Ctr1 Is an Apical Copper Transporter in Mammalian Intestinal Epithelial Cells in Vivo That Is Controlled at the Level of Protein Stability

Copper is an essential trace element that functions in a diverse array of biochemical processes that include mitochondrial respiration, neurotransmitter biogenesis, connective tissue maturation, and reactive oxygen chemistry. The Ctr1 protein is a high-affinity Cu⁺ importer that is structurally and functionally conserved in yeast, plants, fruit flies, and humans and that, in all of these organisms, is localized to the plasma membrane and intracellular vesicles. Although intestinal epithelial cell-specific deletion of Ctr1 in mice demonstrated a critical role for Ctr1 in dietary copper absorption, some controversy exists over the localization of Ctr1 in intestinal epithelial cells in vivo. In this work, we assess the localization of Ctr1 in intestinal epithelial cells through two independent mechanisms. Using immunohistochemistry, we demonstrate that Ctr1 localizes to the apical membrane in intestinal epithelial cells of the mouse, rat, and pig. Moreover, biotinylation of intestinal luminal proteins from mice fed a control or a copper-deficient diet showed elevated levels of both total and apical membrane Ctr1 protein in response to transient dietary copper limitation. Experiments in cultured HEK293T cells demonstrated that alterations in the levels of the glycosylated form of Ctr1 in response to copper availability were a time-dependent, copper-specific posttranslational response. Taken together, these results demonstrate apical localization of Ctr1 in intestinal epithelia across three mammalian species and suggest that increased Ctr1 apical localization in response to dietary copper limitation may represent an adaptive response to homeostatically modulate Ctr1 availability at the site of intestinal copper absorption.     

Further studies on the cell-free synthesis of procollagen-collagen by chick embryo polysomes

Free and membrane bound polysomes were prepared from 8-day-old chick embryos. Both polysome preparations were equally active in protein synthesis but procollagen-collagen synthesis was carried out exclusively by the membrane bound polysomes. The collagenous product was analyzed by DEAE-cellulose chromatography and after hydroxylation with peptidyl proline hydroxylase had a hydroxyproline/proline ratio of 0.77. This suggests that the collagenous product synthesized by the membrane bound polysomes is procollagen.     

ANDROMONOECY AND VARIATION IN PHENOTYPIC GENDER OF PASSIFLORA INCARNATA (PASSIFLORACEAE)

We studied the perennial vine Passiflora incarnata Linnaeus (Passifloraceae) in Alachua County, Florida, from May to August 1983 to determine the breeding system and investigate seasonal changes in phenotypic gender of individual plants. Passiflora incarnata is an obligate outcrosser, incapable of self-fertilization. The major pollinators were carpenter bees (Xylocopa sp.). The proportion of flowers setting fruit was not limited by pollinator service, but the weight of individual fruits and number of seeds/fruit was increased over naturally pollinated flowers by manually performing cross-pollination. Morphological differences in style position among flowers caused some flowers to function primarily as males and others to function as hermaphrodites. Although some of the flowers that functioned as males set fruit when manually cross-pollinated, the proportion of these male flowers capable of setting fruit was lower than the proportion of hermaphroditic flowers setting fruit when manually cross-pollinated. Further, some male flowers had atrophied ovary and styles and were completely incapable of setting fruit. Passiflora incarnata is thus functionally andromonoecious. The relative production of male versus hermaphroditic flowers varied among individuals and over the course of the flowering season. Unmanipulated plants in the population became increasingly male-biased in floral sex ratios as the reproductive season progressed. We attempted to modify phenotypic gender in experimental plants by limiting the ability of plants in some treatment groups to set fruit. Treatment groups significantly affected production of hermaphroditic flowers, but production of male flowers was not affected by treatment. Treatment did not significantly affect fruit weight, number of seeds per fruit or the percentage of hermaphroditic flowers that successfully matured fruit. These results suggest that andromonoecy in P. incarnata is a mechanism for adjusting allocation of reproductive effort to male and female function, and that maternal investment in this species is regulated primarily by varying production of hermaphroditic flowers.     

