Substrate‐dependent cluster density dynamics of Corynebacterium glutamicum phosphotransferase system permeases

Many bacteria take up carbohydrates by membrane‐integral sugar specific phosphoenolpyruvate‐dependent carbohydrate:phosphotransferase systems (PTS). Although the PTS is centrally involved in regulation of carbon metabolism in different bacteria, little is known about localization and putative oligomerization of the permease subunits (EII). Here, we analyzed localization of the fructose specific PtsF and the glucose specific PtsG transporters, as well as the general components EI and HPr from Corynebacterium glutamicum using widefield and single molecule localization microscopy. PtsF and PtsG form membrane embedded clusters that localize in a punctate pattern. Size, number and fluorescence of the membrane clusters change upon presence or absence of the transported substrate, and a direct influence of EI and HPr was not observed. In presence of the transport substrate, EII clusters significantly increased in size. Photo‐activated localization microscopy data revealed that, in presence of different carbon sources, the number of EII proteins per cluster remains the same, however, the density of these clusters reduces. Our work reveals a simple mechanism for efficient membrane occupancy regulation. Clusters of PTS EII transporters are densely packed in absence of a suitable substrate. In presence of a transported substrate, the EII proteins in individual clusters occupy larger membrane areas.     

beta-xylosidase activity and expression of a beta-xylosidase gene during strawberry fruit ripening.

Strawberry fruit shows a marked softening during ripening and the process is associated with an increment of pectin solubility and a reduction of the molecular mass of hemicelluloses. In this work, we report the activity of beta-xylosidase and the expression of a beta-xylosidase gene in strawberry fruit. We have cloned a cDNA fragment encoding a putative beta-xylosidase (FaXyl1) from a cDNA library obtained from ripe strawberry fruit. The analysis of the deduced amino acid sequence revealed that FaXyl1 is closely related to other beta-xylosidases from higher plants. The expression of FaXyl1 was strongly associated to the receptacle tissue although a low expression level was detected in achenes and ovaries. The accumulation of FaXyl1 mRNA is ripening-related, starting in white fruit, reaching the maximum at 25-50% red fruit and decreasing thereafter. The total beta-xylosidase enzyme activity was detected in all ripening stages with the maximum in 25-50% red fruit. The low activity level detected in immature stages, where no expression of FaXyl1 was found, suggests the presence of other beta-xylosidases-like genes. Both the expression of FcaXyl1 and the total beta-xylosidase activity were down regulated by auxins, as occurs for most of the ripening-related processes in strawberry fruit. A putative role of FaXyl1 and beta-xylosidase is discussed.     

Effect of Acute Administration of l-Tyrosine on Oxidative Stress Parameters in Brain of Young Rats

Tyrosinemia type II, also known as Richner–Hanhart syndrome, is an autosomal recessive inborn error of metabolism caused by a deficiency of hepatic cytosolic tyrosine aminotransferase, and is associated with neurologic and development difficulties in numerous patients. Considering that the mechanisms underlying the neurological dysfunction in hypertyrosinemic patients are poorly known and that studies demonstrated that high concentrations of tyrosine provoke oxidative stress in vitro and in vivo in the cerebral cortex of rats, in the present study we investigate the oxidative stress parameters (enzymatic antioxidant defenses, thiobarbituric acid-reactive substances and protein carbonyl content) in cerebellum, hippocampus and striatum of 30-old-day rats after acute administration of l-tyrosine. Our results demonstrated that the acute administration of l-tyrosine increased the thiobarbituric acid reactive species levels in hippocampus and the carbonyl levels in cerebellum, hippocampus and striatum. In addition, acute administration of l-tyrosine significantly decreased superoxide dismutase activity in cerebellum, hippocampus and striatum, while catalase was increased in striatum. In conclusion, the oxidative stress may contribute, along with other mechanisms, to the neurological dysfunction characteristic of hypertyrosinemia and the administration of antioxidants may be considered as a potential adjuvant therapy for tyrosinemia, especially type II.     

