The peptide recognized by HLA-A68.2-restricted, squamous cell carcinoma of the lung-specific cytotoxic T lymphocytes is derived from a mutated elongation factor 2 gene

The identification of naturally processed tumor peptides that can stimulate a tumor-specific, CTL response is crucial to the development of a vaccine-based, immunotherapeutic approach to cancer treatment. One type of cancer in which a tumor-specific, CTL response has been observed is squamous cell carcinoma of the lung. In the system investigated here, the tumor-specific CTLs are HLA-A68.2 restricted. Immunoaffinity chromatography was used to isolate the HLA-A68.2 molecules from the tumor cell line, and peptide was eluted with acid from the HLA-A68.2 molecules and subjected to three rounds of separation by reversed phase-high performance liquid chromatography (RP-HPLC). To determine which fractions contained the peptide recognized by the tumor-specific CTLs, an aliquot of each RP-HPLC fraction was added to the autologous, B-lymphoblastoid cell line, and the cells were then tested as targets for tumor-specific CTLs. After the third round of RP-HPLC, mass spectrometry was used to sequence individual peptide candidates, and a peptide with a m/z of 497 was identified as the active peptide. Collision-activated dissociation of m/z 497 allowed identification of the peptide sequence as ETVSEQSNV. With the exception of a single amino acid difference (glutamic acid versus glutamine as the sixth position in the peptide), this peptide is identical to residues 581 to 589 of elongation factor 2. The PCR was used to amplify the elongation factor 2 gene in both the tumor cells and the autologous B cell line, and DNA sequencing of the products revealed the presence of a heterozygous mutation in the tumor cells that accounts for the difference between the two peptide sequences. Although a similar analysis did not reveal the presence of the mutation in three additional lung cell carcinomas, this does not rule out the possibility that a survey of a larger population of tumor cells would reveal the presence of the mutation at a low frequency. These results demonstrate the utility of this approach for identifying tumor-specific antigens that are the targets of a CTL response.     

Performance and metabolic and endocrine changes with emphasis on glucose metabolism in high-yielding dairy cows with high and low fat content in liver after calving

Elevated liver fat content occurs in high-yielding dairy cows during the transition from pregnancy to lactation after fat mobilization and may affect hepatic glucose metabolism, but the degree of liver fat storage is highly variable. Therefore, we studied metabolic and endocrine changes and hepatic glucose metabolism in cows that markedly differ in liver fat content. Multiparous cows from the same herd with high (HFL; n = 10) and low (LFL; n = 10) liver fat contents (mean of d 1, 10, and 21 after calving for each cow, respectively) were studied from 60 d before expected calving to 56 d in milk. Cows were fed ad libitum and all cows received the same diets. Liver samples were taken on d 1, 10, and 21 after calving; mean fat content (±SEM) in liver of HFL cows was 174 ± 9.6 mg/g, whereas mean liver fat content in LFL cows was 77 ± 3.3 mg/g. Blood samples were taken 20 and 7 d before expected calving and 0, 7, 14, 28, and 56 d after calving to measure plasma concentrations of nonesterified fatty acids, β-hydroxybutyrate, glucose, insulin, glucagon, insulin-like growth factor-I, and leptin. In liver, glycogen content as well as mRNA levels of phosphoenolpyruvate carboxykinase, pyruvate carboxylase, glucose-6-phosphatase, and glucose transporter were measured by quantitative real-time PCR. Back fat thickness decreased and dry matter intake increased with onset of lactation, and back fat thickness was higher but dry matter intake was lower in HFL than in LFL. Energy-corrected milk yield did not differ between groups, but milk fat content was higher and lactose content was lower in HFL than LFL at the beginning of lactation. Energy balance was more negative in HFL than in LFL. Plasma nonesterified fatty acids and β-hydroxybutyrate concentrations increased and plasma glucose concentration tended to decrease more in HFL than LFL with onset of lactation. Glucagon to insulin ratios increased more in HFL than LFL with onset of lactation. Hepatic glycogen content was higher in LFL than HFL, whereas mRNA levels of glucose-6-phosphatase and pyruvate carboxylase were higher in HFL than in LFL, and cytosolic phosphoenolpyruvate carboxykinase mRNA level increased similarly after parturition in both groups. In conclusion, an elevated liver fat content was related to greater fat mobilization and reduced feed intake and was associated with effects on hepatic glucose metabolism. As environment and feeding management were the same, individual cow factors were responsible for differences in energy metabolism during the transition period.     

