Osteoporosis is a complex disease that affects >10 million people in the United States and results in 1.5 million fractures annually. In addition, the high prevalence of osteopenia (low bone mass) in the general population places a large number of people at risk for developing the disease. In an effort to identify genetic factors influencing bone density, we characterized a family that includes individuals who possess exceptionally dense bones but are otherwise phenotypically normal. This high-bone-mass trait (HBM) was originally localized by linkage analysis to chromosome 11q12-13. We refined the interval by extending the pedigree and genotyping additional markers. A systematic search for mutations that segregated with the HBM phenotype uncovered an amino acid change, in a predicted beta-propeller module of the low-density lipoprotein receptor-related protein 5 (LRP5), that results in the HBM phenotype. During analysis of >1,000 individuals, this mutation was observed only in affected individuals from the HBM kindred. By use of in situ hybridization to rat tibia, expression of LRP5 was detected in areas of bone involved in remodeling. Our findings suggest that the HBM mutation confers a unique osteogenic activity in bone remodeling, and this understanding may facilitate the development of novel therapies for the treatment of osteoporosis.

Immunoreceptors such as the high affinity IgE receptor, Fc⑀RI, and T-cell receptor-associated proteins share a common motif, the immunoreceptor tyrosine-based activation motif (ITAM). We used the yeast tribrid system to identify downstream effectors of the phosphorylated Fc⑀RI ITAM-containing subunits ␤ and ␥. One novel cDNA was isolated that encodes a protein that is phosphorylated on tyrosine, contains a Src-homology 2 (SH2) domain, inositolpolyphosphate 5-phosphatase activity, three NXXY motifs, several proline-rich regions, and is called SHIP. Mutation of the conserved tyrosine or leucine residues within the Fc⑀RI ␤ or ␥ ITAMs eliminates SHIP binding and indicates that the SHIP-ITAM interaction is specific. SHIP also binds to ITAMs from the CD3 complex and T cell receptor chain in vitro. SHIP protein possesses both phosphatidylinositol-3,4,5-trisphosphate 5-phosphatase and inositol-1,3,4,5-tetrakisphosphate 5-phosphatase activity. Phosphorylation of SHIP by a protein-tyrosine kinase, Lck, results in a reduction in enzyme activity. Fc⑀RI activation induces the association of several tyrosine phosphoproteins with SHIP. SHIP is constitutively tyrosine-phosphorylated and associated with Shc and Grb2. These data suggest that SHIP may serve as a multifunctional linker protein in receptor activation.The aggregation of immunoreceptors by antigen initiates a complex response leading to cellular activation (1). Receptors on T-, B-, and mast cells each contain subunits with similar primary amino acid sequence within their cytosolic domains, comprising the immunoreceptor-based tyrosine activation motif (ITAM), 1 whose consensus is (D/E)X 2 YXX(L/I)X 6 -8 YXX(L/I) (2, 3). Both tyrosine residues within the ITAM are rapidly phosphorylated by protein kinases after receptor aggregation. The bisphosphorylated ITAM then binds directly to cytosolic tyrosine kinases such as Syk in B-cells and mast cells and ZAP70 in T-cells, thereby activating their tyrosine kinase activity (4, 5). In mast cells, the Fc⑀RI subunits ␤ and ␥ each possess a single ITAM, which, when bisphosphorylated on tyrosine, binds to Syk (6 -8).We have used a novel genetic approach, the yeast tribrid system (8), to isolate cDNAs that encode proteins that interact with the tyrosine-phosphorylated Fc⑀RI ␥ ITAM. The yeast two-hybrid system facilitates the study of protein-protein interactions but is limited to the investigation of proteins that are properly expressed and modified in the host, Saccharomyces cerevisiae. S. cerevisiae does not employ tyrosine phosphorylation as a major regulatory modification of proteins (9, 10). This limits the utility of the two-hybrid system, especially in the area of signal transduction, where tyrosine phosphorylation is a critical component of the process. In order to study protein-protein interactions that are dependent on tyrosine phosphorylation or on other post-translational or allosteric modifications, the yeast tribrid system was developed (8, 11).In the yeast tribrid system, a third plasmid is introduced, which directs th...

