Temtamy preaxial brachydactyly syndrome is caused by loss-of-function mutations in chondroitin synthase 1, a potential target of BMP signaling

Altered Bone Morphogenetic Protein (BMP) signaling leads to multiple developmental defects, including brachydactyly and deafness. Here we identify chondroitin synthase 1 (CHSY1) as a potential mediator of BMP effects. We show that loss of human CHSY1 function causes autosomal-recessive Temtamy preaxial brachydactyly syndrome (TPBS), mainly characterized by limb malformations, short stature, and hearing loss. After mapping the TPBS locus to chromosome 15q26-qterm, we identified causative mutations in five consanguineous TPBS families. In zebrafish, antisense-mediated chsy1 knockdown causes defects in multiple developmental processes, some of which are likely to also be causative in the etiology of TPBS. In the inner ears of zebrafish larvae, chsy1 is expressed similarly to the BMP inhibitor dan and in a complementary fashion to bmp2b. Furthermore, unrestricted Bmp2b signaling or loss of Dan activity leads to reduced chsy1 expression and, during epithelial morphogenesis, defects similar to those that occur upon Chsy1 inactivation, indicating that Bmp signaling affects inner-ear development by repressing chsy1. In addition, we obtained strikingly similar zebrafish phenotypes after chsy1 overexpression, which might explain why, in humans, brachydactyly can be caused by mutations leading either to loss or to gain of BMP signaling.     

Seasonal variation in foliar carbon exchange in Pinus radiata and Populus deltoides: respiration acclimates fully to changes in temperature but photosynthesis does not

We investigated seasonal variation in dark respiration and photosynthesis by measuring gas exchange characteristics on Pinus radiata and Populus deltoides under field conditions each month for 1 year. The field site in the South Island of New Zealand is characterized by large day-to-day and seasonal changes in air temperature. The rate of foliar respiration at a base temperature of 10 °C ( R ₁₀) in both pine and poplar was found to be greater during autumn and winter and displayed a strong downward adjustment in warmer months. The sensitivity of instantaneous leaf respiration to a 10 °C increase in temperature ( Q ₁₀) was also greater during the winter period. The net effect of this strong acclimation was that the long-term temperature response of respiration was essentially flat over a wide range of ambient temperatures. Seasonal changes in photosynthesis were sensitive to temperature but largely independent of leaf nitrogen concentration or stomatal conductance. Over the range of day time growth temperatures (5–32 °C), we did not observe strong evidence of photosynthetic acclimation to temperature, and the long-term responses of photosynthetic parameters to ambient temperature were similar to previously published instantaneous responses. The ratio of foliar respiration to photosynthetic capacity ( R _d/ A _sat) was significantly greater in winter than in spring/summer. This indicates that there is little likelihood that respiration would be stimulated significantly in either of these species with moderate increases in temperature – in fact net carbon uptake was favoured at moderately higher temperatures. Model calculations demonstrate that failing to account for strong thermal acclimation of leaf respiration influences determinations of leaf carbon exchange significantly, especially for the evergreen conifer.     

The essential role of centrosomal NDE1 in human cerebral cortex neurogenesis

We investigated three families whose offspring had extreme microcephaly at birth and profound mental retardation. Brain scans and postmortem data showed that affected individuals had brains less than 10% of expected size (≤10 standard deviation) and that in addition to a massive reduction in neuron production they displayed partially deficient cortical lamination (microlissencephaly). Other body systems were apparently unaffected and overall growth was normal. We found two distinct homozygous mutations of NDE1, c.83+1G>T (p.Ala29GlnfsX114) in a Turkish family and c.684_685del (p.Pro229TrpfsX85) in two families of Pakistani origin. Using patient cells, we found that c.83+1G>T led to the use of a novel splice site and to a frameshift after NDE1 exon 2. Transfection of tagged NDE1 constructs showed that the c.684_685del mutation resulted in a NDE1 that was unable to localize to the centrosome. By staining a patient-derived cell line that carried the c.83+1G>T mutation, we found that this endogeneously expressed mutated protein equally failed to localize to the centrosome. By examining human and mouse embryonic brains, we determined that NDE1 is highly expressed in neuroepithelial cells of the developing cerebral cortex, particularly at the centrosome. We show that NDE1 accumulates on the mitotic spindle of apical neural precursors in early neurogenesis. Thus, NDE1 deficiency causes both a severe failure of neurogenesis and a deficiency in cortical lamination. Our data further highlight the importance of the centrosome in multiple aspects of neurodevelopment.     

Screening Peptide Bead Libraries for Enzyme Inhibitors

Recent developments in the technology of solid-phase peptide synthesis have made it possible to create libraries of peptides mounted on solid supports, which may then be screened for biological activity. By inserting such a randomized library region into a synthetic peptide mounted on resin beads, a full range of potentially useful mutants may be screened simultaneously. This article describes the use of this approach to generate variants of CMTI-I, a conformationally constrained proteinaceous inhibitor of trypsin and chymotrypsin. The result is a library containing mutants for the P′₁-P′₄ reactive-loop sequence of CMTI-I. This is equilibrated with a fluorescentiy labeled target enzyme (in this case human leukocyte elastase), which binds to those beads containing the most potent inhibitors. These are identified and isolated. The method utilizes the conformational rigidity of CMTI-I to improve inhibitory activity. By this means the most potent inhibitors of HLE may be distinguished and then synthesized on a preparative scale for kinetic analysis. This approach may be applied directly to other serine proteinases.     

