FLOWERING REPRESSOR AAA+ ATPase 1 is a novel regulator of perennial flowering in Arabis alpina.

Arabis alpina is a polycarpic perennial, in which PERPETUAL FLOWERING1 (PEP1) regulates flowering and perennial traits in a vernalization-dependent manner. Mutagenesis screens of the pep1 mutant established the role of other flowering time regulators in PEP1-parallel pathways. Here we characterized three allelic enhancers of pep1 (eop002, 085 and 091) which flower early. We mapped the causal mutations and complemented mutants with the identified gene. Using quantitative reverse transcriptase PCR and reporter lines, we determined the protein spatiotemporal expression patterns and localization within the cell. We also characterized its role in Arabidopsis thaliana using CRISPR and in A. alpina by introgressing mutant alleles into a wild-type background. These mutants carried lesions in an AAA+ ATPase of unknown function, FLOWERING REPRESSOR AAA+ ATPase 1 (AaFRAT1). AaFRAT1 was detected in the vasculature of young leaf primordia and the rib zone of flowering shoot apical meristems. At the subcellular level, AaFRAT1 was localized at the interphase between the endoplasmic reticulum and peroxisomes. Introgression lines carrying Aafrat1 alleles required less vernalization to flower and reduced number of vegetative axillary branches. By contrast, A. thaliana CRISPR lines showed weak flowering phenotypes. AaFRAT1 contributes to flowering time regulation and the perennial growth habit of A. alpina.

Oxygen-Glucose Deprivation (OGD) Modulates the Unfolded Protein Response (UPR) and Inflicts Autophagy in a PC12 Hypoxia Cell Line Model.

Hypoxia is the lack of sufficient oxygenation of tissue, imposing severe stress upon cells. It is a major feature of many pathological conditions such as stroke, traumatic brain injury, cerebral hemorrhage, perinatal asphyxia and can lead to cell death due to energy depletion and increased free radical generation. The present study investigates the effect of hypoxia on the unfolded protein response of the cell (UPR), utilizing a 16-h oxygen-glucose deprivation protocol (OGD) in a PC12 cell line model. Expression of glucose-regulated protein 78 (GRP78) and glucose-regulated protein 94 (GRP94), key players of the UPR, was studied along with the expression of glucose-regulated protein 75 (GRP75), heat shock cognate 70 (HSC70), and glyceraldehyde 3-phosphate dehydrogenase, all with respect to the cell death mechanism(s). Cells subjected to OGD displayed upregulation of GRP78 and GRP94 and concurrent downregulation of GRP75. These findings were accompanied with minimal apoptotic cell death and induction of autophagy. The above observation warrants further investigation to elucidate whether autophagy acts as a pro-survival mechanism that upon severe and prolonged hypoxia acts as a concerted cell response leading to cell death. In our OGD model, hypoxia modulates UPR and induces autophagy.

Low Dose Administration of Glutamate Triggers a Non-Apoptotic, Autophagic Response in PC12 Cells.

BACKGROUND/AIMS: Increasing amounts of the neurotransmitter glutamate are associated with excitotoxicity, a phenomenon related both to homeostatic processes and neurodegenerative diseases such as multiple sclerosis. METHODS: PC12 cells (rat pheochromocytoma) were treated with various concentrations of the non-essential amino acid glutamate for 0.5-24 hours. The effect of glutamate on cell morphology was monitored with electron microscopy and haematoxylin-eosin staining. Cell survival was calculated with the MTT assay. Expression analysis of chaperones associated with the observed phenotype was performed using either Western Blotting at the protein level or qRT-PCR at the mRNA level. RESULTS: Administration of glutamate in PC12 cells in doses as low as 10 µM causes an up-regulation of GRP78, GRP94 and HSC70 protein levels, while their mRNA levels show the opposite kinetics. At the same time, GAPDH and GRP75 show reduced protein levels, irrespective of their transcriptional rate. On a cellular level, low concentrations of glutamate induce an autophagy-mediated pro-survival phenotype, which is further supported by induction of the autophagic marker LC3. CONCLUSION: The findings in the present study underline a discrete effect of glutamate on neuronal cell fate depending on its concentration. It was also shown that a low dose of glutamate orchestrates a unique expression signature of various chaperones and induces cell autophagy, which acts in a neuroprotective fashion.

