± 9 months. We stratified people into two groups by fasting plasma glucose (FPG) in Period I. The cutoff point between the lower FPG and higher FPG was 100 mg/dL. In regression analysis, serum alanine aminotransferase (ALT) (p < 0.001), FLUS (p < 0.001) and γ-glutamyl transpeptidase (GGTP) (p = 0.022) in Period I were independently associated with FIRI in Period II, whereas in all participants FPG was not. ALT (p < 0.001) and GGTP (p = 0.001) were also independently associated with UAE in people with FPG < 100 mg/dL in Period II. CONCLUSIONS: Some NAFLD markers were associated with FIRI and UAE independently of fasting plasma glucose.

A long-term study of the effects of SLC12A1 homozygous mutation (g.62382825G>A, p.Pro372Leu) in Japanese Black cattle.

Recessive missense mutation in the solute carrier family 12, member 1 (SLC12A1) gene (g.62382825G>A) is associated with hydrallantois, which is the accumulation of fluid in the allantoic cavity of a pregnant animal, and usually causes fetal death in Japanese Black cattle. However, the symptoms of a homozygote with this mutation that do not result in fetal death have not previously been tracked and evaluated. In the present study, we observed a homozygote with the SLC12A1 risk allele over a long-term period. The calf did not show any obvious clinical symptoms, although it did exhibit a slight growth retardation that accompanied mild calciuria. At 28 months of age, the homozygote showed renal dysfunction, which in turn resulted in hydronephrosis. The time course of the symptoms was consistent with the phenotype of Bartter syndrome in humans. Additionally, the risk heterozygous genotype did not any effects on carcass traits, which indicates that eliminating the risk allele would not have any unfavorable effects. Therefore, we emphasize that both the fetal- and late-stage symptoms associated with the SLC12A1 risk allele compromise animal welfare, and consequently may result in severe economic losses for individual farmers if the SLC12A1 risk allele is not eliminated from the population.

Left ventricular global longitudinal strain calculated from manually traced endocardial border lengths utilizing the images for routine ejection fraction measurement by biplane method of disks.

PURPOSE: The purpose of this study was to test whether the fractional change in the endocardial border length between end-diastole and end-systole as manually traced in left ventricular ejection fraction (LVEF) measurement using the biplane method of disks (MOD) was consistent with the global longitudinal strain derived from speckle-tracking echocardiography. METHODS: For 105 patients who underwent echocardiography, two- and four-chamber images with manually traced endocardial lines for LVEF measurement by MOD were stored. LV endocardial lengths at end-diastole and at end-systole were measured on both images to calculate the fractional length changes, which were averaged (GLSMOD). Speckle-tracking analysis was performed to measure global longitudinal strains in the apical two- and four-chamber and long-axis images, and the three values were averaged (GLSSTE) according to the ASE and EACVI guidelines. RESULTS: There was no significant difference between GLSMOD and GLSSTE. GLSMOD correlated well with GLSSTE (r = 0.81, p < 0.001), and there was no fixed bias in the Bland-Altman analysis. The intraclass correlations for the intra- and inter-observer comparisons for GLSSTE were excellent, and those for GLSMOD were adequate. CONCLUSION: The fractional LV endocardial border length change, GLSMOD, showed sufficient agreement with GLSSTE to justify its use as a substitute for the STE-derived global longitudinal strain.

Right ventricular basal inflow and outflow tract diameters overestimate right ventricular size in subjects with sigmoid-shaped interventricular septum: a study using three-dimensional echocardiography.

