Properties and Characteristics of Multi-Layered Films from Tilapia Skin Gelatin and Poly(Lactic Acid)

Multi-layered films based on tilapia skin gelatin and poly(lactic acid) (PLA) were characterized, in comparison with the control gelatin and PLA films. Three different layers of multi-layered films (PLA/Gelatin/PLA) were visualized by scanning electron microscopic (SEM) analysis. The synergetic effect of lamination was evidenced by the increased mechanical properties (P < 0.05). Multi-layered films had higher water vapor barrier property and water resistance, compared to control gelatin film (P < 0.05). Gelatin films showed increased lightness (L*) with coincidental decrease in total color difference (?E*) in the presence of PLA layers (P < 0.05). Transparency and solubility of films decreased with increasing ratio of PLA (P < 0.05). In addition, multi-layered films showed the enhanced hydrophobicity and thermal stability as evidenced by increased water contact angle and degradation temperature, respectively. Thus, PLA/Gelatin/PLA multi-layered film with improved water vapour barrier property could serve as bio-degradable packaging material for wider applications.     

PHYLOGEOGRAPHY OF THE ASIAN ELEPHANT (ELEPHAS MAXIMUS) BASED ON MITOCHONDRIAL DNA

Abstract Populations of the Asian elephant (Elephas maximus) have been reduced in size and become highly fragmented during the past 3000 to 4000 years. Historical records reveal elephant dispersal by humans via trade and war. How have these anthropogenic impacts affected genetic variation and structure of Asian elephant populations? We sequenced mitochondrial DNA (mtDNA) to assay genetic variation and phylogeography across much of the Asian elephant's range. Initially we compare cytochrome b sequences (cyt b) between nine Asian and five African elephants and use the fossil‐based age of their separation (～5 million years ago) to obtain a rate of about 0.013 (95% CI = 0.011–0.018) corrected sequence divergence per million years. We also assess variation in part of the mtDNA control region (CR) and adjacent tRNA genes in 57 Asian elephants from seven countries (Sri Lanka, India, Nepal, Myanmar, Thailand, Malaysia, and Indonesia). Asian elephants have typical levels of mtDNA variation, and coalescence analyses suggest their populations were growing in the late Pleistocene. Reconstructed phylogenies reveal two major clades (A and B) differing on average by HKY85/Γ‐corrected distances of 0.020 for cyt b and 0.050 for the CR segment (corresponding to a coalescence time based on our cyt b rate of ～1.2 million years). Individuals of both major clades exist in all locations but Indonesia and Malaysia. Most elephants from Malaysia and all from Indonesia are in well‐supported, basal clades within clade A, thus supporting their status as evolutionarily significant units (ESUs). The proportion of clade A individuals decreases to the north, which could result from retention and subsequent loss of ancient lineages in long‐term stable populations or, perhaps more likely, via recent mixing of two expanding populations that were isolated in the mid‐Pleistocene. The distribution of clade A individuals appears to have been impacted by human trade in elephants among Myanmar, Sri Lanka, and India, and the subspecies and ESU statuses of Sri Lankan elephants are not supported by molecular data.     

In vitro conversion of phytol to phytanic acid in rat liver: subcellular distribution of activity and chemical characterization of intermediates using a new bromination technique

The enzymatic conversion of phytol to phytanic acid has been demonstrated in vitro in rat liver. Subcellular fractionation indicated that the mitochondrial fraction possessed the highest activity. Substantial activity was also present in the microsomal fraction. A new bromination-thin-layer chromatography procedure was developed to separate the phytol-dihydrophytol mixture and this procedure was applied to identify, characterize and quantitate the metabolites of phytol-phytanate conversion, i.e., phytanic acid, phytenic acid and dihydrophytol. Phytanic and phytenic acids were formed in the ratio 100:7.4. The conversion of phytol to phytenic acid was in the range 2-3%. No dihydrophytol was detected over boiled, acidified, or no-enzyme controls. The presence of phytenic acid and the absence of dihydrophytol in the incubation mixture confirm the previous in vivo studies and suggest that phytenic acid may be an intermediate in phytol-phytanate conversion.     

