Precursor Pool Size Research Articles

Arabidopsis ACYL CARRIER PROTEIN4 and RHOMBOID LIKE10 act independently in chloroplast phosphatidate synthesis.

ACYL CARRIER PROTEIN4 (ACP4) is the most abundant ACP isoform in Arabidopsis (Arabidopsis thaliana) leaves and acts as a scaffold for de novo fatty acid biosynthesis and as a substrate for acyl-ACP-utilizing enzymes. Recently, ACP4 was found to interact with a protein-designated plastid RHOMBOID LIKE10 (RBL10) that affects chloroplast monogalactosyldiacylglycerol (MGDG) biosynthesis, but the cellular function of this interaction remains to be explored. Here, we generated and characterized acp4 rbl10 double mutants to explore whether ACP4 and RBL10 directly interact in influencing chloroplast lipid metabolism. Alterations in the content and molecular species of chloroplast lipids such as MGDG and phosphatidylglycerol were observed in the acp4 and rbl10 mutants, which are likely associated with the changes in the size and profiles of diacylglycerol (DAG), phosphatidic acid (PA), and acyl-ACP precursor pools. ACP4 contributed to the size and profile of the acyl-ACP pool and interacted with acyl-ACP-utilizing enzymes, as expected for its role in fatty acid biosynthesis and chloroplast lipid assembly. RBL10 appeared to be involved in the conversion of PA to DAG precursors for MGDG biosynthesis as evidenced by the increased 34:x PA and decreased 34:x DAG in the rbl10 mutant and the slow turnover of radiolabeled PA in isolated chloroplasts fed with [14C] acetate. Interestingly, the impaired PA turnover in rbl10 was partially reversed in the acp4 rbl10 double mutant. Collectively, this study shows that ACP4 and RBL10 affect chloroplast lipid biosynthesis by modulating substrate precursor pools and appear to act independently.

EyeVolve, a modular PYTHON based model for simulating developmental eye type diversification.

Vision is among the oldest and arguably most important sensory modalities for animals to interact with their external environment. Although many different eye types exist within the animal kingdom, mounting evidence indicates that the genetic networks required for visual system formation and function are relatively well conserved between species. This raises the question as to how common developmental programs are modified in functionally different eye types. Here, we approached this issue through EyeVolve, an open-source PYTHON-based model that recapitulates eye development based on developmental principles originally identified in Drosophila melanogaster. Proof-of-principle experiments showed that this program’s animated timeline successfully simulates early eye tissue expansion, neurogenesis, and pigment cell formation, sequentially transitioning from a disorganized pool of progenitor cells to a highly organized lattice of photoreceptor clusters wrapped with support cells. Further, tweaking just five parameters (precursor pool size, founder cell distance and placement from edge, photoreceptor subtype number, and cell death decisions) predicted a multitude of visual system layouts, reminiscent of the varied eye types found in larval and adult arthropods. This suggests that there are universal underlying mechanisms that can explain much of the existing arthropod eye diversity. Thus, EyeVolve sheds light on common principles of eye development and provides a new computational system for generating specific testable predictions about how development gives rise to diverse visual systems from a commonly specified neuroepithelial ground plan.

Increased Fatty Acid Synthesis and Catabolism Supports Metastatic Breast Cancer Cell Migration