Analyzing localized corrosion in lon-implanted metals via XPS/AES

Acombined electrochemical/surface analysis approach coupled with the use of ion-implanted samples has been successfully used to study anodic processes relevant to intergranular stress corrosion cracking (IGSCC). Interpretation of the data and applying it to an understanding of IGSCC varies in complexity and difficulty with the system under study. In some cases, there is a direct correspondence between composition and corrosion rates while, in others, the chemistry is more complex and secondary processes become dominant under some conditions. Nevertheless, the use of this approach has proved valuable, at the very least providing a qualitative understanding of the interplay between oxidation of a metal and the chemistry of segregants at grain boundaries that has forwarded modeling efforts on IGSCC. For more information, contact C.F. Windisch, Jr., Pacific Northwest National Laboratory, MS K2-44 Box 999, Richland, WA 99352; (509)375-6538; fax (509) 375-2186; e-mail cf.windisch@pnl.gov     

Translational Diffusion and Interaction of a Photoreceptor and Its Cognate Transducer Observed in Giant Unilamellar Vesicles by Using Dual‐Focus FCS

In order to monitor membrane–protein binding in lipid bilayers at physiological protein concentrations, we employed the recently developed dual‐focus fluorescence correlation spectroscopy (2fFCS) technique. In a case study on a photoreceptor consisting of seven transmembrane helices and its cognate transducer (two transmembrane helices), the lateral diffusion for these integral membrane proteins was analyzed in giant unilamellar vesicles (GUVs). The two‐dimensional diffusion coefficients of both separately diffusing proteins differ significantly, with D=2.2×10^?8 cm² s^?1 for the photoreceptor and with D=4.1×10^?8 cm² s^?1 for the transducer. In GUVs with both membrane proteins present together, we observed significantly smaller diffusion coefficients for labelled transducer molecules; this indicates the presence of larger diffusing units and therefore intermolecular protein binding. Based on the phenomenological dependence of diffusion coefficients on the molecule's cylindrical radius, we are able to estimate the degree of membrane protein binding on a quantitative level.     

Raman and XPS characterization of fuel-cladding interactions using miniature specimens

A combination of laser Raman spectroscopy and X-ray photoelectron spectroscopy was applied in a study of fuel-cladding chemical interactions on miniature oxide-coated HT-9 disks at elevated temperature. The experiments were intended as a preliminary step toward the development of a quick-screening technique for candidate alloys for cladding materials and actinide-based mixed oxide fuel mixtures. The results indicated that laser Raman spectroscopy was capable of determining the major oxides on HT-9 and how they changed in composition due to heating. However, X-ray photoelectron spectroscopy was necessary to identify the role of the metallic phases and provide depth resolution. Using the two techniques the kinetics of chromia growth were shown to be affected by the presence of an applied oxide coating. A single replacement reaction involving residual reduced metal within the coating was also identified.     

High-fat dairy product consumption increases Δ9c’ 11t−18∶2 (rumenic acid) and total lipid concentrations of human milk

Conjugated octadecadienoic acids (18∶2’ conjugated linoleic acids) have been shown to be anticarcinogenic and may influence growth and nutrient partitioning. The Δ9c’ 11t−18∶2 isomer (rumenic acid’ RA) is most common in both food sources and human tissues. To determine if maternal diet can influence milk RA concentration’ breastfeeding women (n=16) were enrolled in a 3-wk crossover study. Women initially consumed minimal amounts of food containing RA during week 1’ then were assigned randomly to consume diets rich in high-fat dairy foods (and thus RA) during week 2 or 3. Milk was collected by complete breast expression twice during each experimental week. Current and chronic RA intakes were estimated by 3-d dietary records and food frequency question-naires’ respectively. Estimated chronic RA intakes ranged from 49 to 659 mg/d. Dietary RA intake was greater during the high compared to the low dairy period (291±75 vs. 15±24 mg/d’ respectively; P<0.0001). Milk contained more RA during the high than the low dairy period (13.5±0.1 vs. 8.2±0.4 μmol/g lipid’ respectively; P<0.0001). Milk lipid concentration was influenced by diet’ such that lipid concentration was greater during the high than the low dairy period (46.6±5.0 vs. 38.3±1.6 mg/g milk’ respectively; P<0.05). Additionally’ multiple regression analyses suggested that body mass index was the primary predictor of milk RA and lipid concentrations. In summary’ these data indicate that both lipid and RA concentrations of human milk can be influenced by diet. Presented in part at the 1997 meeting of the Federation of American Sociieties for Experimental Biology’ FASEB J. 11’ A239.     