Chronic massive rotator cuff tear in rats: in vivo evaluation of muscle force and three-dimensional histologic analysis.

BACKGROUND: Massive rotator cuff tear repair is frequently complicated by unsatisfactory clinical results due to possible tendon retraction, muscle atrophy, and fatty degeneration. The objective of this study was the development of a chronic massive tear in a rat model and the evaluation of the muscle force in vivo and of the histologic changes in a 3- dimensional manner. METHODS: To simulate massive rotator cuff tears, both the supraspinatus (SS) and the infraspinatus (IS) tendons were surgically detached from the right humerus of 15 male adult Sprague-Dawley rats. Twelve weeks postoperatively, all animals underwent isometric tension recordings of both the SS and IS muscles. Histologic analysis and image deconvolution processing were performed to estimate the presence and the distribution of atrophy in 3 dimensions. RESULTS: An overall 30% and 35% reduction in muscle force of the SS and IS muscles, respectively, was observed compared with the left uninjured shoulder (P < .005). Histologic analysis revealed that the degeneration and the fatty infiltration were more evident near the tendon and at the dorsal side in both muscle groups. CONCLUSIONS: These results show that functional impairment of SS and IS muscles after chronic massive tendon tears could be attributed to the decrease in muscle force production during their repair on the greater tuberosity and, second, to the comparatively greater degeneration of their dorsal part.

Proteases inhibition assessment on PC12 and NGF treated cells after oxygen and glucose deprivation reveals a distinct role for aspartyl proteases.

Hypoxia is a severe stressful condition and induces cell death leading to neuronal loss both to the developing and adult nervous system. Central theme to cellular death is the activation of different classes of proteases such as caspases calpains and cathepsins. In the present study we investigated the involvement of these proteases, in the hypoxia-induced PC12 cell death. Rat PC12 is a model cell line for experimentation relevant to the nervous system and several protocols have been developed for either lethal hypoxia (oxygen and glucose deprivation OGD) or ischemic preconditioning (IPS). Nerve Growth Factor (NGF) treated PC12 differentiate to a sympathetic phenotype, expressing neurites and excitability. Lethal hypoxia was established by exposing undifferentiated and NGF-treated PC12 cells to a mixture of N(2)/CO(2) (93:5%) in DMEM depleted of glucose and sodium pyruvate for 16 h. The involvement of caspases, calpains and lysosomal cathepsins D and E to the cell death induced by lethal OGD was investigated employing protease specific inhibitors such as z-VAD-fmk for the caspases, MDL28170 for the calpains and pepstatin A for the cathepsins D and E. Our findings show that pepstatin A provides statistically significant protection from cell death of both naive and NGF treated PC12 cells exposed to lethal OGD. We propose that apart from the established processes of apoptosis and necrosis that are integral components of lethal OGD, the activation of cathepsins D and E launches additional cell death pathways in which these proteases are key partners.

Soleus muscle force following downhill running in ovariectomized rats treated with estrogen.

