Phosphorylation and microtubule association of the Opitz syndrome protein mid-1 is regulated by protein phosphatase 2A via binding to the regulatory subunit α4

Opitz syndrome (OS) is a human genetic disease characterized by deformities such as cleft palate that are attributable to defects in embryonic development at the midline. Gene mapping has identified OS mutations within a protein called Mid1. Wild-type Mid1 predominantly colocalizes with microtubules, in contrast to mutant versions of Mid1 that appear clustered in the cytosol. Using yeast two-hybrid screening, we found that the α4-subunit of protein phosphatases 2A/4/6 binds Mid1. Epitope-tagged α4 coimmunoprecipitated endogenous or coexpressed Mid1 from COS7 cells, and this required only the conserved C-terminal region of α4. Localization of Mid1 and α4 was influenced by one another in transiently transfected cells. Mid1 could recruit α4 onto microtubules, and high levels of α4 could displace Mid1 into the cytosol. Metabolic 32P labeling of cells showed that Mid1 is a phosphoprotein, and coexpression of full-length α4 decreased Mid1 phosphorylation, indicative of a functional interaction. Association of green fluorescent protein–Mid1 with microtubules in living cells was perturbed by inhibitors of MAP kinase activation. The conclusion is that Mid1 association with microtubules, which seems important for normal midline development, is regulated by dynamic phosphorylation involving MAP kinase and protein phosphatase that is targeted specifically to Mid1 by α4. Human birth defects may result from environmental or genetic disruption of this regulatory cycle.

Rate-Limiting Steps in Selenium Assimilation and Volatilization by Indian Mustard1

Se can be accumulated by plants and volatilized to dimethylselenide, providing an attractive technology for Se phytoremediation. To determine the rate-limiting steps in Se volatilization from selenate and selenite, time- and concentration-dependent kinetics of Se accumulation and volatilization were studied in Indian mustard (Brassica juncea). Time-dependent kinetic studies showed that selenate was taken up 2-fold faster than selenite. Selenate was rapidly translocated to the shoot, away from the root, the site of volatilization, whereas only approximately 10% of the selenite was translocated. For both selenate- and selenite-supplied plants, Se accumulation and volatilization increased linearly with external Se concentration up to 20 μm; volatilization rates were also linearly correlated with root Se concentrations. Se-volatilization rates were 2- to 3-fold higher from plants supplied with selenite compared with selenate. Se speciation by x-ray absorption spectroscopy revealed that selenite-supplied plants accumulated organic Se, most likely selenomethionine, whereas selenate-supplied plants accumulated selenate. Our data suggest that Se volatilization from selenate is limited by the rate of selenate reduction, as well as by the availability of Se in roots, as influenced by uptake and translocation. Se volatilization from selenite may be limited by selenite uptake and by the conversion of selenomethionine to dimethylselenide.

Accuracy of lesion bypass by yeast and human DNA polymerase η

DNA polymerase η (Polη) functions in the error-free bypass of UV-induced DNA lesions, and a defect in Polη in humans causes the cancer-prone syndrome, the variant form of xeroderma pigmentosum. Both yeast and human Polη replicate through a cis-syn thymine-thymine dimer (TT dimer) by inserting two As opposite the two Ts of the dimer. Polη, however, is a low-fidelity enzyme, and it misinserts nucleotides with a frequency of ≈ 10−2 to 10−3 opposite the two Ts of the TT dimer as well as opposite the undamaged template bases. This low fidelity of nucleotide insertion seems to conflict with the role of Polη in the error-free bypass of UV lesions. To resolve this issue, we have examined the ability of human and yeast Polη to extend from paired and mispaired primer termini opposite a TT dimer by using steady-state kinetic assays. We find that Polη extends from mispaired primer termini on damaged and undamaged DNAs with a frequency of ≈ 10−2 to 10−3 relative to paired primer termini. Thus, after the incorporation of an incorrect nucleotide, Polη would dissociate from the DNA rather than extend from the mispair. The resulting primer-terminal mispair then could be subject to proofreading by a 3′→5′ exonuclease. Replication through a TT dimer by Polη then would be more accurate than that predicted from the fidelity of nucleotide incorporation alone.

Chaperonin-facilitated protein folding: optimization of rate and yield by an iterative annealing mechanism.

We develop a heuristic model for chaperonin-facilitated protein folding, the iterative annealing mechanism, based on theoretical descriptions of "rugged" conformational free energy landscapes for protein folding, and on experimental evidence that (i) folding proceeds by a nucleation mechanism whereby correct and incorrect nucleation lead to fast and slow folding kinetics, respectively, and (ii) chaperonins optimize the rate and yield of protein folding by an active ATP-dependent process. The chaperonins GroEL and GroES catalyze the folding of ribulose bisphosphate carboxylase at a rate proportional to the GroEL concentration. Kinetically trapped folding-incompetent conformers of ribulose bisphosphate carboxylase are converted to the native state in a reaction involving multiple rounds of quantized ATP hydrolysis by GroEL. We propose that chaperonins optimize protein folding by an iterative annealing mechanism; they repeatedly bind kinetically trapped conformers, randomly disrupt their structure, and release them in less folded states, allowing substrate proteins multiple opportunities to find pathways leading to the most thermodynamically stable state. By this mechanism, chaperonins greatly expand the range of environmental conditions in which folding to the native state is possible. We suggest that the development of this device for optimizing protein folding was an early and significant evolutionary event.

