Fine mapping of the Autosomal Dominant Split Hand/Split Foot Locus on Chromosome 7, Band q21.3-q22.1

Split hand/split foot (SHFD) is a human developmental defect characterized by missing digits, fusion of remaining digits, and a deep median cleft in the hands and feet. Cytogenetic studies of deletions and translocations associated with this disorder have indicated that an autosomal dominant split hand/split foot locus (gene SHFD1) maps to 7q21-q22. To characterize the SHFD1 locus, somatic cell hybrid lines were constructed from cytogenetically abnormal individuals with SHFD. Molecular analysis resulted in the localization of 93 DNA markers to one of 10 intervals surrounding the SHFD1 locus. The translocation breakpoints in four SHFD patients were encompassed by the smallest region of overlap among the SHFD-associated deletions. The order of DNA markers in the SHFD1 critical region has been defined as PON–D7S812–SHFD1–D7S811–ASNS. One DNA marker, D7S811, detected altered restriction enzyme fragments in three patients with translocations when examined by pulsed-field gel electro-phoresis (PFGE). These data map SHFD1, a gene that is crucial for human limb differentiation, to a small interval in the q21.3-q22.1 region of human chromosome 7.     

Excess SeqA prolongs sequestration of oriC and delays nucleoid segregation and cell division

Following initiation of chromosomal replication in Escherichia coli, newly initiated origins (oriCs) are prevented from further initiations by a mechanism termed sequestration. During the sequestration period (which lasts about one-third of a cell cycle), the origins remain hemimethylated. The SeqA protein binds hemimethylated oriC in vitro. In vivo, the absence of SeqA causes overinitiation and strongly reduces the duration of hemimethylation. The pattern of immunostained SeqA complexes in vivo suggests that SeqA has a role in organizing hemimethylated DNA at the replication forks. We have examined the effects of overexpressing SeqA under different cellular conditions. Our data demonstrate that excess SeqA significantly increases the time oriC is hemimethylated following initiation of replication. In some cells, sequestration continued for more than one generation and resulted in inhibition of primary initiation. SeqA overproduction also interfered with the segregation of sister nucleoids and caused a delay in cell division. These results suggest that SeqA's function in regulation of replication initiation is linked to chromosome segregation and possibly cell division.     

The effects of MHC gene dosage and allelic variation on T cell receptor selection

In a T cell receptor transgenic mouse model of thymic selection, the efficiency of selection of the transgenic alpha beta heterodimer is significantly enhanced in animals that express higher densities of the relevant major histocompatibility complex molecule (I-Ek/b). These results imply that there is a stochastic component to positive selection in the thymus. Allelic variants of the original selecting I-Ek molecule are either less efficient (E alpha k:E beta b) or incapable (E alpha k:E beta s and I-Ed) of mediating the selection of transgenic alpha beta + T cells. Two of these three I-E variants appear to differ from I-Ek in amino acid residues of the peptide binding site and not in residues capable of contacting the T cell receptor, suggesting that specific peptides, or conformations of peptides, play a role in positive selection. In contrast, mice transgenic for only the beta chain of this T cell receptor show selection for CD4+ T cells in the presence of all four I-E variants tested.     

UV-induced apoptosis is mediated independent of caspase-9 in MCF-7 cells: a model for cytochrome c resistance

