Objective We have taken a positional approach to assign the spontaneous squiggle tail ( squig ) mutation in mice to a specific gene defect. Results A large panel of backcross mice was produced and characterized to map squig to high genetic resolution on mouse Chromosome (Chr) 11. Two overlapping candidate genes that co-localized with squig ( Meox1 , for mesenchyme homeobox 1; and Gm11551 , which encodes a lncRNA located entirely within the first intron of Meox1 ) were fully sequenced to discover any squig -specific defects. This analysis revealed a 3195 bp deletion that includes all of Meox1 , Exon 1 but does not disrupt Gm11551 . We recommend that the squig mutation be renamed Meox1 squig , and suggest that this variant may offer an appropriate animal model for Klippel-Feil syndrome 2 (KFS2) in humans.

The spontaneous, curly whiskers mutation (abbreviated cw) generates kinky, brittle vibrissae in homozygous mice. Although cw has been mapped to the centromeric end of mouse Chromosome 9, no particular gene has been causally implicated, and this lack of genetic assignment has stymied cw's complete molecular and functional analysis. As a foundation for its positional cloning, we have fine-mapped cw to a small, 0.57 Mb interval that contains only three skin-expressed genes, including hephaestin-like 1 (Hephl1), which encodes a membrane-bound, multi-copper ferroxidase. Sequence analysis of all Hephl1 coding regions in cw/cw mutants revealed a single-base-pair substitution that alters Hephl1 mRNA splicing, and is specific to the cw allele, only. Sequence analysis of a second, independent, re-mutation to curly whiskers (that we verified by complementation testing with cw and have designated cw 2J ) revealed a distinct defect in Hephl1 (a frame-shifting, single-base-pair insertion) that is specific to cw 2J . The results presented strongly suggest that defects in the Hephl1 gene are the molecular basis of the classical, curly-whiskers mutant phenotypes.

Background: The recessive rough fur mutation (ruf)-named for the unkempt, greasy appearance of the hair coat in homozygotes-has previously been mapped on mouse Chromosome 9. However, the assignment of ruf to a particular gene is needed to facilitate a complete molecular analysis of the mutant phenotype. Results: To establish a more refined location for ruf (as a basis for positional cloning) DNA isolated from a large backcross family was typed for microsatellite and single-nucleotide markers on Chromosome 9. This analysis restricted the location of ruf between sites that flank only four genes known to be expressed in skin, one of which-Mpzl3, for myelin protein zero-like 3-generates a similar hair phenotype in mice homozygous for engineered and spontaneous null-alleles. A cross between ruf mutants and mice heterozygous for the MpzI3rc mutation (which controls a recessive phenotype called rough coat) produced offspring that displayed matted, damp-looking fur, indicating that ruf is a mutant allele of Mpzl3. However, sequence analysis of the Mpzl3 promoter, exons and splice junctions revealed no mutant-specific DNA defect. Conclusion: The results presented indicate that ruf is a mutant allele of Mpzl3. With a genetic assignment in hand, the rough fur variant can now be more fully characterized to advance our understanding of Mpzl3's role in normal skin development and function, hepatic triglyceride synthesis, weight regulation, energy and glucose homeostasis; and to model-related human disorders.

The pseudoautosomal (PA) region of the mammalian genome is the region of the X and Y chromosomes that shares extensive DNA sequence homology and is of special interest because it may play an essential role during male meiosis. We have identified three telomere-related restriction fragments from the PA region of the mouse genome, using an oligonucleotide probe composed of the mammalian telomere consensus sequence TTAGGG. PA assignment of two C57BL/6J-derived fragments was initially suggested by anal- ysis of DNAs from progeny sired by C57BL/6J males carrying the rearranged Y chromosome, Y*: the hybridization intensity of both fragments was concordant with the sex-chromosome complement of the offspring. Further analysis indicated that both fragments were present in female and male F1 mice regardless of the sex of their C57BL/6J parent-a criterion for autosomal or PA linkage. Both fragments were closely linked to each other and located on the X chromosome distal to amelogenin (Amg)-in agreement with X or PA linkage. Con- firmation of the PA derivation of these fragments was accom- plished by following their segregation in a cross involving XY* males mated to DBA/2J females. A similar experiment iden- tified a third PA-derived restriction fragment of LT/SvEi origin. Identification of PA-derived telomere-related restric- tion fragments will enable further genetic analysis of this region of the mouse genome.

