Inhibition of a Low Km GTPase Activity in Rat Striatum by Calmodulin

In rat striatum, the activation of adenylate cyclase by the endogenous Ca2+-binding protein, calmodulin, is additive with that of GTP but is not additive with that of the nonhydrolyzable GTP analog, guanosine-5'-(beta, gamma-imido)triphosphate (GppNHp). One possible mechanism for this difference could be an effect of calmodulin on GTPase activity which has been demonstrated to "turn-off" adenylate cyclase activity. We examined the effects of Ca2+ and calmodulin on GTPase activity in EGTA-washed rat striatal particulate fractions depleted of Ca2+ and calmodulin. Calmodulin inhibited GTP hydrolysis at concentrations of 10(-9)-10(-6) M but had no effect on the hydrolysis of 10(-5) and 10(-6) M GTP, suggesting that calmodulin inhibited a low Km GTPase activity. The inhibition of GTPase activity by calmodulin was Ca2+-dependent and was maximal at 0.12 microM free Ca2+. Maximal inhibition by calmodulin was 40% in the presence of 10(-7) M GTP. The IC50 for calmodulin was 100 nM. In five tissues tested, calmodulin inhibited GTP hydrolysis only in those tissues where it could also activate adenylate cyclase. Calmodulin could affect the activation of adenylate cyclase by GTP in the presence of 3,4-dihydroxyphenylethylamine (DA, dopamine). Calmodulin decreased by nearly 10-fold the concentration of GTP required to provide maximal stimulation of adenylate cyclase activity by DA in the striatal membranes. The characteristics of the effect of calmodulin on GTPase activity with respect to Ca2+ and calmodulin dependence and tissue specificity parallel those of the activation of adenylate cyclase by calmodulin, suggesting that the two activities are closely related.(ABSTRACT TRUNCATED AT 250 WORDS)     

Responses of sawgrass and spikerush to variation in hydrologic drivers and salinity in Southern Everglades marshes

Aboveground net primary production (ANPP) by the dominant macrophyte and plant community composition are related to the changing hydrologic environment and to salinity in the southern Everglades, FL, USA. We present a new non-destructive ANPP technique that is applicable to any continuously growing herbaceous system. Data from 16 sites, collected from 1998 to 2004, were used to investigate how hydrology and salinity controlled sawgrass (Cladium jamaicense Crantz.) ANPP. Sawgrass live biomass showed little seasonal variation and annual means ranged from 89 to 639 gdw m⁻². Mortality rates were 20–35% of live biomass per 2 month sampling interval, for biomass turnover rates of 1.3–2.5 per year. Production by C. jamaicense was manifest primarily as biomass turnover, not as biomass accumulation. Rates typically ranged from 300 to 750 gdw m⁻² year⁻¹, but exceeded 1000 gdw m⁻² year⁻¹ at one site and were as high as 750 gdw m⁻² year⁻¹ at estuarine ecotone sites. Production was negatively related to mean annual water depth, hydroperiod, and to a variable combining the two (depth-days). As water depths and hydroperiods increased in our southern Everglades study area, sawgrass ANPP declined. Because a primary restoration goal is to increase water depths and hydroperiods for some regions of the Everglades, we investigated how the plant community responded to this decline in sawgrass ANPP. Spikerush (Eleocharis sp.) was the next most prominent component of this community at our sites, and 39% of the variability in sawgrass ANPP was explained by a negative relationship with mean annual water depth, hydroperiod, and Eleocharis sp. density the following year. Sawgrass ANPP at estuarine ecotone sites responded negatively to salinity, and rates of production were slow to recover after high salinity years. Our results suggest that ecologists, managers, and the public should not necessarily interpret a decline in sawgrass that may result from hydrologic restoration as a negative phenomenon.     

Spatial and temporal patterns of aboveground net primary productivity (ANPP) along two freshwater-estuarine transects in the Florida Coastal Everglades

