A radioimmunoassay for the Gm(b0) allotype of human immunoglobulins

A radioimmunoassay for the human allotype Gm(b⁰) which provides a sensitive and quantitative measurement of the level of this IgG3 genetic marker has been developed. The assay system can detect 15 nanograms of Gm(b⁰) IgG3 protein and is not inhibited by immunoglobulins of other allotypes and isotypes. Using this assay, good correlation was found between IgG3 and Gm(b⁰) levels in homozygous Gm(f, b⁰) sera and gene dosage effects could be confirmed. The correlation between Gm(b⁰) levels and IgG3 in Negroid Gm(a, b⁰) sera was not as good. This reduced correlation has been attributed to antigen differences in the IgG3 Gm markers characteristic of some Negroid Gm(a, b⁰) sera.     

Specific selection of cytotoxic effector cells: the generation of cytotoxic T cells in rat thoracic duct lymphocyte populations positively or negatively selected for reactivity to specific strong histocompatibility alloantigens.

These studies consider the generation of cytotoxic T lymphocytes (CTL) from precursors (CTLP) present in rat thoracic duct lymphocytes after stimulation with strong alloantigens. Also, they explore the relationship between CTLP and "initiator" (I) lymphocytes responsible for specific GVH and MLI reactions. Positively selected TDL populations prepared in bulk MLI cultures show enriched GVH and MLI reactivity for the selecting major histocompatibility complex (MHC) haplotype, but no cytotoxic activity, raising the possibility that I and CTLP may belong to different subpopulations, and the latter failed to differentiate or to survive under these culture conditions. Restimulation of these cells in Marbrook culture vessels with the original priming haplotype under conditions suitable for generating killer cells in vitro resulted in greatly increased specific CTL activity with accelerated kinetics soon after priming and normal kinetics later. These findings indicate that "memory" killer cells can be generated in a previously stimulated lymphocyte population that had no overt cytotoxic activity. Restimulation with third party haplotypes failed to give CTL activity either to specific or to third party targets. Negatively selected TDL populations prepared by "filtration" through x-irradiated F1 rats, depleted of specific GVH and MLI responses, were also depleted of the ability to generate CTL in Marbrook cultures stimulated with the selecting haplotype. Stimulation with third party haplotypes, or with both third party and specific haplotypes together, gave CTL effective only against the third party target.     

The acetylene reduction technique as an assay for nitrogenase activity in the methane oxidizing bacterium Methylococcus capsulatus strain bath

The use of acetylene as a convenient assay substrate for nitrogenase in methane oxidising bacteria is complicated by the observation that it is a potent inhibitor of the methane monooxygenase enzyme in both whole cells and cell-free extracts. If the cells were provided with alternative oxidisable carbon substrates other than methane then nitrogen fixing cells would reduce acetylene to ethylene. Hydrogen gas also served as an oxidisable substrate in the assay. Nitrous oxide, which is reduced by nitrogenase to N₂ and H₂O, was not an inhibitor of methane monooxygenase function and could be used as a convenient assay substrate for nitrogenase. Reduction of both substrates by whole cells showed similar response to oxygen in the assay system and in this respect Methylococcus resembles other free living nitrogen fixing aerobes.     

Prostaglandin synthesis by rheumatoid synovium and its stimulation by colchicine

The synthesis of prostaglandins by rheumatoid synovial tissue in organ culture was studied utilizing radioimmunoassay, with antisera to PGB₁, PGF_1α and PGF_2α. It was established that PGE₂ and PGF_2α were the major prostaglandins formed by analyses of culture media with the two antisera to PGF, before and after alkali treatment. Indomethacin at 5 μg/ml suppressed prostaglandin synthesis, usually to <1% of control cultures. Colchicine, 0.1 μg/ml resulted in marked stimulation of prostaglandin synthesis, in some cases over 10 fold. It is suggested, because of the colchicine effect, that the state of the microtubules may regulate the rate of prostaglandin biosynthesis. It is possible that prostaglandin E₂ produced by rheumatoid synovia may contribute to the pathogenesis of the inflammatory reaction and lead to destruction of juxta-articular bone in rheumatoid arthritis.     

The cornified envelope of terminally differentiated human epidermal keratinocytes consists of cross-linked protein

A small proportion of the protein of stratum corneum of human epidermal callus is insoluble even when boiled in solutions containing sodium dodecylsulfate and a reducing agent. This protein is present in the cornified envelope, a structure located beneath the plasma membrane. When cornified envelopes were dissolved by exhaustive proteolytic digestion and the products analyzed by chromatography, approximately 18% of the total lysine residues were found as the cross-linking dipeptide ?-(γ-glutamyl) lysine.Labeled cornified envelope protein was synthesized by human epidermal keratinocytes allowed to differentiate terminally in culture. The extent of cross-linking, determined from the proportion of radioactive lysine in ?-(γ-glutamyl) lysine after exhaustive proteolysis, was similar to that in stratum corneum. The properties of the cornified envelopes (insolubility in detergent and reducing agents, and solubility following proteolytic digestion) are readily explained by a structure consisting of a cross-linked protein lattice.     

