Co-evolutionary networks of genes and cellular processes across fungal species

Background  

The introduction of measures such as evolutionary rate and propensity for gene loss have significantly advanced our knowledge of the evolutionary history and selection forces acting upon individual genes and cellular processes.     

Isolation, characterization, and properties of a trypsin-chymotrypsin inhibitor from amaranth seeds

A trypsin-chymotrypsin inhibitor was isolated from the seeds of amaranth—a highly nutritious protein source. The purification of the inhibitor (AmI) was carried out by affinity chromatography on trypsin-Sepharose and by HPLC. AmI is a single-chain protein of 8 kD, as determined by electrophoresis on SDS-polyacrylamide gels and by gel exclusion on Sephadex G-50 column. It is stable at neutral and alkalinepH and is relatively thermostable. AmI inhibits trypsin and chymotrypsin from the digestive system of insects such asTribolium castaneum andLocusta migratoria, supporting the hypothesis that inhibitors may have evolved as defense mechanisms of seeds against insects. AmI lost its inhibitory activities when submitted to limited proteolysis with trypsin, while limited proteolysis with chymotrypsin had almost no effect. The partial amino acid sequence of 45 amino acids from the amino terminus of AmI differs significantly from the known sequences of legume-seed and cereal-grain protease inhibitor families. Differences in the chemistry at the inhibitory site(s) and in the amino acid sequence of AmI in comparison to that of other cereal and legume inhibitors suggest that AmI is a member of a new family of serine protease inhibitors. AmI was found to inhibit the anchorage-independent growth of MCF-7 breast cancer cells, suggesting that AmI may have anticarcinogenic activity.     

A conformational change in the N terminus of SLC38A9 signals mTORC1 activation

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Comparison of five physiological parameters to assess the vitality of the lichen Ramalina lacera exposed to air pollution

To determine the environmental impact of industrial activity, we examined and compared the following parameters of physiological integrity in the epiphytic fruticose lichen Ramalina lacera. (1) Potential quantum yield of PSII expressed as the chlorophyll fluorescence ratio F_v/F_m. (2) Spectral reflectance expressed as values of normalized difference vegetation index (NDVI). (3) Production of ethylene. (4) Electrolyte leakage brought about by degrading cell membranes in terms of electric conductivity. (5) The ratio A_435nm/A_415nm indicating the disintegration of chlorophyll. The amounts of Ba, Cu, K, Ni, S, V and Zn contained in the lichen thallus were measured to quantify the degree of pollution. Some of the lichen‐carrying twigs collected at a relatively unpolluted control site were resuspended on the original trees as controls. Other lichens were transplanted to 19 biomonitoring sites. Transplanted lichens in polluted sites contained higher amounts of Ba, Ni, S, V and Zn than lichens in the control site and in most of the rural sites upon an exposure period of 8 months. Statistical analysis revealed negative correlations between F_v/F_m ratios and Ba contents of transplants. High Ba, Cu, Ni and Zn contents correlated negatively with NDVI values. NDVI values correlated with A_435nm/A_415nm, F_v/F_m and K. The ratio A_435nm/A_415nm correlated positively with K and negatively with Ba, Cu and Zn. The production of ethylene correlated positively with Cu and Ba and negatively with A_435nm/A_415nm, F_v/F_m and NDVI. Electric conductivity values correlated positively with Ba, Cu, Na, S, V and Zn and negatively with NDVI. Both elemental content and physiological alterations in transplants of R. lacera point to a high degree of contamination in the Haifa Bay region, which is polluted by fuel‐oil combustion apart from other industrial activities. The present work suggests that in our specific study area, the most sensitive parameter to assess the vitality of the lichen thallus is electric conductivity whereas ethylene production is less sensitive.     

STORAGE OF SEROTONIN AND SEROTONIN BINDING PROTEIN IN SYNAPTIC VESICLES

We have used the newly introduced method of De Lorenzo & Freedman (1978) for isolating synaptic vesicles to determine if such vesicles contain both serotonin (5-HT) and serotonin binding protein (SBP). Two fractions were obtained. A 55, 000 g fraction was morphologically heterogeneous and contained coated vesicles. A 135, 0000 vesicle (dia. 51.3 nm) fraction was homogeneous in ultra-structure and contained no coated vesicles. The specific activity of SBP in this fraction was much higher than that in the supernatant. Unlike SBP, very little lactic dehydrogenase activity appeared in the 135, 000 g fraction. Qualitative and quantitative differences were observed between the polypeptide profiles of soluble proteins extracted from the vesicles and supernatant proteins on SDS gels. Therefore, entrapment of cytosol in the vesicles of the 135, 000 g fraction was minimal. The 5-HT concentration of the 135, 000 g vesicles was 5.5 ng/mg protein and in the supernatant, 11.3 ng/mg protein. The ATP concentration in the 135, 000 g vesicle fraction was only 0.8 ng/mg Pr. Rabbit spinal cords were transected in order to determine if SBP is moved proximo-distally in axons by rapid axonal transport as would be predicted for a constituent of synaptic vesicles. SBP accumulated above the cut at a rate consistent with fast transport (78 mm/day). SBP activity fell caudal to the point of transection and there was no evidence, such as an accumulation below the lesion, that might indicate retrograde transport of SBP. These experiments indicate that SBP is probably synthesized in the cell bodies of serotonergic neurons and some is rapidly transported down axons to be stored in terminals in vesicles.     

