Aging in the post-genomic era: simple or complex?

A report on the Third Genetic Effects on Aging Meeting, The Jackson Laboratory, Bar Harbor, Maine, August 4-8, 2000.     

Influence of two glutamine-containing dipeptides on growth of mammalian cells

Summary The instability of the amino acid glutamine prompted us to investigate substitute compounds appropriate for culture conditions. The effect of two glutamine-containing dipeptides, alanylglutamine (Ala-Gln) and glycylglutamine (Gly-Gln), on the growth behavior of a hematopoietic cell line in culture (K562) was investigated. Growth rates and [³H]thymidine incorporation rates of cells cultivated in sterile-filtrated media, containing glutamine (Gln) or Ala-Gln or Gly-Gln, were not statistically different. Although heat-sterilization of media containing Gln caused approximately 95% decomposition of the Gln, both dipeptides remained unaltered. Consequently, cell growth was drastically decreased when autoclaved free Gln-containing media were used, but growth was unaffected in the presence of autoclaved dipeptides. Both Ala-Gln and Gly-Gln have an advantage over free Gln as growth factors for cell culture due to the stability of the dipeptides during both autoclaving and storage; the biological activity, however, is comparable.     

A modular DNA signal translator for the controlled release of a protein by an aptamer

Owing to the intimate linkage of sequence and structure in nucleic acids, DNA is an extremely attractive molecule for the development of molecular devices, in particular when a combination of information processing and chemomechanical tasks is desired. Many of the previously demonstrated devices are driven by hybridization between DNA ‘effector’ strands and specific recognition sequences on the device. For applications it is of great interest to link several of such molecular devices together within artificial reaction cascades. Often it will not be possible to choose DNA sequences freely, e.g. when functional nucleic acids such as aptamers are used. In such cases translation of an arbitrary ‘input’ sequence into a desired effector sequence may be required. Here we demonstrate a molecular ‘translator’ for information encoded in DNA and show how it can be used to control the release of a protein by an aptamer using an arbitrarily chosen DNA input strand. The function of the translator is based on branch migration and the action of the endonuclease FokI. The modular design of the translator facilitates the adaptation of the device to various input or output sequences.     

Transcriptional response to alcohol exposure in Drosophila melanogaster

Background  

Alcoholism presents widespread social and human health problems. Alcohol sensitivity, the development of tolerance to alcohol and susceptibility to addiction vary in the population. Genetic factors that predispose to alcoholism remain largely unknown due to extensive genetic and environmental variation in human populations. Drosophila, however, allows studies on genetically identical individuals in controlled environments. Although addiction to alcohol has not been demonstrated in Drosophila, flies show responses to alcohol exposure that resemble human intoxication, including hyperactivity, loss of postural control, sedation, and exposure-dependent development of tolerance.     

A rheumatoid factor paradox: inhibition of rituximab effector function

Introduction

Rituximab (RTX) therapy of rheumatoid arthritis (RA) exhibits enhanced effectiveness in seropositive patients. Using patient sera, we tested if this improved efficacy was associated with enhanced RTX mediated complement-dependent cytotoxicity (RTX-CDC).

Methods

We developed an in vitro assay for RTX-CDC using patient sera and the Daudi human B cell line. Using propidium iodide uptake and flow cytometry, we compared RTX-CDC with rheumatoid factor (RF)+ sera relative to normal volunteer, non-RA and RF- sera. Additional studies examined mixing studies of RF+ and RF- sera, as well as the effect of monoclonal IgA or IgM RF. Finally, the effect of RF on RTX mediated trogocytosis of normal B cells was evaluated.

Results

Using human sera, addition of RTX resulted in rapid and profound (> 50%) Daudi cell death that was complement dependent. Surprisingly, RF+ patient sera exhibited reduced RTX-CDC relative to RF- sera, with an inverse relationship of RTX-CDC and RF titer. Mixing studies indicated the presence of an inhibitor of RTX-CDC in RF+ sera. The addition of monoclonal IgM or IgA RF to RF- sera markedly inhibited RTX-CDC. This effect was specific for RF binding to the Fc portion of RTX as it was not apparent with the F(ab)'' domains of RTX engineered onto IgG3 heavy chain. RF also modestly inhibited RTX mediated trogocytosis.

Conclusions

Contrary to expectations, RF+ sera exhibits reduced RTX-CDC due to the presence of RF. The enhanced efficacy of RTX in seropositive RA patients cannot be attributed to improved B cell depletion through CDC. This result indicates that high RF levels may potentially modulate the efficacy of any therapeutic monoclonal antibody dependent on Fc effector function.     

Quantitative analysis of the nanopore translocation dynamics of simple structured polynucleotides

Nanopore translocation experiments are increasingly applied to probe the secondary structures of RNA and DNA molecules. Here, we report two vital steps toward establishing nanopore translocation as a tool for the systematic and quantitative analysis of polynucleotide folding: 1), Using α-hemolysin pores and a diverse set of different DNA hairpins, we demonstrate that backward nanopore force spectroscopy is particularly well suited for quantitative analysis. In contrast to forward translocation from the vestibule side of the pore, backward translocation times do not appear to be significantly affected by pore-DNA interactions. 2), We develop and verify experimentally a versatile mesoscopic theoretical framework for the quantitative analysis of translocation experiments with structured polynucleotides. The underlying model is based on sequence-dependent free energy landscapes constructed using the known thermodynamic parameters for polynucleotide basepairing. This approach limits the adjustable parameters to a small set of sequence-independent parameters. After parameter calibration, the theoretical model predicts the translocation dynamics of new sequences. These predictions can be leveraged to generate a baseline expectation even for more complicated structures where the assumptions underlying the one-dimensional free energy landscape may no longer be satisfied. Taken together, backward translocation through α-hemolysin pores combined with mesoscopic theoretical modeling is a promising approach for label-free single-molecule analysis of DNA and RNA folding.     

The genetic basis of alcoholism: multiple phenotypes,many genes,complex networks

Alcoholism is a significant public health problem. A picture of the genetic architecture underlying alcohol-related phenotypes is emerging from genome-wide association studies and work on genetically tractable model organisms.     

Microsatellite evolution in congeneric mammals: domestic and bighorn sheep

We compared genotypes at eight (AC)n microsatellite loci in domestic sheep (Ovis aries) and wild Rocky Mountain bighorn sheep (O. canadensis). The domestic sheep had greater genetic variation, higher allele-size variances, and larger allele sizes than the wild sheep. Accumulating evidence from higher taxonomic comparisons shows that these parameters are biased if microsatellite loci are selected in one taxon and used in another. Our results demonstrate similar biases between congeneric species. We compared standard measures of genetic variation, differentiation, and distance within and between species (H, D, FST) to newer measures based on allele-size variance (SW, SB, RST). The size-based distances better detected species-level divergence, but standard measures better distinguished allopatric populations. Empirical calibration of these measures at the subspecies level is needed to establish their useful ranges.