Several mammals--including sheep, mice, cows, goats, pigs, rabbits, cats, a mule, a horse and a litter of three rats--have been cloned by transfer of a nucleus from a somatic cell into an egg cell (oocyte) that has had its nucleus removed. This technology has not so far been successful in dogs because of the difficulty of maturing canine oocytes in vitro. Here we describe the cloning of two Afghan hounds by nuclear transfer from adult skin cells into oocytes that had matured in vivo. Together with detailed sequence information generated by the canine-genome project, the ability to clone dogs by somatic-cell nuclear transfer should help to determine genetic and environmental contributions to the diverse biological and behavioural traits associated with the many different canine breeds.

The 110-kDa heat shock protein (hsp110) has long been recognized as one of the primary heat shock proteins in mammalian cells. It belongs to a recently described protein family that is a significantly diverged subgroup of the hsp70 family and has been found in organisms as diverse as yeast and mammals. We describe here the first analysis of the ability of hsp110 to protect cellular and molecular targets from heat damage. It was observed that the overexpression in vivo of hsp110 conferred substantial heat resistance to both Rat-1 and HeLa cells. In vitro heat denaturation and refolding assays demonstrate that hsp110 is highly efficient in selectively recognizing denatured proteins and maintaining them in a soluble, folding-competent state and is significantly more efficient in performing this function than is hsc70. hsp110-bound proteins can then be refolded by the addition of rabbit reticulocyte lysate or hsc70 and Hdj-1, whereas Hdj-1 does not itself function as a co-chaperone in folding with hsp110. hsp110 is one of the principal molecular chaperones of mammalian cells and represents a newly identified component of the primary protection/repair pathway for denatured proteins and thermotolerance expression in vivo.It has been long recognized that the major heat shock proteins (hsps) 1 of mammalian cells are observed at 28, 70, 90, and 110 kDa (1-3) and other hsp families, e.g. hsp60 and hsp40, have been subsequently identified. All of these stress protein groups have been intensively studied, excluding the hsp110 species. The cloning of hsp110 from hamster, mouse, yeast, arabadopsis, and a variety of other species has been recently described (4 -11, 29, 30). Moreover, as is the case with the hsp70 family, multiple members of the hsp110 family have also been found in individual organisms (8 -11). These studies indicate that hsp110 is a significantly enlarged and diverged relative of the hsp70 family of proteins but also includes unique sequence components. The notable constitutive expression and stress inducibility of hsp110 is highly suggestive of a major role in unstressed cells as well as in the heat shock response and the expression of thermotolerance (1, 2). A description of the heat shock response in eucaryotes is not possible without an understanding of the roles played by this major stress protein.We describe here an analysis of the characteristics of hsp110, both in vivo and in vitro. EXPERIMENTAL PROCEDURESPurification of Recombinant His-tagged HSP110 -cDNA for hsp110 was cloned into pRSET vector (Invitrogen), resulting in introduction of a His 6 -(enterokinase recognition sequence)-Arg-Ser tag to the amino terminus of hsp110 (pRSET-hsp110). pRSET-hsp110 was transformed into Escherichia coli strain JM109(DE3) cells. The transformant containing pRSET-hsp110 was grown at 37°C in LB medium with ampicillin until the OD reached 0.6, when the expression of His-hsp110 was induced by the addition of 0.4 mM isopropyl-1-thio-␤-D-galactopyranoside during further incubation at 30°C for 5 h. Cells were lysed in 20 ...

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Genetic defects in the dystrophin-associated protein complex (DAPC) are responsible for a variety of pathological conditions including muscular dystrophy, cardiomyopathy, and vasospasm. Conserved DAPC components from humans to Caenorhabditis elegans suggest a similar molecular function. C. elegans DAPC mutants exhibit a unique locomotory deficit resulting from prolonged muscle excitation and contraction. Here we show that the C. elegans DAPC is essential for proper localization of SLO-1, the large conductance, voltage-, and calcium-dependent potassium (BK) channel, which conducts a major outward rectifying current in muscle under the normal physiological condition. Through analysis of mutants with the same phenotype as the DAPC mutants, we identified the novel islo-1 gene that encodes a protein with two predicted transmembrane domains. We demonstrate that ISLO-1 acts as a novel adapter molecule that links the DAPC to SLO-1 in muscle. We show that a defect in either the DAPC or ISLO-1 disrupts normal SLO-1 localization in muscle. Consistent with observations that SLO-1 requires a high calcium concentration for full activation, we find that SLO-1 is localized near L-type calcium channels in muscle, thereby providing a mechanism coupling calcium influx with the outward rectifying current. Our results indicate that the DAPC modulates muscle excitability by localizing the SLO-1 channel to calcium-rich regions of C. elegans muscle.

Background/AimsThe proper treatment of subclinical hypothyroidism and the normal range of serum thyroid stimulating hormone (TSH) concentration are intensely debated. However, few reports have investigated TSH concentrations in Asian ethnic groups. Therefore, the present study was designed to define the TSH reference range in a Korean population and to investigate the metabolic significance of TSH concentration.MethodsWe enrolled patients who underwent medical examination at the CHA Bundang Medical Center. Anthropometric data were evaluated, and serum TSH, free T4, and lipid profiles were assayed.ResultsA total of 7,270 subjects were included. Mean TSH concentration of the study population was 1.82 ± 0.95 mU/L, and we observed a sex-related difference in TSH concentration (male, 1.67 ± 0.87 mU/L; female, 2.02 ± 1.01 mU/L; p < 0.01). When the 2.5 and 97.5 percentiles were calculated, 95% TSH reference limits were 0.52-4.29 mU/L. TSH concentration was higher in elderly subjects, during winter, in postmenopausal women, and in obese males. Moreover, TSH showed significantly positive correlations with serum total cholesterol, triglyceride, and low density lipoprotein cholesterol regardless of sex, age, season, obesity, or menopausal status (all p < 0.01). Finally, TSH concentration was positively related to the prevalence of metabolic syndrome.ConclusionsWe demonstrated the association between TSH concentration within the normal reference range and serum lipid levels. TSH concentration varies according to sex, age, season, and body mass index (only in males). Moreover, high normal TSH levels were significantly associated with an increased prevalence of metabolic syndrome, which may be of importance when evaluating subjects with high normal TSH concentration.