Loss of CpSRP54 function leads to a truncated light-harvesting antenna size in Chlamydomonas reinhardtii

The Chlamydomonas reinhardtii truncated light-harvesting antenna 4 (tla4) DNA transposon mutant has a pale green phenotype, a lower chlorophyll (Chl) per cell and a higher Chl a/b ratio in comparison with the wild type. It required a higher light intensity for the saturation of photosynthesis and displayed a greater per chlorophyll light-saturated rate of oxygen evolution than the wild type. The Chl antenna size of the photosystems in the tla4 mutant was only about 65% of that measured in the wild type. Molecular genetic analysis revealed that a single plasmid DNA insertion disrupted two genes on chromosome 11 of the mutant. A complementation study identified the “chloroplast signal recognition particle 54” gene (CpSRP54), as the lesion causing the tla4 phenotype. Disruption of this gene resulted in partial failure to assemble and, therefore, lower levels of light-harvesting Chl-binding proteins in the C. reinhardtii thylakoids. A comparative in silico 3-D structure-modeling analysis revealed that the M-domain of the CpSRP54 of C. reinhardtii possesses a more extended finger loop structure, due to different amino acid composition, as compared to that of the Arabidopsis CpSRP54. The work demonstrated that CpSRP54 deletion in microalgae can serve to generate tla mutants with a markedly smaller photosystem Chl antenna size, improved solar energy conversion efficiency, and photosynthetic productivity in high-density cultures under bright sunlight conditions.     

Loss of murine Na+/myo-inositol cotransporter leads to brain myo-inositol depletion and central apnea

myo-Inositol (Ins) and its polyphosphoinositide derivatives that are important in membrane signaling have long been held to play a special role in brain metabolism. As polyphosphoinositides turn over rapidly and are exceptionally abundant in nervous tissue, high Ins levels in the range of 2-15 mm that have been observed in brain may be necessary to maintain the rates of phosphoinositide synthesis in diverse membrane locations within neurons. Cellular concentration gradients of this magnitude indicate a dependence on active Ins transport, especially at the time of growth and differentiation. The Na(+)/myo-inositol cotransporter (SMIT1 or SLC5A3) gene is highly expressed prenatally in the central nervous system and placenta. To gain more insight into brain Ins metabolism, while ascertaining the importance of SMIT1 as a transporter, we generated mice with a homozygous targeted deletion of this gene. Newborn SMIT1(-/-) animals have no evidence of SMIT1 mRNA, a 92% reduction in the level of brain Ins, an 84% reduction in whole body Ins, and expire shortly after birth due to hypoventilation. Gross pathologic and light microscopic examinations of each organ, as well as the placenta, of embryonic day 18.5 fetuses at near term gestation were normal. Based on [(3)H]acetate incorporation into phospholipids of lung tissue explants, immunostaining of lung tissue for surfactant protein A, B, and C, and electron microscopic examination of alveolar cells, there was no evidence of abnormal pulmonary surfactant production by type 2 pneumocytes in lung. Although no histologic lesions were detected in the nervous system, electrophysiological studies of the brainstem pre-B?tzinger respiratory control center demonstrated an abnormal rhythm discharge with periods of central apnea. The cause of death can be explained by the regulatory defect in brainstem control of ventilation. This model demonstrates the critical importance of SMIT1 in the developing nervous system. The high affinity SMIT1 transporter is responsible for the Ins concentration gradient in the murine fetal-placental unit.     

Oocyte-directed depletion of connexin43 using the Cre-LoxP system leads to subfertility in female mice

A T-DNA insertion mutant of FUSCA3 (fus3-T) in Arabidopsis thaliana exhibits several of the expected deleterious effects on seed development, but not the formation of brown seeds, a colouration which results from the accumulation of large amounts of anthocyanin. A detailed phenotypic comparison between fus3-T and a known splice point mutant (fus3-3) revealed that the seeds from both mutants do not enter dormancy and can be rescued at an immature stage. Without rescue, mature fus3-3 seeds are non-viable, whereas those of fus3-T suffer only a slight loss in their germinability. A series of comparisons between the two mutants uncovered differences with respect to conditional lethality, in histological and sub-cellular features, and in the relative amounts of various storage compounds and metabolites present, leading to a further dissection of developmental processes in seeds and a partial reinterpretation of the complex seed phenotype. FUS3 function is now known to be restricted to the acquisition of embryo-dependent seed dormancy, the determination of cotyledonary cell identity, and the synthesis and accumulation of storage compounds. Based on DNA binding studies, a model is presented which can explain the differences between the mutant alleles. The fus3-T lesion is responsible for loss of function only, while the fus3-3 mutation induces various pleiotropic effects conditioned by a truncation gene product causing severe mis-differentiation.     

