Glial-cell-line-derived neurotrophic factor (GDNF) is a potent survival factor for dopaminergic neurons and motor neurons in culture. It also protects these neurons from degeneration in vitro, and improves symptoms like Parkinson's disease induced pharmacologically in rodents and monkeys. Thus GDNF might have beneficial effects in the treatment of Parkinson's disease and amyotrophic lateral sclerosis. To examine the physiological role of GDNF in the development of the mammalian nervous system, we have generated mice defective in GDNF expression by using homologous recombination in embryonic stem cells to delete each of its two coding exons. GDNF-null mice, regardless of their targeted mutation, display complete renal agencies owing to lack of induction of the ureteric bud, an early step in kidney development. These mice also have no enteric neurons, which probably explains the observed pyloric stenosis and dilation of their duodenum. However, ablation of the GDNF gene does not affect the differentiation and survival of dopaminergic neurons, at least during embryonic development.

MicroRNAs (miRNAs) are small, endogenously expressed RNAs that regulate mRNAs post-transcriptionally. The class of miRNA genes, like other gene classes, should experience birth, death and persistence of its members. We carried out deep sequencing of miRNAs from three species of Drosophila, and obtained 107,000 sequences that map to no fewer than 300 loci that were not previously known. We observe a large class of miRNA genes that are evolutionarily young, with a rate of birth of 12 new genes per million years (Myr). Most of these new miRNAs originated from non-miRNA sequences. Among the new genes, we estimate that 96% disappeared quickly in the course of evolution; only 4% of new miRNA genes were retained by natural selection. Furthermore, only 60% of these retained genes became integrated into the transcriptome in the long run (60 Myr). This small fraction (2.5%) of surviving miRNAs may later on become moderately or highly expressed. Our results suggest that there is a high birth rate of new miRNA genes, accompanied by a comparably high death rate. The estimated net gain of long-lived miRNA genes, which is not strongly affected by either the depth or the breadth (number of tissues) of sequencing, is 0.3 genes per Myr in Drosophila.

Mice heterozygous for targeted disruption of Pthrp exhibit, by 3 months of age, diminished bone volume and skeletal microarchitectural changes indicative of advanced osteoporosis. Impaired bone formation arising from decreased BM precursor cell recruitment and increased apoptotic death of osteoblastic cells was identified as the underlying mechanism for low bone mass. The osteoporotic phenotype was recapitulated in mice with osteoblast-specific targeted disruption of Pthrp, generated using Cre-LoxP technology, and defective bone formation was reaffirmed as the underlying etiology. Daily administration of the 1-34 amino-terminal fragment of parathyroid hormone (PTH 1-34) to Pthrp +/-mice resulted in profound improvement in all parameters of skeletal microarchitecture, surpassing the improvement observed in treated WT littermates. These findings establish a pivotal role for osteoblast-derived PTH-related protein (PTHrP) as a potent endogenous bone anabolic factor that potentiates bone formation by altering osteoblast recruitment and survival and whose level of expression in the bone microenvironment influences the therapeutic efficacy of exogenous PTH 1-34.

IntroductionEndogenous parathyroid hormone (PTH) functions to maintain normal extracellular calcium levels in the adult in part by enhancing osteoclastic bone resorption and liberating calcium from the adult skeleton. In contrast, exogenous PTH has been shown to exert significant skeletal anabolic effects in the adult when administered intermittently as a pharmacologic agent (1)(2)(3)(4). No physiologic role for PTH on increasing bone formation has yet been demonstrated, and its role in fetal skeletal development is unknown. In contrast, PTHrelated peptide (PTHrP) is known to play a critical and nonredundant role in fetal endochondral bone formation. Endochondral bone formation, in which bone is generated within a cartilage primordium, is a critical component of vertebrate skeletal development (5). Cells committed to the chondrogenic lineage progress through stages of proliferation, differentiation, hypertrophy, and apoptosis. Vascular invasion and degradation of calcified cartilage matrix then occurs, followed by secretion of trabecular bone matrix by invading osteoblasts. This complex process requires the coordinated activity of growth factors, hormones, proteases, and matrix molecules. Targeted disruption of the PTHrP gene results in lethal dyschondroplasia, caused mainly by a reduction of chondrocyte proliferation in the epiphyseal growth plate (6) and accelerated maturation of chondrocytes to hypertrophy (7). Both PTH and PTHrP interact at a common G protein-linked receptor termed the type I PTH/PTHrP receptor (PTHR). Ablation of the PTHR has been reported to simulate the effect of PTHrP ablation on chondrocyte differentiation (although more slowly) and to delay cartilage matrix mineral deposition, decrease vascular invasion of cartilage, and reduce trabecular bone formation in the primary spongiosa, alterations not seen after PTHrP ablation (8). These alterations were apparently partially alleviated when both PTHrP and the receptor were deficient. The mechanism for these effects, however, remained unclear.To assess the role of PTH in modulating skeletal development in the fetus and any potential interaction of PTH and PTHrP, we analyzed tissues of newborn mice homozygous for targeted ablation of the genes encoding PTH (PTH -/-), PTHrP (PTHrP -/-), and both PTH and PTHrP (PTH -/-, PTHrP -/-) and compared these to each other and to wild-type mice. MethodsMouse models. Mice carrying a disrupted PTH gene (PTH -/-mice) or a disrupted PTHrP gene (PTHrP -/-mice) were derived by homologous recombination in embryonic stem cells (9, 10). To generate PTH -/-mice, the murine PTH gene (GenBank accession numbers AF066074 and AF066075), which has an organizational structure and exon-intron boundaries identical to the human, bovine, and rat PTH genes (11), was introduced into the pPNT vector (12), replacing the entire coding Parathyroid hormone (PTH) is a potent pharmacologic inducer of new bone formation, but no physiologic anabolic effect of PTH on adult bone has been described. We investigated the role of PTH in fetal ...