Sigmoid-shaped ventricular septum (SS), a frequently encountered minor abnormality in echocardiographic examinations of the elderly, may have some influence on RV shape. We aimed to determine the influence of SS on the accuracy of the 6 RV linear diameter measurements in the light of three-dimensional echocardiographic (3DE) RV volume. The aorto-septal angle (ASA) was measured in the parasternal long-axis view using two-dimensional echocardiography (2DE) as an index of SS in 70 patients without major cardiac abnormalities who were subdivided into 35 with SS (ASA ≤ 120°) and 35 without SS (NSS). We measured RV end-diastolic volume (RVEDV) using 3DE; in addition, using 2DE, we measured basal RV diameter, mid-cavity diameter, longitudinal diameter and end-diastolic area in the apical four-chamber view; proximal RV outflow tract (RVOT) diameter in the parasternal long-axis view; and proximal and distal RVOT diameters in the parasternal short-axis view. RVEDV did not differ between the SS and NSS groups. The SS group had greater basal RV diameter and proximal and distal RVOT diameters than the NSS group. RV mid-cavity diameter, longitudinal diameter, and end-diastolic area did not differ between the groups. Among the 2DE parameters of RV size, RV end-diastolic area was most strongly correlated with RVEDV (r = 0.67), followed by RV mid-cavity diameter (r = 0.58). When SS is present, the echocardiographic basal RV diameter and RVOT diameters overestimate RV size, and the measurement of RV end-diastolic area and mid-cavity diameter more correctly reflect 3D RV volume.

Furosemide loading test in a case of homozygous solute carrier family 12, member 1 (SLC12A1) mutation (g.62382825G>A, p.Pro372Leu) in Japanese Black cattle.

Hydrallantois is the excessive accumulation of fluid in the allantoic cavity in a pregnant animal and is associated with fetal death. We recently identified a recessive missense mutation in the solute carrier family 12, member 1 (SLC12A1) gene (g.62382825G>A, p.Pro372Leu) that is associated with hydrallantois in Japanese Black cattle. Unexpectedly, we found a case of the homozygous risk-allele for SLC12A1 in a calf, using a PCR-based direct DNA sequencing test. The homozygote was outwardly healthy up to 3 months of age and the mother did not exhibit any clinical symptoms of hydrallantois. In order to validate these observations, we performed confirmation tests for the genotype and a diuretic loading test using furosemide, which inhibits the transporter activity of the SLC12A1 protein. The results showed that the calf was really homozygous for the risk-allele. In the homozygous calf, administration of furosemide did not alter urinary Na+ or Cl- levels, in contrast to the heterozygote and wild-type calves in which these were significantly increased. These results demonstrate that the SLC12A1 (g.62382825G>A, p.Pro372Leu) is a hypomorphic or loss-of-function mutation and the hydrallantois with this mutation shows incomplete penetrance in Japanese Black cattle.

Analysis of the Japanese Diabetes Risk Score and fatty liver markers for incident diabetes in a Japanese cohort.

AIMS: We examined the effectiveness of the Japanese Diabetes Risk Score (JPDRISC) and fatty liver markers for predicting incident diabetes. METHODS: We created the JPDRISC. The study periods I and II were January 2007 to May 2009 and June 2009 to December 2011, respectively. A total of 2084 people (1389 men, 695 women; mean age: 46 years) were included. People with diabetes in the Period I and those with ethanol intake >140 g/week were excluded. A total of 1515 people were included. Fatty liver using ultrasonography scores (FLUS) were assigned. RESULTS: The mean observation period was 26.3 months, and 24 people had developed diabetes between the Periods I and II. In logistic regression analysis, the JPDRISC (OR=1.197, 95% C.I.: 1.062-1.350, p=0.003) and FLUS (OR=2.591, 95% C.I.: 1.411-4.758, p=0.002) in the Period I were independent determinants of incident diabetes. In receiver operating characteristic analysis, sensitivity and specificity for incident diabetes were 0.885 and 0.536, respectively, in people with both FLUS≥1 and the total JPDRISC≥6 in the Period I. The sensitivity was better than the JPDRISC alone (sensitivity 0.696) and FLUS alone (sensitivity 0.750). CONCLUSIONS: JPDRISC and FLUS were independently associated with incident diabetes and their combination is useful.

MEK inhibitor for gastric cancer with MEK1 gene mutations.