Distinguishing the ORFs from the ELFs: short bacterial genes and the annotation of genomes

A substantial fraction of hypothetical open reading frames (ORFs) in completely sequenced bacterial genomes are short, suggesting that many are not genes but random stretches of DNA. Although it is not feasible to authenticate the coding capacity of all such regions experimentally, comparisons of ORFs in related genomes can expose those that encode functional proteins.     

Genome‐wide association study identifies loci associated with liability to alcohol and drug dependence that is associated with variability in reward‐related ventral striatum activity in African‐ and European‐Americans

Genetic influences on alcohol and drug dependence partially overlap, however, specific loci underlying this overlap remain unclear. We conducted a genome‐wide association study (GWAS) of a phenotype representing alcohol or illicit drug dependence (ANYDEP) among 7291 European‐Americans (EA; 2927 cases) and 3132 African‐Americans (AA: 1315 cases) participating in the family‐based Collaborative Study on the Genetics of Alcoholism. ANYDEP was heritable (h ² in EA = 0.60, AA = 0.37). The AA GWAS identified three regions with genome‐wide significant (GWS; P < 5E‐08) single nucleotide polymorphisms (SNPs) on chromosomes 3 (rs34066662, rs58801820) and 13 (rs75168521, rs78886294), and an insertion‐deletion on chromosome 5 (chr5:141988181). No polymorphisms reached GWS in the EA. One GWS region (chromosome 1: rs1890881) emerged from a trans‐ancestral meta‐analysis (EA + AA) of ANYDEP, and was attributable to alcohol dependence in both samples. Four genes (AA: CRKL, DZIP3, SBK3; EA: P2RX6) and four sets of genes were significantly enriched within biological pathways for hemostasis and signal transduction. GWS signals did not replicate in two independent samples but there was weak evidence for association between rs1890881 and alcohol intake in the UK Biobank. Among 118 AA and 481 EA individuals from the Duke Neurogenetics Study, rs75168521 and rs1890881 genotypes were associated with variability in reward‐related ventral striatum activation. This study identified novel loci for substance dependence and provides preliminary evidence that these variants are also associated with individual differences in neural reward reactivity. Gene discovery efforts in non‐European samples with distinct patterns of substance use may lead to the identification of novel ancestry‐specific genetic markers of risk.     

A New Statistic to Evaluate Imputation Reliability

Background

As the amount of data from genome wide association studies grows dramatically, many interesting scientific questions require imputation to combine or expand datasets. However, there are two situations for which imputation has been problematic: (1) polymorphisms with low minor allele frequency (MAF), and (2) datasets where subjects are genotyped on different platforms. Traditional measures of imputation cannot effectively address these problems.

Methodology/Principal Findings

We introduce a new statistic, the imputation quality score (IQS). In order to differentiate between well-imputed and poorly-imputed single nucleotide polymorphisms (SNPs), IQS adjusts the concordance between imputed and genotyped SNPs for chance. We first evaluated IQS in relation to minor allele frequency. Using a sample of subjects genotyped on the Illumina 1 M array, we extracted those SNPs that were also on the Illumina 550 K array and imputed them to the full set of the 1 M SNPs. As expected, the average IQS value drops dramatically with a decrease in minor allele frequency, indicating that IQS appropriately adjusts for minor allele frequency. We then evaluated whether IQS can filter poorly-imputed SNPs in situations where cases and controls are genotyped on different platforms. Randomly dividing the data into “cases” and “controls”, we extracted the Illumina 550 K SNPs from the cases and imputed the remaining Illumina 1 M SNPs. The initial Q-Q plot for the test of association between cases and controls was grossly distorted (λ = 1.15) and had 4016 false positives, reflecting imputation error. After filtering out SNPs with IQS<0.9, the Q-Q plot was acceptable and there were no longer false positives. We then evaluated the robustness of IQS computed independently on the two halves of the data. In both European Americans and African Americans the correlation was >0.99 demonstrating that a database of IQS values from common imputations could be used as an effective filter to combine data genotyped on different platforms.

Conclusions/Significance

IQS effectively differentiates well-imputed and poorly-imputed SNPs. It is particularly useful for SNPs with low minor allele frequency and when datasets are genotyped on different platforms.