Modulation of Intracellular Calcium Levels by Calcium Lactate Affects Colon Cancer Cell Motility through Calcium-Dependent Calpain

Cancer cell motility is a key phenomenon regulating invasion and metastasis. Focal adhesion kinase (FAK) plays a major role in cellular adhesion and metastasis of various cancers. The relationship between dietary supplementation of calcium and colon cancer has been extensively investigated. However, the effect of calcium (Ca²⁺) supplementation on calpain-FAK-motility is not clearly understood. We sought to identify the mechanism of FAK cleavage through Ca²⁺ bound lactate (CaLa), its downstream signaling and role in the motility of human colon cancer cells. We found that treating HCT116 and HT-29 cells with CaLa immediately increased the intracellular Ca²⁺ (iCa²⁺) levels for a prolonged period of time. Ca²⁺ influx induced cleavage of FAK into an N-terminal FAK (FERM domain) in a dose-dependent manner. Phosphorylated FAK (p-FAK) was also cleaved in to its p-N-terminal FAK. CaLa increased colon cancer cells motility. Calpeptin, a calpain inhibitor, reversed the effects of CaLa on FAK and pFAK cleavage in both cancer cell lines. The cleaved FAK translocates into the nucleus and modulates p53 stability through MDM2-associated ubiquitination. CaLa-induced Ca²⁺ influx increased the motility of colon cancer cells was mediated by calpain activity through FAK and pFAK protein destabilization. In conclusion, these results suggest that careful consideration may be given in deciding dietary Ca²⁺ supplementation to patient undergoing treatment for metastatic cancer.     

Direct Evidence for Changes in the Resistance of Legume Root Nodules to O2 Diffusion

Indirect evidence suggests that legumes can adjust rapidly theresistance of their root nodules to O₂ diffusion. Here we describeexperiments using O₂ specific micro-electrodes and dark fieldmicroscopy to study directly the operation of this diffusionbarrier. The O₂ concentration sensed by the electrode decreasedsharply in the region of the inner cortex and was less than1.0 mmol m^–3 throughout the infected tissue in nodulesof both pea (Pisum sativum) and french bean (Phaseolus vulgaris).In a number of experiments the ambient O₂ concentration wasincreased to 40% while the electrode tip was just inside theinner cortex. In 13 out of 21 cases the O₂ concentration atthis position either remained low and unchanged or increasedirreversibly to near ambient values. In the remaining casesthe O₂ concentration increased after 1 to 2.5 min and then decreasedto its former value. These results are ascribed to an increasein resistance of the barrier in response to increased O₂ fluxinto the nodule. It was shown microscopically that air spacesboth at the boundary between the infected zone and the innercortex, and within the infected zone started to disappear 3min after nodules were exposed to high ambient O₂ concentrationsand had disappeared completely after 8 min. These spaces werenot changed by exposure of the nodule for 10 min to either N₂or air. Key words: Oxygen, root nodules, air spaces     

Evolution of the 28S ribosomal RNA gene in anurans: regions of variability and their phylogenetic implications

Fifteen restriction sites were mapped to the 28S ribosomal RNA gene of individuals representing 54 species of frogs, two species of salamanders, a caecilian, and a lungfish. Eight of these sites were present in all species examined, and two were found in all but one species. Alignment of these conserved restriction sites revealed, among anuran 28S rRNA genes, five regions of major length variation that correspond to four of 12 previously identified divergent domains of this gene. One of the divergent domains (DD8) consists of two regions of length variation separated by a short segment that is conserved at least throughout tetrapods. Most of the insertions, deletions, and restriction-site variations identified in the 28S gene will require sequence-level analysis for a detailed reconstruction of their history. However, an insertion in DD9 that is coextensive with frogs in the suborder Neobatrachia, a BstEII site that is limited to representatives of two leptodactylid subfamilies, and a deletion in DD10 that is found only in three ranoid genera are probably synapomorphies.      

Levels of polyamines, glutathione and glutathione-spermidine conjugates during growth of the insect trypanosomatid Crithidia fasciculata

Levels of the polyamines spermidine and putrescine and the major intracellular thiols glutathione (GSH), glutathionylspermidine (GSH-SPD) and dihydrotrypanothione [bis-(glutathionyl)spermidine); T[SH]2] were measured by high performance liquid chromatography throughout the growth cycle of the insect trypanosomatid Crithidia fasciculata. The amount of total spermidine, putrescine and glutathione (free and conjugated to spermidine) was found to be elevated during growth. Of the total spermidine, 30 to 50% was found conjugated to glutathione during the exponential growth phase, increasing to 60 to 70% at stationary phase. T[SH]2 was the principal intracellular thiol during exponential growth (12.1 to 17.4 nmol per 10(8) cells), whereas GSH-SPD was the major thiol in stationary phase (26.2 nmol per 10(8) cells). GSH levels changed little during the growth cycle and represented a constant proportion (10 to 12%) of the total intracellular glutathione. On dilution of stationary phase cells into fresh medium, a rapid decrease in GSH-SPD levels was observed to be associated with synthesis of T[SH]2. This process reached 90% completion by 15 min, with steady state achieved by 120 min. As the total spermidine and glutathione pools did not increase during this interval, it could be calculated that this rapid redistribution of metabolites resulted in the release of 13 nmol per 10(8) cells unconjugated spermidine without de novo synthesis. This mechanism for rapidly elevating the intracellular concentration of free spermidine may be advantageous to this organism in rapidly adapting to favourable growth conditions.     

