Induction of autophagy by drug-resistant esophageal cancer cells promotes their survival and recovery following treatment with chemotherapeutics

We investigated the cell-death mechanisms induced in esophageal cancer cells in response to the chemotherapeutic drugs, 5-fluorouracil (5-FU) and cisplatin. Chemosensitive cell lines exhibited apoptosis whereas chemoresistant populations exhibited autophagy and a morphology resembling type II programmed cell death (PCD). Cell populations that respond with autophagy are more resistant and will recover following withdrawal of the chemotherapeutic agents. Specific inhibition of early autophagy induction with siRNA targeted to Beclin 1 and ATG7 significantly enhanced the effect of 5-FU and reduced the recovery of drug-treated cells. Pharmacological inhibitors of autophagy were evaluated for their ability to improve chemotherapeutic effect. The PtdIns 3-kinase inhibitor 3-methyladenine did not enhance the cytotoxicity of 5-FU. Disruption of lysosomal activity with bafilomycin A 1 or chloroquine caused extensive vesicular accumulation but did not improve chemotherapeutic effect. These observations suggest that an autophagic response to chemotherapy is a survival mechanism that promotes chemoresistance and recovery and that selective inhibition of autophagy regulators has the potential to improve chemotherapeutic regimes. Currently available indirect inhibitors of autophagy are, however, ineffective at modulating chemosensitivity in these esophageal cancer cell lines.     

Synergistic anti-cancer effects via co-delivery of TNF-related apoptosis-inducing ligand (TRAIL/Apo2L) and doxorubicin using micellar nanoparticles

The use of small molecule drugs in cancer chemotherapy has mostly been limited by dose-dependent toxicity and development of drug resistance resulting from repeated administrations. To overcome such problems, efforts have been made to develop drug delivery systems that can bear multiple therapeutic agents in one system. The purpose of this study is to deliver human tumor necrosis factor (TNF)-related apoptosis-inducing ligand (Apo2L/TRAIL) and doxorubicin (Dox, an anti-cancer drug) with micellar nanoparticles self-assembled from a biodegradable cationic copolymer P(MDS-co-CES) to achieve synergistic cytotoxic effects in cancer cells. Exogenously expressed TRAIL using recombinant methods shows great potential in cancer therapy as it induces cell death selectively in cancer cells with limited toxicity to normal tissues. Dox-loaded nanoparticles and TRAIL formed stable nanocomplexes with a size of ～ 225 nm and zeta potential of ～ 70 mV. Effects of nanocomplexes on both wild type and TRAIL-resistant SW480 colorectal carcinoma cells were investigated. The assemblies of Dox and TRAIL with P(MDS-co-CES) nanoparticles were efficiently delivered to cancer cells. Receptor-blocking studies showed that the nanocomplexes entered cells via death receptor-mediated endocytosis. Synergism in cell death induction was analysed by the isobologram method to study drug interactions. Cytotoxicity of the nanocomplexes to non-cancerous cells was significantly lower than cancerous cells. Anti-proliferative effects of nanocomplexes were retained in remaining cancer cells in long-term cultures after treatment with the nanocomplexes. In summary, this Dox and TRAIL co-delivery system can be a promising candidate for cancer treatment.     

