Muscle-specific deletion of carnitine acetyltransferase compromises glucose tolerance and metabolic flexibility

The concept of "metabolic inflexibility" was first introduced to describe the failure of insulin-resistant human subjects to appropriately adjust mitochondrial fuel selection in response to nutritional cues. This phenomenon has since gained increasing recognition as a core component of the metabolic syndrome, but the underlying mechanisms have remained elusive. Here, we identify an essential role for the mitochondrial matrix enzyme, carnitine acetyltransferase (CrAT), in regulating substrate switching and glucose tolerance. By converting acetyl-CoA to its membrane permeant acetylcarnitine ester, CrAT regulates mitochondrial and intracellular carbon trafficking. Studies in muscle-specific Crat knockout mice, primary human skeletal myocytes, and human subjects undergoing L-carnitine supplementation support a model wherein CrAT combats nutrient stress, promotes metabolic flexibility, and enhances insulin action by permitting mitochondrial efflux of excess acetyl moieties that otherwise inhibit key regulatory enzymes such as pyruvate dehydrogenase. These findings offer therapeutically relevant insights into the molecular basis of metabolic inflexibility.     

Rapid nanoparticle-mediated monitoring of bacterial metabolic activity and assessment of antimicrobial susceptibility in blood with magnetic relaxation

Considering the increased incidence of bacterial infections and the emergence of multidrug resistant bacteria at the global level, we designed superparamagnetic iron oxide nanoparticles as nanosensors for the assessment of antimicrobial susceptibility through magnetic relaxation. In this report, we demonstrate that iron oxide nanosensors, either dextran-coated supplemented with Con A or silica-coated conjugated directly to Con A, can be used for the fast (1) quantification of polysaccharides, (2) assessment of metabolic activity and (3) determination of antimicrobial susceptibility in blood. The use of these polysaccharide nanosensors in the determination of antimicrobial susceptibility in the clinic or the field, and the utilization of these nanoprobes in pharmaceutical R&D are anticipated.     

Atypical Ca<Superscript>2+</Superscript>-independent,raw-starch hydrolysing α-amylase from <Emphasis Type="Italic">Bacillus</Emphasis> sp. GRE1: characterization and gene isolation

Bacillus sp. GRE1 isolated from an Ethiopian hyperthermal spring produced raw-starch digesting, Ca²⁺-independent thermostable α-amylase. Enzyme production in shake flask experiments using optimum nutrient supplements and environmental conditions was 2,360 U l⁻¹. Gel filtration chromatography yielded a purification factor of 33.6-fold and a recovery of 46.5%. The apparent molecular weight of the enzyme was 55 kDa as determined by SDS-PAGE. Presence or absence of Ca²⁺ produced similar temperature optima of 65–70°C. The optimum pH was in the range of 5.5–6.0. The enzyme maintained 50% of its original activity after 45 min of incubation at 80°C and was stable at a pH range of 5.0–9.0. The V _max and K _m values for soluble starch were 42 mg reducing sugar min⁻¹ and 4.98 mg starch ml⁻¹, respectively. Strong inhibitors of enzyme activity included Cu²⁺, Zn²⁺ and Fe²⁺. The enzyme coding gene and the deduced protein translation revealed a characteristic but markedly atypical homology to Bacillus species α-amylase sequences. The enzyme hydrolyzed wheat, corn and tapioca starch granules efficiently below their gelatinization temperatures. Rather than the higher oligosaccharides normally produced by Bacillus α-amylases operating at high temperatures, maltose was the major hydrolysis product with the present enzyme.     

Biophysical analyses of human resistin: oligomer formation suggests novel biological function

Resistin, a small secreted peptide initially identified as a link between obesity and diabetes in mice, was shown to be involved in mediating inflammation in humans. We had shown earlier that recombinant human resistin has a tendency to form aggregates by formation of inter/intramolecular disulfide linkages and that it undergoes a concentration-dependent conformational change in secondary structure from alpha-helical to beta-sheet form. Here we report that this change in secondary structural conformation is due to the increase in the oligomeric form of human resistin as a function of protein concentration. Gel filtration analysis under different conditions further demonstrated that recombinant human resistin exists as a mixture of oligomer and trimer but is converted to a mixture of monomer and oligomer in the presence of 100 mM NaCl. We show that while the trimeric form of human resistin is stable to urea-induced denaturation, it is highly susceptible to NaCl and NaF, indicating the importance of ionic interactions in stabilization of trimer. In addition, urea was able to destabilize the oligomers indicating the involvement of hydrophobic interactions in oligomerization. Ionic as well as hydrophobic interactions stabilize the monomeric human resistin. Our data suggest that human resistin exists predominantly as oligomer and trimer in vitro. The oligomeric form of human resistin shows more potent effect on stimulation of proinflammatory cytokines. Therefore, it is very tempting to propose that the structural conformation of resistin may be involved in maintaining the very fine balance in regulation of macrophage function for successful response to a variety of pathological conditions.     

