Identifying DNA polymorphisms in human TCRA/D variable genes by direct sequencing of PCR products

 The T-cell receptor (TCR) is a highly variable molecule composed of two polypeptide chains that recognize antigenic peptides in the context of major histocompatibility complex (MHC) molecules. In this study, we describe a sequence-based search for germline polymorphisms in the variable (V) gene segments of the human TCRA/D locus. Thirty different V gene segments were amplified from six to eight unrelated individuals and sequenced from low melting point agarose. Twenty-seven polymorphisms were identified in 15 V gene segments. These polymorphisms are mainly single nucleotide substitutions, but an insertion/deletion polymorphism and a single dinucleotide repeat with variable length were also seen. Of the 15 sequence variations found in the coding regions, six are silent and nine encode amino acid changes. All of the amino acid changes are found at non-conserved residues, frequently in the hypervariable regions, where they may influence MHC and/or peptide recognition. Therefore, it is possible that germline variations in TCR genes could influence an individual’s immune response, and may also contribute to susceptibility to diseases such as autoimmunity. Received: 9 January 1996 / Revised: 22 February 1996     

Oxide-Based Solid-State Batteries: A Perspective on Composite Cathode Architecture

The garnet-type phase Li₇La₃Zr₂O₁₂ (LLZO) attracts significant attention as an oxide solid electrolyte to enable safe and robust solid-state batteries (SSBs) with potentially high energy density. However, while significant progress has been made in demonstrating compatibility with Li metal, integrating LLZO into composite cathodes remains a challenge. The current perspective focuses on the critical issues that need to be addressed to achieve the ultimate goal of an all-solid-state LLZO-based battery that delivers safety, durability, and pack-level performance characteristics that are unobtainable with state-of-the-art Li-ion batteries. This perspective complements existing reviews of solid/solid interfaces with more emphasis on understanding numerous homo- and heteroionic interfaces in a pure oxide-based SSB and the various phenomena that accompany the evolution of the chemical, electrochemical, structural, morphological, and mechanical properties of those interfaces during processing and operation. Finally, the insights gained from a comprehensive literature survey of LLZO–cathode interfaces are used to guide efforts for the development of LLZO-based SSBs.     

Comparison of semiautomatic digitizer-tablet and simple point counting performance in morphometry

A comparison is made between the efficiency of an opto-manual digitizer-tablet (MOP-AM 03) and simple point counting in the analysis of drawings of basal cells in the normal and pathologically altered chonchal epithelium. The analysis included estimation of the areas of cellular, nuclear and nucleolar sectional profiles as well as the cell basal width, i.e. the length of cell profile attachment to the basement membrane. The morphometric results obtained with the two methods are virtually identical. The efficiency of point counting is somewhat higher than that of the digitizer, but the prices of the two sets of equipment differ by one or two orders of magnitude. In general, the unquestionable measuring precision of the digitizer is unlikely to be of much impact in most biological studies due to the inevitable biological variation between individuals under study and the large variation which is always added at the level of single features by random sectioning.     

Inositol tetrakisphosphates as second messengers induce Ca(++)-dependent chloride currents in Xenopus laevis oocytes.

Microinjection of inositol 1,3,4,5-tetrakisphosphate or inositol 1,4,5-trisphosphate induced distinct chloride membrane currents in defolliculated Xenopus laevis oocytes. To decide whether these Cl(-)-currents were due to the injected compounds or their metabolic products, [3H]Ins(1,3,4,5)P4 or [3H]Ins(1,4,5)P3 were injected into oocytes and their metabolites were analyzed by HPLC. Our results indicate that Ins(1,3,4,5)P4 itself or its metabolite Ins(1,3,4,6)P4 is able to induce Cl(-)-membrane currents, most likely by increasing the cytosolic Ca(++)-concentration.     