Lipid accumulation is positively associated with breast cancer aggressiveness and poorer clinical outcomes. Despite this observation, mechanisms underlying the increase and role of stored lipid in breast cancer progression remain incompletely understood. The accumulation of triacylglycerol (TAG) is dependent on the balance of uptake, synthesis, and utilization of fatty acids (FAs). In the current study, we utilized non‐metastatic MCF10A‐ras and metastatic MCF10CA1a human breast cancer cells to determine differences in TAG storage and catabolism for sustaining migration—a critical step in the metastatic cascade. Our results demonstrate that MCF10CA1a cells have 90% more TAG than MCF10A‐ras (TAG Assay and Transmission Electron Microscopy), although no significant difference in 14C‐palmitate uptake. MCF10CA1a cells have greater FA synthesis as shown by greater incorporation of 13C‐acetate (60%), 13C‐glucose (65%), and 13C‐glutamine (50%) into palmitate compared to the MCF10A‐ras cells (LC‐MS/MS), as well as higher protein levels of FA synthase (FASN). Additionally, MCF10CA1a cells display greater flux of uniformly‐labeled 13C‐glucose and 13C‐glutamine to the FA synthesis precursor, citrate, and lower intracellular citrate pool size compared to MCF10A‐ras. In addition to increased FA anabolism, MCF10CA1a cells rely on FA oxidation (FAO) for cellular migration compared to MCF10A‐ras cells, as determined by inhibiting the rate‐limiting enzyme of FAO, carnitine palmitoyltransferase 1, with etomoxir (transwell and wound‐healing assays). Pretreatment with the FASN inhibitor TVB‐3166 (3 d) significantly reduced TAG levels and subsequent migration by 67% compared to vehicle‐treated cells. Similarly, pretreatment with inhibitors of the TAG‐synthesizing enzymes diacylglycerol O‐acyltransferase 1 and 2 reduced TAG accumulation and subsequent migration (75%) of MCF10CA1a cells compared to vehicle. Together, the data suggest that FA synthesis may contribute to TAG storage necessary to sustain breast cancer migration. To test this hypothesis, we inhibited the initial enzyme of the TAG lipolysis pathway, adipose triacylglycerol lipase (ATGL; inhibitor ATGListatin), and measured migration. Inhibition of ATGL alone reduced MCF10CA1a cell migration (20% decrease compared to vehicle), and addition of the FAO inhibitor (etomoxir) provided no further effect. Our study indicates that metastatic MCF10CA1a cells accumulate FAs by increasing de novo lipogenesis, store them as TAG, and that catabolism of these stores drives FAO‐dependent migration.

Metabolomics for industrial fermentation.

Metabolomics is essential to understand the metabolism and identify engineering targets to improve the performances of strains and bioprocesses. Although numerous metabolomics techniques have been developed and applied to various organisms, the metabolome of Saccharopolyspora erythraea, a native producer of erythromycin, had never been studied. The 2017 best paper of Bioprocess and Biosystems Engineering reports examination of three methods for quenching and extraction to analyze the intracellular metabolome of S. erythraea, and identified the most reliable methods for studying different groups of the metabolites. Subsequent studies on the dynamics of the intracellular metabolome of S. erythraea during the fed-batch fermentation identified a positive correlation between the specific erythromycin production rate and the pool size of intracellular propionyl-CoA and other precursors of erythromycin. A series of follow-up studies, such as demonstrating the applicability of the quenching/extraction methods in other related antibiotic producers, demonstrating the generality of the best matches between the quenching/extraction methods and the metabolite groups, and combining metabolomics approaches with the fluxomics and systems metabolic engineering approaches, will facilitate the metabolomics studies on important antibiotic producers, enable standardization of the quenching/extraction protocols, and improve the performance of the antibiotic production with deeper insight into their metabolism.

Recycling of Overactivated Acyls by a Type II Thioesterase during Calcimycin Biosynthesis in Streptomyces chartreusis NRRL 3882.

Type II thioesterases typically function as editing enzymes, removing acyl groups that have been misconjugated to acyl carrier proteins during polyketide secondary metabolite biosynthesis as a consequence of biosynthetic errors. Streptomyces chartreusis NRRL 3882 produces the pyrrole polyether ionophoric antibiotic, and we have identified the presence of a putative type II thioesterase-like sequence, calG, within the biosynthetic gene cluster involved in the antibiotic's synthesis. However, targeted gene mutagenesis experiments in which calG was inactivated in the organism did not lead to a decrease in calcimycin production but rather reduced the strain's production of its biosynthetic precursor, cezomycin. Results from in vitro activity assays of purified, recombinant CalG protein indicated that it was involved in the hydrolysis of cezomycin coenzyme A (cezomycin-CoA), as well as other acyl CoAs, but was not active toward 3-S-N-acetylcysteamine (SNAC; the mimic of the polyketide chain-releasing precursor). Further investigation of the enzyme's activity showed that it possessed a cezomycin-CoA hydrolysis Km of 0.67 mM and a kcat of 17.77 min-1 and was significantly inhibited by the presence of Mn2+ and Fe2+ divalent cations. Interestingly, when S. chartreusis NRRL 3882 was cultured in the presence of inorganic nitrite, NaNO2, it was observed that the production of calcimycin rather than cezomycin was promoted. Also, supplementation of S. chartreusis NRRL 3882 growth medium with the divalent cations Ca2+, Mg2+, Mn2+, and Fe2+ had a similar effect. Taken together, these observations suggest that CalG is not responsible for megasynthase polyketide precursor chain release during the synthesis of calcimycin or for retaining the catalytic efficiency of the megasynthase enzyme complex as is supposed to be the function for type II thioesterases. Rather, our results suggest that CalG is a dedicated thioesterase that prevents the accumulation of cezomycin-CoA when intracellular nitrogen is limited, an apparently new and previously unreported function of type II thioesterases.IMPORTANCE Type II thioesterases (TEIIs) are generally regarded as being responsible for removing aberrant acyl groups that block polyketide production, thereby maintaining the efficiency of the megasynthase involved in this class of secondary metabolites' biosynthesis. Specifically, this class of enzyme is believed to be involved in editing misprimed precursors, controlling initial units, providing key intermediates, and releasing final synthetic products in the biosynthesis of this class of secondary metabolites. Our results indicate that the putative TEII CalG present in the calcimycin (A23187)-producing organism Streptomyces chartreusis NRRL 3882 is not important either for the retention of catalytic efficiency of, or for the release of the product compound from, the megasynthase involved in calcimycin biosynthesis. Rather, the enzyme is involved in regulating/controlling the pool size of the calcimycin biosynthetic precursor, cezomycin, by hydrolysis of its CoA derivative. This novel function of CalG suggests a possible additional activity for enzymes belonging to the TEII protein family and promotes better understanding of the overall biosynthetic mechanisms involved in the production of this class of secondary metabolites.

