Regeneration in the auditory system of nymphal and adult bush crickets Tettigonia viridissima

Regeneration and reestablishment of synaptic connections is an important topic in neurobiological research. In the present study, the regeneration of auditory afferents and the accompanying effects in the central nervous system are investigated in nymphs and adults of the bush cricket Tettigonia viridissima L. (Orthoptera: Tettigoniidae). In all animals in which the tympanal nerve is crushed, neuronal tracing shows a regrowth of the afferents into the prothoracic ganglion. This regeneration is seen in both adult and nymphal stages and starts 10–15 days after nerve crushing. Physiological recordings from the leg nerve indicate a recovery of tympanal fibres and a formation of functional connections to interneurones in the same time range. Electrophysiological recordings from the neck connective suggest additional contralateral sprouting of interneurones and the formation of aberrant connections. The regeneration processes of the tympanal nerve in nymphal stages and adults appear to be similar.     

Human NUDT22 Is a UDP-Glucose/Galactose Hydrolase Exhibiting a Unique Structural Fold

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DNA compaction by the bacteriophage protein Cox studied on the single DNA molecule level using nanofluidic channels

The Cox protein from bacteriophage P2 forms oligomeric filaments and it has been proposed that DNA can be wound up around these filaments, similar to how histones condense DNA. We here use fluorescence microscopy to study single DNA–Cox complexes in nanofluidic channels and compare how the Cox homologs from phages P2 and WΦ affect DNA. By measuring the extension of nanoconfined DNA in absence and presence of Cox we show that the protein compacts DNA and that the binding is highly cooperative, in agreement with the model of a Cox filament around which DNA is wrapped. Furthermore, comparing microscopy images for the wild-type P2 Cox protein and two mutants allows us to discriminate between compaction due to filament formation and compaction by monomeric Cox. P2 and WΦ Cox have similar effects on the physical properties of DNA and the subtle, but significant, differences in DNA binding are due to differences in binding affinity rather than binding mode. The presented work highlights the use of single DNA molecule studies to confirm structural predictions from X-ray crystallography. It also shows how a small protein by oligomerization can have great impact on the organization of DNA and thereby fulfill multiple regulatory functions.     

Neonatal calves develop airflow limitation due to chronic hypobaric hypoxia

Neonates and infants presenting with pulmonary hypertension and chronic hypoxia often exhibit airway obstruction. To investigate this association, we utilized a system in which neonatal calves are exposed to chronic hypobaric hypoxia and develop severe pulmonary hypertension. For the present study, one of each pair of six age-matched pairs of neonatal calves was continuously exposed to hypobaric hypoxia at 4,500 m (CH); the other remained at 1,500 m. At 2 wk of age, mean pulmonary arterial pressure (MPAP), dynamic lung compliance (Cdyn), resistance (RL), and static respiratory system compliance (Crs) were measured at 4,500 m in both CH and control calves exposed acutely to hypoxia (C). These measurements were repeated after cumulative administrations of nebulized methacholine (MCh). Tissues were removed for histological examination and assessment of bronchial ring contractility to MCh and KCl. After 2 wk of hypobaric hypoxia, MPAP (C 35 +/- 1.7 vs. CH 120 +/- 7 mmHg, P less than 0.001) and RL (C 2.64 +/- 0.16 vs CH 4.99 +/- 0.47 cmH2O.l-1s, P less than 0.001) increased. Cdyn (C 0.100 +/- 0.01 vs. CH 0.082 +/- 0.007 l/cmH2O) and Crs (CH 0.46 +/- 0.003 vs. C 0.59 +/- 0.009 l/cmH2O) were not significantly different. Compared with airways of C calves, airways of CH animals did not exhibit in vivo or in vitro MCh hyperresponsiveness; however, in vitro contractility to KCl of airways from CH animals was significantly increased. Histologically, airways from the CH calves showed increases in airway fibrous tissue and smooth muscle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Severe pulmonary hypertension and arterial adventitial changes in newborn calves at 4,300 m

Some human newborns have a syndrome characterized by irreversible pulmonary hypertension and severe hypoxemia and by medial hypertrophy and adventitial thickening of pulmonary arteries. We considered that newborn calves made severely hypoxic might reproduce features of the human disease. When 2-day-old calves were placed at 4,300 m simulated altitude, pulmonary arterial pressure was increased and could be reversed by 100% O2. However, after 2 wk at 4,300 m, pulmonary arterial pressures were suprasystemic and there was right-to-left shunting probably through the foramen ovale and a patent but restrictive ductus arteriosus. Suprasystemic pulmonary pressure and hypoxemia persisted with 100% O2 breathing. Morphometrical examination of the lung arteries showed a markedly thickened adventitia with cellular proliferation and collagen and elastin deposition. There was increased medial thickness and distal muscularization of the pulmonary arteries associated with decreased luminal diameter. The rapid development of severe pulmonary hypertension and poor responsiveness to O2 was associated with increased arterial wall thickness, particularly involving the adventitia. Thus the pulmonary arterial circulation in these calves, which were placed at high altitude for 2 wk, exhibited features resembling persistent pulmonary hypertension in newborn infants.     

