Localization of 1,25-(OH)2D3-responsive alkaline phosphatase in osteoblast-like cells (ROS 17/2.8, MG 63, and MC 3T3) and growth cartilage cells in culture

Previous studies have shown 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3)-responsive alkaline phosphatase in cultured growth zone cartilage chondrocytes is localized in extracellular matrix vesicles (MV). Since osteoblast-like cells also have 1,25-(OH)2D3-responsive alkaline phosphatase, this study determined whether the 1,25-(OH)2D3-responsive enzyme activity is localized to MV produced by these cells as well. Osteoblast-like cells from rat (ROS 17/2.8), mouse (MC 3T3), human (MG 63), and rat growth zone cartilage were cultured in Dulbecco's modified Eagle's medium containing 10(-7)-10(-12) M 1,25-(OH)2D3. Alkaline phosphatase total activity and specific activity were measured in the cell layer, MV, and plasma membrane (PM) fractions. MV and PM purity were verified by electron microscopy and MV alkaline phosphatase specific activity compared to PM (MV versus PM: ROS 17/2.8 6 x; MG 63, 5.5 x; MC 3T3, 33 x; GC, 2 x). There was a dose-dependent stimulation of MV alkaline phosphatase (5- to 15-fold increase at 10(-7)-10(-9) M) in all cell types in response to the 1,25-(OH)2D3. The PM enzyme was stimulated in a parallel fashion in the osteoblast cultures. No effect of 1,25-(OH)2D3 was observed in growth cartilage PM. Although MV accounted for less than 20% of the total activity they contributed 50% of the increase in alkaline phosphatase activity in the cell layer in response to 1,25-(OH)2D3 and MV specific activity was enriched 10 times over that of the cell layer. These are common features of MV produced by cells which calcify their matrix and suggest that hormonal regulation of MV enzymes may be important in primary calcification.     

Cultivation of <Emphasis Type="Italic">Gracilaria verrucosa</Emphasis> (Huds) Papenfuss in Chilika Lake for livelihood generation in coastal areas of Orissa State

The agarophyte red alga Gracilaria verrucosa occurs widely in Chilika Lake, one of the RAMSAR wetland sites in India. The lake is situated in the extreme southeast corner of Orissa between latitudes 19°28′ and 19°54′ N and longitudes 85°06′ and 85°35′ E. The natural biomass production is not sufficient for the agar industry, and the only alternative is to maximize the production of the seaweed through mass cultivation by seaweed farming. To elucidate important aspects of the growth and development of G. verrucosa, experimental field cultivation was undertaken at Langaleswar and Samal sites of Chilika Lake using ropes and raft methods during March to August, 2009. After 30 days of cultivation a maximum 15- and 13.8-fold increase in biomass in raft culture and rope culture, respectively, was observed at Langaleswar and an 11.6- and 11.0-fold increase in biomass at Samal. Environmental parameters such as temperature, salinity, pH, transparency, DO, conductivity, nitrate, and phosphate were monitored at both stations, and the influence of environmental parameters is discussed.     

A Mutation in the LDL Receptor–Related Protein 5 Gene Results in the Autosomal Dominant High–Bone-Mass Trait

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 β-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.     

Grooming using rotating brushes as a proactive method to control ship hull fouling

Grooming may be defined as the frequent and gentle cleaning of a ship hull coating, when it is in port or idle, to prevent the establishment of fouling. This study assessed the effectiveness of grooming with a five-headed rotating brush system on epoxy, ablative copper and two silicone fouling release (FR) coatings. These coatings were placed under static immersion at Port Canaveral, FL on a weekly and biweekly frequency. The results showed that grooming reduced fouling on all surfaces and was able to prevent fouling on the ablative copper and FR coatings when performed weekly. It was concluded that the grooming tool used for these tests was sufficient to remove biofilm and most hard fouling. However, when fouling pressure increased or when grooming was performed less frequently, insufficient forces were imparted by the brush to remove all of the established hard fouling organisms.     