IntroductionEndogenous parathyroid hormone (PTH) functions to maintain normal extracellular calcium levels in the adult in part by enhancing osteoclastic bone resorption and liberating calcium from the adult skeleton. In contrast, exogenous PTH has been shown to exert significant skeletal anabolic effects in the adult when administered intermittently as a pharmacologic agent (1)(2)(3)(4). No physiologic role for PTH on increasing bone formation has yet been demonstrated, and its role in fetal skeletal development is unknown. In contrast, PTHrelated peptide (PTHrP) is known to play a critical and nonredundant role in fetal endochondral bone formation. Endochondral bone formation, in which bone is generated within a cartilage primordium, is a critical component of vertebrate skeletal development (5). Cells committed to the chondrogenic lineage progress through stages of proliferation, differentiation, hypertrophy, and apoptosis. Vascular invasion and degradation of calcified cartilage matrix then occurs, followed by secretion of trabecular bone matrix by invading osteoblasts. This complex process requires the coordinated activity of growth factors, hormones, proteases, and matrix molecules. Targeted disruption of the PTHrP gene results in lethal dyschondroplasia, caused mainly by a reduction of chondrocyte proliferation in the epiphyseal growth plate (6) and accelerated maturation of chondrocytes to hypertrophy (7). Both PTH and PTHrP interact at a common G protein-linked receptor termed the type I PTH/PTHrP receptor (PTHR). Ablation of the PTHR has been reported to simulate the effect of PTHrP ablation on chondrocyte differentiation (although more slowly) and to delay cartilage matrix mineral deposition, decrease vascular invasion of cartilage, and reduce trabecular bone formation in the primary spongiosa, alterations not seen after PTHrP ablation (8). These alterations were apparently partially alleviated when both PTHrP and the receptor were deficient. The mechanism for these effects, however, remained unclear.To assess the role of PTH in modulating skeletal development in the fetus and any potential interaction of PTH and PTHrP, we analyzed tissues of newborn mice homozygous for targeted ablation of the genes encoding PTH (PTH -/-), PTHrP (PTHrP -/-), and both PTH and PTHrP (PTH -/-, PTHrP -/-) and compared these to each other and to wild-type mice. MethodsMouse models. Mice carrying a disrupted PTH gene (PTH -/-mice) or a disrupted PTHrP gene (PTHrP -/-mice) were derived by homologous recombination in embryonic stem cells (9, 10). To generate PTH -/-mice, the murine PTH gene (GenBank accession numbers AF066074 and AF066075), which has an organizational structure and exon-intron boundaries identical to the human, bovine, and rat PTH genes (11), was introduced into the pPNT vector (12), replacing the entire coding Parathyroid hormone (PTH) is a potent pharmacologic inducer of new bone formation, but no physiologic anabolic effect of PTH on adult bone has been described. We investigated the role of PTH in fetal ...

Parathyroid hormone (PTH) plays a central role in the regulation of serum calcium and phosphorus homeostasis, while parathyroid hormone-related protein (PTHrP) has important developmental roles. Both peptides signal through the same G protein-coupled receptor, the PTH/PTHrP or PTH type 1 receptor (PTH1R). PTHrP, normally a secreted protein, also contains a nuclear localization signal (NLS) that in vitro imparts functionality to the protein at the level of the nucleus. We investigated this functionality in vivo by introducing a premature termination codon in Pthrp in ES cells and generating mice that express PTHrP (1-84), a truncated form of the protein that is missing the NLS and the C-terminal region of the protein but can still signal through its cell surface receptor. Mice homozygous for the knock-in mutation (Pthrp KI) displayed retarded growth, early senescence, and malnutrition leading postnatally to their rapid demise. Decreased cellular proliferative capacity and increased apoptosis in multiple tissues including bone and bone marrow cells were associated with altered expression and subcellular distribution of the senescenceassociated tumor suppressor proteins p16 INK4a and p21 and the oncogenes Cyclin D, pRb, and Bmi-1. These findings provide in vivo experimental proof that substantiates the biologic relevance of the NLS and C-terminal portion of PTHrP, a polypeptide ligand that signals mainly via a cell surface G protein-coupled receptor.ageing ͉ nucleus ͉ osteoporosis ͉ PTHrP ͉ senescence

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Ergosterol, an important component of the fungal cell membrane, is not only essential for fungal growth and development but also very important for adaptation to stress in fungi. Ergosterol is also a direct precursor for steroid drugs. The biosynthesis of ergosterol can be divided into three modules: mevalonate, farnesyl pyrophosphate (farnesyl-PP) and ergosterol biosynthesis. The regulation of ergosterol content is mainly achieved by feedback regulation of ergosterol synthase activity through transcription, translation and posttranslational modification. The synthesis of HMG-CoA, catalyzed by HMGR, is a major metabolic check point in ergosterol biosynthesis. Excessive sterols can be subsequently stored in lipid droplets or secreted into the extracellular milieu by esterification or acetylation to avoid toxic effects. As sterols are insoluble, the intracellular transport of ergosterol in cells requires transporters. In recent years, great progress has been made in understanding ergosterol biosynthesis and its regulation in . However, few reviews have focused on these studies, especially the regulation of biosynthesis and intracellular transport. Therefore, this review summarizes recent research progress on the physiological functions, biosynthesis, regulation of biosynthesis and intracellular transportation of ergosterol in. .