Amplification of CRAC current by STIM1 and CRACM1 (Orai1)

Depletion of intracellular calcium stores activates store-operated calcium entry across the plasma membrane in many cells. STIM1, the putative calcium sensor in the endoplasmic reticulum, and the calcium release-activated calcium (CRAC) modulator CRACM1 (also known as Orai1) in the plasma membrane have recently been shown to be essential for controlling the store-operated CRAC current (I(CRAC)). However, individual overexpression of either protein fails to significantly amplify I(CRAC). Here, we show that STIM1 and CRACM1 interact functionally. Overexpression of both proteins greatly potentiates I(CRAC), suggesting that STIM1 and CRACM1 mutually limit store-operated currents and that CRACM1 may be the long-sought CRAC channel.     

Nutrient uptake and utilization can be used to select viable day 7 bovine blastocysts after cryopreservation

The ability of individual bovine blastocysts to survive freezing and thawing procedures was assessed by measuring glucose and pyruvate uptake and lactate production immediately before and after cryopreservation. Using glucose and pyruvate uptake and lactate production it was not possible to determine, prior to freezing, which blastocysts would be viable after thawing. However, in the 5 hr immediately after thawing, those blastocysts which expanded their blastocoel had significantly greater glucose and pyruvate uptake and lactate production (P < 0.01) than those embryos which failed to develop after a 14 hr overnight incubation. Interestingly, after thawing, two distinct populations of blastocysts existed with respect to glucose uptake and lactate production, indicating that it is possible to identify those blastocysts immediately after thawing which will reexpand. In contrast, there was a considerable degree of overlap in pyruvate uptakes between the viable and nonviable groups of embryos, indicating that this parameter could not be used to select viable embryos after thawing. There was an increase in the calculated oxidation of carbohydrates after thawing, consistent with a partial uncoupling of the inner mitochondrial membrane. In conclusion, glucose uptake and lactate production can be used to select prospectively viable blastocysts immediately after thawing, indicating that glycolysis is a major energy-generating pathway for the embryo at this time. © 1996 Wiley-Liss, Inc.     

Effect of prostaglandin E2 and tumor necrosis factor alpha on the VEGF-receptor system expression in cultured porcine luteal cells

The purpose of the present study was to investigate the effects of prostaglandin (PG) E(2) and tumor necrosis factor (TNF) alpha on expression of vascular endothelial growth factor (VEGF) and its receptors, fms-like tyrosine kinase (Flt-1) and fetal liver kinase-1/kinase insert domain-containing receptor (Flk-1/KDR), in cultured porcine luteal cells. Real-time PCR was used for quantification of VEGF and its receptors mRNA, whereas VEGF release by luteal cells was determined by radioimmunoassay (RIA). Only the highest dose of PGE(2) (1 microM) after 6 hr of incubation stimulated VEGF release by luteal cells collected in the mid-luteal phase (P < 0.05). Moreover, PGE(2) (100 nM, 1 microM) significantly stimulated VEGF secretion by luteal cells in the late phase and during pregnancy on Days 10-12 (P < 0.05). Elevated mRNA expression of both VEGF 120 and VEGF 164 isoforms was found in luteal cells cultured with PGE(2). The lack of an effect of PGE(2) on VEGF receptors mRNA expression was observed. TNFalpha was able to significantly stimulate VEGF release from cells obtained in the mid- and late luteal phase or during early pregnancy. All tested doses enhanced mRNA levels of VEGF 120 isoform, but not VEGF 164. Additionally, TNFalpha was able to decrease Flk-1/KDR mRNA expression, whereas Flt-1 mRNA levels were not affected. These results indicated that PGE(2) and TNFalpha influenced VEGF ligand-receptor system expression in porcine luteal cells and may therefore play an important role in regulation of luteal functions during the estrous cycle and pregnancy in pigs.     

CIP2A oncoprotein controls cell growth and autophagy through mTORC1 activation

mTORC1 (mammalian target of rapamycin complex 1) integrates information regarding availability of nutrients and energy to coordinate protein synthesis and autophagy. Using ribonucleic acid interference screens for autophagy-regulating phosphatases in human breast cancer cells, we identify CIP2A (cancerous inhibitor of PP2A [protein phosphatase 2A]) as a key modulator of mTORC1 and autophagy. CIP2A associates with mTORC1 and acts as an allosteric inhibitor of mTORC1-associated PP2A, thereby enhancing mTORC1-dependent growth signaling and inhibiting autophagy. This regulatory circuit is reversed by ubiquitination and p62/SQSTM1-dependent autophagic degradation of CIP2A and subsequent inhibition of mTORC1 activity. Consistent with CIP2A’s reported ability to protect c-Myc against proteasome-mediated degradation, autophagic degradation of CIP2A upon mTORC1 inhibition leads to destabilization of c-Myc. These data characterize CIP2A as a distinct regulator of mTORC1 and reveals mTORC1-dependent control of CIP2A degradation as a mechanism that links mTORC1 activity with c-Myc stability to coordinate cellular metabolism, growth, and proliferation.     