Loss of the nutrient sensor TAS1R3 leads to reduced bone resorption

The taste receptor type 1 (TAS1R) family of heterotrimeric G protein-coupled receptors participates in monitoring energy and nutrient status. TAS1R member 3 (TAS1R3) is a bi-functional protein that recognizes amino acids such as L-glycine and L-glutamate or sweet molecules such as sucrose and fructose when dimerized with TAS1R member 1 (TAS1R1) or TAS1R member 2 (TAS1R2), respectively. It was recently reported that deletion of TAS1R3 expression in Tas1R3 mutant mice leads to increased cortical bone mass but the underlying cellular mechanism leading to this phenotype remains unclear. Here, we independently corroborate the increased thickness of cortical bone in femurs of 20-week-old male Tas1R3 mutant mice and confirm that Tas1R3 is expressed in the bone environment. Tas1R3 is expressed in undifferentiated bone marrow stromal cells (BMSCs) in vitro and its expression is maintained during BMP2-induced osteogenic differentiation. However, levels of the bone formation marker procollagen type I N-terminal propeptide (PINP) are unchanged in the serum of 20-week-old Tas1R3 mutant mice as compared to controls. In contrast, levels of the bone resorption marker collagen type I C-telopeptide are reduced greater than 60% in Tas1R3 mutant mice. Consistent with this, Tas1R3 and its putative signaling partner Tas1R2 are expressed in primary osteoclasts and their expression levels positively correlate with differentiation status. Collectively, these findings suggest that high bone mass in Tas1R3 mutant mice is due to uncoupled bone remodeling with reduced osteoclast function and provide rationale for future experiments examining the cell-type-dependent role for TAS1R family members in nutrient sensing in postnatal bone remodeling.     

Loss of the mitochondrial lipid cardiolipin leads to decreased glutathione synthesis

Previous studies demonstrated that loss of CL in the yeast mutant crd1Δ leads to perturbation of mitochondrial iron‑sulfur (FeS) cluster biogenesis, resulting in decreased activity of mitochondrial and cytosolic Fe-S-requiring enzymes, including aconitase and sulfite reductase. In the current study, we show that crd1Δ cells exhibit decreased levels of glutamate and cysteine and are deficient in the essential antioxidant, glutathione, a tripeptide of glutamate, cysteine, and glycine. Glutathione is the most abundant non-protein thiol essential for maintaining intracellular redox potential in almost all eukaryotes, including yeast. Consistent with glutathione deficiency, the growth defect of crd1Δ cells at elevated temperature was rescued by supplementation of glutathione or glutamate and cysteine. Sensitivity to the oxidants iron (FeSO₄) and hydrogen peroxide (H₂O₂), was rescued by supplementation of glutathione. The decreased intracellular glutathione concentration in crd1Δ was restored by supplementation of glutamate and cysteine, but not by overexpressing YAP1, an activator of expression of glutathione biosynthetic enzymes. These findings show for the first time that CL plays a critical role in regulating intracellular glutathione metabolism.     