Seasonal plant water uptake patterns in the saline southeast Everglades ecotone

The purpose of this study was to determine the seasonal water use patterns of dominant macrophytes coexisting in the coastal Everglades ecotone. We measured the stable isotope signatures in plant xylem water of Rhizophora mangle, Cladium jamaicense, and Sesuvium portulacastrum during the dry (DS) and wet (WS) seasons in the estuarine ecotone along Taylor River in Everglades National Park, FL, USA. Shallow soilwater and deeper groundwater salinity was also measured to extrapolate the salinity encountered by plants at their rooting zone. Average soil water oxygen isotope ratios (δ ¹⁸O) was enriched (4.8 ± 0.2‰) in the DS relative to the WS (0.0 ± 0.1‰), but groundwater δ ¹⁸O remained constant between seasons (DS: 2.2 ± 0.4‰; WS: 2.1 ± 0.1‰). There was an inversion in interstitial salinity patterns across the soil profile between seasons. In the DS, shallow water was euhaline [i.e., 43 practical salinity units (PSU)] while groundwater was less saline (18 PSU). In the WS, however, shallow water was fresh (i.e., 0 PSU) but groundwater remained brackish (14 PSU). All plants utilized 100% (shallow) freshwater during the WS, but in the DS R. mangle switched to a soil–groundwater mix (δ 55% groundwater) while C. jamaicense and S. portulacastrum continued to use euhaline shallow water. In the DS, based on δ ¹⁸O data, the roots of R. mangle roots were exposed to salinities of 25.4 ± 1.4 PSU, less saline than either C. jamaicense (39.1 ± 2.2 PSU) or S. portulacastrum (38.6 ± 2.5 PSU). Although the salinity tolerance of C. jamaicense is not known, it is unlikely that long-term exposure to high salinity is conducive to the persistence of this freshwater marsh sedge. This study increases our ecological understanding of how water uptake patterns of individual plants can contribute to ecosystem levels changes, not only in the southeast saline Everglades, but also in estuaries in general in response to global sea level rise and human-induced changes in freshwater flows.     

Carbon demand,utilization, and metabolic diversity of bacterioplankton in the frontal regimes of the Indian sector of the Southern Ocean

Bacterial production, respiration and metabolic diversity were measured up to 120 m depth in the Sub-Antarctic Front (SAF) and Polar Fronts I and II (PFI and PFII) of the Indian Ocean sector of the Southern Ocean during 2010 Austral Summer. Prokaryotic cell count was maximum at PFI and PFII (~109 cells L−1) and minimum at SAF (~107 cells L−1). Furthermore, integrated bacterial production was higher at PFI (1.07 mg C m−2 h−1) and PFII (0.72 mg C m−2 h−1) compared to SAF (0.61 mg C m−2 h−1). At PFII, integrated bacterial growth efficiency was higher (8.96) compared to PFI (7.42) and SAF (7.17), signifying that the net contribution of PFII to the microbial loop could be relatively pronounced. Enhanced cell numbers and production at polar fronts indicate that the dissolved organic matter could be converted to secondary biomass through the microbial loop. However, integrated bacterial respiration rate at PFII (0.83 mg C m−2 h−1) was lower than that at PFI (1.84 mg C m−2 h−1) resulting in higher growth efficiency at PFII. Metabolic flexibility at SAF was clearly brought about by utilization of carboxylic acids like D-malic acid and itaconic acid, and carbohydrates like N-acetyl D-glucosamine, D-cellobiose and D-lactose. Utilization of amino acids like glycyl L-glutamic acid and L-threonine, and an amine, phenylethylamine, was critical in determining the metabolic variability at PFI. PFII hosted microbes that utilized phenolic compounds (2-hydroxy benzoic acid and 4-hydroxy benzoic acid) and polymers (like Tween 80). Utilization of polyols over carbohydrates in polar waters indicates a niche with lesser influence of the Antarctic melt waters on the bacterioplankton metabolism.     

Co-option of an oral-aboral patterning mechanism to control left-right differentiation: the direct-developing sea urchin Heliocidaris erythrogramma is sinistralized, not ventralized, by NiCl2