Erratum to “Helminth communities in the burrowing toad, Rhinella fernandezae, from Northeastern Argentina” by Monika Inés Hamann, Arturo Ignacio Kehr & Cynthya Elizabeth González

The original version of the article was published in Biologia 68 (6): 1155–1163 (2013), DOI: 10.2478/s11756-013-0272-5. Unfortunately, the original version of this article contains a mistake in Table 1 and in left collumn, line 8 on page 1157. Here we display the corrected version of the table and text.     

Rücken‐WNT für die Primärachse der Tiere

Axis formation in animals The last common ancestor of Cnidaria and Bilateria likely used the WNT/β‐Catenin pathway in a regionalized fashion to establish its primary, anterior‐posterior axis. Unexpectedly, the morphological head of Cnidaria corresponds to the rear end of Bilateria. Moreover, annelids use the WNT/β‐Catenin system for early, local and binary decisions, and insects developed a completely unrelated pathway. They use Bicoid (Drosophila) – or Hunchback/Orthodenticle (Tribolium) – to control axis formation. Nevertheless, WNT functions are essential during the segmentation phase in insects and in ancestral insects as well as in other arthropods during formation of posterior structures. In summary, the WNT/β‐Catenin system is an essential part of the molecular tool kit, which helped to establish the unique features of animals.     

Application of 13C-[2] - and 13C-[1,2] acetate in metabolic labelling studies of yeast and insect cells

The advantage of using ¹³C-labelled glucose in metabolic studies is that it is an important carbon and energy source for almost all biotechnologically and medically important organisms. On the other hand, the disadvantage is its relatively high cost in the labelling experiments. Looking for cheaper alternatives we found that ¹³C-[2] acetate or ¹³C-[1,2] acetate is a prospective compound for such experiments. Acetate is well incorporated by many organisms, including mammalian and insect cell cultures as preferred source of acetyl-CoA. Our experimental results using ¹³C NMR demonstrated that acetate was efficiently incorporated into glutamate and alanine secreted by the insect cell culture. Using D-stat culture of Saccharomyces uvarum on glucose/¹³C-acetate mineral media we demonstrated that the labelling patterns of proteinogenic amino acids can be well predicted on the basis of specific substrate consumption rates using the modified scheme of yeast metabolism and stoichiometric modelling. According to this scheme aspartate and alanine in S. uvarum under the experimental conditions used is synthesised in the mitochondria. Synthesis of alanine in the mitochondria was also demonstrated for Spodoptera frugiperda. For both organisms malic enzyme was also operative. For S. uvarum it was shown that the activity of malic enzyme is sufficient for supporting the mitochondrial biosynthetic reactions with NADPH.     

Effect of hyperhomocysteinemia on rat cardiac sarcoplasmic reticulum

Molecular and Cellular Biochemistry - Increased concentration of plasma homocysteine (Hcy) is an independent risk factor of cardiovascular disease, yet the mechanism by which hyperhomocysteinemia...     

Molecular identification, immunolocalization, and functional activity of a vacuolar-type H+-ATPase in bovine rumen epithelium

In this study, we have studied the expression, localization, and functionality of vacuolar-type H⁺-ATPase (vH⁺-ATPase) and Na⁺/K⁺-ATPase in the bovine rumen epithelium. Compared with the intracellular pH (pH_i) of control rumen epithelial cells (REC; 7.06 ± 0.07), application of inhibitors selective for vH⁺-ATPase (foliomycin) and Na⁺/K⁺-ATPase (ouabain) reduced pH_i by 0.10 ± 0.03 and 0.18 ± 0.03 pH-units, respectively, thereby verifying the existence of both functional proteins. Results from qRT-PCR and immunoblotting clearly confirm the expression of vH⁺-ATPase B subunit in REC. However, the amount of Na⁺/K⁺-ATPase mRNA and protein is tenfold and 11-fold of those of vH⁺-ATPase subunit B, respectively, reflecting a lower overall abundance of the latter in REC. Na⁺/K⁺-ATPase immunostaining has revealed the protein in the plasma membrane of all REC from the stratum basale to stratum granulosum, with the highest abundance in basal cells. In contrast, the vH⁺-ATPase B subunit has been detected in groups of cells only, mainly localized in the stratum spinosum and stratum granulosum of the epithelium. Furthermore, vH⁺-ATPase has been detected in the cell membrane and in intracellular pools. Thus, functional vacuolar-type H⁺ pumps are expressed in REC and probably play a role in the adaptation of epithelial transport processes.