Efficient recycling of nutrients in modern and past hypersaline environments.

The biogeochemistry of hypersaline environments is strongly influenced by changes in biological processes and physicochemical parameters. Although massive evaporation events have occurred repeatedly throughout Earth history, their biogeochemical cycles and global impact remain poorly understood. Here, we provide the first nitrogen isotopic data for nutrients and chloropigments from modern shallow hypersaline environments (solar salterns, Trapani, Italy) and apply the obtained insights to δ15N signatures of the Messinian salinity crisis (MSC) in the late Miocene. Concentrations and δ15N of chlorophyll a, bacteriochlorophyll a, nitrate, and ammonium in benthic microbial mats indicate that inhibition of nitrification suppresses denitrification and anammox, resulting in efficient ammonium recycling within the mats and high primary productivity. We also suggest that the release of 15N-depleted NH3(gas) with increasing salinity enriches ammonium 15N in surface brine (≈34.0‰). Such elevated δ15N is also recorded in geoporphyrins isolated from sediments of the MSC peak (≈20‰), reflecting ammonium supply sufficient for sustaining phototrophic primary production. We propose that efficient nutrient supply combined with frequent bottom-water anoxia and capping of organic-rich sediments by evaporites of the Mediterranean MSC could have contributed to atmospheric CO2 reduction during the late Miocene.

Global iron connections between desert dust, ocean biogeochemistry, and climate.

The environmental conditions of Earth, including the climate, are determined by physical, chemical, biological, and human interactions that transform and transport materials and energy. This is the "Earth system": a highly complex entity characterized by multiple nonlinear responses and thresholds, with linkages between disparate components. One important part of this system is the iron cycle, in which iron-containing soil dust is transported from land through the atmosphere to the oceans, affecting ocean biogeochemistry and hence having feedback effects on climate and dust production. Here we review the key components of this cycle, identifying critical uncertainties and priorities for future research.

Role of osteopontin in bone remodeling caused by mechanical stress.

Changes in the number and proportion of osteopontin mRNA (Opn) expressing osteocytes and osteoclasts caused by the mechanical stress applied during experimental tooth movement were examined in the present study. Opn expression was detected in the osteocytes on the pressure side at the early stage, and gradually spread to those on the tension side and also to the osteoblasts and bone-lining cells in the alveolar bone. Only 3.3% of the osteocytes located on the pressure side expressed Opn in the interradicular septum of control rats; in contrast, the value was increased to 87.5% at 48 h after the initiation of tooth movement. These results indicate that these cells responded to mechanical stress loaded on the bone with expression of the osteopontin gene. Following the increased expression of Opn in these cells, a 17-fold greater number of osteoclasts compared with the control and numerous resorption pits were observed on the pressure side of the alveolar bone. Injection of arginine-glycine-aspartic acid-serine peptide but not that of arginine-glycine-glutamic acid-serine peptide strongly inhibited the increase in the number of osteoclasts. Furthermore, an in vitro migration assay demonstrated the chemotactic activity of osteopontin (OPN) on the precursor of osteoclasts. Our study strongly suggests that OPN is an important factor triggering bone remodeling caused by mechanical stress.

Transcriptional regulation of osteopontin gene in vivo by PEBP2alphaA/CBFA1 and ETS1 in the skeletal tissues.

Osteopontin (Opn) and polyoma enhancer-binding protein (PEBP) 2alphaA/core binding factor (CBFA) 1 have been suggested to play important roles in ossification. The overlapping localization of opn and PEBP2alphaA/CBFA1 mRNA, and the marked decrease of opn mRNA expression in PEBP2alphaA knockout mice, indicated that the transcription of opn gene was controlled by PEBP2alphaA. In the present study, we determined the direct regulation of PEBP2alphaA on the opn promoter activity. Opn promoter activity was markedly enhanced by PEBP2alphaA and ETS1 in a synergistic manner. The synergistic effect was diminished when either the PEBP2alphaA or ETS1 binding site was mutated, or the spatial arrangement of these sites was mutated by a 4-nt insertion. The distance between these sites was important for transactivation but not protein-DNA binding. The direct interaction between PEBP2alphaA and ETS1 was depended on protein-DNA binding. These results suggested that the specific spatial arrangement of both sites and direct interaction between PEBP2alphaA and ETS1, were essential for promoter function. Furthermore, endogenous opn mRNA was decreased with the introduction of dominant negative PEBP2alphaA to MC3T3/E1 cells expressing endogenous PEBP2alphaA, ETS1 and opn. These findings suggest that PEBP2alphaA and ETS1 cooperate in vivo to regulate expression of the opn gene in the skeletal tissue. Cell type-specific regulation of Opn gene expression will also be discussed.

Expression of bone matrix proteins mRNA during distraction osteogenesis.