Mouse ovarian follicles secrete factors affecting the growth and development of like-sized ovarian follicles in vitro

A series of experiments have been carried out to determine whether follicles secrete factors able to affect the growth and development of other, like-sized follicles. Late preantral mouse ovarian follicles were either cocultured or cultured in media conditioned by previously cultured follicles. In particular, the experiments examined whether follicles do secrete such factors, whether the level of FSH in the culture media can affect that process, and what the nature of such secretory factor(s) might be. First, pairs of follicles were cocultured across a polycarbonate membrane containing pores. This showed that communication between the follicles resulted in the stimulation of growth and that the stimulation was due, at least in part, to the production of secretory factor(s). In subsequent experiments, follicles were cultured in media that had been preconditioned by previously cultured follicles. The concentration of FSH in the cultures determined the effect of the conditioned media: conditioned media was stimulatory to follicle growth when levels of FSH remained high throughout the culture, but inhibitory when FSH levels were dropped midway through the cultures. Heat inactivation removed this inhibitory effect, showing that the factor was likely to be a protein; addition of follistatin to the conditioned media did not alter its effect, indicating that the factor was unlikely to be activin. We have shown through a series of culture experiments that mouse follicles secrete factor(s) that can affect the development of other like-sized follicles when cultured from the late preantral to Graafian stages. Furthermore, we have shown that the effect (or production) of such factors is dependent on the FSH environment of the follicles.     

Sediment phosphorus cycling in a large shallow lake: spatio-temporal variation in phosphorus pools and release

Sediment and water column phosphorus fractions were recorded monthly for one year (April 2004–April 2005) in a shallow lake recovering from nutrient pollution (Loch Leven, Scotland). Equilibrium phosphate concentration (EPC0) and gross sediment phosphorus (P) release rates were estimated from laboratory experiments. Pore water and organic P pools were lowest during warm water periods whereas bottom water P was lowest during cold water periods. Reductant-soluble, organic, metal oxide-adsorbed, residual and sediment total phosphorus pools all varied significantly with overlying water depth. Short-term, high magnitude, redox initiated P release events occurred in late summer and winter as a result of anoxic sediment conditions. Lower magnitude long-term release conditions were maintained for most of the year, most likely as a result of organic P cycling and maintenance of high concentration gradients between the pore and bottom water P pools. Estimates of summer P uptake/release rates, across an intact sediment-water interface, suggested that maximum gross internal release was ~12 mg SRP m⁻² lake surface area d⁻¹ with EPC0 values ranging between 180 and 270 μg P L⁻¹. This study highlights the biological mediation of internal loading in shallow eutrophic lakes, and in particular, the role of sediment algae in decreasing, and sediment bacteria in enhancing, sediment P release.     

The long-term (1979–2005) effects of the North Atlantic Oscillation on wind-induced wave mixing in Loch Leven (Scotland)

We report on long-term covariation (1979–2005) between indices of the North Atlantic Oscillation (NAO) and wind speed and direction in Loch Leven. The effects of the observed variations in wind speed and direction were combined to produce modelled wave-mixed depths (Zc). Positive correlations were observed between seasonal and annual wind speeds and westerly frequency and indices of the NAO that are in line with general perception: positive NAO was correlated with stronger, more westerly winds and these correlations were strongest in winter and spring. Correlations between NAO and estimates of Zc were strongest in the most westerly exposed site in spring (r ² = 0.701; Zc_spring versus spring NAO index). On average, over a 25-year period Zc was deeper in spring and shallower in summer. Major anomalies from the 25-year seasonal means were observed in 1982, 1979, and 1991. Annual average Zc was low in the late 1970s and early 1980s (shallowest average annual Zc of 1.0 m (1984)), high in the late 1980s and early 1990s (deepest average annual Zc of 1.9 m (1990)) and moderate in recent years (up to 2005). This study has major implications for our understanding of potential climate change drivers and the related responses of shallow lake ecosystems, including alterations to littoral habitat quality and benthic–pelagic coupling.     