Larval dorsoventral (DV) and left-right (LR) axial patterning unfold progressively in sea urchin development, leading to commitment of the major embryonic regions by the gastrula stage. The direct-developing sea urchin Heliocidaris erythrogramma has lost oral-aboral differentiation along the DV axis but has accelerated vestibular ectoderm development on the left side. NiCl(2) radializes indirect-developing sea urchins by shifting cells toward a ventral fate (oral ectoderm). We treated embryos of H. erythrogramma and the indirect-developing H. tuberculata with NiCl(2). H. tuberculata was ventralized exactly like other indirect developers, establishing that basic patterning mechanisms are conserved in this genus. H. erythrogramma was also radialized; timing, dosage response, and some morphological features were similar to those in other sea urchins. Ectodermal explant and recombination experiments demonstrate that the effect of nickel is autonomous to the ectoderm, another feature in common with indirect developers. However, H. erythrogramma is distinctly sinistralized rather than ventralized, its cells shifting toward a left-side fate (vestibular ectoderm). This geometric contrast in the midst of pervasive functional similarity suggests that nickel-sensitive processes in H. erythrogramma axial patterning, homologous to those in indirect developers, have been redeployed, and hence co-opted, from their ancestral role in DV axis determination to a new role in LR axis determination. We discuss DV and LR axial patterning and their evolutionary transformation.     

Biosynthesis of the anti‐diabetic metabolite montbretin A: glucosylation of the central intermediate mini‐MbA

Type 2 diabetes (T2D) affects over 320 million people worldwide. Healthy lifestyles, improved drugs and effective nutraceuticals are different components of a response against the growing T2D epidemic. The specialized metabolite montbretin A (MbA) is being developed for treatment of T2D and obesity due to its unique pharmacological activity as a highly effective and selective inhibitor of the human pancreatic α‐amylase. MbA is an acylated flavonol glycoside found in small amounts in montbretia (Crocosmia × crocosmiiflora) corms. MbA cannot be obtained in sufficient quantities for drug development from its natural source or by chemical synthesis. To overcome these limitations through metabolic engineering, we are investigating the genes and enzymes of MbA biosynthesis. We previously reported the first three steps of MbA biosynthesis from myricetin to myricetin 3‐O‐(6′‐O‐caffeoyl)‐glucosyl rhamnoside (mini‐MbA). Here, we describe the sequence of reactions from mini‐MbA to MbA, and the discovery and characterization of the gene and enzyme responsible for the glucosylation of mini‐MbA. The UDP‐dependent glucosyltransferase CcUGT3 (UGT703E1) catalyzes the 1,2‐glucosylation of mini‐MbA to produce myricetin 3‐O‐(glucosyl‐6′‐O‐caffeoyl)‐glucosyl rhamnoside. Co‐expression of CcUGT3 with genes for myricetin and mini‐MbA biosynthesis in Nicotiana benthamiana validated its biological function and expanded the set of genes available for metabolic engineering of MbA.     

Enhanced circulating half-life and antitumor activity of a site-specific pegylated interferon-alpha protein therapeutic

Recombinant interferon alpha-2 (IFN-alpha2) has proven useful for treating a variety of human cancers and viral diseases. IFN-alpha2 has a short circulating half-life in vivo, which necessitates daily or thrice weekly administration to patients. It is possible to extend the circulating half-life of IFN-alpha2 by random modification of lysine residues in the protein with polyethylene glycol (PEG); however, such preparations have heterogeneous structures and low specific activities, and may not provide optimal therapeutic benefits to patients. A long-acting, site-specific, monoPEGylated IFN-alpha2 protein has now been created by targeted attachment of a 20 kDa or a 40 kDa maleimide-PEG to a cysteine analogue of IFN-alpha2, M111C. In vitro bioactivities of the purified 20 kDa and 40 kDa PEG-M111C proteins were within 2- to 3-fold of those of wild type IFN-alpha2 and 7- to 10-fold better than that of a 40 kDa PEG IFN-alpha2 protein created using nontargeted, amine-PEGylation methodology. The 20 kDa and 40 kDa PEG-M111C proteins demonstrated 26- to 38-fold longer half-lives, respectively, than IFN-alpha2 following subcutaneous administration to rats. The 20 kDa PEG M111C protein inhibited growth of human NIH:OVCAR-3 cells transplanted into nude mice by >90%, as measured by tumor size, tumor weight, and number of animals with detectable tumors at necropsy, and was significantly more effective than a comparable dose of IFN-alpha2. These data extend our previous findings that bioactivity of IFN-alpha2 can be largely preserved by targeted attachment of PEG moieties to nonessential sites in the protein and provide evidence that site-specific PEGylated IFN-alpha2 proteins possess enhanced tumoricidal properties in vivo.     