We present here a 4-year dataset (2001–2004) on the spatial and temporal patterns of aboveground net primary production (ANPP) by dominant primary producers (sawgrass, periphyton, mangroves, and seagrasses) along two transects in the oligotrophic Florida Everglades coastal landscape. The 17 sites of the Florida Coastal Everglades Long Term Ecological Research (FCE LTER) program are located along fresh-estuarine gradients in Shark River Slough (SRS) and Taylor River/C-111/Florida Bay (TS/Ph) basins that drain the western and southern Everglades, respectively. Within the SRS basin, sawgrass and periphyton ANPP did not differ significantly among sites but mangrove ANPP was highest at the site nearest the Gulf of Mexico. In the southern Everglades transect, there was a productivity peak in sawgrass and periphyton at the upper estuarine ecotone within Taylor River but no trends were observed in the C-111 Basin for either primary producer. Over the 4 years, average sawgrass ANPP in both basins ranged from 255 to 606 g m⁻² year⁻¹. Average periphyton productivity at SRS and TS/Ph was 17–68 g C m⁻² year⁻¹ and 342–10371 g C m⁻² year⁻¹, respectively. Mangrove productivity ranged from 340 g m⁻² year⁻¹ at Taylor River to 2208 g m⁻² year⁻¹ at the lower estuarine Shark River site. Average Thalassia testudinum productivity ranged from 91 to 396 g m⁻² year⁻¹ and was 4-fold greater at the site nearest the Gulf of Mexico than in eastern Florida Bay. There were no differences in periphyton productivity at Florida Bay. Interannual comparisons revealed no significant differences within each primary producer at either SRS or TS/Ph with the exception of sawgrass at SRS and the C−111 Basin. Future research will address difficulties in assessing and comparing ANPP of different primary producers along gradients as well as the significance of belowground production to the total productivity of this ecosystem.     

Methods for improving the efficiency of estimating total osteon density in the human anterior mid-diaphyseal femur

In order to preserve whole bone integrity and minimize destruction, paleohistologists often rely on histomorphometric data obtained from small areas (1.5–50 mm²) sampled within the anterior mid-diaphyseal femur. Because bone exhibits significant histological variation, the validity of results based on such sampling is questionable. The accuracy of various subareas (columns, rows, squares approximating dimensions and locations assessed by paleohistologists) in predicting total osteon density in the anterior mid-diaphyseal femur is assessed in the present study. Thirty-five specimens (12.7 mm wide, 100 μm thick, average area 56.7 mm²) were chosen at random from a skeletal population of 94 Inuits and Pueblo agriculturists. The specimens were photographed and enlarged; an acetate grid (12 columns, 10 rows, 120 squares, square = 1 mm² of bone surface) was superimposed over the photograph; and secondary osteons and fragments were identified. Alternate columns (50% total area, T.Ar) predicted over 98% of entire section total osteon density. Two column combinations (15% T.Ar), separated by at least one column, predicted 91 to 95% of total osteon density. Individual column (8% T.Ar) predictability ranged from 48 to 86%. Two row combination (32 to 40% T.Ar) predictability values ranged from 86 to 95%. Individual rows (<1 to 20% T.Ar) predicted from 45 to 92% of total variation. Combinations of squares approximating areas and locations assessed by other paleohistologists ranged in predictability values from 80 to 94%. The results demonstrate that subareas of as little as 15% predict 95% of variation in total osteon density in the entire anterior mid-diaphyseal femoral section. A minimization of histological area evaluated without the loss of accuracy allows for a minimization of time invested in data collection and the utilization of partially damaged specimens. Am J Phys Anthropol 107:13–24, 1998. © 1998 Wiley-Liss, Inc.     

Effects of disrupted beta1-integrin function on the skeletal response to short-term hindlimb unloading in mice.

The study was designed to determine whether beta1-integrin plays a role in mediating the acute skeletal response to mechanical unloading. Transgenic (TG) mice were generated to express a dominant negative form of beta1-integrin under the control of the osteocalcin promoter, which targets expression of the transgene to mature osteoblasts. At 63 days of age, wild-type (WT) and TG mice were subjected to hindlimb unloading by tail suspension for 1 wk. Pair-fed, normally loaded WT and TG mice served as age-matched controls. Bone samples from each mouse were processed for quantitative bone histomorphometry and biomechanical testing. The skeletal phenotype of TG mice was characterized by lower cancellous bone mass in the distal femoral metaphysis (-52%) and lumbar vertebral body (-20%), reduced curvature of the proximal tibia (-20%), and decreased bone strength (-20%) and stiffness (-23%) of the femoral diaphysis with relatively normal indexes of cancellous bone turnover. Hindlimb unloading for only 1 wk induced a 10% decline in tibial curvature and a 30% loss of cancellous bone in the distal femur due to a combination of increased bone resorption and decreased bone formation in both WT and TG mice. However, the strength and stiffness of the femoral diaphysis were unaffected by short-term hindlimb unloading in both genotypes. The observed increase in osteoclast surface was greater in unloaded TG mice (92%) than in unloaded WT mice (52%). Cancellous bone formation rate was decreased in unloaded WT (-29%) and TG (-15%) mice, but, in contrast to osteoclast surface, the genotype by loading interaction was not statistically significant. The results indicate that altered integrin function in mature osteoblasts may enhance the osteoclastic response to mechanical unloading but that it does not have a major effect on the development of cancellous osteopenia in mice during the early stages of hindlimb unloading.     