The prognosis for patients with unresectable advanced or recurrent gastric cancer remains poor. The identification of additional oncogenes with influences similar to those of epidermal growth factor receptor gene mutations, upon which the growth of cancer cells is dependent, is needed. In this study, we evaluated sensitivity to MEK inhibitors (GSK1120212 and PD0325901) in several gastric cancer cell lines in vitro and found three poorly differentiated gastric cancer cell lines that were hypersensitive to the inhibitors. The sequence analyses in these three cell lines revealed that one cell line had a novel MEK1 mutation, while the other two had previously reported KRAS and MEK1 mutations, respectively; the gene statuses of the other resistant cell lines were all wild-type. Experiments using MEK1 expression vectors demonstrated that the MEK1 mutations induced the phosphorylation of ERK1/2 and had a transforming potential, enhancing the tumorigenicity. The MEK inhibitor dramatically reduced the phosphorylation of ERK1/2 and induced apoptosis in the cell lines with MEK1 mutations. In vivo, tumor growth was also dramatically decreased by an inhibitor. One of the 46 gastric cancer clinical samples that were examined had a MEK1 mutation; this tumor had a poorly differentiated histology. Considering the addiction of cancer cells to active MEK1 mutations for proliferation, gastric cancer with such oncogenic MEK1 mutations might be suitable for targeted therapy with MEK inhibitors.

Fatty liver and serum cholinesterase are independently correlated with HbA1c levels: cross-sectional analysis of 5384 people.

OBJECTIVES: To examine the association between glycosylated haemoglobin (HbA1c) and fatty liver markers. METHODS: This cross-sectional analysis stratified subjects into quintiles based on HbA1c. Fatty liver using ultrasonography scores (FLUS) were assigned as follows: 2 points, moderate or severe fatty liver; 1 point, mild fatty liver; and 0 points, normal liver. Subjects with viral hepatitis, alcohol intake >175 g/week or receiving hypoglycaemic treatment were excluded. RESULTS: The study included 5384 subjects. Serum cholinesterase (ChE) and FLUS showed a significant graded increase with increasing HbA1c. In linear regression analysis stratified by body mass index (BMI) and age, ChE and FLUS were significantly associated with lower (1 + 2) and higher (3 + 4 + 5) HbA1c quintiles, respectively, independent of BMI and age. CONCLUSIONS: The findings show that both ChE and FLUS are significantly correlated with HbA1c, independent of BMI and age.

Roles of conserved Arg(72) and Tyr(71) in the ascorbate-specific transmembrane electron transfer catalyzed by Zea mays cytochrome b561.

Cytochromes b561, novel transmembrane electron transport proteins residing in eukaryotic cells, have a number of common features including six transmembrane α-helices and two heme ligation sites. Our recent studies on recombinant Zea mays cytochrome b561 suggested that concerted proton/electron transfer mechanism was functioning in plant cytochromes b561 as well and that conserved Lys(83) on a cytosolic loop had important roles for ascorbate-binding and a succeeding electron transfer. In the present study, we conducted site-directed mutagenesis analyses on conserved Arg(72) and Tyr(71). Removal of a positive charge at Arg(72) did not affect significantly on the final heme reduction level with ascorbate as reductant. However, characteristic pH-dependent initial time-lag upon electron acceptance from ascorbate was completely lost for R72A and R72E mutants. Substitution of Tyr(71) with Ala or Phe affected both on the final heme reduction level and on the pH-dependent initial time-lag, causing acceleration of the electron transfer. These observations were interpreted as existence of specific interactions of Tyr(71) and Arg(72) with ascorbate. However, their mechanistic roles were distinctly different from that of Lys(83), as exemplified by K83A/Y71A double mutant, and might be related for expelling of monodehydroascorbate radical from the substrate-binding site to prevent a back-flow of electrons.

Functional characterization of the recombinant human tumour suppressor 101F6 protein, a cytochrome b(561) homologue.

