Synthesis,characterization, and use of 2-[(2H(9))butoxy]acetic acid and 2-(3-methylbutoxy)acetic acid as an internal standard and an instrument performance surrogate,respectively, for the gas chromatographic-mass spectrometric determination of 2-butoxyacetic acid,a human metabolite of 2-butoxyethanol

2-[(2H(9))Butoxy]acetic acid and 2-(3-methylbutoxy)acetic acid were synthesized, mixed with 2-butoxyacetic acid, and separated by capillary gas chromatography on a fused-silica column with a length of 50 m, inside diameter of 0.200 mm, and a "free fatty acid phase" wall coating of 0.3 microm film. 2-[(2H(9))Butoxy]acetic acid, 2-butoxyacetic acid, and 2-(3-methylbutoxy)acetic acid were baseline resolved at retention times of 13.55, 13.78, and 15.20 min; 2-(3-methylbutoxy)acetic acid having a peak efficiency of 360,000. Mass spectrometric detection using selected ion monitoring at m/z 66, 57, and 71 showed linear analytical responses from 0.04 ng to at least 200 ng with a limit of detection of 0.04 ng for 2-butoxyacetic acid.     

A novel dynein light intermediate chain colocalizes with the retrograde motor for intraflagellar transport at sites of axoneme assembly in chlamydomonas and Mammalian cells

The assembly of cilia and flagella depends on bidirectional intraflagellar transport (IFT). Anterograde IFT is driven by kinesin II, whereas retrograde IFT requires cytoplasmic dynein 1b (cDHC1b). Little is known about how cDHC1b interacts with its cargoes or how it is regulated. Recent work identified a novel dynein light intermediate chain (D2LIC) that colocalized with the mammalian cDHC1b homolog DHC2 in the centrosomal region of cultured cells. To see whether the LIC might play a role in IFT, we characterized the gene encoding the Chlamydomonas homolog of D2LIC and found its expression is up-regulated in response to deflagellation. We show that the LIC subunit copurifies with cDHC1b during flagellar isolation, dynein extraction, sucrose density centrifugation, and immunoprecipitation. Immunocytochemistry reveals that the LIC colocalizes with cDHC1b in the basal body region and along the length of flagella in wild-type cells. Localization of the complex is altered in a collection of retrograde IFT and length control mutants, which suggests that the affected gene products directly or indirectly regulate cDHC1b activity. The mammalian DHC2 and D2LIC also colocalize in the apical cytoplasm and axonemes of ciliated epithelia in the lung, brain, and efferent duct. These studies, together with the identification of an LIC mutation, xbx-1(ok279), which disrupts retrograde IFT in Caenorhabditis elegans, indicate that the novel LIC is a component of the cDHC1b/DHC2 retrograde IFT motor in a variety of organisms.     

RHAMM is a centrosomal protein that interacts with dynein and maintains spindle pole stability

The receptor for hyaluronan-mediated motility (RHAMM), an acidic coiled coil protein, has previously been characterized as a cell surface receptor for hyaluronan, and a microtubule-associated intracellular hyaluronan binding protein. In this study, we demonstrate that a subset of cellular RHAMM localizes to the centrosome and functions in the maintenance of spindle integrity. We confirm a previous study showing that the amino terminus of RHAMM interacts with microtubules and further demonstrate that a separate carboxy-terminal domain is required for centrosomal targeting. This motif overlaps the defined hyaluronan binding domain and bears 72% identity to the dynein interaction domain of Xklp2. RHAMM antibodies coimmunprecipitate dynein IC from Xenopus and HeLa extracts. Deregulation of RHAMM expression inhibits mitotic progression and affects spindle architecture. Structure, localization, and function, along with phylogenetic analysis, suggests that RHAMM may be a new member of the TACC family. Thus, we demonstrate a novel centrosomal localization and mitotic spindle-stabilizing function for RHAMM. Moreover, we provide a potential mechanism for this function in that RHAMM may cross-link centrosomal microtubules, through a direct interaction with microtubules and an association with dynein.     

Body composition in Division I football players

This study assessed body composition of Division I football players (n = 69) and compared the findings with previously reported data to ascertain whether the increase in player total body mass that has been observed over the past 10 years has been accompanied by an increase in body fat. Body composition was determined by hydrostatic weighing and the measurement of skinfold thicknesses. Total body mass, skinfold thicknesses, and body fat were greater in the current players than in players in studies conducted in the early 1980s and early 1990s. Body fat varied significantly across playing position, with the defensive backs, offensive backs, and receivers being the leanest and the offensive linemen and tight ends the most fat. There was no significant relationship between body composition and playing year or scholarship status, nor were any differences observed between ethnic groups. Of important clinical relevance was the finding that the linemen (offensive, defensive) and tight ends were on average greater than 25% body fat, the borderline for obesity in this age group. Much of this fat was deposited in the abdominal region, a significant finding when one considers the high correlation between abdominal obesity and ischemic heart disease and stroke. The current findings suggest that more attention needs to be given to the nature of the increase in body mass being achieved by today's football player to minimize long-term negative health consequences, and the findings reemphasize the need identified in earlier studies of the importance of detraining programs for these athletes.     