FGF2 deficit during development leads to specific neuronal cell loss in the enteric nervous system

The largest part of the peripheral nervous system is the enteric nervous system (ENS). It consists of an intricate network of several enteric neuronal subclasses with distinct phenotypes and functions within the gut wall. The generation of these enteric phenotypes is dependent upon appropriate neurotrophic support during development. Glial cell line-derived neurotrophic factor (GDNF) and fibroblast growth factor-2 (FGF2) play an important role in the differentiation and function of the ENS. A lack of GDNF or its receptor (Ret) causes intestinal aganglionosis in mice, while fibroblast growth factor receptor signaling antagonist is identified as regulating proteins in the GDNF/Ret signaling in the developing ENS. Primary myenteric plexus cultures and wholemount preparations of wild type (WT) and FGF2-knockout mice were used to analyze distinct enteric subpopulations. Fractal dimension (D) as a measure of self-similarity is an excellent tool to analyze complex geometric shape and was applied to classify the subclasses of enteric neurons concerning their individual morphology. As a consequence of a detailed analysis of subpopulation variations, wholemount preparations were stained for the calcium binding proteins calbindin and calretinin. The fractal analysis showed a reliable consistence of subgroups with different fractal dimensions (D) in each culture investigated. Seven different neuronal subtypes could be differentiated according to a rising D. Within the same D, the neurite length revealed significant differences between wild type and FGF2-knockout cultures, while the subclass distribution was also altered. Depending on the morphological characteristics, the reduced subgroup was supposed to be a secretomotor neuronal type, which could be confirmed by calbindin and calretinin staining of the wholemount preparations. These revealed a reduction up to 40 % of calbindin-positive neurons in the FGF2-knockout mouse. We therefore consider FGF2 playing a more important role in the fine-tuning of the ENS during development as previously assumed.     

Loss of Fgfr2 leads to partial XY sex reversal

In mammals, sex is determined in the bipotential embryonic gonad by a balanced network of gene actions which when altered causes disorders of sexual development (DSD, formerly known as intersex). In the XY gonad, presumptive Sertoli cells begin to differentiate when SRY up-regulates SOX9, which in turn activates FGF9 and PGDS to maintain its own expression. This study identifies a new and essential component of FGF signaling in sex determination. Fgfr2 mutant XY mice on a mixed 129/C57BL6 genetic background had either normal testes, or developed ovotestes, with predominantly testicular tissue. However, backcrossing to C57BL6 mice resulted in a wide range of gonadal phenotypes, from hypoplastic testes to ovotestes with predominantly ovarian tissue, similar to Fgf9 knockout mice. Since typical male-specific FGF9-binding to the coelomic epithelium was abolished in Fgfr2 mutant XY gonads, these results suggest that FGFR2 acts as the receptor for FGF9. Pgds and SOX9 remained expressed within the testicular portions of Fgfr2 mutant ovotestes, suggesting that the Prostaglandin pathway acts independently of FGFR2 to maintain SOX9 expression. We could further demonstrate that double-heterozygous Fgfr2/Sox9 knockout mice developed ovotestes, demonstrating that both Fgfr2 and Sox9 can act as modifier intersex genes in the heterozygous state. In summary, we provide evidence that FGFR2 is important for male sex determination in mice, thereby rendering human FGFR2 a candidate gene for unsolved DSD cases such as 10q26 deletions.     

Loss of ATRX leads to chromosome cohesion and congression defects

Alpha thalassemia/mental retardation X linked (ATRX) is a switch/sucrose nonfermenting-type ATPase localized at pericentromeric heterochromatin in mouse and human cells. Human ATRX mutations give rise to mental retardation syndromes characterized by developmental delay, facial dysmorphisms, cognitive deficits, and microcephaly and the loss of ATRX in the mouse brain leads to reduced cortical size. We find that ATRX is required for normal mitotic progression in human cultured cells and in neuroprogenitors. Using live cell imaging, we show that the transition from prometaphase to metaphase is prolonged in ATRX-depleted cells and is accompanied by defective sister chromatid cohesion and congression at the metaphase plate. We also demonstrate that loss of ATRX in the embryonic mouse brain induces mitotic defects in neuroprogenitors in vivo with evidence of abnormal chromosome congression and segregation. These findings reveal that ATRX contributes to chromosome dynamics during mitosis and provide a possible cellular explanation for reduced cortical size and abnormal brain development associated with ATRX deficiency.     

