Anti‐tumor effect of SLPI on mammary but not colon tumor growth

Secretory leukocyte protease inhibitor (SLPI) is a serine protease inhibitor that was related to cancer development and metastasis dissemination on several types of tumors. However, it is not known the effect of SLPI on mammary and colon tumors. The aim of this study was to examine the effect of SLPI on mammary and colon tumor growth. The effect of SLPI was tested on in vitro cell apoptosis and in vivo tumor growth experiments. SLPI over‐expressing human and murine mammary and colon tumor cells were generated by gene transfection. The administration of murine mammary tumor cells over‐expressing high levels of SLPI did not develop tumors in mice. On the contrary, the administration of murine colon tumor cells over‐expressing SLPI, developed faster tumors than control cells. Intratumoral, but not intraperitoneal administration of SLPI, delayed the growth of tumors and increased the survival of mammary but not colon tumor bearing mice. In vitro culture of mammary tumor cell lines treated with SLPI, and SLPI producer clones were more prone to apoptosis than control cells, mainly under serum deprivation culture conditions. Herein we demonstrated that SLPI induces the apoptosis of mammary tumor cells in vitro and decreases the mammary but not colon tumor growth in vivo. Therefore, SLPI may be a new potential therapeutic tool for certain tumors, such as mammary tumors. J. Cell. Physiol. 228: 469–475, 2013. © 2012 Wiley Periodicals, Inc.     

Nutrient induced fluorescence transients (NIFTs) provide a rapid measure of P and C (co-)limitation in a green alga

Nutrient Induced Fluorescence Transients (NIFTs) have been shown to be a possible way of testing for the limiting nutrient in algal populations. In this study we tested the hypothesis that NIFTs can be used to detect a (co-)limitation for inorganic phosphorus (Pi) and CO₂ in the green alga Chlamydomonas acidophila and that the magnitude of the NIFTs can be related to cellular P:C ratios. We show a co-limitation response for Pi and CO₂ via traditional nutrient enrichment experiments in natural phytoplankton populations dominated by C. acidophila. We measured NIFT responses after a Pi- or a CO₂-spike in C. acidophila batch cultures at various stages of Pi and inorganic C limitation. Significant NIFTs were observed in response to spikes in both nutrients. The NIFT response to a Pi-spike showed a strong negative correlation with cellular P:C ratio that was pronounced below 3 mmol P: mol C (equivalent to 0.2 pg P cell^–1). Both cellular P and C content influenced the extent of the Pi-NIFT response. The NIFT response to a CO₂-spike correlated to low CO₂ culturing conditions and also had a negative correlation with cellular P content. A secondary response within the Pi-NIFT response was related to the CO₂ concentration and potentially reflected co-limitation. In conclusion, NIFTs provided a quick and reliable method to detect the growth-limiting nutrient in an extremophile green alga, under Pi-, CO_2- and Pi/CO₂ (co-)limited growth conditions.     

Specific targeted integration of kanamycin resistance-associated nonselectable DNA in the genome of the yeast Saccharomyces cerevisiae

Sophisticated genome manipulation requires the possibility to modify any intergenic or intragenic DNA sequence at will, without leaving large amounts of undesired vector DNA at the site of alteration. To this end, a series of vectors was developed from a previous gene knockout plasmid system to integrate nonselectable foreign DNA at any desired genomic location in yeast, with a minimum amount of residual plasmid DNA. These vectors have two mutated Flp recognition targets (FRT) sequences flanking the KanMX4 gene and multiple sites for subcloning the DNA fragment to be integrated. The selectable marker can be recycled by Flp site-specific excision between the identical FRTs, thereby allowing the integration of further DNA fragments. With this system, the NLS-tetR-GFP and DsRed genes were successfully integrated at the thr1 locus, and the RVB1 gene was tagged at the C-terminus with the V5-epitope-6-histidine tag. This plasmid system provides for a new molecular tool to integrate any DNA fragment at any genome location in [cir+] yeast strains. Moreover, the system can be extrapolated to other eukaryotic cells in which the FLP/FRT system functions efficiently.     