Distraction osteogenesis is a recently advanced principle of bone lengthening in which a bone separated by osteotomy is subjected to slow progressive distraction using an external fixation device. Appropriate mechanical tension-stress is believed not to break the callus but rather to stimulate osteogenesis. To study the molecular features of this process, the expression and localization of the mRNAs encoding osteopontin (OPN), osteocalcin (OC), matrix Gla protein (MGP), osteonectin (ON), and collagen type I and I during distraction osteogenesis were examined by in situ hybridization and Northern blot analysis. The process can be divided into three distinct phases: the lag phase for 7 days between osteotomy and the beginning of distraction, the distraction phase for 21 days, and the consolidation phase for several weeks. The histologic and molecular events taking place during the lag phase were similar to those observed in fracture healing. The osteotomy site was surrounded by external callus consisting of hyaline cartilage. As distraction started at the rate of 0.25 mm/12 h, the cartilaginous callus was elongated, deformed, and eventually separated into proximal and distal segments. The chondrocytes were stretched along the tension vector and became fibroblast-like in shape. Although morphologically these cells were distinguishable from osteogenic cells, they expressed OPN, OC, and alkaline phosphatase mRNAs. As distraction advanced, the cartilaginous callus was progressively replaced by bony callus by endochondral ossification and thereafter new bone was formed directly by intramembranous ossification. OPN mRNA was detected in preosteoblasts and osteoblasts at the boundary between fibrous tissue and new bone. ON, MGP, and OC mRNAs appeared early in the differentiation stage. The variety of cell types expressing mRNA encoding bone matrix proteins in distraction osteogenesis was much greater than that detected in the embryonic bone formation and fracture healing process. Moreover, the levels of OPN, ON, MGP, and OC mRNA expression markedly increased during the distraction phase. These results suggested that mechanical tension-stress modulates cell shape and phenotype, and stimulates the expression of the mRNA for bone matrix proteins.

Differential in situ expression of alpha2(XI) collagen mRNA isoforms in the developing mouse.

Type XI collagen is an essential structural component of the extracellular matrix of cartilage and plays a role in collagen fibril formation and skeletal morphogenesis. The expression of all three type XI collagen genes is not restricted to cartilage. In addition, alternative exon usage seems to increase the structural diversity and functional potential of type XI collagen during development. In order to investigate type XI collagen expression during development, we have examined alpha2(XI) and alpha1(XI) collagen genes by in situ hybridization in mice. Transcripts of the alpha2(XI) collagen gene were first detected in the notochord of mouse embryos after 11.5 days of gestation. Subsequently, alpha2(XI) mRNA was mainly found in the cartilaginous tissues of the developing limbs and axial skeleton together with transcripts of the alpha1(XI) gene. The alpha2(XI) transcripts seemed to be alternatively spliced isoforms lacking exons 6-8, which code for an acidic domain. Expression of alpha2(XI) outside the cartilage was relatively restricted, whereas expression of the alpha1(XI) gene was widespread. However, expression of alpha2(XI) transcripts containing exons 6-8 was found in non-chondrogenic tissues, including the calvarium and periosteum where intramembranous ossification occurs. These results indicate that alpha2(XI) mRNA isoforms are differentially expressed in various tissues during development. In addition, alpha2(XI) mRNA isoforms containing alternative exons are present in osteogenic cells, and their expression may be closely related to the formation of bone or cartilage.

Impaired expression of noncollagenous bone matrix protein mRNAs during fracture healing in ascorbic acid-deficient rats.

In scorbutic patients, fractures are slow to heal because of impaired collagen synthesis. To investigate the influence of impaired collagen synthesis on the differentiation and proliferation of osteogenic and chondrogenic cells, we examined the expression of genes encoding bone matrix proteins, including osteonectin (ON), osteopontin (OPN), osteocalcin (OC), and matrix Gla protein (MGP), as differentiation markers for osteogenic and chondrogenic cells during fracture healing in Osteogenic Disorder Shionogi (ODS) rats, which have a hereditary defect in the ability to synthesize ascorbic acid (Asc). In ODS rats without Asc supplementation, intramembranous ossification was completely inhibited. Although a few fibroblast-like cells expressing ON mRNA were observed, no OPN mRNA-expressing cells were detected. During endochondral ossification, a small amount of metachromatic staining cartilage appeared at the fracture site, but there was no provisional calcification zone in the cartilage. Chondrocytes expressed ON and MGP mRNAs, but not OPN mRNA. When Asc was given to these rats, callus formation was soon detected around the fracture site, while OPN mRNA was expressed by differentiated osteoblasts and hypertrophic chondrocytes. Our data indicate that impaired collagen synthesis due to Asc deficiency inhibited the increase of ON and MGP mRNA-expressing cells as well as the appearance of OPN mRNA-expressing cells. Since OPN is considered to play an important role in normal and pathological mineralization, lack of OPN mRNA expression accompanying impaired collagen synthesis may have a role in defective mineralization and delayed fracture healing in scurvy.

Three modes of ossification during distraction osteogenesis in the rat.

We developed a rat model of limb lengthening to study the basic mechanism of distraction osteogenesis, using a small monolateral external fixator. In 11-week-old male rats we performed a subperiosteal osteotomy in the midshaft of the femur with distraction at 0.25 mm every 12 hours from seven days after operation. Radiological and histological examinations showed a growth zone of constant thickness in the middle of the lengthened segment, with formation of new bone at its proximal and distal ends. Osteogenic cells were arranged longitudinally along the tension vector showing the origin and the fate of individual cells in a single section. Typical endochondral bone formation was prominent in the early stage of distraction, but intramembraneous bone formation became the predominant mechanism of ossification at later stages. We also showed a third mechanism of ossification, 'transchondroid bone formation'. Chondroid bone, a tissue intermediate between bone and cartilage, was formed directly by chondrocyte-like cells, with transition from fibrous tissue to bone occurring gradually and consecutively without capillary invasion. In situ hybridisation using digoxigenin-11-UTP-labelled complementary RNAs showed that the chondroid bone cells temporarily expressed type-II collagen mRNA. They did not show the classical morphological characteristics of chondrocytes, but were assumed to be young chondrocytes undergoing further differentiation into bone-forming cells. We found at least three different modes of ossification during bone lengthening by distraction osteogenesis. We believe that this is the first report of such a rat model, and have shown the validity of in situ hybridisation techniques for the study of the cellular and molecular mechanisms involved in distraction osteogenesis.