The Significance of the Bifunctional Kinase/Phosphatase Activities of Diphosphoinositol Pentakisphosphate Kinases (PPIP5Ks) for Coupling Inositol Pyrophosphate Cell Signaling to Cellular Phosphate Homeostasis

Proteins responsible for Pi homeostasis are critical for all life. In Saccharomyces cerevisiae, extracellular [Pi] is "sensed" by the inositol-hexakisphosphate kinase (IP6K) that synthesizes the intracellular inositol pyrophosphate 5-diphosphoinositol 1,2,3,4,6-pentakisphosphate (5-InsP7) as follows: during a period of Pi starvation, there is a decline in cellular [ATP]; the unusually low affinity of IP6Ks for ATP compels 5-InsP7 levels to fall in parallel (Azevedo, C., and Saiardi, A. (2017) Trends. Biochem. Sci. 42, 219-231. Hitherto, such Pi sensing has not been documented in metazoans. Here, using a human intestinal epithelial cell line (HCT116), we show that levels of both 5-InsP7 and ATP decrease upon [Pi] starvation and subsequently recover during Pi replenishment. However, a separate inositol pyrophosphate, 1,5-bisdiphosphoinositol 2,3,4,6-tetrakisphosphate (InsP8), reacts more dramatically (i.e. with a wider dynamic range and greater sensitivity). To understand this novel InsP8 response, we characterized kinetic properties of the bifunctional 5-InsP7 kinase/InsP8 phosphatase activities of full-length diphosphoinositol pentakisphosphate kinases (PPIP5Ks). These data fulfil previously published criteria for any bifunctional kinase/phosphatase to exhibit concentration robustness, permitting levels of the kinase product (InsP8 in this case) to fluctuate independently of varying precursor (i.e. 5-InsP7) pool size. Moreover, we report that InsP8 phosphatase activities of PPIP5Ks are strongly inhibited by Pi (40-90% within the 0-1 mm range). For PPIP5K2, Pi sensing by InsP8 is amplified by a 2-fold activation of 5-InsP7 kinase activity by Pi within the 0-5 mm range. Overall, our data reveal mechanisms that can contribute to specificity in inositol pyrophosphate signaling, regulating InsP8 turnover independently of 5-InsP7, in response to fluctuations in extracellular supply of a key nutrient.

Modeling the flux of metabolites in the juvenile hormone biosynthesis pathway using generalized additive models and ordinary differential equations.

Juvenile hormone (JH) regulates development and reproductive maturation in insects. The corpora allata (CA) from female adult mosquitoes synthesize fluctuating levels of JH, which have been linked to the ovarian development and are influenced by nutritional signals. The rate of JH biosynthesis is controlled by the rate of flux of isoprenoids in the pathway, which is the outcome of a complex interplay of changes in precursor pools and enzyme levels. A comprehensive study of the changes in enzymatic activities and precursor pool sizes have been previously reported for the mosquito Aedes aegypti JH biosynthesis pathway. In the present studies, we used two different quantitative approaches to describe and predict how changes in the individual metabolic reactions in the pathway affect JH synthesis. First, we constructed generalized additive models (GAMs) that described the association between changes in specific metabolite concentrations with changes in enzymatic activities and substrate concentrations. Changes in substrate concentrations explained 50% or more of the model deviances in 7 of the 13 metabolic steps analyzed. Addition of information on enzymatic activities almost always improved the fitness of GAMs built solely based on substrate concentrations. GAMs were validated using experimental data that were not included when the model was built. In addition, a system of ordinary differential equations (ODE) was developed to describe the instantaneous changes in metabolites as a function of the levels of enzymatic catalytic activities. The results demonstrated the ability of the models to predict changes in the flux of metabolites in the JH pathway, and can be used in the future to design and validate experimental manipulations of JH synthesis.