Purpose:The present investigation examined the physiological and performance effects of lower-body compression garments (LBCG) during a one-hour cycling time-trial in well-trained cyclists.Methods:Twelve well-trained male cyclists ([mean ± SD] age: 20.5 ± 3.6 years; height: 177.5 ± 4.9 cm; body mass: 70.5 ± 7.5 kg; VO2max: 55.2 ± 6.8 mL·kg−1·min−1) volunteered for the study. Each subject completed two randomly ordered stepwise incremental tests and two randomly ordered one-hour time trials (1HTT) wearing either full-length SportSkins Classic LBCG or underwear briefs (control). Blood lactate concentration ([BLa−]), heart rate (HR), oxygen consumption (VO2) and muscle oxygenation (mOxy) were recorded throughout each test. Indicators of cycling endurance performance were anaerobic threshold (AnT) and VO2max values from the incremental test, and mean power (W), peak power (W), and total work (kJ) from the 1HTT Magnitude-based inferences were used to determine if LBCG demonstrated any performance and/or physiological benefits.Results:A likely practically significant increase (86%:12%:2%; η2 = 0.6) in power output at AnT was observed in the LBCG condition (CONT: 245.9 ± 55.7 W; LBCG: 259.8 ± 44.6 W). Further, a possible practically significant improvement (78%:19%:3%; η2 = 0.6) was reported in muscle oxygenation economy (W·%mOxy−1) across the 1HTT (mOxy: CONT: 52.2 ± 12.2%; LBCG: 57.3 ± 8.2%).Conclusions:The present results demonstrated limited physiological benefits and no performance enhancement through wearing LBCG during a cycling time trial.

Protein-protein interactions are often dependent on the post-translational modification of one component of a complex. To facilitate the study of these interactions in signal transduction, we have developed the yeast tribrid system, a modification of the yeast two-hybrid system. We demonstrate that the interactions are dependent upon the presence of a tyrosine kinase, an SH2 domain and a tyrosine containing substrate. Using the gamma subunit of the high-affinity IgE receptor, Fc epsilon RI, this approach has been used to isolate a novel SH2-containing family member. The mRNA encoding this novel protein is differentially expressed in rat tissues. The yeast tribrid system can be readily adapted for the characterization of novel tyrosine kinases or substrates, as well as the study of protein-protein interactions which involve other post-translational modifications.

Dehydration in athletes alters cardiovascular and thermoregulatory function and may inhibit endurance exercise capacity if fluid loss exceeds 2% of bodyweight (BW). If this level of dehydration cannot be prevented when starting from a state of euhydration, then athletes may create a state of hyperhydration by consuming extra fluid prior to exercise. From this hyperhydrated situation, individuals have a greater capacity to tolerate fluid loss before becoming dehydrated. Furthermore, excess pre-exercise fluid intake enhances thermoregulatory ability, as well as increasing plasma volume to maintain cardiac output. However, hyperhydrating before exercise is difficult, because a large fluid intake is typically accompanied by diuresis. Glycerol-containing beverages create an osmotic gradient in the circulation favouring fluid retention, thereby facilitating hyperhydration and protecting against dehydration. Many studies have shown that increases in body water by 1 L or more are achievable through glycerol hyperhydration. This article analyses the evidence for glycerol use in facilitating hyperhydration and rehydration, and provides guidelines for athletes wishing to use this compound. An analysis of the studies in this area indicates that endurance athletes intending to hyperhydrate with glycerol should ingest glycerol 1.2 g/kg BW in 26 mL/kg BW of fluid over a period of 60 minutes, 30 minutes prior to exercise. The effects of glycerol on total body water when used during rehydration are less well defined, due to the limited studies conducted. However, ingesting glycerol 0.125 g/kg BW in a volume equal to 5 mL/kg BW during exercise will delay dehydration, while adding glycerol 1.0 g/kg BW to each 1.5 L of fluid consumed following exercise will accelerate the restoration of plasma volume. Side effects from glycerol ingestion are rare, but include nausea, gastrointestinal discomfort and light-headedness. In summary, glycerol ingestion before, during or following exercise is likely to improve the hydration state of the endurance athlete.