Binding of simple carbohydrates and some N-acetyllactosamine-containing oligosaccharides to Erythrina cristagalli agglutinin as followed with a fluorescent indicator ligand

Erythrina cristagalli agglutinin, a dimeric lectin [J. L. Iglesias, et al. (1982) Eur. J. Biochem.123, 247–252] was shown by equilibrium dialysis to be bivalent for 4-methylumbelliferyl-β-d-galactoside. Upon binding to the lectin, this ligand showed a difference absorption spectrum with two maxima (at 322 and 336 nm) of equal intensity (Δ? = 1.2 × 10³m^?1 cm^?1). A similar spectrum with a comparable value of Δ? was obtained with 4-methylumbelliferyl-N-acetyl-β-d-galactosaminide. Binding of methyl-α-d-galactoside, lactose, and N-acetyllactosamine all produced small but equally intense protein difference spectra with a maximum (Δ? = 2.8 × 10² M^?1 cm^?1) at 291.6 nm. Upon binding of N-dansyl-d-galactosamine to the lectin, there was a fivefold increase in fluorescence intensity of this ligand. The association constant for N-dansyl-d-galactosamine was caused by a very favorable ΔS° of the dansyl group without affecting the strictly carbohydrate-specific character of binding. N-Dansyl-d-galactosamine was employed as a fluorescent indicator ligand in substitution titrations. This involved the use of simple carbohydrates, N-acetyllactosamine, and oligosaccharides which occur in the carbohydrate units of N-glycoproteins; the latter were Gal(β → 4)GlcNAc(β1 → 2)Man, Gal(β1 → 4)GlcNAc(β1 → 6)Man, and Gal(β1 → 4)GlcNAc(β1 → 6)[Gal(β1 → 4)GlcNAc(β1 → 2)]Man. The titrations were performed at two temperatures to determine the thermodynamic parameters. In the series N-acetyl-d-galactosamine, methyl-α-d-galactoside, and lactose, ?ΔH° increased from 24 to 41 kJ mol^?1; it increased further for N-acetyllactosamine and then remained unchanged for the N-acetyllactosamine-containing oligosaccharides (55 ± 1 kJ mol^?1). This indicated that the site specifically accommodated the disaccharide structure with an important contribution of the 2-acetamido group in the penultimate sugar. Beyond this, no additional contacts seemed to be formed. This conclusion also followed from considerations of ΔS° values which became more unfavorable in the above series (?23 to ?101 ± 4 J mol^?1 K^?1); the most negative value of ΔS° was observed with N-acetyllactosamine and the three N-acetyllactosamine-containing oligosaccharides.     

Patterns of female dominance in Propithecus diadema edwardsi of Ranomafana National Park, Madagascar

Many lemur species are characterized by some form of female dominance, ranging from female feeding priority to complete female dominance, although this is a rare trait in primates and other mammals. The status of the Milne-Edwards' sifaka (Propithecus diadema edwardsi), a diurnal lemur, is ambiguous. Some short-term studies have found little or no aggression. The aim of the current, long-term study was to quantify the intersexual-dominance patterns of this sifaka. The distribution, outcome, and context of aggressive interactions were studied in four groups of wild sifakas. The majority of intersexual aggressive interactions were decided, with the loser expressing submissive behavior. Intersexual aggressive interactions occurred in all social contexts, and within all social contexts the females won the vast majority (92.7-96.0%) of aggressive interactions. While aggression rates were low (0.22/hr), this evidence suggests female dominance. We propose that female dominance exists because it provides a fitness advantage to both males and females.