NOD1 ligand administration restores optimal steady-state hematopoiesis in germ-free mice

Abstract The microbiota is required for optimal hematopoiesis. When compared to specific-pathogen free (SPF) mice, germ free (GF) animals display decreased numbers of hematopoietic stem cells (HSC), common myeloid progenitors (CMP) and common lymphoid progenitors (CLP) in bone marrow. Muropeptides produced by both gram-negative and gram-positive bacteria are important agonists of the innate immune response and are recognized by the intracellular pattern-recognition receptors NOD1 and NOD2. In the present study, we examined both direct and indirect effects of NOD signaling on hematopoietic homeostasis. While stimulation with NOD1 or NOD2 ligands had no effect on survival/proliferation of HSC, CMP and CLP in vitro, exposure of bone marrow-derived stroma cells (BMSC) to NOD1, but not NOD2, ligand induced expression of the differentiation factors SCF, IL-7, IL-3, Thpo and IL-6. Interestingly LPS, which is known to act directly on hematopoietic precursors, selectively induced only IL-6 in BMSC. To test the effects of NOD1 ligand in vivo, we compared groups of GF animals with and without peroral NOD1 ligand administration. NOD1 ligand treatment restored to normal levels serum concentrations of SCF, Thpo and IL-6, as well as the numbers of HSC, CMP and CLP in bone marrow. Taken together, these findings demonstrate that NOD1 ligand induces BMSC to produce cytokines that regulate the size of the major hematopoietic precursor pools. Thus, NOD1 signaling appears to be a major factor underlying the requirement for the microbiota in the maintenance of steady-state hematopoiesis. This work was supported by the Intramural Research Program of the National Institute of Allergy and Infectious diseases.

High-level production of ethylmalonyl-CoA pathway-derived dicarboxylic acids by Methylobacterium extorquens under cobalt-deficient conditions and by polyhydroxybutyrate negative strains

Bio-based production of dicarboxylic acids is an emerging research field with remarkable progress during the last decades. The recently established synthesis of the ethylmalonyl-CoA pathway (EMCP)-derived dicarboxylic acids, mesaconic acid and (2S)-methylsuccinic acid, from the alternative carbon source methanol (Sonntag et al., Appl Microbiol Biotechnol 98:4533-4544, 2014) gave a proof of concept for the sustainable production of hitherto biotechnologically inaccessible monomers. In this study, substantial optimizations of the process by different approaches are presented. Abolishment of mesaconic and (2S)-methylsuccinic acid reuptake from culture supernatant and a productivity increase were achieved by 30-fold decreased sodium ion availability in culture medium. Undesired flux from EMCP into polyhydroxybutyrate (PHB) cycle was hindered by the knockout of polyhydroxyalkanoate synthase phaC which was concomitant with 5-fold increased product concentrations. However, frequently occurring suppressors of strain ΔphaC lost their beneficial properties probably due to redirected channeling of acetyl-CoA. Pool sizes of the product precursors were increased by exploiting the presence of two cobalt-dependent mutases in the EMCP: Fine-tuned growth-limiting cobalt concentrations led to 16-fold accumulation of mesaconyl- and (2S)-methylsuccinyl-CoA which in turn resulted in 6-fold increased concentrations of mesaconic and (2S)-methylsuccinic acids, with a combined titer of 0.65 g/l, representing a yield of 0.17 g/g methanol. This work represents an important step toward an industrially relevant production of ethylmalonyl-CoA pathway-derived dicarboxylic acids and the generation of a stable PHB synthesis negative Methylobacterium extorquens strain.

