Methods of compliance evaluation for ocean outfall design and analysis.

Sewage discharge from an ocean outfall is subject to water quality standards, which are often stated in probabilistic terms. Monte Carlo simulation (MCS) has been used in the past to evaluate the ability of a designed outfall to meet water quality standards or compliance guidelines associated with sewage discharges. In this study, simpler and less computer-intensive probabilistic methods are considered. The probabilistic methods evaluated are the popular mean first-order second-moment (MFOSM) and the advance first-order second-moment (AFOSM) methods. Available data from the Spaniard's Bay Outfall located on the east coast of New-foundland, Canada, were used as inputs for a case study. Both methods were compared with results given by MCS. It was found that AFOSM gave a good approximation of the failure probability for total coliform concentration at points remote from the outfall. However, MFOSM was found to be better when considering only the initial dilutions between the discharge point and the surface. Reasons for the different results may be the difference in complexity of the performance function in both cases. This study does not recommend the use of AFOSM for failure analysis in ocean outfall design and analysis because the analysis requires computational efforts similar to MCS. With the advancement of computer technology, simulation techniques, available software, and its flexibility in handling complex situations, MCS is still the best choice for failure analysis of ocean outfalls when data or estimates on the parameters involved are available or can be assumed.

Availability and characterization of transgenic and knockout mice with behavioral manifestations: where to look and what to search for.

Mice altered by transgenesis or gene targeting ("knockouts") have increasingly been employed as alternative effective tools in elucidating the genetic basis of neurophysiology and behavior. Standardization of specific behavioral paradigms and phenotyping strategies will ensure that these behavioral mouse mutants offer robust models for evaluating the efficacy of novel therapeutics in the treatment of hereditary neurological disorders. The Induced Mutant Resource (IMR) at The Jackson Laboratory (Bar Harbor, Maine, USA) imports, cryopreserves, develops, maintains, and distributes to the research community biomedically valuable stocks of transgenic and targeted mutant mice. Information on behavioral and neurological strains-including a phenotypic synopsis, husbandry requirements, strain availability, and genetic typing protocols-is available through the IMR database (http://www.jax.org/resources/documents/imr/). A current catalog of available strains is readily accessible via the JAX Mice Web site at http://jaxmice.jax.org/index.shtml. In addition, The Jackson Laboratory is now home to TBASE (http://tbase.jax.org/), a comprehensive, community database whose primary focus is on mouse knockouts. TBASE accommodates an exhaustive bibliographical resource for transgenic and knockout mice and provides a detailed phenotypic characterization of numerous behavioral knockouts that is primarily extracted from the literature. Concerted efforts to merge the two resources into a new, schematically reformed database are underway.

Transgenic and knockout databases: behavioral profiles of mouse mutants.

Genetically engineered strains of mice, modified by transgenesis or gene targeting ("knockouts") are being generated at an impressive rate and used, among other areas, as premiere research tools in deciphering the genetic basis of behavior. As behavioral phenotyping strategies continue to evolve, characterization of these "designer" mice will provide models to evaluate the efficacy of new pharmacological and gene therapy treatments in human hereditary diseases. Reported behavioral profiles include aberrant social, reproductive, and parental behaviors, learning and memory deficits, feeding disorders, aggression, anxiety-related behaviors, pain/analgesia, and altered responses to antidepressants, antipsychotics, ethanol, and psychostimulant drugs of abuse. The Induced Mutant Resource (IMR) at The Jackson Laboratory (TJL, Bar Harbor, ME, USA) imports, cryopreserves, develops, maintains, and distributes biomedically important stocks of transgenic and targeted mutant mice to the research community. Information on neurological/behavioral strains--including behavioral performance, husbandry requirements, strain availability, and genetic typing protocols--is provided through the IMR database (http://www.jax.org/resources/documents/imr/). A catalog of available strains is readily accessible via the JAX Mice website at http://jaxmice.jax.org/index.shtml. In addition, TJL is now host to TBASE (http://tbase.jax.org/), a comprehensive, public-domain database with primary emphasis on mouse knockouts. TBASE contains an exhaustive list of knockout-related citations and provides an extensive phenotypic characterization of numerous behavioral mutants that is extracted directly from the literature. Present efforts to merge the two resources into a novel, schematically enhanced database, provisionally named Transgenic and Targeted Mutation Database (TTMD), will be briefly discussed.

Genotypic and phenotypic characterization of six new recombinant congenic strains derived from NOD/Shi and CBA/J genomes.