Candidate human tumour suppressor gene product, 101F6 protein, is a highly hydrophobic transmembrane protein and a member of cytochrome b(561) family. Purified 101F6 protein expressed in Pichia pastoris cells showed visible absorption spectra similar but distinct from those of cytochrome b(561). Haem content analysis indicated presence of two haems B per molecule. Midpoint potentials of the purified protein were found as +109 and +26 mV for two haems, slightly lower than those for bovine chromaffin granule or plant Zea mays cytochromes b(561). Electron paramagnetic resonance (EPR) spectra in oxidized state at 5 K showed only a highly anisotropic low-spin (HALS) signal at g(z) = 3.75. However, at 15 and 20 K, another HALS-type signal appeared at g(z) = 3.65 being overlapped with that of g(z) = 3.75. The rhombic EPR signal at g(z) = 3.16 previously seen in other cytochromes b(561) was not observed, suggesting distinct haem environments. Absence of the inhibition in the electron transfer from ascorbate by a treatment of 101F6 protein with diethylpyrocarbonate showed a remarkable contrast from those of other cytochromes b(561) where the 'concerted H(+)/e(-) transfer mechanism' at the cytosolic haem centre was blocked by specific Nε-carbethoxylation of haem-coordinating imidazole, suggesting that 101F6 protein might accept electrons via a mechanism distinct from other cytochromes b(561).

Selective synthesis and crystal structure of [10]cycloparaphenylene.

[10]Cycloparaphenylene ([10]CPP) was selectively synthesized in four steps in 13% overall yield from commercially available 4,4'-diiodobiphenyl by using mono-I-Sn exchange, Sn-Pt transmetalation, I-Pd exchange, and subsequent oxidative coupling reactions. The single-crystal X-ray structure of [10]CPP is described.

Interaction of modified tail-anchored proteins with liposomes: effect of extensions of hydrophilic segment at the COOH-terminus of holo-cytochromes b5.

A group of membrane proteins having a single COOH-terminal hydrophobic domain capable of post-translational insertion into lipid bilayer is known as tail-anchored (TA) proteins. To clarify the insertion mechanism of the TA-domain of human cytochrome b(5) (Hcytb5) into ER membranes, we produced and purified various membrane-bound forms of Hcytb5 with their heme b-bound, in which various truncated forms of NH(2)-terminal bovine opsin sequence were appended at the COOH-terminus of the native form. We analyzed the integration of the TA-domains of these forms onto protein-free liposomes. The integration occurred efficiently even in the presence of a small amount of sodium cholate and, once incorporated, such proteoliposomes were very stable. The mode of the integration was further analyzed by treatment of the proteoliposomes with trypsin either on the extravesicular side or on the luminal side. LC-MS analyses of the trypsin digests obtained from the proteoliposomes indicated that most of the C-terminal hydrophilic segment of the native Hcytb5 were exposed towards the lumen of the vesicles and, further, a significant part of the population of the extended C-terminal hydrophilic segments of the modified Hcytb5 were exposed in the lumen as well, suggesting efficient translocation ability of the TA-domain without any assistance from other protein factors. Present results opened a route for the use of the C-terminal TA-domain as a convenient tool for the transport of proteins as well as short peptides into artificial liposomes.

[What the Japan Cycling Federation hopes to accomplish in cooperation with pharmacists in doping and implementing effective anti-doping controls?].

Today the number of international cycling races has increased markedly compared with the past, and inevitably there are many riders traveling all over the world to participate in these events. It is inevitable that riders often become exposed to many more drugs at the cycling venues or through the internet where a wide selection of drugs is available. According to Union Cycliste Internationale (UCI) regulations, riders must report to a Doping Control Officer (DCO) any medications taken in the 72 h preceding a race. High level international teams are very familiar with these regulations and submit these reports with little problem. Lower level teams with less expertise, however, tend to have much more trouble submitting these reports. The close cooperation with pharmacists is believed to promoting the healthy development of the sport. To improve anti-doping controls, I strongly believe that good communication among staff during the events to exchange information smoothly and also good education through regular seminars and symposia for the staff involved are definitely needed. In other words, we have to develop human resources to build up a firm foundation to constantly generate future leaders.

