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.     

Stimulated indole alkaloid release from Catharanthus roseus immobilized cultures. Initial studies.

Vacuolar sequestration of valuable secondary metabolites remains the major limitation to the use of immobilization technology for large scale plant-cell-based bioprocesses, which otherwise may be a more efficient culture system than suspension for this biomass. In this initial study, the release of indole alkaloids produced by immobilized Catharanthus roseus cells cultured in Zenk's Alkaloid Production Medium was evaluated. Unstimulated alkaloid release in immobilized cultures reached levels of 10 to 50% of total production or 3 to 100% of known alkaloid content (30 to 4700 micrograms l-1), which was higher than that found for suspension cultures of the cell line used (10 to 25% of total production) without apparent cell lysis. Modifications of the medium pH value of immobilized cultures were explored in order to improve this release. Periodical additions of acid (HCl 0.1 N) or base (KOH 0.1 N) solutions (2% v/v) to different cultures resulted in rapid (less than 3 h) and transient variations in extracellular pH value from 5.5 to 4.3, and 5.8 to 8.5, respectively. In both cases, these variations provoked significant increase in total alkaloid (from approximately 5-10 mg l-1 to 15 mg l-1), ajmalicine (from 0 to approximately 0.29 mg l-1) and serpentine (from 0 to approximately 0.20 mg l-1) release, without apparent cell lysis or decrease in the culture viability. This product release was estimated to represent 100% of alkaloids produced.     

Glycine betaine and proline are the principal compatible solutes ofStaphylococcus aureus

The foodborne pathogenStaphylococcus aureus is distinguished by its ability to grow within environments of extremely high osmolarity (e.g., foods with low water activity values). In the present study, we examined the accumulation of intracellular organic solutes withinS. aureus strain ATCC 12600 when cells were grown in a complex medium containing high concentrations of NaCl. Consistent with previous reports [Measures JC (1975) Nature 257:398–400; Koujima I, et al. (1978) Appl Environ Microbiol 35:467–470; and Anderson CB, Witter LD (1982) Appl Environ Microbiol 43:1501–1503], intracellular proline was found to accumulate to high concentrations. However, NMR spectroscopy of cell extracts revealed glycine betaine to be the predominant intracellular organic solute accumulated within cells grown at high osmolarity. In additional experiments, we examined the growth rate ofS. aureus in a defined medium of high osmolarity and found it to be stimulated significantly by the presence of either exogenous proline or glycine betaine. Highest growth rates were obtained when the defined medium was supplemented with glycine betaine.     

The fermentation process for l-phenylalanine production using an auxotrophic regulatory mutant of Escherichia coli

Summary A process for l-phenylalanine production was studied using a tyrosine auxotrophic regulatory mutant of Escherichia coli, resistant to both -2-thienyl-dl-alanine and p-fluoro-dl-phenylalanine. Fermentations were carried out in a 30-1 fermentor with intermittent feeding of glucose plus phosphate. The mutant accumulated l-phenylalanine in the fermentation broth up to 15 g/l at pH 7.0 and 33°C. Column chromatography on a strong cation exchanger was employed as the most effective step in the purification of l-phenyl-alanine from the broth. This step brought about 4-fold concentration of the product with 96% recovery.     

Pilot-scale production of l-phenylalanine from d-glucose

Effects of inoculum size and total sugar content on both l-phenylalanine productivity and titre have been investigated using a tyrosine auxotrophic regulatory mutant of Escherichia coli. Fermentations were carried out in a 500 litre pilot fermenter with intermittent feeding of d-glucose plus phosphate. It was found that the productivity was not greatly affected by inoculum size. However, the l-phenylalanine titre was significantly affected by total sugar content. Relatively high productivities of up to 0.35–0.40 g l-phenylalanine l^?1 h^?1 have been achieved at l-phenylalanine titres of 14–15 g l^?1.     

Partial characterization of anEscherichia coll strain harboring a xylanase encoding plasmid

Summary The plasmid pRH271, harboring a xylanase gene cioned fromBacilius subtilis, has been transferred into a mutant ofE. coli SK2284 which allowed the release of part of the xylanase in the culture supernatant. Kinetic parameters of this recombinantE. coll strain were determined in microscale batch culture with and without the selective pressure of antibiotics. No significant difference in µ_max was observed for the nontransformedE. coli strain when compared to the recombinant strain. However, K₅ values for glucose were two times higher in the case of the recombinant strain. Preliminary study of xylanase production in a large batch farmenter was also described.     

Anatomical variation of orthotropic elastic moduli of the proximal human tibia

The anatomical variation of orthotropic elastic moduli of the cancellous bone from three human proximal tibiae was investigated using an ultrasonic technique. With this technique, it was possible to measure three orthogonal elastic moduli and three shear moduli from cubic specimens of cancellous bone as small as 8 mm per side. Correlation with mechanical tensile testing has shown this technique to offer a precise measure of cancellous modulus (Eten = 0.94Eult + 144.6 MPa, r2 = 0.96, n = 34). The cancellous bone of the proximal tibia was found to be very inhomogeneous, with the axial modulus ranging between 340 and 3350 MPa. A course map is presented, showing measured Young's moduli as a function of anatomical position. The anisotropy of the cancellous bone, determined by the relative differences between the three orthogonal moduli, was shown to be relatively constant over the entire range of cancellous densities tested. The relationship between the axial elastic modulus and the apparent density was found to be approximately linear, as reported by others for proximal tibial cancellous bone.     

Comparison of lodging safety factor of untreated and succinic Acid 2,2-dimethylhydrazide-treated shoots of mulberry tree

This study examined the lodging resistance of mulberry tree (Morus bombycis Koidz. cv Kenmochi) shoots treated or not treated with succinic acid 2,2-dimethylhydrazide (SADH). The lodging safety factor, an indicator of lodging resistance, was defined as the ratio of critical lodging load to the leaf fresh weight observed, provided that the distribution of the critical lodging load along the stem was similar to that of the leaf fresh weight observed. The critical lodging load was experimentally estimated by loading weights onto the stems. In the untreated trees, the lodging safety factor was maintained at about 3.2. In the SADH-treated trees, the stem elongation was inhibited to about 80% of that in the untreated trees, and the percentage of shoot dry matter partitioned into the leaves was always larger than that of the untreated trees. This dwarfing of the stem caused by SADH increased the critical lodging load supported by the unit stem dry weight, while this large investment of materials in leaves increased the leaf fresh weight supported by the unit stem dry weight. Since the increments canceled each other, the lodging safety factor of the SADH-treated shoots was similar to that of the untreated ones. These results suggest that the shoot formation of the mulberry tree is controlled to maintain the lodging safety factor at a constant level.