CXCL12/CXCR4 signaling in the osteoblast regulates the mesenchymal stem cell and osteoclast lineage populations

The chemokine CXCL12 and its receptor CXCR4 play a key role in regulation of hematopoietic stem cells and cell migratory function during morphogenesis. Osteoblasts express both the ligand and the receptor, but little is known about the role of CXCL12-CXCR4 signaling in maintaining skeletal homeostasis. Using Cre-Lox technology to delete CXCR4 in mature osteoblasts in mice, we show here a significant decrease in bone mass and alterations in cancellous bone structure. CXCR4 gene ablation increased the number of colony-forming units (CFU), CFU-positive for alkaline phosphatase (CFU-AP(+)), and mineralizing nodules in bone marrow stromal cell (BMSC) cultures. The adipocyte precursor population decreased in BMSCs harvested from the KO animals. The nonadherent population of BMSCs harvested from the long bone diaphysis of KO animals formed more osteoclasts, a finding that was associated with increased circulatory levels of pyridinoline, a marker of bone resorption. Our data show that osteoblast-specific CXCR4 deletion has profound effects on the mesenchymal stem cell pool and allocation to the osteoblastic and adipocytic cell lineages. They also show that CXCL12/CXCR4 signaling in the mature osteoblast can feedback to regulate the osteoclast precursor pool size and play a multifunctional role in regulating bone formation and resorption.

Simulated spaceflight produces a rapid and sustained loss of osteoprogenitors and an acute but transitory rise of osteoclast precursors in two genetic strains of mice

Loss of skeletal weight bearing or skeletal unloading as occurs during spaceflight inhibits bone formation and stimulates bone resorption. These are associated with a decline in the osteoblast (Ob.S/BS) and an increase in the osteoclast (Oc.S/BS) bone surfaces. To determine the temporal relationship between changes in the bone cells and their marrow precursor pools during sustained unloading, and whether genetic background influences these relationships, we used the hindlimb unloading model to induce bone loss in two strains of mice known to respond to load and having significantly different cancellous bone volumes (C57BL/6 and DBA/2 male mice). Skeletal unloading caused a progressive decline in bone volume that was accompanied by strain-specific changes in Ob.S/BS and Oc.S/BS. These were associated with a sustained reduction in the osteoprogenitor population and a dramatic but transient increase in the osteoclast precursor pool size in both strains. The results reveal that bone adaptation to skeletal unloading involves similar rapid changes in the osteoblast and osteoclast progenitor populations in both strains of mice but striking differences in Oc.S/BS dynamics, BFR, and cancellous bone structure. These strain-specific differences suggest that genetics plays an important role in determining the osteoblast and osteoclast populations on the bone surface and the dynamics of bone loss in response to skeletal unloading.

Enhanced isoprene emission capacity and altered light responsiveness in aspen grown under elevated atmospheric CO2 concentration

AbstractControversial evidence of CO2‐responsiveness of isoprene emission has been reported in the literature with the response ranging from inhibition to enhancement, but the reasons for such differences are not understood. We studied isoprene emission characteristics of hybrid aspen (Populus tremula x P. tremuloides) grown under ambient (380 μmol mol−1) and elevated (780 μmol mol−1) [CO2] to test the hypothesis that growth [CO2] effects on isoprene emission are driven by modifications in substrate pool size, reflecting altered light use efficiency for isoprene synthesis. A novel in vivo method for estimation of the pool size of the immediate isoprene precursor, dimethylallyldiphosphate (DMADP) and the activity of isoprene synthase was used. Growth at elevated [CO2] resulted in greater leaf thickness, more advanced development of mesophyll and moderately increased photosynthetic capacity due to morphological “upregulation”, but isoprene emission rate under growth light and temperature was not significantly different among ambient‐ and elevated‐[CO2]‐grown plants independent of whether measured at 380 μmol mol−1 or 780 μmol mol−1 CO2. However, DMADP pool size was significantly less in elevated‐[CO2]‐grown plants, but this was compensated by increased isoprene synthase activity. Analysis of CO2 and light response curves of isoprene emission demonstrated that the [CO2] for maximum isoprene emission was shifted to lower [CO2] in elevated‐[CO2]‐grown plants. The light‐saturated isoprene emission rate (Imax,Q) was greater, but the quantum efficiency at given Imax,Q was less in elevated‐[CO2]‐grown plants, especially at higher CO2 measurement concentration, reflecting stronger DMADP limitation at lower light and higher [CO2]. These results collectively demonstrate important shifts in light and CO2‐responsiveness of isoprene emission in elevated‐[CO2]‐acclimated plants that need consideration in modeling isoprene emissions in future climates.