Heterozygous Reelin Mutations Cause Autosomal-Dominant Lateral Temporal Epilepsy

Autosomal-dominant lateral temporal epilepsy (ADLTE) is a genetic epilepsy syndrome clinically characterized by focal seizures with prominent auditory symptoms. ADLTE is genetically heterogeneous, and mutations in LGI1 account for fewer than 50% of affected families. Here, we report the identification of causal mutations in reelin (RELN) in seven ADLTE-affected families without LGI1 mutations. We initially investigated 13 ADLTE-affected families by performing SNP-array linkage analysis and whole-exome sequencing and identified three heterozygous missense mutations co-segregating with the syndrome. Subsequent analysis of 15 small ADLTE-affected families revealed four additional missense mutations. 3D modeling predicted that all mutations have structural effects on protein-domain folding. Overall, RELN mutations occurred in 7/40 (17.5%) ADLTE-affected families. RELN encodes a secreted protein, Reelin, which has important functions in both the developing and adult brain and is also found in the blood serum. We show that ADLTE-related mutations significantly decrease serum levels of Reelin, suggesting an inhibitory effect of mutations on protein secretion. We also show that Reelin and LGI1 co-localize in a subset of rat brain neurons, supporting an involvement of both proteins in a common molecular pathway underlying ADLTE. Homozygous RELN mutations are known to cause lissencephaly with cerebellar hypoplasia. Our findings extend the spectrum of neurological disorders associated with RELN mutations and establish a link between RELN and LGI1, which play key regulatory roles in both the developing and adult brain.     

Synthesis and anti-aromatase activity of some new steroidal D-lactones

Starting from D-seco derivatives of 5-androstene 1-3, the D-homo lactones, 4 and 5, were synthesized. By the Oppenauer oxidation and/or by dehydration of 4 and 5 with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) or 2,3,5,6-tetrachloro-1,4-benzoquinone (chloranil), the corresponding D-lactones 6-12 were obtained. The structures of 6 and 10 were unambiguously proved by the appropriate X-ray structural analysis. Anti-aromatase assay showed that tested compounds possess inhibition potency, however, two to four times smaller (IC50 from 0.2 to 0.7 microM, respectively) in comparison to aminoglutethimide (AG).     

Effect of pH and ionic strength on the cytolytic toxin Cyt1A: a fluorescence spectroscopy study

Cyt1A is a cytolytic toxin produced by Bacillus thuringiensis var. israelensis. Due to its toxicity in vivo against mosquitoes and black flies, it is used as an environmentally friendly insecticide, although its mode of action is not completely understood. The toxin is membrane-active, but its membrane-bound conformation is unknown. In the absence of direct structural data, fluorescence spectroscopy was used to obtain indirect information on Cyt1A conformation changes in the environment mimicking the vicinity of the lipid membrane (lower pH and increased ionic strength). With decreasing pH, Cyt1A's surface hydrophobicity increased, which is consistent with an increased interaction with model membranes at low pH values, as observed previously. The pK(a) value of this conformation change is 4.4+/-0.1. Intrinsic tryptophan fluorescence decreased with decreasing pH, and the pK(a) value was the same as the one determined with synthetic probes. The protein has two types of hydrophobic binding sites, and at low pH these sites bind more probe molecules (bis-ANS) with a higher affinity than at pH 7.4. When bound to the lipid, the toxin exhibited conformation similar to the molten-globule state and showed some characteristics also observed at low pH. However, the conformation of the lipid-bound toxin did not depend on pH. Neutral salts like NaCl and KCl induced conformational changes at neutral pH, but not at low pH. These changes were most probably due to specific interactions of the salt ions with the charged amino acids on the protein surface rather than due to general effects such as Hofmeister and Debye-Hückel. Our results might contribute to elucidating the mode of action of Cyt1A, and perhaps also to improving the formulation of the insecticidal preparations.     

Heavy metal accumulation of Danube river aquatic plants — indication of chemical contamination

In this paper, the ecological status of a section of the Danube River flowing through Serbia from Bezdan to Djerdap was evalutated. Using the chemical composition of water, sediment samples from the littoral zone and dominant aquatic macrophytes, the level of chemical pollution was ascertained. Chemical analyses of the water and sediment indicated that the tributaries flowing into the Danube significantly influenced the chemical load of the water and as a direct consequence, the sediment. The concentration of heavy metals including Cu, Mn and Cd found in plants of the Potamogeton genus, further indicated significant chemical pollution, establishing a clear link between the chemical composition of plant tissues and the chemical composition of water and sediment. This paper therefore describes how the chemical composition of aquatic plants can be used as a reliable indicator for heavy metal pollution of aquatic ecosystems.     