Ecological attributes recorded in stable isotope ratios of arboreal prosimian hair

Carbon and nitrogen stable isotope ratios were measured in hair samples from two species of Galago from Gedi Ruins National Monument in eastern Kenya and from Lepilemur leucopus from Beza Mahafaly Special Reserve in southern Madagascar. Forest structure was generally similar in the two areas but average rainfall was lower in Madagascar. Species average ¹³C values varied with feeding height in the forest canopy and with average rainfall level as expected from reported variation in plant ¹³C values. G. garnettii, which feeds higher in the forest canopy, had less negative ¹³C values than G. zanzibaricus, which spends more time below 5 m. L. leucopus, from a drought-afflicted forest, had less negative hair ¹³C values than the two galago species. The values within the Lepilemur sample showed a positive linear relation with percent dependence on a CAM tree species and with xeric conditions within the species reserve. Nitrogen stable isotope ratios varied with trophic level of feeding and with time spent feeding on leguminous plants. The insectivorous galagos had significantly more positive ¹⁵N values than the folivorous L. leucopus. Within the Lepilemur sample, ¹⁵N values varied inversely with the percent of feeding time spent on leguminous plants. The range of ¹⁵N and ¹³C values in each of the prosimian species is larger than reported for animals fed monotonous diets and for New World monkey species. The monkey species feed as groups of individuals whereas the prosimians have solitary feeding habits. The ranges in the prosimian species apparently reflect the greater variation in diet among individual prosimians compared to individual monkeys. The isotope data reported here are equivalent, on average, to those reported for other arboreal species from similar forest habitats and with similar dietary habits. This supports the use of such data for paleoecological reconstruction of forest and woodland systems and diet reconstruction of extinct primate populations and species. Received: 18 April 1997 / Accepted: 11 August 1997     

Enhanced expression of Fas Ligand (FasL) in the lower airways of patients with fibrotic interstitial lung diseases (ILDs)

The exact role of FasL, and particularly its soluble and membrane-bound forms, in the development of chronic ILDs and lung fibrosis has not been extensively explored. We aimed at analyzing membrane-bound FasL expression on alveolar macrophages (AM) and lymphocytes (AL) as well as soluble FasL (sFasL) levels in bronchoalveolar lavage (BAL) from ILDs patients, incl. pulmonary sarcoidosis (PS), hypersensitivity pneumonitis (HP), silicosis, asbestosis, idiopathic pulmonary fibrosis (IPF), nonspecific interstitial pneumonia (NSIP), and healthy subjects (n = 89, 12, 7, 8, 23, 6, 17, respectively). In IPF, significantly increased percentage of AM FasL(+) and CD8(+)FasL(+) cells as well as sFasL levels in BAL were found. Increased sFasL levels were also observed in HP. NSIP and asbestosis were characterized by higher AM FasL(+) relative number; CD8(+)FasL(+) population was expanded in asbestosis only. There was a significant decline in AL FasL(+) percentage in PS and HP. Vital capacity was negatively correlated with sFasL levels, AM FasL(+) and CD8(+)FasL(+) cell relative count. CD4(+)FasL(+) and CD8(+)FasL(+) percentage strongly correlated with BAL neutrophilia, an unfavorable prognostic factor in lung fibrosis. The concurrent comparative BAL analysis of FasL expression indicates that FasL(+) AM and AL (mainly Tc cells) comprise an important element of the fibrotic process, mostly in IPF. FasL might play a crucial role in other fibrosis-complicated ILDs, like NSIP and asbestosis.     