Ethical and legal dilemmas arising during predictive testing for adult-onset disease: the experience of Huntington disease.

The goal of predictive testing is to modify the risk for currently healthy individuals to develop a genetic disease in the future. Such testing using polymorphic DNA markers has had major application in Huntington disease. The Canadian Collaborative Study of Predictive Testing for Huntington Disease has been guided by major principles of medical ethics, including autonomy, beneficence, confidentiality, and justice. Numerous ethical and legal dilemmas have arisen in this program, challenging these principles and occasionally casting them into conflict. The present report describes these dilemmas and offers our approach to resolving them. These issues will have relevance to predictive-testing programs for other adult-onset disorders.     

Interaction of pyrimethamine, cycloguanil, WR99210 and their analogues with Plasmodium falciparum dihydrofolate reductase: structural basis of antifolate resistance

The nature of the interactions between Plasmodium falciparum dihydrofolate reductase (pfDHFR) and antimalarial antifolates, i.e., pyrimethamine (Pyr), cycloguanil (Cyc) and WR99210 including some of their analogues, was investigated by molecular modeling in conjunction with the determination of the inhibition constants (Ki). A three-dimensional structural model of pfDHFR was constructed using multiple sequence alignment and homology modeling procedures, followed by extensive molecular dynamics calculations. Mutations at amino acid residues 16 and 108 known to be associated with antifolate resistance were introduced into the structure, and the interactions of the inhibitors with the enzymes were assessed by docking and molecular dynamics for both wild-type and mutant DHFRs. The Ki values of a number of analogues tested support the validity of the model. A 'steric constraint' hypothesis is proposed to explain the structural basis of the antifolate resistance.     

Huntington's disease: prediction and prevention

The identification of a DNA restriction fragment length polymorphism closely linked to Huntington's disease on the short arm of chromosome 4 has for the first time allowed presymptomatic prediction to be undertaken in first-degree relatives at risk. The late and variable onset of this dominantly inherited disorder makes such prediction a powerful and potentially valuable aid in genetic counselling, but in the absence of effective therapy there are serious ethical reservations concerning such a predictive test. The new developments have stimulated an active and informative debate among professionals and family members on whether and how predictive tests should be used. Guidelines have emerged which should be useful not only for Huntington's disease, but for other serious late-onset neurogenetic disorders. Meanwhile, studies in Wales and elsewhere have not only confirmed the original linkage but have excluded multi-locus heterogeneity as a significant problem. Genetic prediction for the individual at risk remains critically dependent on a suitable family structure, present in only a minority of families in Wales. A more feasible alternative for most families is prenatal exclusion, which can allow risk prediction for a pregnancy without altering the situation for the person at risk. This approach has already been applied in Wales; the experience gained will be useful in full prediction, which is currently being introduced.     

Mutations in ZDHHC9, which encodes a palmitoyltransferase of NRAS and HRAS, cause X-linked mental retardation associated with a Marfanoid habitus

We have identified one frameshift mutation, one splice-site mutation, and two missense mutations in highly conserved residues in ZDHHC9 at Xq26.1 in 4 of 250 families with X-linked mental retardation (XLMR). In three of the families, the mental retardation phenotype is associated with a Marfanoid habitus, although none of the affected individuals meets the Ghent criteria for Marfan syndrome. ZDHHC9 is a palmitoyltransferase that catalyzes the posttranslational modification of NRAS and HRAS. The degree of palmitoylation determines the temporal and spatial location of these proteins in the plasma membrane and Golgi complex. The finding of mutations in ZDHHC9 suggests that alterations in the concentrations and cellular distribution of target proteins are sufficient to cause disease. This is the first XLMR gene to be reported that encodes a posttranslational modification enzyme, palmitoyltransferase. Furthermore, now that the first palmitoyltransferase that causes mental retardation has been identified, defects in other palmitoylation transferases become good candidates for causing other mental retardation syndromes.     

Predictive thresholds for forecasting the compatibility of <Emphasis Type="Italic">Forficula auricularia</Emphasis> and <Emphasis Type="Italic">Aphelinus mali</Emphasis> as biological control agents against woolly apple aphid in apple orchards

The woolly apple aphid (WAA), Eriosoma lanigerum (Hausmann) (Hemiptera: Aphididae) is a well-known pest of apple orchards world-wide. Several studies have demonstrated variable control of WAA populations by the European earwig, Forficula auricularia (L.) (Dermaptera: Forficulidae) and the WAA parasitoid Aphelinus mali (Halderman) (Hymenoptera: Aphelinidae). We examine whether a beneficial interaction between F. auricularia and A. mali exists and calculate optimal numbers for each species to maintain WAA infestations below acceptable levels. We demonstrate that trees possessing >14 earwigs per trunk trap per week within the first seven weeks post-blossom contained WAA infestations well below acceptable levels. Where these earwig thresholds were not met, a first generation of A.mali greater than 0.5 wasps per tree was required. If these beneficial insect targets were not met, severe WAA infestations occurred. Our findings suggest that if F. auricularia and A. mali numbers exceed these thresholds chemical intervention may not be required.