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.     

Genetic diversity and population structure of the gizzard shad,Konosirus punctatus (Clupeidae,Pisces), in Korean waters based on mitochondrial DNA control region sequences

In this study, we clarified the population structure of the gizzard shad, Konosirus punctatus, in Korean waters. We analyzed 896 base pairs of the mitochondrial DNA control region in 182 individuals, which were sampled from eight localities between the East Sea and the Yellow Sea. The haplotype diversity (h) was very high (0.9662–1.0000) but the nucleotide diversity (π) was very low (0.0061–0.0434). A neighbor-joining tree showed that the population clustered into two reciprocal monophyletic groups, lineages A and B. Lineage A is distributed on all coasts of Korea, from the Yellow Sea to the East Sea, declining to the east, whereas lineage B is distributed in the East Sea and Korea Strait, disappearing completely from middle Korea Strait to the west. Analysis of molecular variance showed strong structuring (F _ST = 0.856; P < 0.0001) between the two lineages. Neutrality tests and mismatch distribution analyses showed that a recent rapid expansion event occurred only in lineage A. Our results suggest that the management unit of the Korean gizzard shad may be divided in two, lineage A and lineage B.     

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.     

Variations in and Inheritance of NS-Glycoprotein in Self-Incompatible Brassica campestris L.

The stigma of Brassica species contain NS-glycoproteins thatexhibit a high degree of structural homology to the S-glycoproteinsof self-incompatibility. Inheritance of and variations in theNS-glycoprotein were studied with reference to self-incompatibility.The detection of NS-glycoproteins was performed by cross-reactionwith an antiserum raised against a purified NS-glycoprotein.In B. campestris, four isoforms of the NS-glycoprotein weredifferentiated by their pI values, but their molecular weightswere identical to one another. The genes for these isoformsof NS-glycoprotein were controlled by alleles at a single locus,tentatively named the NS allele, which was independent of Salleles at both the protein and the DNA level. Segregation ofF₂ plants with respect to the self-incompatibility behaviorof pollen tubes can be explained by the S allele model, butit appears not to be affected by the NS alleles. NS-glycoproteinswere found in all 21 species of Brassica and its allies examinedto date. The pI values of these glycoproteins varied among differentspecies. In addition to the isoforms of the NS alleles, maturestigmas contained other groups of proteins that reacted weaklywith the antiserum against the NS-glycoprotein. (Received July 30, 1991; Accepted February 21, 1992)     

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.