Genetic variation in Arizona Mexican Americans: estimation and interpretation of admixture proportions

Mexican Americans are a numerous and fast growing ethnic population in the United States. Yet little is known about their genetic structure. Since they are a hybrid, it is of interest to identify their parental populations and to estimate the relative contributions of these groups. This information is relevant to historical, biomedical, and evolutionary concerns. New genetic typings on 730 Arizona Mexican Americans for the HLA-A, HLA-B, ABO, Rh, MNSs, Duffy, Kidd, and Kell loci are presented here and they are used to estimate ancestral contributions. We considered both a dihybrid model with Amerindians and Spaniards as proposed ancestors, and a trihybrid model with Amerindians, Spaniards, and Africans as proposed ancestors. A modified weighted least squares method that allows for linkage disequilibrium was used to estimate ancestral contributions for each model. The following admixture estimates were obtained: Amerindian, 0.29 +/- 0.04; Spaniard, 0.68 +/- 0.05; and African, 0.03 +/- 0.02. The interpretation of these results with respect to Amerindian and Spanish ancestry is straightforward. African ancestry is strongly supported by the presence of a marker of African descent, Fy, despite the fact that the standard error of the estimate is as large as the estimated admixture proportion. An evaluation of the sensitivity of these results to a number of variables is presented: 1) our choices of ancestral allele frequencies, 2) the possibility of selection at HLA and the blood groups, and 3) genetic drift in Mexican Americans.     

Effects of increased hypothalamic leptin gene expression on ovariectomy-induced bone loss in rats

Estrogen deficiency results in accelerated bone turnover with a net increase in bone resorption. Subcutaneous administration of leptin attenuates bone loss in ovariectomized (ovx) rats by reducing bone resorption. However, in addition to its direct beneficial effects, leptin has been reported to have indirect (central nervous system-mediated) antiosteogenic effects on bone, which may limit the efficacy of elevated serum leptin to prevent estrogen deficiency-associated bone loss. The present study evaluated the long-term effects of increased hypothalamic leptin transgene expression, using recombinant adeno-associated virus-leptin (rAAV-Lep) gene therapy, on bone mass, architecture, and cellular endpoints in sexually mature ovx Sprague-Dawley rats. Ovx rats were implanted with cannulae in the 3rd ventricle of the hypothalamus and injected with either rAAV-Lep or rAAV-GFP (control vector encoding green fluorescent protein) and maintained for 10 weeks. Additional controls consisted of ovary-intact rats and ovx rats pair-fed to rAAV-Lep rats. Lumbar vertebrae were analyzed by micro-computed tomography and tibiae by histomorphometry. Cancellous bone volume was lower and osteoclast perimeter, osteoblast perimeter, and bone marrow adipocyte density were greater in ovx rats compared to ovary-intact controls. In contrast, differences among ovx groups were not detected for any endpoint evaluated. In conclusion, whereas estrogen deficiency resulted in marked cancellous osteopenia, increased bone turnover and marrow adiposity, increasing hypothalamic leptin transgene expression in ovx rats had neither detrimental nor beneficial effects on bone mass, architecture, or cellular endpoints. These findings demonstrate that the antiresorptive effects of subcutaneous leptin administration in ovx rats are mediated through leptin targets in the periphery.     

Central leptin gene therapy corrects skeletal abnormalities in leptin-deficient ob/ob mice

Skeletal growth is tightly coupled to energy balance via complex and incompletely understood mechanisms. Leptin-deficient ob/ob mice are obese and develop multiple pathologies associated with the metabolic syndrome. Additionally, ob/ob mice have skeletal abnormalities. The objective of this study was to evaluate the effects of leptin deficiency and long duration selective central leptin repletion via recombinant adeno-associated virus-leptin (rAAV-lep) gene therapy on bone in growing ob/ob mice. The ob/ob mice were injected in the hypothalamus with either rAAV-lep or rAAV-GFP (control vector). Treated ob/ob and untreated wild-type (WT) mice were then maintained on a normal diet for 15 weeks. In a second experiment, similarly treated mice along with a group of pair-fed mice were maintained for 30 weeks. Leptin was not detected in blood of either rAAV-lep- or rAAV-GFP-treated mice although rAAV-lep-treated mice displayed leptin transgene expression in the hypothalamus. As expected, rAAV-lep normalized body weight and food intake. Compared to WT mice, rAAV-GFP-treated ob/ob mice had decreased femoral length (by 1.6 mm or 10%, P<0.001), decreased total femur bone volume (by 3.3 mm(3) or 19%, P<0.001), but increased cancellous bone volume in the distal femur (by 0.04 mm(3) or 60%, P<0.09) and lumbar vertebrae (by 0.26 mm(3) or 118%, P<0.001). Treatment with rAAV-lep rescued the ob/ob skeletal phenotype by increasing femoral length and total bone volume, and decreasing femoral and vertebral cancellous bone volume, so that at 15 weeks post-rAAV-lep injection the ob/ob mice no longer differed from WT mice. No further skeletal changes in either the femur or lumbar vertebra were observed at 30 weeks post-rAAV-lep administration. The results suggest that hypothalamic leptin functions as an essential permissive factor for normal bone growth.