Background: A combination of genetic fine-mapping and complementation testing was used previously to assign the juvenile alopecia mutation (abbreviated jal) to the GATA binding protein 3 (Gata3) gene on Chromosome 2 in mice. However, sequence analysis of Gata3 exons (including coding and noncoding regions) revealed no differences between wild type C3H/HeJ and co-isogenic C3H/HeJ-jal/J mutant mice. Results: Using a PCR-based scanning method, here we have tested the hypothesis that jal might result from insertion of a transposable element in or near the Gata3 gene. We show that the jal mutation is specifically associated with an intracisternal A particle (IAP) element of the I Delta 1 subtype that has transposed to Intron 3-4 in the Gata3 gene, and use the same panel of recombinants used previously to fine-map jal to show that this IAP element and jal are located within the same small genetic interval. Conclusion: Transposition of an IAP element of the I Delta 1 subtype into Intron 3-4 of the mutant Gata3(jal) allele is the likely cause of the juvenile alopecia phenotype in mutant mice.

The recessive wellhaarig (we) mutations, named for the wavy coat and curly whiskers they generate in homozygotes, have previously been mapped on mouse Chromosome 2. To further limit the possible location of the we locus, we crossed hybrid (C57BL/6 × AKR)F1, we4J/+ females with AKR, we4J/we4J mutant males to create a large backcross family that was typed for various microsatellite markers and single-nucleotide polymorphisms (SNPs) that distinguish strains AKR and B6. This analysis restricted the location of we4J between sites that flank only one gene known to be expressed in skin: epidermal-type transglutaminase 3 (Tgm3). To test Tgm3 as a candidate for the basis of the wellhaarig phenotype we took two approaches. First, we sequenced all Tgm3 coding regions in mice homozygous for four independent, naturally-occurring wellhaarig alleles (we, weBkr, we3J and we4J) and found distinct defects in three of these mutants. Second, we crossed mice homozygous for an induced mutant allele of Tgm3 (Tgm3Btlr) with mice heterozygous for one of the wellhaarig alleles we possess (we4J or weBkr) to test for complementation. Because the progeny inheriting both a recessive we allele and a recessive Tgm3Btlr allele displayed wavy hair, we conclude that the classic wellhaarig mutations result from defects in Tgm3.

Because of the similar phenotypes they generate and their proximate reported locations on Chromosome 7, we tested the recessive retarded hair growth (rhg) and frizzy (fr) mouse mutations for allelism, but found instead that these defects complement. To discover the molecular basis of rhg, we analyzed a large intraspecific backcross panel that segregated for rhg and restricted this locus to a 0.9 Mb region that includes fewer than ten genes, only five of which have been reported to be expressed in skin. Complementation testing between rhg and a recessive null allele of fibroblast growth factor receptor 2 eliminated Fgfr2 as the possible basis of the retarded hair growth phenotype, but DNA sequencing of another of these candidates, ornithine aminotransferase (Oat), revealed a G to C transversion specifically associated with the rhg allele that would result in a glycine to alanine substitution at residue 353 of the gene product. To test whether this missense mutation might cause the mutant phenotype, we crossed rhg/rhg mice with mice that carried a recessive, perinatal-lethal, null mutation in Oat (designated OatΔ herein). Hybrid offspring that inherited both rhg and OatΔ displayed markedly delayed postnatal growth and hair development, indicating that these two mutations are allelic, and suggesting strongly that the G to C mutation in Oat is responsible for the retarded hair growth phenotype. Comparisons among +/+, +/rhg, rhg/rhg and rhg/OatΔ mice showed plasma ornithine levels and ornithine aminotransferase activities (in liver lysates) consistent with this assignment. Because histology of 7- and 12-month-old rhg/rhg and rhg/OatΔ retinas revealed chorioretinal degeneration similar to that described previously for OatΔ/OatΔ mice, we suggest that the rhg mutant may offer an ideal model for gyrate atrophy of the choroid and retina (GACR) in humans, which is also caused by the substitution of glycine 353 in some families.