Genetic analysis of type 1 diabetes using whole genome approaches.

Whole genome linkage analysis of type 1 diabetes using affected sib pair families and semi-automated genotyping and data capture procedures has shown how type 1 diabetes is inherited. A major proportion of clustering of the disease in families can be accounted for by sharing of alleles at susceptibility loci in the major histocompatibility complex on chromosome 6 (IDDM1) and at a minimum of 11 other loci on nine chromosomes. Primary etiological components of IDDM1, the HLA-DQB1 and -DRB1 class II immune response genes, and of IDDM2, the minisatellite repeat sequence in the 5' regulatory region of the insulin gene on chromosome 11p15, have been identified. Identification of the other loci will involve linkage disequilibrium mapping and sequencing of candidate genes in regions of linkage.

Aod1, the immunoregulatory locus controlling abrogation of tolerance in neonatal thymectomy-induced autoimmune ovarian dysgenesis, maps to mouse chromosome 16.

Mice thymectomized at three days of age (D3Tx) develop during adulthood a variety of organ-specific autoimmune diseases, including autoimmune ovarian dysgenesis (AOD). The phenotypic spectrum of AOD is characterized by the development of anti-ovarian autoantibodies, oophoritis, and atrophy. The D3Tx model of AOD is unique in that disease induction depends exclusively on perturbation of the normal developing immune system, is T-cell-mediated, and is strain specific. For example, D3Tx A/J mice are highly susceptible to AOD, whereas C57BL/6J mice are resistant. After D3Tx, self ovarian antigens, expressed at physiological levels, trigger an autoimmune response capable of eliciting disease. The D3Tx model provides, therefore, the opportunity to focus on the mechanisms of self-tolerance that are relevant to disease pathogenesis. Previous studies indicate that the principal mechanisms involved in AOD susceptibility are genetically controlled and govern developmental processes associated with the induction and maintenance of peripheral tolerance. We report here the mapping of the Aod1 locus to mouse chromosome 16 within a region encoding several loci of immunologic relevance, including scid, Igl1, VpreB, Igll, Igl1r, Mtv6 (Mls-3), Ly-7, Ifnar, and Ifgt.

New birds from central Peru, by John T. Zimmer ... Reports on results of the Captain Marshall Field expeditions ...

v.12:no.4(1924)

Children’s Perceptions of Cohesion

The general purpose of the two studies reported here was to examine perceptions of team cohesiveness in children aged 9 to 12 years. In Study 1, focus groups were used to examine individual perceptions of cohesion from the perspective of group integration – the group as a totality. In Study 2, open-ended questionnaires were used to examine individual perceptions of cohesion from the perspective of individual attractions to the group. The results showed that children as young as nine years understand the phenomenon known as cohesion. They can discuss the group as a totality, the characteristics of cohesive and non-cohesive teams, and identify the major factors attracting them to and maintaining their involvement in the group. Also, the ability to clearly distinguish between task and social cohesion is present. The results provide insight into the world of child sport and emphasise the importance of group cohesion, affiliation, and other social constructs in children’s involvement and adherence to sport groups

Geochemical and Microbiological Studies of Nitrous Oxide Variations within the New NEEM Greenland Ice Core During the Last Glacial Period

Deep polar ice cores provide atmospheric records of nitrous oxide (N₂O) and other trace gases reflecting climate history along with a parallel archive of microbial cells transported with mineral dust, marine and volcanic aerosols from around the globe. Our interdisciplinary study of 32 samples from different depths of the recently drilled NEEM Greenland ice core addressed the question whether the identified microorganisms were capable of post-depositional biological production of N₂O in situ. We used high-resolution geochemical and microbiological approaches to examine the N₂O concentrations, the quantitative distributions of dust, Ca⁺², NH₄⁺ and NO₃⁻ ¡ons related to N cycle pathways, the microbial abundance and diversity at specific NEEM core depths from 1758 m to 1867.8 m. Results showed varying concentrations of N₂O (220 –271.5 ppb). Microbial abundance fluctuated between 3.3 x 10⁴ and 3.3 x 10⁶ cells mL⁻¹ in direct correlation with dust and Ca²⁺ concentrations with higher cell numbers deposited during colder periods. The average values of NH₄⁺ and NO₃⁻ indicated that substrates were available for the microorganisms capable of utilizing them. PCR amplification of selected functional genes involved in bacterial and archaeal nitrification and denitrification was not successful. Sanger and Illumina MiSeq sequence analyses of SSU rRNA genes showed variable representation of Alpha-, Beta- and Gammaproteobacteria, Firmicutes, Actinobacteria, chloroplasts and fungi. The metabolic potential of the dominant genera of Proteobacteria and Firmicutes as possible N₂O producers suggested that denitrification activity may have led to in-situ production and accumulation of N₂O.