Insulin-like growth factor I stimulates elastin synthesis by bovine pulmonary arterial smooth muscle cells

Insulin-like growth factor I stimulates mitogenesis in smooth muscle cells, and upregulates elastin synthesis in embryonic aortic tissue. Increased smooth muscle elastin synthesis may play an important role in vascular remodeling in chronic pulmonary hypertension. Therefore, we studied the effect of IGF-I on elastin and total protein synthesis by pulmonary arterial smooth muscle cells in vitro. Tropoelastin synthesis was measured by enzyme immunoassay, and total protein synthesis was measured by [3H]-leucine incorporation. In addition, the steady-state levels of tropoelastin mRNA were determined by slot blot hybridization. Incubation of confluent cultures with various concentrations of IGF-I resulted in a dose-dependent stimulation of elastin synthesis, with a 2.4-fold increase over control levels at 1000 ng/ml of IGF. The increase in elastin synthesis was reflected by a stimulation of the steady-state levels of tropoelastin mRNA. We conclude that IGF-I has potent elastogenic effects on vascular smooth muscle cells, and speculate that it may contribute to vascular wall remodeling in chronic hypertension.     

Deubiquitinase inhibition by WP1130 leads to ULK1 aggregation and blockade of autophagy

Autophagy represents an intracellular degradation process which is involved in both regular cell homeostasis and disease settings. In recent years, the molecular machinery governing this process has been elucidated. The ULK1 kinase complex consisting of the serine/threonine protein kinase ULK1 and the adapter proteins ATG13, RB1CC1, and ATG101, is centrally involved in the regulation of autophagy initiation. This complex is in turn regulated by the activity of different nutrient- or energy-sensing kinases, including MTOR, AMPK, and AKT. However, next to phosphorylation processes it has been suggested that ubiquitination of ULK1 positively influences ULK1 function. Here we report that the inhibition of deubiquitinases by the compound WP1130 leads to increased ULK1 ubiquitination, the transfer of ULK1 to aggresomes, and the inhibition of ULK1 activity. Additionally, WP1130 can block the autophagic flux. Thus, treatment with WP1130 might represent an efficient tool to inhibit the autophagy-initiating ULK1 complex and autophagy.     

Osteopontin is an endogenous modulator of the constitutively activated phenotype of pulmonary adventitial fibroblasts in hypoxic pulmonary hypertension

Increased cell proliferation and migration, of several cell types are key components of vascular remodeling observed in pulmonary hypertension (PH). Our previous data demonstrate that adventitial fibroblasts isolated from pulmonary arteries of chronically hypoxic hypertensive calves (termed PH-Fibs) exhibit a "constitutively activated" phenotype characterized by high proliferative and migratory potential. Osteopontin (OPN) has been shown to promote several cellular activities including growth and migration in cancer cells. We thus tested the hypothesis that elevated OPN expression confers the "activated" highly proproliferative and promigratory/invasive phenotype of PH-Fibs. Our results demonstrate that, both in vivo and ex vivo, PH-Fibs exhibited increased expression of OPN, as well as its cognate receptors, α(V)β(3) and CD44, compared with control fibroblasts (CO-Fibs). Augmented OPN expression in PH-Fibs corresponded to their high proliferative, migratory, and invasive properties and constitutive activation of ERK1/2 and AKT signaling. OPN silencing via small interfering RNA or sequestering OPN production by specific antibodies led to decreased proliferation, migration, invasion, and attenuated ERK1/2, AKT phosphorylation in PH-Fibs. Furthermore, increasing OPN levels in CO-Fibs via recombinant OPN resulted in significant increases in their proliferative, migratory, and invasive capabilities to the levels resembling those of PH-Fibs. Thus our data suggest OPN as an essential contributor to the activated (highly proliferative, migratory, and proinvasive) phenotype of pulmonary adventitial fibroblasts in hypoxic PH.     

Endocytic pathways regulate Toll-like receptor 4 signaling and link innate and adaptive immunity

Immune responses are initiated when molecules of microbial origin are sensed by the Toll-like receptors (TLRs). We now report the identification of essential molecular components for the trafficking of the lipopolysaccharide (LPS) receptor complex. LPS was endocytosed by a receptor-mediated mechanism dependent on dynamin and clathrin and colocalized with TLR4 on early/sorting endosomes. TLR4 was ubiquitinated and associated with the ubiquitin-binding endosomal sorting protein hepatocyte growth factor-regulated tyrosine kinase substrate, Hrs. Inhibition of endocytosis and endosomal sorting increased LPS signaling. Finally, the LPS receptor complex was sorted to late endosomes/lysosomes for degradation and loading of associated antigens onto HLA class II molecules for presentation to CD4+ T cells. Our results show that endosomal trafficking of the LPS receptor complex is essential for signal termination and LPS-associated antigen presentation, thus controlling both innate and adaptive immunity through TLR4.