A role of autophagy in PTP4A3-driven cancer progression

Autophagy, a “self-eating” cellular process, has dual roles in promoting and suppressing tumor growth, depending on cellular context. PTP4A3/PRL-3, a plasma membrane and endosomal phosphatase, promotes multiple oncogenic processes including cell proliferation, invasion, and cancer metastasis. In this study, we demonstrate that PTP4A3 accumulates in autophagosomes upon inhibition of autophagic degradation. Expression of PTP4A3 enhances PIK3C3-BECN1-dependent autophagosome formation and accelerates LC3-I to LC3-II conversion in an ATG5-dependent manner. PTP4A3 overexpression also enhances the degradation of SQSTM1, a key autophagy substrate. These functions of PTP4A3 are dependent on its catalytic activity and prenylation-dependent membrane association. These results suggest that PTP4A3 functions to promote canonical autophagy flux. Unexpectedly, following autophagy activation, PTP4A3 serves as a novel autophagic substrate, thereby establishing a negative feedback-loop that may be required to fine-tune autophagy activity. Functionally, PTP4A3 utilizes the autophagy pathway to promote cell growth, concomitant with the activation of AKT. Clinically, from the largest ovarian cancer data set (GSE 9899, n = 285) available in GEO, high levels of expression of both PTP4A3 and autophagy genes significantly predict poor prognosis of ovarian cancer patients. These studies reveal a critical role of autophagy in PTP4A3-driven cancer progression, suggesting that autophagy could be a potential Achilles heel to block PTP4A3-mediated tumor progression in stratified patients with high expression of both PTP4A3 and autophagy genes.     

Novel synthetic signal peptides for the periplasmic secretion of green fluorescent protein in Escherichia coli

New secretion vectors containing synthetic signal peptides were constructed to study the periplasmic translocation of green fluorescent protein (GFP) in Escherichia coli. These constructs encode synthetic signal peptides spA and spD fused to the amino terminal end of GFP, and expressed from T7/lac promoter in the BL21DE3 strain by induction with IPTG. The recombinant protein was detected in both the cytoplasmic and periplasmic fractions. Fluorescence analysis revealed that recombinant proteins with signal peptides were not fluorescent, indicating translocation to periplasmic space. In contrast, recombinant proteins without signal peptide were fluorescent. These results indicate that the expressed recombinant proteins were translocated into the periplasm. Therefore, the synthetic signal peptides derived from signal peptides of Bacillus sp. could efficiently secrete the heterologous proteins to the periplasmic space of E. coli.     

Perfringolysin O, a cholesterol-binding cytolysin, as a probe for lipid rafts

Gaining an understanding of the structural and functional roles of cholesterol in membrane lipid rafts is a critical issue in studies on cellular signaling and because of the possible involvement of lipid rafts in various diseases. We have focused on the potential of perfringolysin O (theta-toxin), a cholesterol-binding cytolysin produced by Clostridium perfringens, as a probe for studies on membrane cholesterol. We prepared a protease-nicked and biotinylated derivative of perfringolysin O (BCtheta) that binds selectively to cholesterol in cholesterol-rich microdomains of cell membranes without causing membrane lesions. Since the domains fulfill the criteria of lipid rafts, BCtheta can be used to detect cholesterol-rich lipid rafts. This is in marked contrast to filipin, another cholesterol-binding reagent, which binds indiscriminately to cell cholesterol. Using BCtheta, we are now searching for molecules that localize specifically in cholesterol-rich lipid rafts. Recently, we demonstrated that the C-terminal domain of perfringolysin O, domain 4 (D4), possesses the same binding characteristics as BCtheta. BIAcore analysis showed that D4 binds specifically to cholesterol with the same binding affinity as the full-size toxin. Cell-bound D4 is recovered predominantly from detergent-insoluble, low-density membrane fractions where raft markers, such as cholesterol, flotillin and Src family kinases, are enriched, indicating that D4 also binds selectively to lipid rafts. Furthermore, a green fluorescent protein-D4 fusion protein (GFP-D4) was revealed to be useful for real-time monitoring of cholesterol in lipid rafts in the plasma membrane. In addition, the expression of GFP-D4 in the cytoplasm might allow the investigations of intracellular trafficking of lipid rafts. The simultaneous visualization of lipid rafts in plasma membranes and inside cells might help in gaining a total understanding of the dynamic behavior of lipid rafts.     