The eIF4E RNA regulon promotes the Akt signaling pathway

Eukaryotic initiation factor 4E (eIF4E) promotes cellular proliferation and can rescue cells from apoptotic stimuli such as serum starvation. However, the mechanisms underlying apoptotic rescue are not well understood. In this study, we demonstrate that eIF4E overexpression leads to enhanced survival signaling through Akt and that eIF4E requires Akt1 to rescue serum-deprived fibroblasts. Furthermore, a mutant form of eIF4E (W73A), which is messenger RNA (mRNA) export competent but does not promote translation, rescues cells as readily as wild-type eIF4E. We show that eIF4E mediates Akt activation via up-regulation of Nijmegen breakage syndrome 1 (NBS1), a phosphoinositide-3 kinase-Akt pathway upstream activator. Additionally, eIF4E coordinately up-regulates the expression of downstream effectors of the Akt pathway, thereby amplifying Akt signaling effects. A negative regulator of eIF4E, the promyelocytic leukemia protein (PML), suppresses Akt activation and apoptotic rescue. These PML activities likely arise, at least in part, through its inhibition of eIF4E-mediated NBS1 mRNA export. In summary, eIF4E coordinately regulates gene expression to potentiate Akt activation, an activity required for apoptotic rescue.     

Mutant Parkin impairs mitochondrial function and morphology in human fibroblasts

Background

Mutations in Parkin are the most common cause of autosomal recessive Parkinson disease (PD). The mitochondrially localized E3 ubiquitin-protein ligase Parkin has been reported to be involved in respiratory chain function and mitochondrial dynamics. More recent publications also described a link between Parkin and mitophagy.

Methodology/Principal Findings

In this study, we investigated the impact of Parkin mutations on mitochondrial function and morphology in a human cellular model. Fibroblasts were obtained from three members of an Italian PD family with two mutations in Parkin (homozygous c.1072delT, homozygous delEx7, compound-heterozygous c.1072delT/delEx7), as well as from two relatives without mutations. Furthermore, three unrelated compound-heterozygous patients (delEx3-4/duplEx7-12, delEx4/c.924C>T and delEx1/c.924C>T) and three unrelated age-matched controls were included. Fibroblasts were cultured under basal or paraquat-induced oxidative stress conditions. ATP synthesis rates and cellular levels were detected luminometrically. Activities of complexes I-IV and citrate synthase were measured spectrophotometrically in mitochondrial preparations or cell lysates. The mitochondrial membrane potential was measured with 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide. Oxidative stress levels were investigated with the OxyBlot technique. The mitochondrial network was investigated immunocytochemically and the degree of branching was determined with image processing methods. We observed a decrease in the production and overall concentration of ATP coinciding with increased mitochondrial mass in Parkin-mutant fibroblasts. After an oxidative insult, the membrane potential decreased in patient cells but not in controls. We further determined higher levels of oxidized proteins in the mutants both under basal and stress conditions. The degree of mitochondrial network branching was comparable in mutants and controls under basal conditions and decreased to a similar extent under paraquat-induced stress.

Conclusions

Our results indicate that Parkin mutations cause abnormal mitochondrial function and morphology in non-neuronal human cells.     

CO2 acquisition in Chlamydomonas acidophila is influenced mainly by CO2, not phosphorus,availability

The extremophilic green microalga Chlamydomonas acidophila grows in very acidic waters (pH 2.3–3.4), where CO₂ is the sole inorganic carbon source. Previous work has revealed that the species can accumulate inorganic carbon (Ci) and exhibits high affinity CO₂ utilization under low-CO₂ (air-equilibrium) conditions, similar to organisms with an active CO₂ concentrating mechanism (CCM), whereas both processes are down-regulated under high CO₂ (4.5 % CO₂) conditions. Responses of this species to phosphorus (Pi)-limited conditions suggested a contrasting regulation of the CCM characteristics. Therefore, we measured external carbonic anhydrase (CA_ext) activities and protein expression (CAH1), the internal pH, Ci accumulation, and CO₂-utilization in cells adapted to high or low CO₂ under Pi-replete and Pi-limited conditions. Results reveal that C. acidophila expressed CA_ext activity and expressed a protein cross-reacting with CAH1 (the CA_ext from Chlamydomonas reinhardtii). Although the function of this CA remains unclear, CA_ext activity and high affinity CO₂ utilization were the highest under low CO₂ conditions. C. acidophila accumulated Ci and expressed the CAH1 protein under all conditions tested, and C. reinhardtii also contained substantial amounts of CAH1 protein under Pi-limitation. In conclusion, Ci utilization is optimized in C. acidophila under ecologically relevant conditions, which may enable optimal survival in its extreme Ci- and Pi-limited habitat. The exact physiological and biochemical acclimation remains to be further studied.