Direct electrochemical analyses of human cytochromes b5 with a mutated heme pocket showed a good correlation between their midpoint and half wave potentials.

BACKGROUND: Cytochrome b5 performs central roles in various biological electron transfer reactions, where difference in the redox potential of two reactant proteins provides the driving force. Redox potentials of cytochromes b5 span a very wide range of ~400 mV, in which surface charge and hydrophobicity around the heme moiety are proposed to have crucial roles based on previous site-directed mutagenesis analyses. METHODS: Effects of mutations at conserved hydrophobic amino acid residues consisting of the heme pocket of cytochrome b5 were analyzed by EPR and electrochemical methods. Cyclic voltammetry of the heme-binding domain of human cytochrome b5 (HLMWb5) and its site-directed mutants was conducted using a gold electrode pre-treated with ß-mercarptopropionic acid by inclusion of positively-charged poly-L-lysine. On the other hand, static midpoint potentials were measured under a similar condition. RESULTS: Titration of HLMWb5 with poly-L-lysine indicated that half-wave potential up-shifted to -19.5 mV when the concentration reached to form a complex. On the other hand, midpoint potentials of -3.2 and +16.5 mV were obtained for HLMWb5 in the absence and presence of poly-L-lysine, respectively, by a spectroscopic electrochemical titration, suggesting that positive charges introduced by binding of poly-L-lysine around an exposed heme propionate resulted in a positive shift of the potential. Analyses on the five site-specific mutants showed a good correlation between the half-wave and the midpoint potentials, in which the former were 16~32 mV more negative than the latter, suggesting that both binding of poly-L-lysine and hydrophobicity around the heme moiety regulate the overall redox potentials. CONCLUSIONS: Present study showed that simultaneous measurements of the midpoint and the half-wave potentials could be a good evaluating methodology for the analyses of static and dynamic redox properties of various hemoproteins including cytochrome b5. The potentials might be modulated by a gross conformational change in the tertiary structure, by a slight change in the local structure, or by a change in the hydrophobicity around the heme moiety as found for the interaction with poly-L-lysine. Therefore, the system consisting of cytochrome b5 and its partner proteins or peptides might be a good paradigm for studying the biological electron transfer reactions.

Importance of the conserved lysine 83 residue of Zea mays cytochrome b(561) for ascorbate-specific transmembrane electron transfer as revealed by site-directed mutagenesis studies.

Cytochromes b(561), a novel class of transmembrane electron transport proteins residing in a large variety of eukaryotic cells, have a number of common structural features including six hydrophobic transmembrane alpha-helices and two heme ligation sites. We found that recombinant Zea mays cytochrome b(561) obtained by a heterologous expression system using yeast Pichia pastoris cells could utilize the ascorbate/mondehydroascorbate radical as a physiological electron donor/acceptor. We found further that a concerted proton/electron transfer mechanism might be operative in Z. mays cytochrome b(561) as well upon the electron acceptance from ascorbate to the cytosolic heme center. The well-conserved Lys(83) residue in a cytosolic loop was found to have a very important role(s) for the binding of ascorbate and the succeeding electron transfer via electrostatic interactions based on the analyses of three site-specific mutants, K83A, K83E, and K83D. Further, unusual behavior of the K83A mutant in pulse radiolysis experiments indicated that Lys(83) might also be responsible for the intramolecular electron transfer to the intravesicular heme. On the other hand, pulse radiolysis experiments on two site-specific mutants, S118A and W122A, for the well-conserved residues in the putative monodehydroascorbate radical binding site showed that their electron transfer activities to the monodehydroascorbate radical were very similar to those of the wild-type protein, indicating that Ser(118) and Trp(122) do not have major roles for the redox events on the intravesicular side.

