Homeobox Genes, Retinoic Acid and the Development and Evolution of Dual Body Plans in the Ascidian Herdmania curvata

Ascidians, along with other urochordates, are the most evolutionarydistant group from vertebrates to display definitive chordate-specificcharacters, such as a notochord, dorsal hollow nerve cord, pharynxand endostyle. Most solitary ascidians have a biphasic lifehistory that has partitioned the development of these charactersbetween a planktonic microscopic tadpole larva (notochord anddorsal nerve cord) and a larger sessile adult (pharynx and endostyle).Very little is known of the molecular axial patterning processesoperating during ascidian postlarval development. Two axialpatterning homeobox genes Otx and Cdx are expressed in a spatiallyrestricted manner along the ascidian anteroposterior axis duringembryogenesis and postlarval development (i.e., metamorphosis).Comparisons of these patterns with those of homologous cephalochordateand vertebrate genes suggest that the novel ascidian biphasicbody plan was not accompanied by a deployment of these genesinto new pathways but by a heterochronic shift in tissue-specificexpression. Studies examining the role of all-trans retinoicacid (RA) in axial patterning in chordates also contribute toour understanding of the role of homeobox genes in the developmentof larval and adult ascidian body plans. Our studies demonstratethat RA does not regulate axial patterning in the developingascidian larval neuroaxis in a manner homologous to that foundin vertebrates. Although RA may regulate the expression of someascidian homeobox genes, ectopic application of RA does notappear to alter the morphology of the larval CNS. However, treatmentwith similar or lower concentrations of RA, have a profoundeffect on postlarval development and the juvenile body plan.These changes are correlated to a dramatic reduction of Otxexpression. Through these RA-induced effects we infer that whileRA may regulate the expression of some homeobox genes duringembryogenesis it has a far more dramatic impact on postlarvaldevelopment where regulative processes predominate.     

Nitrogen fixation in coniferous bark litter

Summary Non-symbiotic heterotrophic N₂ fixation in coniferous bark litter was investigated with the acetylene reduction assay under aerobic and anaerobic conditions. The litter studied was composed essentially of bark, of pH 5 and a C/N ratio of 101; the ratio of available C to available N, which governs N₂ fixation, was considerably higher. The rate of N₂ fixation was estimated as 2.5–4.4 g N. g^–1 dry wt. day^–1. Nitrogenase activity was still evident after seven months of incubation under aerobic conditions. The N₂-ase activity was O₂ dependent: under anaerobic conditions no N₂-ase activity was found unless a fermentable C source was added. The importance of N₂ fixation in N-poor litter for the maintenance of soil fertility is emphasized.     

Development and specificity of inhibitory terminal arborizations in the central nervous system

This study examined the morphological development of single inhibitory arborizations in the gerbil central auditory brain stem. Using a brain slice preparation, neurons of the medial nucleus of the trapezoid body (MNTB) were filled with horseradish peroxidase (HRP), and their complete arborizations were analyzed along the tonotopic axis of the lateral superior olive (LSO). The projections in neonatal animals displayed well-defined arbors that were ordered appropriately within the LSO. It was evident from the axonal pathways that the MNTB afferents could correct for projection errors after reaching the postsynaptic population. As development progressed, a number of arbors established diffuse or inappropriate projections within the LSO. These immature arborizations were no longer apparent by 18–25 days postnatal. The anatomical specificity of arbors at 12–13 and 18–25 days was quantified by measuring the distance that terminal boutons spread across the frequency axis. There was a significant reduction of this distance in older animals. In addition, there was a significant reduction in the mean number of boutons per arbor between 12–13 days and 18–25 days. The maximum nucleus cross-sectional area continued to increase through 15–16 days, indicating that the refined arbors occupied an even smaller fraction of the postsynaptic structure. Taken together, these observations suggest that central inhibitory arbors form exuberant contacts that must be eliminated during development.     

An in situ assay for induced diphtheria-toxin-resistant mutants of diploid human fibroblasts

The sensitivity of diploid human fibroblasts to the cytotoxic effects of diphtheria toxin (DT) depended on the cell growth status. Exponentially growing cells treated with 10^?3-1 lethal flocculating units (LF) of DT/ml for 4 days survived with a frequency of 4 × 10^?4. However, the DT-resistant phenotype of colonies isolated under these conditions was not stable. When the growth of the cells had been arrested by confluence or deprivation of serum growth factors prior to treatment with DT (4 days, 10^?3-0.6 LF/ml), the survival decreased to 2 × 10^?6 and the resistance of isolated colonies was stable. An in situ assay for induced DT-resistant mutants was developed in order to avoid problems associated with the possible reduced viability of the mutants relative to that of wild-type cells. A reproducible and linear dose response was obtained for the induction of DT-resistant mutants by ethylnitrosourea. The mutants were induced with high frequency by this compound (e.g., 10^?3 mutants/viable cell at a 37% survival dose); complete expression of the mutant phenotype occurred after 6 generations of growth under nonselective conditions. Isolated mutant colonies showed stable resistance to DT and were cross-resistant to Pseudomonas aeruginosa exotoxin A.     