The ovariectomized (OVX) rat model was used to investigate the effects of estrogen treatment on soleus muscle functionality in situ following muscle injury induced by downhill running. Fifty immature, 24- to 26-d-old, OVX rats were randomly assigned to 5 separate experimental groups: sedentary controls (OVX-Sed), placebo-treated and studied immediately after exercise (OVX-Pb0), placebo-treated and studied 72 h after exercise (OVX-Pb72), estradiol-treated and studied immediately after exercise (OVX-Ed0), and estradiol-treated and studied 72 h after exercise (OVX-Ed72). At the age of 9 weeks, under ketamine and xylazine anesthesia i.p., the rats were subcutaneously implanted with either placebo or 17beta-estradiol-impregnated pellets (0.05 mg/pellet, 3 week release). Treatment with 17beta-estradiol increased the estradiol plasma levels in OVX animals to those normally seen during the proestrous cycle of normal animals. Three weeks after the implantation the rats were subjected to a 90 min intermittent downhill running protocol. Our results indicate that the exercise protocol used in the study induced injury in the soleus muscle, as it was detected by the significant reduction in unfused (stimulation at 10, 20, and 40 Hz) and maximal (Po) tetanic force, as well as the decreased ability of the soleus muscle to maintain tension (stimulation at 40 Hz for 3 min) in OVX-Pb0 and OVX-Pb72 placebo-treated animals subjected to downhill running (injured muscles) as compared with OVX-Sed control rats (uninjured muscle). Estradiol replacement in OVX rats partially protected the soleus muscle from the injury normally induced by downhill running. As compared with the OVX-Pb0 and OVX-Pb72 placebo-treated groups, the soleus muscles of OVX-Ed0 and OVX-Ed72 estradiol-treated rats were capable of producing significantly greater unfused tetanic force and had an increased ability to maintain tension after fatigue. However, estrogen at the dose administered did not prevent the decrease in maximal tetanic force. We postulate that the protective effect of estrogens on muscle strength may be related to the ability of estrogen hormones to attenuate the E--C coupling failure and (or) the disorganization of the contractile apparatus associated with eccentric exercise through a mechanism or mechanisms yet to be fully understood.

Planejamento cirúrgico dos implantes dentários / Surgical planning of dental implants with computed tomography

Encontramos na literatura pesquisada, diversas técnicas radiográficas empregadas com finalidade de diagnóstico e planejamento cirúrgico de implantes dentários; tais como as radiografias periapicais; oclusais; panorâmicas; teleradiografias laterais; tomografias convencionais e computadorizadas. Através de uma minuciosa revisão bibliográfica, concluímos que a tomografia computadorizada associada a programas de reformatação de imagens, é uma técnica eficiente e precisa para avaliar um local proposto para a colocação de implantes dentários. A imagem da estrutura óssea pode ser analisada por completo sem sobreposição, com o mínimo de magnificação, o que na implantodontia é fundamental

Morphological, histochemical, and interstitial pressure changes in the tibialis anterior muscle before and after aortofemoral bypass in patients with peripheral arterial occlusive disease.

BACKGROUND: Morphological and electrophysiological studies of ischemic muscles in peripheral arterial disease disclosed evidence of denervation and fibre atrophy. The purpose of the present study is to describe morphological changes in ischemic muscles before and after reperfusion surgery in patients with peripheral occlusive arterial disease, and to provide an insight into the effect of reperfusion on the histochemistry of the reperfused muscle. METHODS: Muscle biopsies were obtained from the tibialis anterior of 9 patients with chronic peripheral arterial occlusive disease of the lower extremities, before and after aortofemoral bypass, in order to evaluate the extent and type of muscle fibre changes during ischemia and after revascularization. Fibre type content and muscle fibre areas were quantified using standard histological and histochemical methods and morphometric analysis. Each patient underwent concentric needle electromyography, nerve conduction velocity studies, and interstitial pressure measurements. RESULTS: Preoperatively all patients showed muscle fibre atrophy of both types, type II fibre area being more affected. The mean fibre cross sectional area of type I was 3,745 microm2 and of type II 4,654 microm2. Fibre-type grouping, great variation in fibre size and angular fibres were indicative of chronic dennervation-reinnervation, in the absence of any clinical evidence of a neuropathic process. Seven days after the reperfusion the areas of both fibre types were even more reduced, being 3,086 microm2 for type I and 4,009 microm2 for type II, the proportion of type I fibres, and the interstitial pressure of tibialis anterior were increased. CONCLUSIONS: The findings suggest that chronic ischemia of the leg muscles causes compensatory histochemical changes in muscle fibres resulting from muscle hypoxia, and chronic dennervation-reinnervation changes, resulting possibly from ischemic neuropathy. Reperfusion seems to bring the oxidative capacity of the previously ischemic muscle closer to normal.