ERRATUM: Effects of Loading on the Biomechanical Behavior of Molars of Homo,Pan, and Pongo. Am J Phys Anthropol 109:211–227.

ERRATUM: Macho GA and Spears IR. 1999. Effects of Loading on the Biomechanical Behavior of Molars of Homo, Pan, and Pongo. Am J Phys Anthropol 109:211–227. The correct title of the article is given above. The word “biochemical” should be read as “biomechanical.”     

Gonadotropin induction of ovulation and corpus luteum formation in young estrogen receptor-alpha knockout mice

Estrogen receptor-alpha (ERalpha) knockout (ERalphaKO) female mice are infertile. Initially, they exhibit normal follicular development, but by 4-5 wk of age, they begin to develop hemorrhagic ovarian cysts. Follicles in adult ERalphaKO female mice progress to the graafian stage, but there are no corpora lutea (CL). To test whether ERalpha is required for ovarian folliculogenesis, ovulation, and CL formation, eCG and hCG were used to ovulate 3- to 5-wk-old ERalphaKO and wild-type (WT) sibling mice. Gonadotropin administration resulted in ovulation in both ERalphaKO and WT mice. Gonadotropin-treated ERalphaKO females that ovulated produced 7.09 +/- 0.77 oocytes per mouse, whereas gonadotropin-treated WT female mice had 16.17 +/- 0.84 oocytes. Surprisingly, ruptured ERalphaKO ovarian follicles developed into CL that had normal morphology. Gonadotropin-treated ERalphaKO mice had 3-fold higher concentrations of serum progesterone than did control ERalphaKO mice that had been administered saline rather than gonadotropins. Thus, the CL in gonadotropin-treated ERalphaKO mice appeared to be steroidogenically functional. On the basis of these findings, ovarian folliculogenesis, ovulation, and CL formation can occur in the absence of ERalpha, although to a lesser extent than in WT mice.     

Effects of loading on the biochemical behavior of molars of Homo,Pan, and Pongo

In a previous study, we found systematic differences in the biomechanical behavior of modern human molars using finite element stress analyses (FESA), which led us to propose that molars are adapted to differently-directed loads depending on their position within the mouth (Spears and Macho [1998] Am. J. Phys. Anthropol. 106:467–482 ). While the FESA results thus derived have not been verified experimentally, such an interpretation seemed reasonable. To refine the model previously presented, this study assessed the effects of 1) food particle size on the biomechanical behavior of molars, and those of 2) differences in morphology, particularly enamel thickness, on stress distribution. In order to appraise the evolutionary significance of the findings, the FESA results for modern humans were subsequently compared with those obtained for molars of one individual of Pan and Pongo, respectively. Bearing in mind limitations imposed by the FESA models created and analyzed in this study, constant cleavage-type loads and cuspal tip loads at different directions were employed on all teeth: this facilitated comparisons of patterns of stress distribution across molars and species. In Pan and Homo, cleavage-type loads exerted by big food particles tended to be better dissipated anteriorly than posteriorly, although trends in Pongo were less clear-cut. Furthermore, similar to modern humans, the buccal cusps of mandibular molars appeared to be able to dissipate the loads associated with a pestle-type action, while maxillary molars were better designed to dissipate the loads which would result if they acted as mortars against which the food is crushed/ground. While increases in enamel thickness lowered the overall stress values in teeth only slightly, changes in outer morphology could have a more profound effect on these stress levels. Overall, Pan appeared to be most generalized, while Homo and Pongo showed a number of unique specializations, which are in accordance with what is currently understood about their respective masticatory apparatus and dietary niche. Am J Phys Anthropol 109:211–227, 1999.© 1999 Wiley-Liss, Inc.