The LS locus of pea encodes the gibberellin biosynthesis enzyme ent-kaurene synthase A

Gibberellins (GAs) are hormones required for several aspects of plant development, including internode elongation and seed development in pea (Pisum sativum L.). The first committed step in the GA biosynthesis pathway is the conversion of geranylgeranyl diphosphate (GGDP) to ent-kaurene via copalyl diphosphate (CDP). These two reactions are catalyzed by the cyclases ent-kaurene synthase A (KSA) and ent-kaurene synthase B (KSB), respectively. Previous genetic and biochemical analysis of the GA-responsive ls-1 mutant of pea suggested that GA levels are reduced in a developmental- and organ-specific manner due to reduced GA biosynthesis. Analysis of cell-free enzyme preparations from WT and ls-1 embryos at contact point reveals that ls-1 reduces the activity of KSA but not KSB. To characterize the ls-1 mutation in more detail, a cDNA coding for a pea KSA was cloned and shown to be encoded by the LS locus. The ls-1 mutation results from an intronic G to A substitution that causes impaired RNA splicing. To determine the activity of the KSAs encoded by the LS and ls-1 alleles, a new in vitro assay for combined KSA and KSB activity has been developed using the KSB gene of pumpkin. Using recombinant WT KSA and KSB fusion proteins, GGDP is converted to ent-kaurene in vitro. Based on the sequence of RT-PCR products, three different truncated KSA proteins are predicted to exist in ls-1 plants. The most abundant mutant KSA protein does not possess detectable activity in vitro. Nevertheless, the ls-1 allele is not null and is able to encode at least a partially functional KSA since a more severe ls allele has been identified. The ls-1 mutation has played a key role in identifying a role for GAs in pea seed development in the first few days after fertilization, but not in older seeds. KSA expression in seeds is developmentally regulated and parallels overall GA biosynthesis, suggesting that KSA expression may play an important role in the regulation of GA biosynthesis and seed development.     

Evolutionary response to size-selective mortality in an exploited fish population

Many collapsed fish populations have failed to recover after a decade or more with little fishing. This may reflect evolutionary change in response to the highly selective mortality imposed by fisheries. Recent experimental work has demonstrated a rapid genetic change in growth rate in response to size-selective harvesting of laboratory fish populations. Here, we use a 30-year time-series of back-calculated lengths-at-age to test for a genetic response to size-selective mortality in the wild in a heavily exploited population of Atlantic cod (Gadus morhua). Controlling for the effects of density- and temperature-dependent growth, the change in mean length of 4-year-old cod between offspring and their parental cohorts was positively correlated with the estimated selection differential experienced by the parental cohorts between this age and spawning. This result supports the hypothesis that there have been genetic changes in growth in this population in response to size-selective fishing. Such changes may account for the continued small size-at-age in this population despite good conditions for growth and little fishing for over a decade. This study highlights the need for management regimes that take into account the evolutionary consequences of fishing.     

Phenylacetylglycine,a putative biomarker of phospholipidosis: Its origins and relevance to phospholipid accumulation using amiodarone treated rats as a model

Amiodarone was given to male Sprague–Dawley rats at a dose of 150 mg kg^?1 day^?1 for 7 consecutive days to induce phospholipidosis in the lungs of treated rats. Amiodarone was given alone or concurrently with phenobarbitone. Animals given amiodarone had raised total phospholipid in serum, lung and lymphocytes, and elevated lyso(bis)phosphatidic acid (LBPA) in all tissues. Urinary and plasma phenylacetylglycine (PAG) and hepatic portal:aortal phenylacetate (PA) ratio were increased, whereas hepatic phenylalanine hydroxylase (PAH) activity and plasma phenylalanine:tyrosine ratio were not affected. Phenobarbitone treatment increased hepatic total P450 content and induced 7-pentoxyres-orufin O-dealkylatian (PROD) activity, as expected, but had no effect on any other biochemical parameter. Plasma amiodarone concentration was reduced in rats co-administered both drugs and phospholipid accumulation in target tissues was attenuated compared with rats treated with amiodarone alone. However, phenobarbitone co-administration failed to alter the magnitude of response with regards to urinary PAG excretion and plasma concentration of its precursors after amiodarone treatment. Increased intestinal absorption of PAG precursors probably resulted in the raised urinary PAG after amiodarone treatment. Urinary PAG correlated weakly with serum, lymphocyte and lung phospholipids. However, urinary PAG excretion was similar in rats dosed solely with amiodarone or in combination with phenobarbitone, despite the fact that the degree of phospholipid accumulation was far less in rats given the combined treatment. Nevertheless, urinary PAG was raised only in animals exhibiting abnormal phospholipid accumulation in target tissues and may thus be useful as a surrogate biomarker for phospholipidosis.     