Transgenic crops: Present status and future developments

Microalgae are rapidly growing, low-input requiring, sun light-utilizing microorganisms capable of converting carbon dioxide into various natural products, a major portion of which are hydrocarbons. Their cellular compartmentalization and photosynthetic apparatus depend on robust turnover of two hydrocarbon classes, isoprenoids and acyl-lipids. This review summarizes the current understanding of algal hydrocarbon metabolism, including carbon partitioning capacities, the localization and size of precursor pools, environmental effects on flux distribution, and limiting factors towards efficient (heterologous) hydrocarbon production. Questions and challenges regarding our knowledge of algal hydrocarbon metabolism as well as guidelines for systematic engineering are presented. Recent engineering achievements indicate fundamental plasticity in the (heterologous) hydrocarbon metabolism of green algae while highlighting their potential as renewable sources of these products.

Effect of Reduced c‐Kit Signaling on Bone Marrow Adiposity

c-Kit (CD117) is required for normal differentiation of osteoblasts from bone marrow stromal cells and for normal bone formation. Osteoblasts and adipocytes originate from a common progenitor cell, and a reciprocal relationship in differentiation of the two lineages is often observed. Therefore, the effects of abnormal c-kit signaling on bone marrow adiposity and adipocyte precursor pool size were evaluated in mouse strains with loss of function mutations in kit receptor or kit ligand. Additionally, to determine whether short-duration pharmacological disruption of kit signaling influences bone marrow adiposity, we administered the kit receptor antagonist gleevec (imatinib mesilate) for 1 week to middle aged (13-month-old) male rats known to have high levels of bone marrow fat. Compared to wild-type littermates, adipocytes were absent and adipocyte precursors greatly reduced in bone marrow from kit receptor-deficient Kit(W/W-ν) mice. Administration of secreted kit ligand to membrane-associated kit ligand-deficient Kit(Sl/Sl-d) mice was ineffective in inducing bone marrow adipogenesis. These findings suggest that activation of kit receptor by the membrane-associated form of kit ligand is required for kit signaling to promote bone marrow adipogenesis in mice. Rats treated with gleevec had lower adipocyte density compared to age-matched controls, suggesting that kit signaling is required to maintain normal bone marrow adiposity. Taken together, our results indicate that c-Kit signaling plays an important but previously unsuspected role in regulating bone marrow adiposity.

Growth hormone regulates the balance between bone formation and bone marrow adiposity

Cancellous bone decreases and bone marrow fat content increases with age. Osteoblasts and adipocytes are derived from a common precursor, and growth hormone (GH), a key hormone in integration of energy metabolism, regulates the differentiation and function of both cell lineages. Since an age-related decline in GH is associated with bone loss, we investigated the relationship between GH and bone marrow adiposity in hypophysectomized (HYPOX) rats and in mice with defects in GH signaling. HYPOX dramatically reduced body weight gain, bone growth and mineralizing perimeter, serum insulin-like growth factor 1 (IGF-1) levels, and mRNA levels for IGF-1 in liver and bone. Despite reduced body mass and adipocyte precursor pool size, HYPOX resulted in a dramatic increase in bone lipid levels, as reflected by increased bone marrow adiposity and bone triglyceride and cholesterol content. GH replacement normalized bone marrow adiposity and precursor pool size, as well as mineralizing perimeter in HYPOX rats. In contrast, 17β -estradiol, IGF-1, thyroxine, and cortisone were ineffective. Parathyroid hormone (PTH) reversed the inhibitory effects of HYPOX on mineralizing perimeter but had no effect on adiposity. Finally, bone marrow adiposity was increased in mice deficient in GH and IGF-1 but not in mice deficient in serum IGF-1. Taken together, our findings indicate that the reciprocal changes in bone and fat mass in GH signaling-deficient rodents are not directly coupled with one another. Rather, GH enhances adipocyte as well as osteoblast precursor pool size. However, GH increases osteoblast differentiation while suppressing bone marrow lipid accumulation. © 2010 American Society for Bone and Mineral Research

Production of IL-7 is increased in ovariectomized mice, but not RANKL mRNA expression by osteoblasts/stromal cells in bone, and IL-7 enhances generation of osteoclast precursors in vitro