Abstract. The NUF2 gene of the yeast Saccharomyces cerevisiae encodes an essential 53-kd protein with a high content of potential coiled-coil structure similar to myosin. Nuf2 is associated with the spindle pole body (SPB) as determined by coimmunofluorescence with known SPB proteins. Nuf2 appears to be localized to the intranuclear region and is a candidate for a protein involved in SPB separation. The nuclear associati0n of Nuf2 can be disrupted, in part, by 1 M salt but not by the detergent Triton X-100. All Nuf2 can be removed from nuclei by 8 M urea extraction. In this regard, Nuf2 is similar to other SPB-associated proteins including Nufl/SPCll0, also a coiled-coil protein. Temperature-sensitive alleles of NUF2 were generated within the coiled-coil region of Nuf2 and such NUF2 mutant cells rapidly arrest after temperature shift with a single undivided or partially divided nucleus in the bud neck, a shortened mitotic spindle and their DNA fully replicated. In sum, Nuf2 is a protein associated with the SPB that is critical for nuclear division. Anti-Nuf2 antibodies also recognize a mammalian 73-kd protein and display centrosome staining of mammalian tissue culture cells suggesting the presence of a protein with similar function. THE process of nuclear division in the yeast Saccharomyces cerevisiae requires a large number of gene products, many of which have been characterized extensively. The yeast microtubule organizing center, the spindie pole body (SPB) ~, is a large, trilaminar structure embedded in the nuclear envelope (Byers and Goetsch, 1974). Its behavior during the cell-cycle is morphologically well studied (Byers and Goetsch, 1975). At mitosis, the SPB duplicates, with two SPBs initially located side-by-side within the nuclear envelope; this stage is termed a monopolar spindle. The SPBs then separate, establishing a bipolar mitotic spindle across the interior of the nucleus. The duplicated SPB is oriented toward the daughter bud, and the spindle elongates as the nucleus is separated and enters the bud. At its maximum, the bipolar spindle extends from within the daughter bud through the bud neck into the mother cell. The mitotic spindle is responsible for correctly distributing the chromosomal complement to both the mother and daughter cell. Mutants in genes responsible for spindle formation and maintenance often display increases in chromosome loss

Abstract. A variety of peptides can mediate the localization of proteins to the nucleus. We have identified yeast proteins of 70 and 59 kD that bind to nuclear localization peptides of SV-40 T antigen, Xenopus nucleoplasmin, and the yeast proteins Gal4 and histone H2B. These proteins are assayed by the binding of peptide-albumin conjugates to proteins immobilized on nitrocellulose filters. These binding proteins fractionate with nuclei and are extractable with salt but not detergent. Radiolabeled peptide-albumin conjugates also bind to isolated nuclei; the binding is saturable and can be extracted with salt. Different nuclear localization peptides compete with each other, implying that a single class of proteins is responsible for their recognition. The 70-and 59-kD proteins have the properties expected for a receptor that would act to direct proteins to the nucleus. istinct set of proteins is localized to the nucleus. By one model, transport of proteins from the cytoplasm into the nucleus is triggered by specific interaction of a short amino acid sequence (termed a nuclear localization sequence [NLS]') within the transported protein and a receptor, perhaps at the nuclear pore. This model is supported by the existence of discrete nuclear localization sequences within nuclear proteins (Dingwall et al., 1982;Kalderon et al., 1984a;Lanford and Butel, 1984;Silver et al., 1984;Hall et al., 1984). These sequences are necessary for specific transport across the nuclear envelope and are sufficient to cause nonnuclear proteins to enter the nucleus.Recent evidence argues for the existence of an apparatus that would recognize nuclear localization sequences and transport proteins into the nucleus. (a) Uptake of proteins into Xenopus oocyte nuclei is saturable (Goldfarb et al., 1986); (b) Depletion of ATP blocks nuclear protein accumulation both in vivo (Newmeyer et al., 1986a) and in vitro (Markland et al., 1987;Newmeyer and Forbes, 1988), consistent with specific transport requiring energy. (c) Nuclear protein uptake can be separated into at least two steps: binding at the nuclear envelope followed by ATP-dependent trans= location through the pore Newmeyer and Frobes, 1988).Genetically or chemically conjugated peptides (derived from nuclear proteins) direct nonnuclear proteins to the nucleus (for example, see Kalderon et al., 1984b;Silver et al., 1984; Laaford et al., 1986). These peptides contain many basic amino acids, but otherwise have little sequence similarity. One of the best characterized nuclear localization sequences is PKKKRKV, found in SV-40 T antigen between amino acids 126 and 132 (Kalderon et al., 1984b; Roberts et all., 1987). We have previously shown that the SV-40 T antigen NLS functions in the yeast, Saccharomyces cerevisiae, to direct normally cytoplasmic proteins to the nucleus (Nel- son and Silver, 1989). Moreover, a single amino acid change in the SV-40 T antigen NLS reduces its function in animal cells (Kalderon et al., 1984a;Lanford and Butel, 1984), as well as in yeast (Nelson and Silver, 1989)....

The findings suggest that running intensity varies between and within halves; however, the index used will influence both the magnitude and the temporal identification of peak periods.