Ewe and lamb contact at lambing is influenced by both shelter type and birth number

A significant number of lambs born each year in Australia die within 72 h of birth. Periods of high wind, combined with rain and low temperatures can lead to marked increases in the mortality level. Under these climatic conditions mortality levels may be reduced with the provision of shelter. This study used contact loggers to compare interactions between ewes with twin lambs across two shelter types (Hessian and shrubs), while also comparing ewes with single and twin lambs in a single shelter type (Hessian). The contact loggers record the time of the initial contact (within approximately 4 to 5 m) between collared animals and the duration of each contact. Contact levels between ewes immediately after lambing were only 10% of the initial levels (1 h/day). For single-born lambs, lambs averaged 11 h contact per day with their mother, while for twinborn lambs, each lamb averaged 9.25 h/day with its mother and 14.7 h/day with its sibling. The level of contact between ewes and each of their offspring in the Hessian was 24% lower (P < 0.05) for ewes with twin lambs than with singles. For ewes with twin lambs the level of contact was 17% lower (P < 0.05) in the Hessian shelter compared with shrub shelter. We conclude that shelter type and birth number can affect the level of contact between ewes and their offspring.     

Complex regulation of the lactase-phlorizin hydrolase promoter by GATA-4

Lactase-phlorizin hydrolase (LPH), a marker of intestinal differentiation, is expressed in absorptive enterocytes on small intestinal villi in a tightly regulated pattern along the proximal-distal axis. The LPH promoter contains binding sites that mediate activation by members of the GATA-4, -5, and -6 subfamily, but little is known about their individual contribution to LPH regulation in vivo. Here, we show that GATA-4 is the principal GATA factor from adult mouse intestinal epithelial cells that binds to the mouse LPH promoter, and its expression is highly correlated with that of LPH mRNA in jejunum and ileum. GATA-4 cooperates with hepatocyte nuclear factor (HNF)-1alpha to synergistically activate the LPH promoter by a mechanism identical to that previously characterized for GATA-5/HNF-1alpha, requiring physical association between GATA-4 and HNF-1alpha and intact HNF-1 binding sites on the LPH promoter. GATA-4 also activates the LPH promoter independently of HNF-1alpha, in contrast to GATA-5, which is unable to activate the LPH promoter in the absence of HNF-1alpha. GATA-4-specific activation requires intact GATA binding sites on the LPH promoter and was mapped by domain-swapping experiments to the zinc finger and basic regions. However, the difference in the capacity between GATA-4 and GATA-5 to activate the LPH promoter was not due to a difference in affinity for binding to GATA binding sites on the LPH promoter. These data indicate that GATA-4 is a key regulator of LPH gene expression that may function through an evolutionarily conserved mechanism involving cooperativity with an HNF-1alpha and/or a GATA-specific pathway independent of HNF-1alpha.     

Potential sites of bioactive gibberellin production during reproductive growth in Arabidopsis

Gibberellin 3-oxidase (GA3ox) catalyzes the final step in the synthesis of bioactive gibberellins (GAs). We examined the expression patterns of all four GA3ox genes in Arabidopsis thaliana by promoter-beta-glucuronidase gene fusions and by quantitative RT-PCR and defined their physiological roles by characterizing single, double, and triple mutants. In developing flowers, GA3ox genes are only expressed in stamen filaments, anthers, and flower receptacles. Mutant plants that lack both GA3ox1 and GA3ox3 functions displayed stamen and petal defects, indicating that these two genes are important for GA production in the flower. Our data suggest that de novo synthesis of active GAs is necessary for stamen development in early flowers and that bioactive GAs made in the stamens and/or flower receptacles are transported to petals to promote their growth. In developing siliques, GA3ox1 is mainly expressed in the replums, funiculi, and the silique receptacles, whereas the other GA3ox genes are only expressed in developing seeds. Active GAs appear to be transported from the seed endosperm to the surrounding maternal tissues where they promote growth. The immediate upregulation of GA3ox1 and GA3ox4 after anthesis suggests that pollination and/or fertilization is a prerequisite for de novo GA biosynthesis in fruit, which in turn promotes initial elongation of the silique.