Phosphorylation states greatly regulate the activity and gating properties of Cav3.1 T-type Ca2+ channels

Ca_v3.1 T-type Ca²⁺ channels play pivotal roles in neuronal low-threshold spikes, visceral pain, and pacemaker activity. Phosphorylation has been reported to potently regulate the activity and gating properties of Ca_v3.1 channels. However, systematic identification of phosphorylation sites (phosphosites) in Ca_v3.1 channel has been poorly investigated. In this work, we analyzed rat Ca_v3.1 protein expressed in HEK-293 cells by mass spectrometry, identified 30 phosphosites located at the cytoplasmic regions, and illustrated them as a Ca_v3.1 phosphorylation map which includes the reported mouse Ca_v3.1 phosphosites. Site-directed mutagenesis of the phosphosites to Ala residues and functional analysis of the phospho-silent Ca_v3.1 mutants expressed in Xenopus oocytes showed that the phospho-silent mutation of the N-terminal Ser18 reduced its current amplitude with accelerated current kinetics and negatively shifted channel availability. Remarkably, the phospho-silent mutations of the C-terminal Ser residues (Ser1924, Ser2001, Ser2163, Ser2166, or Ser2189) greatly reduced their current amplitude without altering the voltage-dependent gating properties. In contrast, the phosphomimetic Asp mutations of Ca_v3.1 on the N- and C-terminal Ser residues reversed the effects of the phospho-silent mutations. Collectively, these findings demonstrate that the multiple phosphosites of Ca_v3.1 at the N- and C-terminal regions play crucial roles in the regulation of the channel activity and voltage-dependent gating properties.     

Microtubule-assisted mechanism for toxisome assembly in Fusarium graminearum

In Fusarium graminearum, a trichothecene biosynthetic complex known as the toxisome forms ovoid and spherical structures in the remodelled endoplasmic reticulum (ER) under mycotoxin-inducing conditions. Previous studies also demonstrated that disruption of actin and tubulin results in a significant decrease in deoxynivalenol (DON) biosynthesis in F. graminearum. However, the functional association between the toxisome and microtubule components has not been clearly defined. In this study we tested the hypothesis that the microtubule network provides key support for toxisome assembly and thus facilitates DON biosynthesis. Through fluorescent live cell imaging, knockout mutant generation, and protein–protein interaction assays, we determined that two of the four F. graminearum tubulins, α₁ and β₂ tubulins, are indispensable for DON production. We also showed that these two tubulins are directly associated. When the α₁–β₂ tubulin heterodimer is disrupted, the metabolic activity of the toxisome is significantly suppressed, which leads to significant DON biosynthesis impairment. Similar phenotypic outcomes were shown when F. graminearum wild type was treated with carbendazim, a fungicide that binds to microtubules and disrupts spindle formation. Based on our results, we propose a model where α₁–β₂ tubulin heterodimer serves as the scaffold for functional toxisome assembly in F. graminearum.     

The adhesion of thin carbon films to metallic substrates.

As part of the development of carbon-coated prosthetic devices, the adhesion of thin carbon films to metallic substrates has been studied. The bond strength of carbon films about 5000 A thick on Ti-6A1-4V and stainless steel was measured in a pull test and found to be greater than 4700 psi. Auger electron spectroscopy showed a reactive film/substrate interface. The ultimate bond strength was found to be dependent on the substrate and the deposition parameters.     

Characterization of non-canonical G beta-like protein FvGbb2 and its relationship with heterotrimeric G proteins in Fusarium verticillioides

Fusarium verticillioides is a fungal pathogen that is responsible for maize ear rot and stalk rot diseases worldwide. The fungus also produces carcinogenic mycotoxins, fumonisins on infested maize. Unfortunately, we still lack clear understanding of how the pathogen responds to host and environmental stimuli to trigger fumonisin biosynthesis. The heterotrimeric G protein complex, consisting of canonical Gα, Gβ and Gγ subunits, is involved in transducing signals from external stimuli to regulate downstream signal transduction pathways. Previously, we demonstrated that Gβ protein FvGbb1 directly impacts fumonisin regulation but not other physiological aspects in F. verticillioides. In this study, we identified and characterized a RACK1 (Receptor for Activated C Kinase 1) homolog FvGbb2 as a putative Gβ-like protein in F. verticillioides. The mutant exhibited severe defects not only in fumonisin biosynthesis but also vegetative growth and conidiation. FvGbb2 was positively associated with carbon source utilization and stress agents but negatively regulated general amino acid control. While FvGbb2 does not interact with canonical G protein subunits, it may associate with diverse proteins in the cytoplasm to regulate vegetative growth, virulence, fumonisin biosynthesis and stress response in F. verticillioides.