Osteoclastogenic cytokines produced by T and B lineage cells and interleukin (IL)-7-induced expansion of the pool size of osteoclast precursors have been suggested to play an important role in acceleration of osteoclastogenesis induced by estrogen deficiency. However, the contribution of increased RANKL produced by osteoblasts/stromal cells to increase osteoclastogenesis in a mouse model of estrogen-deficient osteoporosis and in vitro effects of IL-7 on osteoclast precursor generation remain controversial. Thus, we investigated the effect of ovariectomy (OVX) of mice on production of RANKL, osteoprotegerin (OPG), and IL-7 in bone and the effect of IL-7 on osteoclast precursor generation in vitro. OVX did not significantly stimulate mRNA expressions of RANKL and OPG in whole femurs. Because the epiphysis, but not the femoral shaft (diaphysis) or bone marrow, is the main site of osteoclastogenesis, it is important to specifically analyze mRNA expression by osteoblasts/stromal cells at these parts of the femur. Therefore, we isolated RNA from bone marrow cell-free epiphysis, diaphysis, and flushed-out bone marrow and examined mRNA expression. The results showed no significant changes of RANKL and OPG mRNA expression in any part of the femur. In addition, OVX did not significantly affect RANKL and OPG mRNA expression by the adherent stromal cells isolated from flushed-out bone marrow cells but did stimulate RANKL mRNA expression by B220(+) cells in the nonadherent cell fraction. On the other hand, OVX increased IL-7 mRNA expression in the femur as well as IL-7 concentrations in bone fluid. In cultures of unfractionated bone cells isolated by vigorous agitation of minced whole long bones to release the cells tightly attached to the bone surfaces, but not in cocultures of clonal osteoblasts/stromal cells and flushed-out bone marrow cells, IL-7 stimulated generations of osteoclasts as well as osteoclast precursors. These data suggest that increased RANKL production by osteoblasts/stromal cells is unlikely to play a central role in acceleration of osteoclastogenesis in estrogen deficiency of mice and that IL-7 stimulates osteoclast precursor generation, presumably through an action of IL-7 on the cells attached to bone rather than on cells contained in the bone marrow cell population.

Photosynthesis and substrate supply for isoprene biosynthesis in poplar leaves

Gray poplar leaves emit high amounts of isoprene. In this context, we investigated the degree to which photosynthesis delivers necessary precursors for chloroplast isoprene biosynthesis, and whether this energy-consuming pathway could be involved in protecting the photosynthetic electron transport system. Such protection could result from consumption of a surplus in ATP and NADPH, generated under constricted net assimilation caused by high leaf temperatures and high light intensities. During the course of the day triose phosphate (TP) and dimethylallyl diphosphate (DMADP) concentrations showed pronounced diurnal variations closely related to net assimilation and isoprene emission rates, while other variables, e.g. energy (ATP/ADP) and redox (NADPH/NADP) ratio, as well as phosphoenolpyruvate (PEP) and pyruvate strongly scattered related to changing temperature and light intensities. Intra-day positive correlations were found mainly between leaf concentrations of TP and DMADP, and sucrose, ATP/ADP ratio and net assimilation rates. Under non-saturating light (200–400 μmol photons m −2 s −1), leaf DMADP pools were positively correlated mainly with PEP, starch, and fructose 2,6-bisphosphate (F26BP). Under saturating light, correlations improved and additionally involved sucrose, TP, and the ratio of NADPH/NADP. Study of temperature response curves showed that net assimilation and isoprene emission were negatively correlated to each other. This disconnection was mostly visible by the transient change of DMADP contents with maximum levels at 25 °C. At higher temperatures, declining pools of DMADP, TP and pyruvate indicated that DMADP consumption overcompensated DMADP production resulting in highest isoprene emission rates at declining pool sizes of precursors. In parallel to the reduction of net assimilation increases of NADPH/NADP and ATP/ADP ratios also portended that the MEP pathway dissipates a surplus of ATP and NADPH which cannot be used for carbon reduction under limited CO 2 supply. Taken together we thus assume that DMADP/isoprene biosynthesis constitutes a way to avoid damage to the photosynthetic electron transport chain when CO 2 reduction limits photosynthetic electron transport.

Aging Increases Stromal/Osteoblastic Cell-Induced Osteoclastogenesis and Alters the Osteoclast Precursor Pool in the Mouse