Loss of BRCC3 deubiquitinating enzyme leads to abnormal angiogenesis and is associated with syndromic moyamoya

Moyamoya is a cerebrovascular angiopathy characterized by a progressive stenosis of the terminal part of the intracranial carotid arteries and the compensatory development of abnormal and fragile collateral vessels, also called moyamoya vessels, leading to ischemic and hemorrhagic stroke. Moyamoya angiopathy can either be the sole manifestation of the disease (moyamoya disease) or be associated with various conditions, including neurofibromatosis, Down syndrome, TAAD (autosomal-dominant thoracic aortic aneurysm), and radiotherapy of head tumors (moyamoya syndromes). Its prevalence is ten times higher in Japan than in Europe, and an estimated 6%-12% of moyamoya disease is familial in Japan. The pathophysiological mechanisms of this condition remain obscure. Here, we report on three unrelated families affected with an X-linked moyamoya syndrome characterized by the association of a moyamoya angiopathy, short stature, and a stereotyped facial dysmorphism. Other symptoms include an hypergonadotropic hypogonadism, hypertension, dilated cardiomyopathy, premature coronary heart disease, premature hair graying, and early bilateral acquired cataract. We show that this syndromic moyamoya is caused by Xq28 deletions removing MTCP1/MTCP1NB and BRCC3. We also show that brcc3 morphant zebrafish display angiogenesis defects that are rescued by endothelium-specific expression of brcc3. Altogether, these data strongly suggest that BRCC3, a deubiquitinating enzyme that is part of the cellular BRCA1 and BRISC complexes, is an important player in angiogenesis and that BRCC3 loss-of-function mutations are associated with moyamoya angiopathy.     

Methylglyoxal accumulation by glutathione depletion leads to cell cycle arrest in Dictyostelium

Reduced glutathione (GSH) serves as a primary redox buffer and its depletion causes growth inhibition or apoptosis in many organisms. In Dictyostelium discoideum, the null mutant (gcsA(-)) of gcsA encoding gamma-glutamylcysteine synthetase shows growth arrest and developmental defect when GSH is depleted. To investigate the mechanism by which GSH depletion induces growth arrest, a proteomic analysis was performed and aldose reductase (AlrA) was identified as the most prominently induced protein in gcsA(-) cells. Induction of AlrA was dependent on GSH concentration and was repressed by GSH but not effectively by either the reducing agent such as dithiothreitol or overexpression of superoxide dismutase. Methylglyoxal (MG), a toxic alpha-ketoaldehyde, strongly induced alrA expression and AlrA catalysed MG reduction efficiently. The alrA knockdown gcsA(-) cells (gcsA(-)/alrA(as)) exhibited more decreased growth rate than gcsA(-) cells, whereas the gcsA(-) cells overexpressing alrA (gcsA(-)/alrA(oe)) showed the recovery of growth rate. Interestingly, intracellular MG levels were significantly augmented in gcsA(-)/alrA(as) cells compared with gcsA(-) cells following GSH depletion. By contrast, gcsA(-)/alrA(oe) cells showed repression of MG induction. Furthermore, MG treatment inhibited growth of wild-type KAx3 cells, inducing G1 phase arrest. Thus, our findings suggest that MG accumulated by GSH depletion inhibits cell growth in Dictyostelium.     

Loss of ATM kinase activity leads to embryonic lethality in mice

Ataxia telangiectasia (A-T) mutated (ATM) is a key deoxyribonucleic acid (DNA) damage signaling kinase that regulates DNA repair, cell cycle checkpoints, and apoptosis. The majority of patients with A-T, a cancer-prone neurodegenerative disease, present with null mutations in Atm. To determine whether the functions of ATM are mediated solely by its kinase activity, we generated two mouse models containing single, catalytically inactivating point mutations in Atm. In this paper, we show that, in contrast to Atm-null mice, both D2899A and Q2740P mutations cause early embryonic lethality in mice, without displaying dominant-negative interfering activity. Using conditional deletion, we find that the D2899A mutation in adult mice behaves largely similar to Atm-null cells but shows greater deficiency in homologous recombination (HR) as measured by hypersensitivity to poly (adenosine diphosphate-ribose) polymerase inhibition and increased genomic instability. These results may explain why missense mutations with no detectable kinase activity are rarely found in patients with classical A-T. We propose that ATM kinase-inactive missense mutations, unless otherwise compensated for, interfere with HR during embryogenesis.     