High-frequency adventitious shoots regeneration from leaf of non-heading Chinese cabbage (<Emphasis Type="Italic">Brassica campestris</Emphasis> ssp. <Emphasis Type="Italic">chinensis</Emphasis>) cultured in vitro

The objective of the present work was selection of cultivar and suitable medium for regenerating shoots from leaf segments of non-heading Chinese cabbage. We evaluated six types of supplemented media with 2.0, 5.0 and 10.0 mg l⁻¹ 6-BA; 1.0 and 2.0 mg l⁻¹ TDZ; 0.1, 0.3, 0.5, 0.8 and 1.0 mg l⁻¹NAA; 3.0, 5.0 and 7.5 mg l⁻¹AgNO₃; 0.01 mg l⁻¹ 2–4, D and 4.0 mg l⁻¹ KT for shoot regeneration and six cultivars “Sanchidaye”, “Liuchuandasuomian”, “Qingyou 4”, “Liangbaiye”, “AiKang 5” and “Hanxiao F3”, furthermore for root formation three types of supplemented media with 0.2, 0.3, 0.5 mg l⁻¹ NAA, and for survival rate two types of base media: turf + vermiculite + manure (1:2:0.2) and soil + vermiculite (1:2). Culturing leaf segments on MS medium supplemented with 2 mg l⁻¹ TDZ; 0.5 mg l⁻¹ NAA and 7.5 mg l⁻¹ AgNO₃ gave the highest number of shoots per leaf segment (66) while roots were best formed on the medium supplemented with 0.2 mg l⁻¹ NAA. Survival rate was highest (61.6%) in the turf: vermiculite: manure (1:2:0.2) medium. The highest percentage of responding leaf segments, number of shoots per leaf segment, rooting percentage and survival rate were observed in “Liuchuandasuomian”. The plantlets were transferred to the soil and grown into mature plants in pots. These results could be used for preliminary selections of cultivars to transfer disease resistance (Bt) gene through agrobacterium in non-heading Chinese cabbage.     

Patterns of ion excretion and survival in two stoloniferous arid zone grasses

Desert plants show specific mechanisms to thrive under prevailing harsh conditions. To study the survival mechanism(s) in native desert plant species, Lesser Cholistan desert in Pakistan was surveyed and two potential salt secretory grass species, Aeluropus lagopoides and Ochthochloa compressa , were selected from five saline sites. Both these grasses responded differentially to saline environments by showing specialized mechanisms of survival including excretion of toxic ions through trichomes, vesicular and glandular hairs through leaf surface. In A. lagopoides , salt tolerance was associated with excreted Na⁺ concentration through leaf surface and accumulation of useful ions like Ca²⁺ and K⁺ in the shoot. Contrarily, O. compressa excreted all the ions through leaves without discriminating among toxic or beneficial ions. Results suggested that A. lagopoides was more successfully adapted to saline desert environments than O. compressa by excretion of excessive toxic ions and retention of Ca²⁺ and K⁺ in the shoot. This appears to be an adaptive character of the former species to successfully thrive in harsh desert conditions.     

<Emphasis Type="Italic">Burkholderia cenocepacia</Emphasis>: a new biocatalyst for efficient bioreduction of ezetimibe intermediate

Ezetimibe is a selective acyl-coenzyme A: cholesterol acyltransferase (ACAT) inhibitor used in hypercholesterolemia. Synthesis of ezetimibe requires enantiopure 3-[5-(4-fluorophenyl)-5(S)-hydroxypentanoyl]-4(S)-4-phenyl-1,3-oxazolidin-2-one (FOP alcohol) as a crucial intermediate which is produced by reduction of the corresponding prochiral ketone (FOP dione). A new biocatalyst from acclimatized soil was screened for bioreduction of the above prochiral ketone. The microorganism was identified by 16S mRNA sequencing, as Burkholderia cenocepacia. Various physicochemical conditions were optimized to increase cellmass and enzyme activity. The overall increase in cellmass concentration and enzyme activity was 2.06 and 1.85-fold, respectively. Various reaction conditions, for example pH, temperature, agitation, and cellmass concentration, were optimized for maximum product yield (chiral alcohol) with excellent enantioselectivity. Best reduction was achieved in phosphate buffer (50 mM, pH 8.0) at 40°C (200 rpm) and the yield of enantiopure alcohol from the corresponding prochiral ketone was 54%. This biocatalyst was also used for the reduction of various other prochiral ketones.     

The microscopic biological response of human chondrocytes to bovine bone scaffold

This study was performed to determine the microscopic biological response of human nasal septum chondrocytes and human knee articular chondrocytes placed on a demineralized bovine bone scaffold. Both chondrocytes were cultured and seeded onto the bovine bone scaffold with seeding density of 1 × 105 cells per 100 μl/scaffold and incubated for 1, 2, 5 and 7 days. Proliferation and viability of the cells were measured by mitochondrial dehydrogenase activity (MTT assay), adhesion study was analyzed by scanning electron microscopy and differentiation study was analyzed by immunofluorescence staining and confocal laser scanning electron microscopy. The results showed good proliferation and viability of both chondrocytes on the scaffolds from day 1 to day 7. Both chondrocytes increased in number with time and readily grew on the surface and into the open pores of the scaffold. Immunofluorescence staining demonstrated collagen type II on the scaffolds for both chondrocytes. The results showed good cells proliferation, attachment and maturity of the chondrocytes on the demineralized bovine bone scaffold. The bovine bone being easily resourced, relatively inexpensive and non toxic has good potential for use as a three dimensional construct in cartilage tissue engineering.