Inhibition of electron acceptance from ascorbate by the specific N-carbethoxylations of maize cytochrome b561: a common mechanism for the transmembrane electron transfer in cytochrome b561 protein family.

Cytochromes b(561) constitute a novel class of proteins in eukaryotic cells with a number of highly relevant common features including six transmembrane alpha-helices and two haem groups. Of particular interest is the presence of a large number of plant homologues having putative ascorbate- and monodehydroascorbate radical-binding sites. We conducted a diethylpyrocarbonate-modification study employing Zea mays cytochrome b(561) heterologously expressed in Pichia pastoris cells. Pre-treatment of cytochrome b(561) with diethylpyrocarbonate in oxidized form caused N-carbethoxylation of His(86), His(159) and Lys(83), leading to a drastic inhibition of the electron transfer from ascorbate. The activity was protected by the inclusion of ascorbate during the treatment. However, midpoint potentials of two haem centres did show only slight decreases upon the treatment, suggesting that changes in the midpoint potentials were not the major cause of the inhibition. Present results indicated that Zea mays cytochrome b(561) conducted an ascorbate-specific transmembrane electron transfer by utilizing a concerted H(+)/e(-) transfer mechanism and that the specific N-carbethoxylation of haem axial His(86) that would inhibit the removal of a proton from the bound ascorbate was a major cause of the inhibition. On the other hand, Lys(83) might be important for an initial step(s) of the fast electron acceptance from ascorbate.

Adsorption-induced intramolecular dipole: correlating molecular conformation and interface electronic structure.

The interfaces formed between pentacene (PEN) and perfluoropentacene (PFP) molecules and Cu(111) were studied using photoelectron spectroscopy, X-ray standing wave (XSW), and scanning tunneling microscopy measurements, in conjunction with theoretical modeling. The average carbon bonding distances for PEN and PFP differ strongly, that is, 2.34 A for PEN versus 2.98 A for PFP. An adsorption-induced nonplanar conformation of PFP is suggested by XSW (F atoms 0.1 A above the carbon plane), which causes an intramolecular dipole of approximately 0.5 D. These observations explain why the hole injection barriers at both molecule/metal interfaces are comparable (1.10 eV for PEN and 1.35 eV for PFP) whereas the molecular ionization energies differ significantly (5.00 eV for PEN and 5.85 eV for PFP). Our results show that the hypothesis of charge injection barrier tuning at organic/metal interfaces by adjusting the ionization energy of molecules is not always readily applicable.

Functional expression and characterization of human 101F6 protein, a homologue of cytochrome b561 and a candidate tumor suppressor gene product.

A highly hydrophobic protein with six transmembrane structure that is coded by the candidate tumor suppressor gene 101F6 located in the human chromosome 3p.21.3 and a possible member of the cytochrome b 561 protein family was expressed, purified, and characterized in its functional form for the first time. The protein was heterologously expressed in methylotrophic yeast Pichia pastoris as a fusion protein containing a C-terminal thrombin-specific sequence and an 8-His residue tag. Purification was achieved by ion exchange chromatography on DEAE-Sepharose and affinity chromatography on Ni-NTA-Sepharose. SDS-PAGE analysis revealed a single protein band with an estimated molecular weight of 26 kDa, while Western blot and MALDI-TOF-MS analysis confirmed the presence of the cytochrome b561 specific sequence in the protein. The 101F6 protein was found to be reducible by ascorbate efficiently and to have two midpoint potentials at +89.5 and +13.1 mV, slightly lower than the corresponding values of +155 and +62 mV, respectively, of bovine adrenal cytochrome b 561, despite a lower conservation of the putative ascorbate binding site sequence in the 101F6 protein. The "modified motif 1" sequence unique in 101F6 protein may be responsible for other molecular functions, such as protein-protein interactions, in the endoplasmic membranes.