Stromal/osteoblastic cell expression of RANKL and M-CSF regulates osteoclastogenesis. We show that aging is accompanied by increased RANKL and M-CSF expression, increased stromal/osteoblastic cell-induced osteoclastogenesis, and expansion of the osteoclast precursor pool. These changes correlate with age-related alterations in the relationship between osteoblasts and osteoclasts in cancellous bone. Bone mass is maintained through a balance between osteoblast and osteoclast activity. Osteoblasts regulate the number and activity of osteoclasts through expression of RANKL, osteoprotegerin (OPG), and macrophage-colony stimulation factor (M-CSF). To determine whether age-related changes in stromal/osteoblastic cell expression of RANKL, OPG, and M-CSF are associated with stimulation of osteoclastogenesis and whether the osteoclast precursor pool changes with age, we studied cultures of stromal/osteoblastic cells and osteoclast precursor cells from animals of different ages and examined how aging influences bone cell populations in vivo. Osteoclast precursors from male C57BL/6 mice of 6 weeks (young), 6 months (adult), and 24 months (old) of age were either co-cultured with stromal/osteoblastic cells from young, adult, or old mice or treated with M-CSF, RANKL, and/or OPG. Osteoclast precursor pool size was determined by fluorescence-activated cell sorting (FACS), and osteoclast formation was assessed by measuring the number of multinucleated TRACP(+) cells and pit formation. The levels of mRNA for RANKL, M-CSF, and OPG were determined by quantitative RT-PCR, and transcription was measured by PCR-based run-on assays. Osteoblast and osteoclast numbers in bone were measured by histomorphometry. Osteoclast formation increased dramatically when stromal/osteoblastic cells from old compared with young donors were used to induce osteoclastogenesis. Regardless of the origin of the stromal/osteoblastic cells, the number of osteoclasts formed from the nonadherent population of cells increased with increasing age. Stromal/osteoblastic cell expression of RANKL and M-CSF increased, whereas OPG decreased with aging. Exogenously administered RANKL and M-CSF increased, dose-dependently, osteoclast formation from all donors, but the response was greater in cells from old donors. Osteoclast formation in vitro positively, and the ratio of osteoblasts to osteoclasts in vivo negatively, correlated with the ratio of RANKL to OPG expression in stromal/osteoblastic cells for all ages. The effects of RANKL-induced osteoclastogenesis in vitro were blocked by OPG, suggesting a causal relationship between RANKL expression and osteoclast-inducing potential. The osteoclast precursor pool and expression of RANK and c-fms increased with age. Our results show that aging significantly increases stromal/osteoblastic cell-induced osteoclastogenesis, promotes expansion of the osteoclast precursor pool and alters the relationship between osteoblasts and osteoclasts in cancellous bone.

A role for Gsh1 in the developing striatum and olfactory bulb of Gsh2 mutant mice.

We have examined the role of the two closely related homeobox genes Gsh1 and Gsh2, in the development of the striatum and the olfactory bulb. These two genes are expressed in a partially overlapping pattern by ventricular zone progenitors of the ventral telencephalon. Gsh2 is expressed in both of the ganglionic eminences while Gsh1 is largely confined to the medial ganglionic eminence. Previous studies have shown that Gsh2(-/-) embryos suffer from an early misspecification of precursors in the lateral ganglionic eminence (LGE) leading to disruptions in striatal and olfactory bulb development. This molecular misspecification is present only in early precursor cells while at later stages the molecular identity of these cells appears to be normalized. Concomitant with this normalization, Gsh1 expression is notably expanded in the Gsh2(-/-) LGE. While no obvious defects in striatal or olfactory bulb development were detected in Gsh1(-/-) embryos, Gsh1/2 double homozygous mutants displayed more severe disruptions than were observed in the Gsh2 mutant alone. Accordingly, the molecular identity of LGE precursors in the double mutant is considerably more perturbed than in Gsh2 single mutants. These findings, therefore, demonstrate an important role for Gsh1 in the development of the striatum and olfactory bulb of Gsh2 mutant mice. In addition, our data indicate a role for Gsh genes in controlling the size of the LGE precursor pools, since decreasing copies of Gsh2 and Gsh1 alleles results in a notable decrease in precursor cell number, particularly in the subventricular zone.

Skewed X inactivation and recurrent spontaneous abortion.

Recent studies show that women experiencing recurrent spontaneous abortion exhibit nonrandom X-chromosome inactivation (XCI) more often than in controls. This suggests that genetic factors may be important in explaining the losses in this subset of women. Nonetheless there are a number of possible explanations for this finding and the underlying causes may be heterogeneous. One hypothesis commonly cited is that a mutation on the X chromosome results in both preferential inactivation of the mutated X as well as lethality of male embryos inheriting this mutated X. However, this hypothesis does not explain the increase in chromosome abnormalities observed in the karyotyped losses from women with recurrent pregnancy loss and skewed XCI. This finding leads us to suggest that the mechanism involved may be associated with a reduction in number of ovarian follicles, either due to X mutations affecting oocyte atresia or a restriction in precursor pool size during development.