Characterization of phytol-phytanate conversion activity in rat liver

The enzymatic conversion of phytol to phytanic acid was investigated in rat liver postnuclear and other subcellular fractions using [1-3H]phytol as the substrate. The assay method involved incubation of the substrate with appropriate cofactors and the enzyme source, followed by subjecting the mixture to Folch partition and measuring the radioactivity in the upper layer. The phytol-phytanate conversion activity was present in mitochondrial and microsomal fractions. Cytosol had no activity. In mitochondrial fraction, investigation of cofactor requirements indicated that only NAD was required for activity. Other pyridine nucleotides supported the activity to a lesser extent when compared with NAD. FAD at 1 mM concentration did not support the activity. Bovine serum albumin (0.4 mg/ml) stimulated the activity. The reaction did not require molecular oxygen. From substrate kinetic studies, an apparent Km of 14.3 and 11.1 microM was calculated for phytol in mitochondrial and microsomal fractions, respectively. The amount of tritiated water produced from incubation increased linearly up to 7-8 min. The activity was linear with the amount of mitochondrial and microsomal protein up to 200 and 40 micrograms, respectively. Among the various rat tissue homogenates tested, liver had the highest activity. Spleen and kidney had 8-9% of the activity of liver. Brain possessed negligible activity. Both ethanol and pyrazole had no inhibitory effect on phytol-phytanate conversion. This observation and the absence of activity in cytosol suggests that alcohol dehydrogenase may not be involved in phytol-phytanate conversion.     

A redox-active crosslinker reveals an essential and inhibitable oxidative folding network in the endoplasmic reticulum of malaria parasites

Malaria remains a major global health problem, creating a constant need for research to identify druggable weaknesses in P. falciparum biology. As important components of cellular redox biology, members of the Thioredoxin (Trx) superfamily of proteins have received interest as potential drug targets in Apicomplexans. However, the function and essentiality of endoplasmic reticulum (ER)-localized Trx-domain proteins within P. falciparum has not been investigated. We generated conditional mutants of the protein PfJ2—an ER chaperone and member of the Trx superfamily—and show that it is essential for asexual parasite survival. Using a crosslinker specific for redox-active cysteines, we identified PfJ2 substrates as PfPDI8 and PfPDI11, both members of the Trx superfamily as well, which suggests a redox-regulatory role for PfJ2. Knockdown of these PDIs in PfJ2 conditional mutants show that PfPDI11 may not be essential. However, PfPDI8 is required for asexual growth and our data suggest it may work in a complex with PfJ2 and other ER chaperones. Finally, we show that the redox interactions between these Trx-domain proteins in the parasite ER and their substrates are sensitive to small molecule inhibition. Together these data build a model for how Trx-domain proteins in the P. falciparum ER work together to assist protein folding and demonstrate the suitability of ER-localized Trx-domain proteins for antimalarial drug development.     

Gene Flow Technique for Stage-Structured Populations

The gene flow technique for predicting response to selection with overlapping generations is formulated in terms of demographic parameters for stage-structured population and illustrated with a sequential selection scheme in dairy cows.     

Aberrant Fat Metabolism in Caenorhabditis elegans Mutants with Defects in the Defecation Motor Program

The molecular mechanisms by which dietary fatty acids are absorbed by the intestine, and the way in which the process is regulated are poorly understood. In a genetic screen for mutations affecting fat accumulation in the intestine of Caenorhabditis elegans, nematode worms, we have isolated mutations in the aex-5 gene, which encodes a Kex2/subtilisin-family, Ca²⁺-sensitive proprotein convertase known to be required for maturation of certain neuropeptides, and for a discrete step in an ultradian rhythmic phenomenon called the defecation motor program. We demonstrate that aex-5 mutants have markedly lower steady-state levels of fat in the intestine, and that this defect is associated with a significant reduction in the rate at which labeled fatty acid derivatives are taken up from the intestinal lumen. Other mutations affecting the defecation motor program also affect steady-state levels of triglycerides, suggesting that the program is required per se for the proper accumulation of neutral lipids. Our results suggest that an important function of the defecation motor program in C. elegans is to promote the uptake of an important class of dietary nutrients. They also imply that modulation of the program might be one way in which worms adjust nutrient uptake in response to altered metabolic status.     

SLC36A4 (hPAT4) is a high affinity amino acid transporter when expressed in Xenopus laevis oocytes