Nucleosomal Positioning and Genetic Divergence Study Based on DNA Flexibility Map

Abstract

Based on worm like chain model, DNA structural parameters—tilt, roll and rise, derived from crystallographic database have been used to determine the flexibility of DNA that regulates the nucleosomal translational positioning. Theoretically derived data has been compared to the experimental values available in Ioshikhes and Trifonov's database. The methodology has been extended to determine the flexibility of 18S rRNA genome in eukarya, where yeast shows a distinct difference when compared with mammals like human, mouse and rabbit.     

<Emphasis Type="Italic">In Planta</Emphasis> transformation for conferring salt tolerance to a tissue-culture unresponsive indica rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) cultivar

Many farmer-popular indica rice (Oryza sativa L.) cultivars are recalcitrant to Agrobacterium-mediated transformation through tissue culture and regeneration. In planta transformation using Agrobacterium could therefore be a useful alternative for indica rice. A simple and reproducible in planta protocol with higher transformation efficiencies than earlier reports was established for a recalcitrant indica rice genotype. Agrobacterium tumefaciens containing the salt tolerance-enhancing Pea DNA Helicase45 (PDH45) gene, with the reporter and selectable marker genes, gus-INT (β-glucuronidase with intron) and hygromycin phosphotransferase (hpt), respectively, were used. Overnight-soaked mature embryos were infected and allowed to germinate, flower, and set T₁ seeds. T₀ plants were considered positive for the transgene if the spikelets of one or more of their panicles were positive for gus. Thereafter, selection at T₁ was done by germination in hygromycin and transgenic status re-confirmation by subjecting plantlet DNA/RNA to gene-specific PCR, Southern and semi-quantitative RT-PCR. Additionally, physiological screening under saline stress was done at the T₂ generation. Transformation efficiency was found to be 30–32% at the T₀ generation. Two lines of the in planta transformed seedlings of the recalcitrant rice genotype were shown to be saline tolerant having lower electrolyte leakage, lower Na⁺/K⁺, minimal leaf damage, and higher chlorophyll content under stress, compared to the WT at the T₂ generation.     

Activation of Wnt-β-catenin pathway in basal–parabasal layers of normal cervical epithelium comparable during development of uterine cervical carcinoma

In this study, importance of Wnt-β-catenin pathway in the development of uterine cervical carcinoma was evaluated. For this purpose, the profiles (expression/methylation/deletion) of β-catenin, p-β-catenin (Y654), Wnt3a, and APC were studied in disease free normal cervical epithelium (n = 9), adjacent normal cervical epithelium of primary tumors (n = 70), CIN (n = 28), CACX (n = 102) samples, and two CACX cell lines (HeLa and SiHa). Immunohistochemical analysis revealed high/medium (74–95%) expression of β-catenin/p-β-catenin (Y654) and Wnt3a and low expression (23–26%) of APC in proliferating basal–parabasal layers contrary to differentiated spinous layer in normal cervix irrespective of HPV16 infection. The expression profile of the genes in the basal–parabasal layers did not change significantly during development of CACX. High (66%) promoter methylation of APC was seen in basal–parabasal layers and the cervical lesions (42–69%), unlike in spinous layers (25%). The promoter methylation status of APC was validated by in vitro demethylation experiments using 5-aza-dC in CACX cell lines. However, additional deletion of APC was significantly increased from CIN (12%) to stage I/II (40%) and became comparable in stage III/IV (48%) of the tumor. Patients with alterations (deletion/methylation) of APC and high/medium expression of Wnt3a/β-catenin/p-β-catenin (Y654) showed significantly poor survival. Thus our data indicate that cumulative effect of Wnt3a overexpression and APC inactivation are needed for overexpression of β-catenin during the development of CACX.     

Marine natural pigments as potential sources for therapeutic applications

In recent years, marine natural pigments have emerged as a powerful alternative in the various fields of food, cosmetic, and pharmaceutical industries because of their excellent biocompatibility, bioavailability, safety, and stability. Marine organisms are recognized as a rich source of natural pigments such as chlorophylls, carotenoids, and phycobiliproteins. Numerous studies have shown that marine natural pigments have considerable medicinal potential and promising applications in human health. In this review, we summarize the marine natural pigments as potential sources for therapeutic applications, including: antioxidant, anticancer, antiangiogenic, anti-obesity, anti-inflammatory activities, drug delivery, photothermal therapy (PTT), photodynamic therapy (PDT), photoacoustic imaging (PAI), and wound healing. Marine natural pigments will offer a better platform for future theranostic applications.