Background Mice homozygous for the juvenile alopecia mutation ( jal ) display patches of hair loss that appear as soon as hair develops in the neonatal period and persist throughout life. Although a report initially describing this mouse variant suggested that jal maps to mouse Chromosome 13, our preliminary mapping analysis did not support that claim. Results To map jal to a particular mouse chromosome, we produced a 103-member intraspecific backcross panel that segregated for jal, and typed it for 93 PCR-scorable, microsatellite markers that are located throughout the mouse genome. Only markers from the centromeric tip of Chromosome 2 failed to segregate independently from jal , suggesting that jal resides in that region. To more precisely define jal ’s location, we characterized a second, 374-member backcross panel for the inheritance of five microsatellite markers from proximal Chromosome 2. This analysis restricted jal ’s position between D2Mit359 and D2Mit80 , an interval that includes Il2ra (for interleukin 2 receptor, alpha chain), a gene that is known to be associated with alopecia areata in humans. Complementation testing with an engineered null allele of Il2ra , however, showed that jal is a mutation in a distinct gene. To further refine the location of jal , the 374-member panel was typed for a set of four single-nucleotide markers located between D2Mit359 and D2Mit80 , identifying a 0.55 Mb interval where jal must lie. This span includes ten genes—only one of which, Gata3 (for GATA binding protein 3)—is known to be expressed in skin. Complementation testing between jal and a Gata3 null allele produced doubly heterozygous, phenotypically mutant offspring. Conclusions The results presented indicate that the jal mutation is a mutant allele of the Gata3 gene on mouse Chromosome 2. We therefore recommend that the jal designation be changed to Gata3 jal , and suggest that this mouse variant may provide an animal model for at least some forms of focal alopecia that have their primary defect in the hair follicle and lack an inflammatory component.

Background Mice homozygous for the spontaneous wooly mutation (abbreviated wly ) are recognized as early as 3–4 weeks of age by the rough or matted appearance of their coats. Previous genetic analysis has placed wly in a 5.9 Mb interval on Chromosome 11 that contains over 200 known genes. Assignment of wly to one of these genes is needed in order to provide probes that would ultimately facilitate a complete molecular analysis of that gene’s role in the normal and disrupted development of the mammalian integument. Results Here, a large intraspecific backcross family was used to genetically map wly to a smaller (0.8 Mb) span on mouse Chromosome 11 that includes fewer than 20 genes. DNA sequencing of the coding regions in two of these candidates known to be expressed in skin has revealed a 955 bp, wly -specific deletion. This deletion, which lies within the coordinates of both Slc5a10 [for solute carrier family 5 (sodium/glucose cotransporter), member 10] and Fam83g (for family with sequence similarity 83, member G), alters the splicing of mutant Fam83g transcripts only, and is predicted to result in a severely truncated (probably non-functional) protein product. Conclusion We suggest that this mutation in Fam83g is the likely basis of the mouse wooly phenotype.

Please cite this paper as: The mouse frizzy (fr) and rat ‘hairless’ (frCR) mutations are natural variants of protease serine S1 family member 8 (Prss8). Experimental Dermatology 2010; 19: 527–532.Abstract: We have previously suggested (based on genetic mapping analysis) that the allelic ‘fuzzy’ and ‘hairless’ mutations in the rat are likely orthologues of the mouse frizzy mutation (fr). Here, we analysed three large intraspecific backcross panels that segregated for mouse fr to restrict this locus to a 0.6‐Mb region that includes fewer than 30 genes. DNA sequencing of one of these candidates known to be expressed in skin, protease serine S1 family member 8 (Prss8), revealed a T to A transversion associated with the fr allele that would result in a valine to aspartate substitution at residue 170 in the gene product. To test whether this missense mutation might be the molecular basis of this frizzy variant, we crossed fr/fr mice with mice that carried a recessive perinatal lethal mutation in Prss8. Hybrid offspring that inherited both fr and the Prss8 null allele displayed abnormal hair and skin, showing that these two mutations are allelic, and suggesting strongly that the T to A mutation in Prss8 is responsible for the mutant frizzy phenotype. Sequence analysis of all Prss8 coding regions in the ‘hairless’ rat identified a 12‐bp deletion in the third exon, indicating that mouse fr and the rat ‘hairless’ mutations are indeed orthologues. However, this analysis failed to detect any alterations to Prss8 coding sequences in the allelic ‘fuzzy’ rat variant.

Males homozygous for the mouse male sterility and histoincompatibility (mshi) mutation exhibit small testes and produce no sperm. In addition, mshi generates an "antigen-loss" histoincompatibility barrier, such that homozygous mutants reject skin grafts from wild type co-isogenic BALB/cByJ donors. To facilitate the molecular characterization of the pleiotropic mshi mutation, we genetically mapped mshi into a 0.68 megabasepair region which contains fewer than 10 candidate genes. Complementation testing showed that one of these, Mtap7, is disrupted in mshi mice. Sequence analysis has revealed a 13 kilobasepair deletion in BALB/cByJ-mshi/J mice that begins in Intron 10-11 of Mtap7, and ends less than 2000 base pairs downstream of the wild type gene. Analysis of the mutant cDNA predicts that Mtap7(mshi) encodes a 457 amino acid protein, the first 423 of which are identical to wild type, and the last 34 of which are due to aberrant mRNA splicing with two cryptic exons in the Mtap7 to P04Rik intergenic region. This molecular assignment for the mshi mutation further supports an essential role for microtubule stabilization in spermatogenesis and indicates a new role in allograft transplantation.