Mice lacking the mitochondrial exonuclease MGME1 develop inflammatory kidney disease with glomerular dysfunction

Mitochondrial DNA (mtDNA) maintenance disorders are caused by mutations in ubiquitously expressed nuclear genes and lead to syndromes with variable disease severity and tissue-specific phenotypes. Loss of function mutations in the gene encoding the mitochondrial genome and maintenance exonuclease 1 (MGME1) result in deletions and depletion of mtDNA leading to adult-onset multisystem mitochondrial disease in humans. To better understand the in vivo function of MGME1 and the associated disease pathophysiology, we characterized a Mgme1 mouse knockout model by extensive phenotyping of ageing knockout animals. We show that loss of MGME1 leads to de novo formation of linear deleted mtDNA fragments that are constantly made and degraded. These findings contradict previous proposal that MGME1 is essential for degradation of linear mtDNA fragments and instead support a model where MGME1 has a critical role in completion of mtDNA replication. We report that Mgme1 knockout mice develop a dramatic phenotype as they age and display progressive weight loss, cataract and retinopathy. Surprisingly, aged animals also develop kidney inflammation, glomerular changes and severe chronic progressive nephropathy, consistent with nephrotic syndrome. These findings link the faulty mtDNA synthesis to severe inflammatory disease and thus show that defective mtDNA replication can trigger an immune response that causes age-associated progressive pathology in the kidney.     

Selective pericentromeric heterochromatin dismantling caused by TP53 activation during senescence

Cellular senescence triggers various types of heterochromatin remodeling that contribute to aging. However, the age-related mechanisms that lead to these epigenetic alterations remain elusive. Here, we asked how two key aging hallmarks, telomere shortening and constitutive heterochromatin loss, are mechanistically connected during senescence. We show that, at the onset of senescence, pericentromeric heterochromatin is specifically dismantled consisting of chromatin decondensation, accumulation of DNA breakages, illegitimate recombination and loss of DNA. This process is caused by telomere shortening or genotoxic stress by a sequence of events starting from TP53-dependent downregulation of the telomere protective protein TRF2. The resulting loss of TRF2 at pericentromeres triggers DNA breaks activating ATM, which in turn leads to heterochromatin decondensation by releasing KAP1 and Lamin B1, recombination and satellite DNA excision found in the cytosol associated with cGAS. This TP53–TRF2 axis activates the interferon response and the formation of chromosome rearrangements when the cells escape the senescent growth arrest. Overall, these results reveal the role of TP53 as pericentromeric disassembler and define the basic principles of how a TP53-dependent senescence inducer hierarchically leads to selective pericentromeric dismantling through the downregulation of TRF2.     

Histone deacetylase inhibition decreases preference without affecting aversion for nicotine

Mutations in the parkin gene cause early-onset, autosomal recessive Parkinson's disease. Parkin functions as an E3 ubiquitin ligase to mediate the covalent attachment of ubiquitin monomers or linked chains to protein substrates. Substrate ubiquitination can target proteins for proteasomal degradation or can mediate a number of non-degradative functions. Parkin has been shown to preserve mitochondrial integrity in a number of experimental systems through the regulation of mitochondrial fission. Upon mitochondrial damage, parkin translocates to mitochondria to mediate their selective elimination by autophagic degradation. The mechanism underlying this process remains unclear. Here, we demonstrate that parkin interacts with and selectively mediates the atypical poly-ubiquitination of the mitochondrial fusion factor, mitofusin 1, leading to its enhanced turnover by proteasomal degradation. Our data supports a model whereby the translocation of parkin to damaged mitochondria induces the degradation of mitofusins leading to impaired mitochondrial fusion. This process may serve to selectively isolate damaged mitochondria for their removal by autophagy.     

Urinary CD133+ Extracellular Vesicles Are Decreased in Kidney Transplanted Patients with Slow Graft Function and Vascular Damage

Extracellular vesicles (EVs) present in the urine are mainly released from cells of the nephron and can therefore provide information on kidney function. We here evaluated the presence of vesicles expressing the progenitor marker CD133 in the urine of normal subjects and of patients undergoing renal transplant. We found that EV expressing CD133 were present in the urine of normal subjects, but not of patients with end stage renal disease. The first day after transplant, urinary CD133⁺ EVs were present at low levels, to increase thereafter (at day 7). Urinary CD133⁺ EVs significantly increased in patients with slow graft function in respect to those with early graft function. In patients with a severe pre-transplant vascular damage of the graft, CD133⁺ EVs did not increase at day 7. At variance, the levels of EVs expressing the renal exosomal marker CD24 did not vary in the urine of patients with end stage renal disease or in transplanted patients in respect to controls. Sorted CD133⁺ EVs were found to express glomerular and proximal tubular markers. These data indicate that urinary CD133⁺ EVs are continuously released during the homeostatic turnover of the nephron and may provide information on its function or regenerative potential.     

Analysis of DNA double-strand break response and chromatin structure in mitosis using laser microirradiation

In this study the femtosecond near-IR and nanosecond green lasers are used to induce alterations in mitotic chromosomes. The subsequent double-strand break responses are studied. We show that both lasers are capable of creating comparable chromosomal alterations and that a phase paling observed within 1-2 s of laser exposure is associated with an alteration of chromatin as confirmed by serial section electron microscopy, DAPI, γH2AX and phospho-H3 staining. Additionally, the accumulation of dark material observed using phase contrast light microscopy (indicative of a change in refractive index of the chromatin) ～ 34 s post-laser exposure corresponds spatially to the accumulation of Nbs1, Ku and ubiquitin. This study demonstrates that chromosomes selectively altered in mitosis initiate the DNA damage response within 30 s and that the accumulation of proteins are visually represented by phase-dark material at the irradiation site, allowing us to determine the fate of the damage as cells enter G1. These results occur with two widely different laser systems, making this approach to study DNA damage responses in the mitotic phase generally available to many different labs. Additionally, we present a summary of most of the published laser studies on chromosomes in order to provide a general guide of the lasers and operating parameters used by other laboratories.