COP9 signalosome subunit 7 from Arabidopsis interacts with and regulates the small subunit of ribonucleotide reductase (RNR2)

The COP9 Signalosome protein complex (CSN) is a pleiotropic regulator of plant development and contains eight-subunits. Six of these subunits contain the PCI motif which mediates specific protein interactions necessary for the integrity of the complex. COP9 complex subunit 7 (CSN7) contains an N-terminal PCI motif followed by a C-terminal extension which is also necessary for CSN function. A yeast-interaction trap assay identified the small subunit of ribonucelotide reductase (RNR2) from Arabidopsis as interacting with the C-terminal section of CSN7. This interaction was confirmed in planta by both bimolecular fluorescence complementation and immuoprecipitation assays with endogenous proteins. The subcellular localization of RNR2 was primarily nuclear in meristematic regions, and cytoplasmic in adult cells. RNR2 was constitutively nuclear in csn7 mutant seedlings, and was also primarily nuclear in wild type seedlings following exposure to UV-C. These two results correlate with constitutive expression of several DNA-damage response genes in csn7 mutants, and to increased tolerance of csn7 seedlings to UV-C treatment. We propose that the CSN is a negative regulator of RNR activity in Arabidopsis.     

PI3K/Akt responses to oxytocin stimulation in Caco2BB gut cells

Recently, we discovered oxytocin receptor (OTR) expression in the developing gut villus epithelium that emerges in villus-crypt junctions after weaning. Oxytocin (OT) and OTR regulate many physiological functions in various tissues; however, their function in gut epithelium is unknown. We explored responses of PI3K and Akt phosphoisoforms to OT stimuli in the Caco2BB human gut cell line. In Caco2BB cells, PI3K and pAkt levels peaked at 62.5 nM OT. At higher concentrations, PI3K decreased more gradually than pAkt(S473) suggesting that the pAkt(S473) response is separate from PI3K. At ≤7.8 nM OT, pAkt(T308) increased while pAkt(S473) decreased. Using a specific OTR antagonist, we demonstrated that responses of pAkt(T308) to OT depend on OTR in contrast to the partial OTR-dependence of the pAkt(S473) response. Differential pAkt phosphoisoform responses included pAkt phosphoserine 473 persistently free of phosphothreonine 308. The reduction in PI3K after 62.5 nM OT for 30 min coincided with OTR internalization. The PI3K/Akt activation profile was somewhat different in other cell lines (MCF-7 breast cancer cells, HT29 gut cells), which have PI3K activating mutations, that were examined to establish experimental parameters. In Caco2BB cells, the divergent effects of OT upon pAkt phosphoisoforms suggests separate sub-pathways; pAkt (T308) activation depends on OTR via the PI3K pathway and pAkt(S473) presumably results from its specific kinase mTORC2 (mammalian target of rapamycin complex 2). Thus, OT may modulate gut cell functions downstream of mTOR complexes (e.g., translation control as suggested by others in uterine cells). We will next explore OT-stimulated kinase activities downstream of mTOR related to pAkt phosphoisoforms.     

Mutual interaction of special phytoestrogenic compounds, their synthetic carboxy-derivatives and the less-calcemic vitamin D analog activities in human derived female cultured osteoblasts

Cultured female-derived human bone cells (hObs) responded by different parameters to different phytoestrogenic and vitamin D compounds. Pre- and post-menopausal hObs express ERα and ERβ mRNA with higher abundance of ERα. Pre-treatment with the less-calcemic vitamin D analog JKF 1624F(2)-2 (JKF) upregulated responsiveness to estrogens via modulation of ERs expression. These estrogenic compounds induce the expression and activity of 25 hydroxy-vitamin D(3)-1α hydroxylase (1OHase). We now analyzed the effects of carboxy-genistein (cG), carboxy-biocainin A (cBA) and carboxy-daidzein (cD), of BA, D or G and of licorice derived compounds glabridin (Glb) and glabrene (Gla) and estradiol-17β (E(2)) on DNA synthesis, creatine kinase specific activity (CK), intracellular and membranal E(2) binding and their modulations by JKF in hObs. We also analyzed modulation by phytoestrogenic compounds of 1OHase mRNA expression and activity. We showed that: (1) all compounds stimulated DNA synthesis and CK. (2) JKF and all estrogenic compounds modulated ERα and ERβ mRNA expression. (3) Pre-treatment with JKF increased DNA synthesis and CK responses only to E(2), D, G and Gla. (4) JKF increased the intracellular competitive binding only of E(2), D and G. (5) JKF abolished the membranal binding of all protein-bound estrogens. (6) JKF and all estrogenic compounds except the protein-bound ones up-regulated 1OHase expression and activity. In conclusion phytoestrogens and their analogs increase DNA synthesis and CK, and lead to increased production of 1,25(OH)(2)D(3) in hObs, while pre-treatment with JKF modulates the effect of estrogenic compounds via regulation of ERs mRNA expression in a yet unclear mechanism.