Fatty Acid Incorporation in Normal and Degenerating Rat Sciatic Nerve In Vivo

Abstract: Labeled palmitic acid ([16-¹⁴C]palmitate) (0).5 μCi) was injected into rat sciatic nerves in vivo to characterize thc incorporation of this fatty acid into complex peripheral nerve lipids after time lapses of 1 min to 2 weeks. For the first 30 min after intraneural injection, the label was concentrated in nerve diglycerides. Thereafter, the relative diglyccride label declined rapidly, and phospholipid radioactivity rose steadily. After 120 min, phospholipids contained over 70% of the total lipid radioactivity. Among the phospholipids, phosphatidylcholine had the largest percentage of total phospholipid label, and acylation of lysophosphatidylcholine accounted for approximately 75% of this label. With time, there was conversion of [16-¹⁴C]palmitate to other long-chain fatty acids by elongation and desaturation. Phosphatidic acid was labeled also, suggesting the operation of the de novo biosynthetic mechanism. However, the specific radioactivity of 1,2-diacylglycerol was much higher than that of phosphatidic acid, suggesting phosphorylation of diglycerides by diglyceride kinase. After nerve section and survival of 2 h to 50 days, the injection of [16-¹⁴C]palmitate into the degenerating distal segment revealed an overall decline of phospholipid labeling and a commensurate increase of triglyceride radioactivity. Phosphatidylcholine in degenerating nerve contained a larger percentage of the fatty acid label than that in normal nerve. Almost all of the labeling was due to acylation of lysophosphatidylcholine, implying a much smaller contribution of the de novo pathway. Phosphatidylethanolamine and phosphatidylserine showed a relative loss of radioactivity. The changes were apparent at 1 day, but not at 2 h, suggesting loss of homeostatic control, presumably by interruption of axonal flow. An incidental observation was the stimulation of phosphatidylcholine biosynthesis by acylation of lysophosphatidylcholine in the contralateral unoperated sciatic nerve.     

Rainfall manipulation experiments as simulated by terrestrial biosphere models: Where do we stand?

Changes in rainfall amounts and patterns have been observed and are expected to continue in the near future with potentially significant ecological and societal consequences. Modelling vegetation responses to changes in rainfall is thus crucial to project water and carbon cycles in the future. In this study, we present the results of a new model‐data intercomparison project, where we tested the ability of 10 terrestrial biosphere models to reproduce the observed sensitivity of ecosystem productivity to rainfall changes at 10 sites across the globe, in nine of which, rainfall exclusion and/or irrigation experiments had been performed. The key results are as follows: (a) Inter‐model variation is generally large and model agreement varies with timescales. In severely water‐limited sites, models only agree on the interannual variability of evapotranspiration and to a smaller extent on gross primary productivity. In more mesic sites, model agreement for both water and carbon fluxes is typically higher on fine (daily–monthly) timescales and reduces on longer (seasonal–annual) scales. (b) Models on average overestimate the relationship between ecosystem productivity and mean rainfall amounts across sites (in space) and have a low capacity in reproducing the temporal (interannual) sensitivity of vegetation productivity to annual rainfall at a given site, even though observation uncertainty is comparable to inter‐model variability. (c) Most models reproduced the sign of the observed patterns in productivity changes in rainfall manipulation experiments but had a low capacity in reproducing the observed magnitude of productivity changes. Models better reproduced the observed productivity responses due to rainfall exclusion than addition. (d) All models attribute ecosystem productivity changes to the intensity of vegetation stress and peak leaf area, whereas the impact of the change in growing season length is negligible. The relative contribution of the peak leaf area and vegetation stress intensity was highly variable among models.     

Age-, gender-, and species-dependent mutagenicity in T cells of mice and rats exposed by inhalation to 1,3-butadiene

Experiments were performed: (i) to investigate potential age- and gender-dependent differences in mutagenic responses in T cells following exposures of B6C3F1 mice and F344 rats by inhalation for 2 weeks to 0 or 1250 ppm butadiene (BD), and (ii) to determine if exposures for 2 weeks to 62.5 ppm BD produce a mutagenic effect in female rats. To evaluate the effect of age on mutagenic response, mutant manifestation curves for splenic T cells of female mice exposed at 8-9 weeks of age were defined by measuring Hprt mutant frequencies (MFs) at multiple time points after BD exposure using a T cell cloning assay and comparing the resulting mutagenic potency estimate (calculated as the difference of areas under the mutant manifestation curves of treated versus control animals) to that reported for female mice exposed to BD in the same fashion beginning at 4-5 weeks of age. The shapes of the mutant T cell manifestation curves for spleens were different [e.g., the maximum BD-induced MFs in older mice (8.0+/-1.0 [S.D.]x10(-6)) and younger mice (17.8+/-6.1 x 10(-6)) were observed at 8 and 5 weeks post-exposure, respectively], but the mutagenic burden was the same for both age groups. To assess the effect of gender on mutagenic response, female and male rodents were exposed to BD at 4-5 weeks of age and Hprt MFs were measured when maximum MFs are expected to occur post-exposure. The resulting data demonstrated that the pattern for mutagenic susceptibility from high-level BD exposure is female mice>male mice>female rats>male rats. Exposures of female rats to 62.5 ppm BD caused a minor but significant mutagenic response compared with controls (n=16/group; P=0.03). These results help explain part of the differing outcomes/interpretations of data in earlier Hprt mutation studies in BD-exposed rodents.     