Evidence of a landlocked reproducing population of the marine pejerrey Odontesthes argentinensis (Actinopterygii; Atherinopsidae)

In South America, the order Atheriniformes includes the monophyletic genus Odontesthes with 20 species that inhabit freshwater, estuarine and coastal environments. Pejerrey Odontesthes argentinensis is widely distributed in coastal and estuarine areas of the Atlantic Ocean and is known to foray into estuaries of river systems, particularly in conditions of elevated salinity. However, to our knowledge, a landlocked self-sustaining population has never been recorded. In this study, we examined the pejerrey population of Salada de Pedro Luro Lake (south-east of Buenos Aires Province, Argentina) to clarify its taxonomic identity. An integrative taxonomic analysis based on traditional meristic, landmark-based morphometrics and genetic techniques suggests that the Salada de Pedro Luro pejerrey population represents a novel case of physiological and morphological adaptation of a marine pejerrey species to a landlocked environment and emphasises the environmental plasticity of this group of fishes.     

Revisiting the coupling of fatty acid to phospholipid synthesis in bacteria with FapR regulation

A key aspect in membrane biogenesis is the coordination of fatty acid to phospholipid synthesis rates. In most bacteria, PlsX is the first enzyme of the phosphatidic acid synthesis pathway, the common precursor of all phospholipids. Previously, we proposed that PlsX is a key regulatory point that synchronizes the fatty acid synthase II with phospholipid synthesis in Bacillus subtilis. However, understanding the basis of such coordination mechanism remained a challenge in Gram-positive bacteria. Here, we show that the inhibition of fatty acid and phospholipid synthesis caused by PlsX depletion leads to the accumulation of long-chain acyl-ACPs, the end products of the fatty acid synthase II. Hydrolysis of the acyl-ACP pool by heterologous expression of a cytosolic thioesterase relieves the inhibition of fatty acid synthesis, indicating that acyl-ACPs are feedback inhibitors of this metabolic route. Unexpectedly, inactivation of PlsX triggers a large increase of malonyl-CoA leading to induction of the fap regulon. This finding discards the hypothesis, proposed for B. subtilis and extended to other Gram-positive bacteria, that acyl-ACPs are feedback inhibitors of the acetyl-CoA carboxylase. Finally, we propose that the continuous production of malonyl-CoA during phospholipid synthesis inhibition provides an additional mechanism for fine-tuning the coupling between phospholipid and fatty acid production in bacteria with FapR regulation.     

Variations in the periphytic community structure and dynamics of Lake Nahuel Huapi (Patagonia,Argentina)

Little is known about periphyton community in Patagonia and the influence that urban development has on a particular water body. Lake Nahuel Huapi is located in a National Park with little industrial or agricultural activity in the basin, but it is influenced by urban discharges from San Carlos de Bariloche, a town of 80000 inhabitants located on its south shore. Variations in structure and dynamics of the periphyton community in Lake Nahuel Huapi were studied during 1 year, to examine the influence of urban discharges on this community. Periphyton samples were collected on the shoreline of the lake and evaluated for biomass (as chlorophyll a concentration) and cell density of the main algal groups, at sites with and without urban influence. Results indicated the major influence of pollution from urban sources on periphyton at the most impacted sites, as well as the effect of different local events at each site (degree of contamination, type of coast, wind exposure, etc.). Higher biomass and cell densities at contaminated sites were evident, but the variation of main algal groups through the year was similar in all the sites. Periphyton in Lake Nahuel Huapi proved to be an important bioindicator of urban impacts on this water body.     

EphA2 Mutation in Lung Squamous Cell Carcinoma Promotes Increased Cell Survival,Cell Invasion,Focal Adhesions,and Mammalian Target of Rapamycin Activation