The SLC36 family of transporters consists of four genes, two of which, SLC36A1 and SLC36A2, have been demonstrated to code for human proton-coupled amino acid transporters or hPATs. Here we report the characterization of the fourth member of the family, SLC36A4 or hPAT4, which when expressed in Xenopus laevis oocytes also encodes a plasma membrane amino acid transporter, but one that is not proton-coupled and has a very high substrate affinity for the amino acids proline and tryptophan. hPAT4 in Xenopus oocytes mediated sodium-independent, electroneutral uptake of [(3)H]proline, with the highest rate of uptake when the uptake medium pH was 7.4 and an affinity of 3.13 μM. Tryptophan was also an excellently transported substrate with a similarly high affinity (1.72 μM). Other amino acids that inhibited [(3)H]proline were isoleucine (K(i) 0.23 mM), glutamine (0.43 mM), methionine (0.44 mM), and alanine (1.48 mM), and with lower affinity, glycine, threonine, and cysteine (K(i) >5 mM for all). Of the amino acids directly tested for transport, only proline, tryptophan, and alanine showed significant uptake, whereas glycine and cysteine did not. Of the non-proteogenic amino acids and drugs tested, only sarcosine produced inhibition (K(i) 1.09 mM), whereas γ-aminobutyric acid (GABA), β-alanine, L-Dopa, D-serine, and δ-aminolevulinic acid were without effect on [(3)H]proline uptake. This characterization of hPAT4 as a very high affinity/low capacity non-proton-coupled amino acid transporter raises questions about its physiological role, especially as the transport characteristics of hPAT4 are very similar to the Drosophila orthologue PATH, an amino acid "transceptor" that plays a role in nutrient sensing.     

Zn Binding Disrupts the Asp-Lys Salt Bridge without Altering the Hairpin-Shaped Cross-β Structure of Aβ42 Amyloid Aggregates

Observations like high Zn²⁺ concentrations in senile plaques found in the brains of Alzheimer's patients and evidences emphasizing the role of Zn²⁺ in amyloid-β (Aβ)-induced toxicity have triggered wide interest in understanding the nature of Zn²⁺-Aβ interaction. In vivo and in vitro studies have shown that aggregation kinetics, toxicity, and morphology of Aβ aggregates are perturbed in the presence of Zn²⁺. Structural studies have revealed that Zn²⁺ has a binding site in the N-terminal region of monomeric Aβ, but not much is precisely known about the nature of binding of Zn²⁺ with aggregated forms of Aβ or its effect on the molecular structure of these aggregates. Here, we explore this aspect of the Zn²⁺-Aβ interaction using one- and two-dimensional ¹³C and ¹⁵N solid-state NMR. We find that Zn²⁺ causes major structural changes in the N-terminal and the loop region connecting the two β-sheets. It breaks the salt bridge between the side chains of Asp²³ and Lys²⁸ by driving these residues into nonsalt-bridge-forming conformations. However, the cross-β structure of Aβ₄₂ aggregates remains unperturbed though the fibrillar morphology changes distinctly. We conclude that the salt bridge is not important for defining the characteristic molecular architecture of Aβ₄₂ but is significant for determining its fibrillar morphology and toxicity.     

Subcellular localization of total and activated Src kinase in African American and Caucasian breast cancer

Background

Src, a non-receptor tyrosine kinase is elevated in cancer with expression and activity correlated with cell proliferation, adhesion, survival, motility, metastasis and angiogenesis. There is limited data on Src expression and subcellular localization in breast cancer and no information about expression in racial/ethnic groups.

Methodology/Principal Findings

The present study evaluated Src expression, activity, and subcellular localization in triple negative breast cancer (TNBC) and ERα positive breast cancer (ER+BC), cancer tissue and adjacent normal epithelial ducts, and Caucasian and African American cases. 79 paraffin embedded breast carcinoma cases were obtained from Tulane University Hospital between 2007–2009. 39 cases represented TNBC (33-African Americans, 4-Caucasians, 2-unknowns) and 40 cases represented ER+BC (21-African Americans, 16-Caucasians, 3-unknowns). Immunohistochemistry was used to measure staining distribution and intensity of total Src and activated phospho-SrcY416 (p-Y416Src) in carcinoma tissue and adjacent normal mammary ducts. In TNBC and ER+BC, total Src was significantly higher in cancer compared to adjacent normal ducts (P<0.0001) in both cell membrane and cytoplasm. In membranes, p-Y416Src was elevated in cancer compared to normal ducts. Total Src in the tumor cytoplasm was significantly higher in TNBC compared to ER+BC (P = 0.0028); conversely, p-Y416Src in the tumor cell membranes was higher in TNBC compared to ER+BC (P = 0.0106). Comparison between African American (n = 21) and Caucasian ER+BC (n = 16) revealed no significant difference in expression and localization of total Src and p-Y416Src. TNBC cases positive for lymph node metastasis showed elevated membrane p-Y416Src compared to lymph node negative TNBC (P = 0.027).

Conclusion/Significance

Total Src and p-Y416Src were expressed higher in cancer compared to adjacent normal ducts. Cytoplasmic total Src and membrane p-Y416Src were significantly higher in TNBC compared to ER+BC. TNBC cases with lymph node metastasis showed elevated membrane p-Y416Src. Taken together, Src was elevated in the membrane and cytoplasm of more aggressive TNBC.