The importance of the mitochondria in UV-induced apoptosis has become increasingly apparent. Following DNA damage cytochrome c and other pro-apoptotic factors are released from the mitochondria, allowing for formation of the apoptosome and subsequent cleavage and activation of caspase-9. Active caspase-9 then activates downstream caspases-3 and/or -7, which in turn cleave poly(ADP)-ribose polymerase (PARP) and other down-stream targets, resulting in apoptosis. In an effort to understand the mechanisms of Akt-mediated cell survival in breast cancer, we studied the effects of insulin-like growth factor (IGF)-I treatment on UV-treated MCF-7 human breast cancer cells. Apoptosis was induced in MCF-7 cells after UV treatment, as measured by caspase-7 and PARP cleavage, and IGF-I co-treatment protected against this response. Surprisingly caspase-9 cleavage was unchanged with UV and/or IGF-I treatment. Using MCF-7 cells overexpressing caspase-3 we have shown that resistance of caspase-9 to cleavage was not altered by the expression of caspase-3. Furthermore, overexpression of caspase-9 did not enhance PARP or caspase-7 cleavage after UV treatment. Because caspase-8 was activated with UV treatment alone, we believe that UV-induced apoptosis in MCF-7 cells occurs independently of cytochrome c and caspase-9, supporting the existence of a cytoplasmic inhibitor of cytochrome c in MCF-7 cells. We anticipate that such inhibitors may be overexpressed in cancer cells, allowing for treatment resistance.     

Determination of thiopurine S-methyltransferase activity in erythrocytes using 6-thioguanine as substrate and a non-extraction liquid chromatographic technique

A non-extraction high-performance liquid chromatographic (HPLC) method has been developed for the determination of 6-methylthioguanine (6-MTG), as part of the determination of thiopurine S-methyltransferase activity (TPMT) in erythrocytes. Erythrocyte lysate is added to a glass vial containing substrates and incubation buffer, which is then sealed for the rest of the analysis. Enzyme incubation, sample preparation, and analysis are then undertaken without further sample-handling steps. The need for a solvent extraction step has been overcome by heating the incubate to 85 degrees C to stop the enzyme reaction. The heat inactivation step precipitates protein which upon centrifugation forms a thin film in the bottom of the glass vial enabling the supernatant to be injected directly onto the HPLC system. The assay shows excellent precision and recovery with a within-batch imprecision giving a co-efficient of variation of 2.9% (mean=41.5 nmol 6-MTG/gHb/h, n=10) and 5.1% (mean=12.6 nmol 6-MTG/g Hb/h, n=10). The between-batch imprecision gives a co-efficient of variation of 8.2% (mean=11.1 nmol 6-MTG/gHb/h, n=11) and 7.3% (mean=41.0 nmol 6-MTG/gHb/h, n=16). Determination of the TPMT activity in 120 people shows a range of enzyme activity of 11.3-63.8 nmol 6-MTG/gHb/h with a mean and median activity of 34.8 and 34.2 nmol 6-MTG/gHb/h, respectively. TPMT is increasingly used in clinical practice to ensure optimisation of treatment with thioguanine drugs. This direct HPLC method minimises sample-handling, reduces inherent imprecision, the possibility of laboratory error and with the potential for further automation, makes it ideal for use in a regional referral laboratory.     

Inhibition of Farnesoic Acid Methyl transferase by Sinefungin

Sinefungin inhibited the S-adenosylmethionine-dependent farnesoic acid methyltransferase in a cell-free system containing a homogenate of corpora allata from female locusts, Locusta migratoria. The enzyme catalyzed the penultimate step of juvenile hormone biosynthesis in the insects. Culturing corpora allata in the presence of sinefungin greatly suppressed juvenile hormone production. The following in vivo effects were visible after injection of the inhibitor: increase in mortality and reduction of total haemolymph protein titer and ovary fresh weight, as well as length of terminal oocytes. Attempts to reverse these effects by topical application of the juvenile hormone analog ZR-515 (methoprene) were only partly successful. Therefore, the in vivo effects may be due to a general inhibition of methyltransferase enzymes in the insect. Sinefungin appeared to be of potential interest as the first representative of a new class of insect growth regulators.     