Joint ventures with equity participation by partners bring together parties with different capabilities to achieve a common goal. The expectation typically focuses on synergy that will benefit both parties. BP, the British oil company and the Russian conglomerate TNK formed a joint venture with similar hopes. BP offered its significant market power in the world and technological know-how; TNK brought to the table the vast oil reserves in the steppes of Siberia. In a few short years, the venture started manifesting instability primarily caused by the differences in economic motives and cultural orientations of the partners. This paper traces the chronology of events in the souring relationships between the partners as evidenced in recent times and assesses the instability of the venture.

We have previously shown that the rat fuzzy and Charles River 'hairless' mutations are defects in the same gene on rat Chr 1, and are likely orthologues of the frizzy mutation (fr) on mouse Chr 7. To test the hypothesis that these variants could result from defects in Fgfr2, we crossed fr/fr mice (from the inbred FS/EiJ strain) with mice that carry a recessive lethal mutation in Fgfr2. Mice inheriting both mutations were phenotypically normal, indicating that fr is not an allele of Fgfr2. To genetically map fr, we crossed these hybrid mice, or F(1) mice made by crossing FS/EiJ with the wild-type C57BL/6J or BALB/cBy strains, back to the FS/EiJ strain. The resulting 546 backcross progeny were typed for linked markers to position fr centromeric of Fgfr2, between D7Csu5 and D7Mit165; an interval that contains only 2.7 Mb and fewer than 70 genes. Further characterization of regional recombinants for sequence-level polymorphisms should allow sufficient refinement of fr's location to facilitate an eventual molecular assignment for this classical mutation.

A method for fast and selective determination of the main triterpenic compounds present in olive leaves — oleanolic, ursolic and maslinic acids as triterpenic acids and, uvaol and erythrodiol as triterpenic dialcohols — is reported here. Quantitative isolation of the analytes has been accomplished in 5 min by microwave assistance using ethanol as extractant. Due to the medium polarity of triterpenic acids and dialcohols, different ethanol–water ratios were tested in order to select the optimum extractant composition for their solubilisation. Microwave assistance provided a significant shortening of the leaching time as compared to conventional procedures by maceration, which usually requires at least 5 h. After extraction, determination was carried out by liquid chromatography–tandem mass spectrometry (LC–MS–MS) with a triple quadrupole (qQq) mass detector without any clean-up step prior to chromatographic analysis. Highly selective identification of triterpenes was confirmed by multiple reaction monitoring (MRM) using the most representative transitions from the precursor ion to the different product ions, while the most sensitive transitions were used for MS–MS quantitation. Total analysis performed in 25 min enables the characterization of a fraction with particular interest in the pharmacological area.

Background, Low-density lipoprotein cholesterol (LDL-C) is the primary therapeutic target in the National Cholesterol Education Program Adult Treatment Panel III (ATP III) guidelines. This study tested the hypothesis that ezetimibe/simvastatin, a lipid-lowering agent that inhibits both intestinal cholesterol absorption and cholesterol synthesis, provides greater LDL-C reductions than atorvastatin across dose ranges.Methods This multicenter, double-blind, 6-week parallel-group study randomized 1902 patients with LDL-C above ATP III goal to atorvastatin (10, 20, 40, or 80 mg) or to ezetimibe/simvastatin (10/10, 10/20, 10/40, or 10/80 mg). Patients were stratified by prerandomization LDL-C level.Results At each milligram-eqvivalent statin dose comparison, and averaged across doses, ezetimibe/simvastatin provided greater LDL-C reductions (47%-59%) than atorvastatin (36%-53%). Ezetimibe/simvastatin 10/40 and 10/80 mg also provided significantly greater high-density lipoprotein cholesterol (HDL-C) increases than atorvastatin 40 and 80 mg. Triglyceride reductions were similar for all comparisons. More ezetimibe/simvastatin than atorvastatin patients with coronary heart disease (CHID) or CHID risk equivalents attained the ATP III LDL-C goal of <100 mg/dL and the optional LDL-C target of <70 mg/dL. C-reactive protein reductions were similar between treatment groups. Consecutive elevations in alanine aminotransferase and/or aspartate aminotransferase occurred in significantly more atorvastatin patients than ezetimibe/ simvastatin patients. No myopathy or liver-related adverse events led to study discontinuation with either drug.Conclusions Ezetimibe/simvastatin was more effective than atorvastatin in lowering LDL-C at each dose comparison and provided greater increases in HDL-C at the 40- and 80-mg statin dose. Ezetimibe/simvastatin is a highly efficacious, well-tolerated treatment option for hypercholesterolemic patients.