Glycolipid receptor depletion as an approach to specific antimicrobial therapy

Mucosal pathogens recognize glycoconjugate receptors at the site of infection, and attachment is an essential first step in disease pathogenesis. Inhibition of attachment may prevent disease, and several approaches have been explored. This review discusses the prevention of bacterial attachment and disease by agents that modify the glycosylation of cell surface glycoconjugates. Glycosylation inhibitors were tested in the urinary tract infection model, where P-fimbriated Escherichia coli rely on glycosphingolipid receptors for attachment and tissue attack. N-butyldeoxynojirimycin blocked the expression of glucosylceramide-derived glycosphingolipids and attachment was reduced. Bacterial persistence in the kidneys was impaired and the inflammatory response was abrogated. N-butyldeoxynojirimycin was inactive against strains which failed to engage these receptors, including type 1 fimbriated or nonadhesive strains. In vivo attachment has been successfully prevented by soluble receptor analogues, but there is little clinical experience of such inhibitors. Large-scale synthesis of complex carbohydrates, which could be used as attachment inhibitors, remains a technical challenge. Antibodies to bacterial lectins involved in attachment may be efficient inhibitors, and fimbrial vaccines have been developed. Glycosylation inhibitors have been shown to be safe and efficient in patients with lipid storage disease and might therefore be tested in urinary tract infection. This approach differs from current therapies, including antibiotics, in that it targets the pathogens which recognize these receptors.     

Loss of the maternal imprint in Dnmt3Lmat-/- mice leads to a differentiation defect in the extraembryonic tissue

DNA methylation of the genome is essential for mammalian development and plays crucial roles in a variety of biological processes including genomic imprinting. Although the DNA methyltransferase 3-like (Dnmt3L) protein lacks DNA methylase activity, it is thought to establish the maternal imprint in combination with the functional DNA methyltransferases. Oogenesis apparently proceeds normally in female mice homozygous for a targeted deletion of Dnmt3L, but their heterozygous offspring (Dnmt3L(mat-/-)) die before midgestation due to an imprinting defect. In this study, we show that Dnmt3L is required for the establishment of maternal methylation imprints both in the embryos and the placentae and that the placentae of these embryos develop abnormally. There is a defect in the formation of the labyrinth, reduced formation of the spongiotrophoblast layer, excess trophoblast giant cells and insufficient attachment between the chorion layer and the ectoplacental cone. In addition, we demonstrate arrest of proliferation of the extraembryonic tissue without apoptosis in vivo and a disturbance of the cell fate of Dnmt3L(mat-/-) trophoblastic stem cells in vitro. Furthermore, we report that DNA methylation during oogenesis is essential for the establishment of imprinting Mash2. These findings provide evidence that not only is DNA methylation required for the appropriate maternal imprint in the placenta but that the appropriate imprint is absolutely required for vertebrate placentation.     

Genetic depletion of histamine from the nervous system of Drosophila eliminates specific visual and mechanosensory behavior

The role of histamine as a fast neuro-transmitter of imaginai insect photoreceptors is firmly established. In adult Drosophila, histamine is also found in mechanosensory receptors of cuticular hair sensilla and in a small number of nonreceptor neurons in head and body ganglia. Here we investigate the function of histamine by immunohistochemical and behavioral analysis of mutants deficient in the hdc gene that codes for histidine decarboxylase. The allele hdc ^JK910 appears to be a null mutation, as histamine immunoreactivity is almost entirely eliminated. Homozygous flies are blind in various behavioral paradigms. Mutant larvae, on the other hand, show normal photokinetic responses. Thus, adult Drosophila photoreceptors most likely utilize only a single substance, histamine, as a neurotransmitter, whereas larval photoreceptors apparently employ a different transmitter. With the alleles hdc ^p211 , hdc ^p217 , and hdc ^p218 , variable amounts of histamine are found in photoreceptors and mechanoreceptors, but no histamine could be detected in any of the nonreceptor neurons. These mutants show various degrees of visual and mechanosensory impairment, as determined by quantitative behavioral assays. We conclude that histamine is required for normal function of cuticular hair sensilla and for efficient grooming of the body surface. Thus, in Drosophila, histamine represents a major functional neurotransmitter for mechanosensory receptors.Abbreviations ERG electroretinogram - WT wild type