X-ray photoelectron spectroscopic analyses of corrosion products formed on rock bolt carbon steel in chloride media with bicarbonate and silicate ions

The passivation behavior of Yucca Mountain Repository rock bolt carbon steel in deaerated 3.5% NaCl solution containing SiO₃²⁻ and HCO₃⁻ ions was investigated by potentiodynamic polarization, electrochemical impedance spectroscopy, scanning electron microscopy, and X-ray photoelectron spectroscopic methods. Polarization results indicate that combinations of silicate and bicarbonate anions decrease the passive current density and raise the pitting potential. XPS results indicate the enrichment of silica at passive potentials and the formation of mixed FeCO₃ and silica film at lower potentials. This change in film composition was responsible for the changes in corrosion rate at lower and higher potentials. XPS results also support the thermodynamic data with regard to the occurrence of second oxidation peak observed in the polarization curves to be due to the oxidation of FeCO₃ to Fe₂O₃.     

EPA,but not DHA,decreases mean platelet volume in normal subjects

The first indication of platelet activation is an increase in mean platelet volume (MPV). n−3 FA are known to inhibit platelet function and to reduce the risk for coronary heart disease. The purpose of this study was to determine the effects of FPA and DHA on MPV. Healthy subjects received olive oil placebo for 4 wk and then were randomly assigned to receive 4g of ethyl esters of either safflower oil (n=11), EPA (n=10), or DHA (n=12) for 4 wk. At the end of placebo run-in and treatment periods, MPV (fL; mean±SEM) and platelet count (PLT-CT; 10³/μL blood) were measured in the basal state and after ex vivo stimulation with collagen (10 μg/mL), cold (4°C), and heat (37°C). Unlike DHA, EPA lowered MPV as compared with safflower oil (7.2±0.1 vs. 7.5±0.1 fL; P<0.05) and raised PLT-CT (211±18 vs. 192±18 10³/μL; P<0.05) in the fasting state. Collagen and cold significantly increased MPV whereas heat lowered MPV regardless of treatment. All stimuli decreased PLT-CT. EPA significantly increased platelet EPA (0.2±0.1 vs. 3.3±0.4%) and docosapentaenoic acid (DPA; 2.2±0.3 vs. 2.9±0.3%) concentrations, but not DHA. DHA treatment significantly increased DHA (1.4±0.2 vs. 4.1±0.5%) and DPA (2.0±0.4 vs. 3.0±0.4%) concentrations, but not EPA. In conclusion, EPA, but not DHA, reduces platelet activation, an early step in platelet aggregation.     

Electrocardiographic changes in acute n-butylisocyanate poisoning

The effect of phenobarbital and phenylbutazone treatment on the renal damage induced by the toxic mushroom Cortinarius speciosissimus was studied in female rats. Phenobarbital sodium was given in drinking water (0.05% solution) for 11 days prior to the administration of mushroom. Phenylbutazone was given s.c. in doses of 50 and 100 mg/kg 1 h before the mushroom administration. Homogenized mushroom was given orally by stomach tube at a dose of 250 mg dried mushroom/kg body weight. It was found that the phenobarbital treatment strongly increased the damage induced by C. speciosissimus in the tubules of the kidney cortex but had no effect on the inflammation in the renal outer medullary zone induced by this toxic mushroom. Phenylbutazone treatment had no effect on the renal damage induced by C. speciosissimus.