Recombinant Congenic Strains (RCS) are useful for dissecting complex polygenic traits. Here, we describe genetic and phenotypic characterization of six new RCS generated from outcrosses between NOD/Shi and CBA/LsLt, followed by sib mating of first backcross progeny (to CBA) for 20 generations, whereupon genetic and phenotypic analysis commenced. Four of the RCS were selected on the basis of residual heterozygosity present at F20 in one of the three original RCS. Contrary to expectations for RCS developed at first backcross, all derived at least 50% of the polymorphic markers typed from the NOD parental strain. Development of autoimmune insulin-dependent diabetes mellitus (IDDM) in NOD is a strain-specific characteristic. The major genetic component predisposing NOD mice to IDDM, their H2(g7) haplotype, was present in all RCS. Nevertheless, the presence of variable amounts of CBA genome at non-MHC loci conferred complete resistance in all RCS to spontaneous IDDM development, and rendered them strongly resistant to cyclophosphamide-induced IDDM. Although the RCS more resemble NOD in regard to certain strain-specific characteristics, such as prolificacy, an immunologic phenotype that was significantly reduced when compared to both parental strains was the number of peripheral CD8(+) T cells. Given the genetic characterization presented, these new RCS should prove valuable to investigators interested in studying genes controlling differential susceptibilities distinguishing the NOD and CBA inbred strain backgrounds.

An alternative first exon in the distal end of the erythroid ankyrin gene leads to production of a small isoform containing an NH2-terminal membrane anchor.

Mouse erythroid ankyrin is encoded by the Ank1 gene on Chromosome 8. The best studied isoform is 210 kDa and contains three large functional domains. We have recently reported a small Ank1 isoform (relative mobility 25 kDa) that localizes to the M and Z lines in skeletal muscle. Analyses of cDNA and genomic clones show that three transcripts of 3.5, 2.0, and 1.6 kb code for this protein. The different transcript sizes are due to their 3'-untranslated regions. They are encoded by a new first exon located in intron 39 of the Ank1 gene and three previously described Ank1 exons (40, 41, and 42). The 5'-flanking region contains a putative muscle-specific promoter. The sequence of the first 72 amino acids is novel and is predicted to form a transmembrane helix at the NH2-terminus. Functional testing of the putative transmembrane segment indicates that it acts as a membrane anchor, suggesting that the new Ank1 isoform may play an important role in organizing the contractile apparatus within the cell.

Genetic variation among 129 substrains and its importance for targeted mutagenesis in mice.

Targeted mutagenesis in mice, a powerful tool for the analysis of gene function and human disease, makes extensive use of 129 mouse substrains. Although all are named 129, we document that outcrossing of these substrains, both deliberate and accidental, has lead to extensive genetic variability among substrains and embryonic stem cells derived from them. This clearer understanding of 129 substrain variability allows consideration of its negative impact on targeting technology, including: homologous recombination frequencies, preparation of inbred animals, and availability of appropriate controls. Based on these considerations we suggest a number of recommendations for future experimental design.

Small, membrane-bound, alternatively spliced forms of ankyrin 1 associated with the sarcoplasmic reticulum of mammalian skeletal muscle.

We have recently found that the erythroid ankyrin gene, Ank1, expresses isoforms in mouse skeletal muscle, several of which share COOH-terminal sequence with previously known Ank1 isoforms but have a novel, highly hydrophobic 72-amino acid segment at their NH2 termini. Here, through the use of domain-specific peptide antibodies, we report the presence of the small ankyrins in rat and rabbit skeletal muscle and demonstrate their selective association with the sarcoplasmic reticulum. In frozen sections of rat skeletal muscle, antibodies to the spectrin-binding domain (anti-p65) react only with a 210-kD Ank1 and label the sarcolemma and nuclei, while antibodies to the COOH terminus of the small ankyrin (anti-p6) react with peptides of 20 to 26 kD on immunoblots and decorate the myoplasm in a reticular pattern. Mice homozygous for the normoblastosis mutation (gene symbol nb) are deficient in the 210-kD ankyrin but contain normal levels of the small ankyrins in the myoplasm. In nb/nb skeletal muscle, anti-p65 label is absent from the sarcolemma, whereas anti-p6 label shows the same distribution as in control skeletal muscle. In normal skeletal muscle of the rat, anti-p6 decorates Z lines, as defined by antidesmin distribution, and is also present at M lines where it surrounds the thick myosin filaments. Immunoblots of the proteins isolated with rabbit sarcoplasmic reticulum indicate that the small ankyrins are highly enriched in this fraction. When expressed in transfected HEK 293 cells, the small ankyrins are distributed in a reticular pattern resembling the ER if the NH2-terminal hydrophobic domain is present, but they are uniformly distributed in the cytosol if this domain is absent. These results suggest that the small ankyrins are integral membrane proteins of the sarcoplasmic reticulum. We propose that, unlike the 210-kD form of Ank1, previously localized to the sarcolemma and believed to be a part of the supporting cytoskeleton, the small Ank1 isoforms may stabilize the sarcoplasmic reticulum by linking it to the contractile apparatus.

Semisynthetic cytochrome c.

Horse heart cytochrome c can be split with cyanogen bromide into a heme peptide (residues 1-65) and a nonheme peptide (residues 66-104). In a process involving (i) complex formation between the two fragments and (ii) restoration of the severed peptide linkage, a fully active cytochrome c preparation can be re-formed. Use has been made of this process to couple the heme peptide to peptide 66-104 synthesized by the Merrifield solid-phase procedure. The semisynthetic product formed in this manner is indistinguishable from reconstituted cytochrome c prepared with nonsynthetic peptide 66-104.