Quantitative analysis of N-terminal valine peptide adducts specific for 1,2-epoxy-3-butene

Butadiene (BD) metabolism shows gender, species and concentration dependency, making the extrapolation of animal results to humans complex. BD is metabolized mainly by cytochrome P450 2E1 to three epoxides, 1,2-epoxy-3-butene (EB), 1,2;3,4-diepoxybutane (DEB) and 1,2-epoxy-butanediol (EB-diol). For accurate risk assessment it is important to elucidate species differences in the internal formation of the individual epoxides in order to assign the relative risks associated with their different mutagenic potencies. Analysis of N-terminal globin adducts is a common approach for monitoring the internal formation of BD derived epoxides. Our long term strategy is to develop an LC-MS/MS method for simultaneous detection of all three BD hemoglobin adducts. This approach is modeled after the recently reported immunoaffinity LC-MS/MS method for the cyclic N,N-(2,3-dihydroxy-1,4-butadyil)-valine (pyr-Val, derived from DEB). We report herein the analysis of the EB-derived 2-hydroxyl-3-butenyl-valine peptide (HB-Val). The procedure utilizes trypsin hydrolysis of globin and immunoaffinity (IA) purification of alkylated heptapeptides. Quantitation is based on LC-MS/MS monitoring of the transition from the singly charged molecular ion of HB-Val (1-7) to the a(1) fragment. Human HB-Val (1-11) was synthesized and used for antibody production. As internal standard, the labeled rat-[(13)C(5)(15)N]-Val (1-11) was prepared through direct alkylation of the corresponding peptide with EB. Standards were characterized and quantified by LC-MS/MS and LC-UV. The method was validated with different amounts of human HB-Val standard. The recovery was >75% and coefficient of variation <25%. The LOQ was set to 100 fmol/injection. For a proof of principal experiment, globin samples from male and female rats exposed to 1000 ppm BD for 90 days were analyzed. The amounts of HB-Val present were 268.2+/-56 and 350+/-70 pmol/g (mean+/-S.D.) for males and females, respectively. No HB-Val was detected in controls. These data are much lower compared to previously reported values measured by GC-MS/MS. The difference may be due higher specificity of the LC-MS/MS method to the N-terminal peptide from the alpha-chain versus derivatization of both alpha- and beta-chain by Edman degradation, and possible instability of HB-Val adducts during long term storage (about 10 years) between the analyses. These differences will be resolved by examining recently collected samples, using the same internal standard for parallel analysis by GC-MS/MS and LC-MS/MS. Based on our experience with pyr-Val adduct assay we anticipate that this assay will be suitable for evaluation of HB-Val in multiple species.     

A multimedia tool for the informed consent of patients prior to gastric banding

Objective: Severe obesity is a clear indication for appropriate, effective weight loss therapy. One option is operative intervention, e.g., gastric banding. Risks of the operation and therapeutic alternatives need to be comprehensibly presented to the patient. The literature has shown that better informed consent is obtained using information presented in a multimedia/video‐based format. The current study developed and evaluated a multimedia program aimed at obtaining informed consent from obese patients before gastric banding. Research Methods and Procedure: An interactive multimedia program was developed with information about preoperative examinations, the operation itself, hospital stay, operative risks, alternative therapies, and the pathophysiology and health risks of obesity. Two groups (Group 1, n = 20, mean age 38 years, informed consent attained with conventional document information; Group 2, n = 20, mean age 37 years, informed consent attained with additional multimedia information) were interviewed regarding comprehensibility of the information presented, personal satisfaction, and anxiety levels during the informed consent process. Results: Group 2 showed significantly better (p < 0.05) understanding of the presented information and higher levels of satisfaction with the informed consent process. Anxiety levels did not significantly differ between the two groups. Discussion: Because patient satisfaction with the informed consent process and understanding of the presented information significantly improved, the multimedia program clearly benefits both surgeons and patients. Personal contact from the surgeon remains essential. High volumes of information presented in multimedia format do not alleviate patient anxiety, and personal contact may be beneficial.