Non-small cell lung cancer (NSCLC) has a poor prognosis and improved therapies are needed. Expression of EphA2 is increased in NSCLC metastases. In this study, we investigated EphA2 mutations in NSCLC and examined molecular pathways involved in NSCLC. Tumor and cell line DNA was sequenced. One EphA2 mutation was modeled by expression in BEAS2B cells, and functional and biochemical studies were conducted. A G391R mutation was detected in H2170 and 2/28 squamous cell carcinoma patient samples. EphA2 G391R caused constitutive activation of EphA2 with increased phosphorylation of Src, cortactin, and p130^Cas. Wild-type (WT) and G391R cells had 20 and 40% increased invasiveness; this was attenuated with knockdown of Src, cortactin, or p130^Cas. WT and G391R cells demonstrated a 70% increase in focal adhesion area. Mammalian target of rapamycin (mTOR) phosphorylation was increased in G391R cells with increased survival (55%) compared with WT (30%) and had increased sensitivity to rapamycin. A recurrent EphA2 mutation is present in lung squamous cell carcinoma and increases tumor invasion and survival through activation of focal adhesions and actin cytoskeletal regulatory proteins as well as mTOR. Further study of EphA2 as a therapeutic target is warranted.     

Imbalanced circulating matrix metalloproteinases in polycystic ovary syndrome

Altered levels of matrix metalloproteinases (MMPs) may reflect relevant pathogenetic mechanisms of disease conditions. The objective of this study was to compare the plasma levels of MMPs and tissue inhibitors of MMPs (TIMPs) in polycystic ovary syndrome (PCOS) patients with those found in healthy ovulatory controls and to examine whether the levels of these biomarkers are associated with clinical and biochemical features of this syndrome. Sixty-five healthy ovulatory subjects (controls) and 80 patients with PCOS were include in this study. MMP-2, MMP-8, MMP-9, TIMP-1, TIMP-2 concentrations were measured in plasma samples by gelatin zymography or enzyme-linked immunoassays. MMP-2, MMP-8, MMP-9, and TIMP-1 levels were similar in PCOS patients and in healthy controls (P > 0.05). PCOS patients had lower plasma TIMP-2 levels than healthy controls (P < 0.05). We found higher MMP-2/TIMP-2 and MMP-9/TIMP-1 ratios in PCOS patients than in healthy controls (all P < 0.05). Testosterone levels correlated positively with the MMP-9/TIMP-1 ratio and negatively with TIMP-2 levels (r = 0.26, P < 0.01 and r = -0.21, P = 0.02, respectively). In addition, only testosterone was an independent predictor of TIMP-2 levels (estimate = -0.35, P = 0.04) and the MMP-9/TIMP-1 ratio (estimate = 0.01, P = 0.04). We found evidence indicating that the balance between MMPs and TIMPs in women with PCOS is altered, probably due to androgen excess found in these women.     

New insights into the early biochemical activation of jasmonic acid biosynthesis in leaves

In plants, herbivore attack elicits the rapid accumulation of jasmonic acid (JA) which results from the activation of constitutively expressed biosynthetic enzymes. The molecular mechanisms controlling the activation of JA biosynthesis remain largely unknown however new research has elucidated some of the early regulatory components involved in this process. Nicotiana attenuata plants, a wild tobacco species, responds to fatty acid amino acid conjuguates (FAC) elicitors in the oral secretion of its natural herbivore, Manduca sexta, by triggering specific defense and tolerance responses against it; all of the defense responses known to date require the amplification of the wound-induced JA increase. We recently demonstrated that this FAC-elicited JA burst requires an increased flux of free linolenic acid (18:3) likely originating from the activation of a plastidial glycerolipase (GLA1) which is activated by an abundant FAC found in insect oral secretions, N-linolenoyl-glutamate (18:3-Glu). The lack of accumulation of free 18:3 after elicitation suggests a tight physical association between GLA1 and LOX3 in N. attenuata leaves. In addition, the salicylate-induced protein kinase (SIPK) and the nonexpressor of PR-1 (NPR1) participate in this activation mechanism that controls the supply of 18:3. In contrast, the wound-induced protein kinase (WIPK) does not but instead regulates the conversion of 13(S)-hydroperoxy-18:3 into 12-oxo-phytodienoic acid (OPDA). These results open new perspectives on the complex network of signals and regulatory components inducing the JA biosynthetic pathway.Key words: jasmonic acid, lipase, lipoxygenase, wounding, plant-insect interactions, FAC