Pulmonary bioavailability of ascorbic acid in an ascorbate-synthesising species,the horse

Vitamin C (ascorbic acid) is a non-enzymatic antioxidant important in protecting the lung against oxidative damage and is decreased in lung lining fluid of horses with airway inflammation. To examine possible therapeutic regimens in a species with ascorbate-synthesising capacity, we studied the effects of oral supplementation of two forms of ascorbic acid, (each equivalent to 20 mg ascorbic acid per kg body weight) on the pulmonary and systemic antioxidant status of six healthy ponies in a 3 x 3 Latin square design. Two weeks supplementation with ascorbyl palmitate significantly increased mean plasma ascorbic acid concentrations compared to control (29 +/- 5 and 18 +/- 7 micromol/l, respectively; p < 0.05). Calcium ascorbyl-2-monophosphate, a more stable form of ascorbic acid, also increased mean plasma ascorbic acid concentrations, but not significantly (23 +/- 1 micromol/l; p = 0.07). The concentration of ascorbic acid in bronchoalveolar lavage fluid increased in five out of six ponies following supplementation with either ascorbyl palmitate or calcium ascorbyl-2-monophosphate compared with control (30 +/- 10, 25 +/- 4 and 18 +/- 8 micromol/l, respectively; p < 0.01). Neither supplement altered the concentration of glutathione, uric acid or alpha-tocopherol in plasma or bronchoalveolar lavage fluid. In conclusion, the concentration of lung lining fluid ascorbic acid is increased following ascorbic acid supplementation (20 mg/kg body weight) in an ascorbate-synthesising species.     

Serial in vivo imaging of the targeted migration of human HSV-TK-transduced antigen-specific lymphocytes

New technologies are needed to characterize the migration, survival, and function of antigen-specific T cells in vivo. Here, we demonstrate that Epstein-Barr virus (EBV)--specific T cells transduced with vectors encoding herpes simplex virus-1 thymidine kinase (HSV-TK) selectively accumulate radiolabeled 2'-fluoro-2'-deoxy-1-beta-D-arabinofuranosyl-5-iodouracil (FIAU). After adoptive transfer, HSV-TK+ T cells labeled in vitro or in vivo with [131I]FIAU or [124I]FIAU can be noninvasively tracked in SCID mice bearing human tumor xenografts by serial images obtained by scintigraphy or positron emission tomography (PET), respectively. These T cells selectively accumulate in EBV+ tumors expressing the T cells' restricting HLA allele but not in EBV- or HLA-mismatched tumors. The concentrations of transduced T cells detected in tumors and tissues are closely correlated with the concentrations of label retained at each site. Radiolabeled transduced T cells retain their capacity to eliminate targeted tumors selectively. This technique for imaging the migration of ex vivo-transduced antigen-specific T cells in vivo is informative, nontoxic, and potentially applicable to humans.     

The Lyon and the LINE hypothesis

X-chromosome inactivation (XCI) was first suggested as an explanation for the variegated phenotypes in mice heterozygous for X-linked colour genes or for X-autosome translocations involving autosomal coat colour genes. The effects seen in X-autosome translocations led to the suggestion of an X-inactivation centre (Xic) from which the inactivation was initiated, and this suggestion has led to major advances in understanding. Another feature of X-autosome translocations is incomplete inactivation of the attached autosomal segment, implying that the X-chromosome is enriched in features favouring inactivation. Interspersed repeat elements, and in particular long interspersed elements (LINEs), have been suggested as the relevant enriching features. Recent evidence concerning this hypothesis is discussed.     

Natural freeze-tolerance in hatchling painted turtles?

Hatchlings of the North American painted turtle (Family Emydidae: Chrysemys picta) typically spend their first winter of life inside a shallow, subterranean hibernaculum (the natal nest) where life-threatening conditions of ice and cold commonly occur. Although a popular opinion holds that neonates exploit a tolerance for freezing to survive the rigors of winter, hatchlings are more likely to withstand exposure to ice and cold by avoiding freezing altogether-and to do so without the benefit of an antifreeze. In the interval between hatching by turtles in late summer and the onset of wintery weather in November or December, the integument of the animals becomes highly resistant to the penetration of ice into body compartments from surrounding soil, and the turtles also purge their bodies of catalysts for the formation of ice. These two adjustments, taken together, enable the animals to supercool to temperatures below those that they routinely experience in nature. However, cardiac function in hatchlings is diminished at subzero temperatures, thereby compromising the delivery of oxygen to peripheral tissues and eliciting an increase in reliance by those tissues on anaerobic metabolism for the provision of ATP. The resulting increase in production of lactic acid may disrupt acid/base balance and lead to death even in animals that remain unfrozen. Although an ability to undergo supercooling may be key to survival by overwintering turtles in northerly populations, a similar capacity to resist inoculation and undergo supercooling characterizes animals from a population near the southern limit of distribution, where winters are relatively benign. Thus, the suite of characters enabling hatchlings to withstand exposure to ice and cold may have been acquired prior to the northward dispersal of the species at the end of the Pleistocene, and the characters may not have originated as adaptations specifically to the challenges of winter.