Objective and Perceived Risk Estimates of Travel Modes in the Norwegian Public

The aim of this study was to identify groups of travel mode users, based on objective risk estimates, and examine overall differences in demographic characteristics, perceived risk, worry, perceived control when using travel modes, trust in authorities, and safety motivation. The results were based on a self-completion questionnaire survey about risk perception and travel mode use in a representative sample of the Norwegian public (n = 1864). In addition, aggregate-level data on accidents in transport were used to establish the “objective risk” for various travel modes. The respondents were split into two clusters. The first cluster was characterized by a relatively greater objective risk for accidents related to public travel modes as well as related to being a pedestrian, while the second cluster was characterized by a higher risk level related to motorized private modes of transportation. There was a significant overall difference in the risk estimates among the members of the two clusters. There was also an overall difference in risk perception and other risk-related judgments due to which risk estimate-based cluster the respondents belonged. Associations between objective risk estimates, perceived risk, and worry are discussed in relation to cluster differences in objective risk.     

Predation on artificial,solitary and aggregated wader nests on farmland

Predation rates on artificial wader nests, solitary curlew (Numenius arquata) and lapwing (Vanellus vanellus) nests and lapwing nests in colonies were studied on a farmland site in central Sweden. Predation rates were highest on artificial wader nests, intermediate on solitary curlew and lapwing nests and lowest on lapwing nests in colonies, probably because of active defence of adults at real nests and/or because of selection of nest sites with lower predation risk by breeding birds. A comparison of nests close to (50 m) and far away from (200 m) forest edges revealed no increased predation risk close to edges for any of the studied nest types. Predation risk changed during the season for artificial nests (highest in the middle of May), while predation rates on lapwing and curlew nests were more stable. Artificial nests seem to be inappropriate for measuring actual predation rates and temporal differences in predation rates on real nests, but they might be suitable for use as an index of spatial differences.     

Translocation biosensors to study signal-specific nucleo-cytoplasmic transport, protease activity and protein-protein interactions

Regulated nucleo-cytoplasmic transport is crucial for cellular homeostasis and relies on protein interaction networks. In addition, the spatial division into the nucleus and the cytoplasm marks two intracellular compartments that can easily be distinguished by microscopy. Consequently, combining the rules for regulated nucleo-cytoplasmic transport with autofluorescent proteins, we developed novel cellular biosensors composed of glutathione S-transferase, mutants of green fluorescent protein and rational combinations of nuclear import and export signals. Addition of regulatory sequences resulted in three classes of biosensors applicable for the identification of signal-specific nuclear export and import inhibitors, small molecules that interfere with protease activity and compounds that prevent specific protein-protein interactions in living cells. As a unique feature, our system exploits nuclear accumulation of the cytoplasmic biosensors as the reliable readout for all assays. Efficacy of the biosensors was systematically investigated and also demonstrated by using a fully automated platform for high throughput screening (HTS) microscopy and assay analysis. The introduced modular biosensors not only have the potential to further dissect nucleo-cytoplasmic transport pathways but also to be employed in numerous screening applications for the early stage evaluation of potential drug candidates.     

Cloned cattle derived from a novel zona-free embryo reconstruction system

As the demand for cloned embryos and offspring increases, the need arises for the development of nuclear transfer procedures that are improved in both efficiency and ease of operation. Here, we describe a novel zona-free cloning method that doubles the throughput in cloned bovine embryo production over current procedures and generates viable offspring with the same efficiency. Elements of the procedure include zona-free enucleation without a holding pipette, automated fusion of 5-10 oocyte-donor cell pairs and microdrop in vitro culture. Using this system, zona-free embryos were reconstructed from five independent primary cell lines and cultured either singularly (single-IVC) or as aggregates of three (triple-IVC). Blastocysts of transferable quality were obtained at similar rates from zona-free single-IVC, triple-IVC, and control zona-intact embryos (33%, 25%, and 29%, respectively). In a direct comparison, there was no significant difference in development to live calves at term between single-IVC, triple-IVC, and zona-intact embryos derived from the same adult fibroblast line (10%, 13%, and 15%, respectively). This zona-free cloning method could be straightforward for users of conventional cloning procedures to adopt and may prove a simple, fast, and efficient alternative for nuclear cloning of other species as well.