Biochemistry Insights

In the current study, we used immunoprecipitation and immunoblotting to examine the levels and phosphorylation status of the insulin receptor-beta subunit (IR-β), as well as the down stream target in PI3K pathway, total PKB/Akt as well as their phosphorylated forms. The assessment of FFAs treatment showed no direct and significant effect on the PI3K stimulation, specifically the IR-β in primary hepatic control cells treated with insulin. Cells treated with either oleate or palmitate (360 μM) showed no statistically significant values following insulin stimulation (P > 0.05). To further investigate the effect of both FFAs and high insulin (1 μg), we examined the effects of oleate and palmitate at 360 μM concentration on IR-β as well as PKB. There was no significant difference in the total protein levels and their phosphorylated forms in cells treated with or without oleate or plamitate. Interestingly, IR-β tyrosine phosphorylation showed a similar insignificant effect in vivo and ex vivo hepatic cells treated with oleate or palmitate in comparison to their controls in the fructose fed hamsters.

Incubation of methyl 2-benzamidomethyl-3-oxobutanoate with cultured plant cells of Parthenocissus tricuspidata for 2 days afforded methyl (2R,3S)-2-benzamidomethyl-3-hydroxybutanoate with 100% de and >99% ee in 51% yield. The use of immobilized cells of P. tricuspidata in calcium alginate gel improved the yield of the product (95% yield) with 100% de and >99% ee. The immobilized cells of P. tricuspidata maintained the high potential for the reduction of methyl 2-benzamidomethyl-3-oxobutanoate (85% yield) after 5 times of usage.

Isomerization of amino acids in proteins has recently been identified as a part of the aging process. Increases in D-amino acids as a consequence of isomerization influence the function and structure of proteins. Senescence-related pulmonary diseases, such as chronic obstructive pulmonary disease, are thought to be caused by reductions of lung function with age. We hypothesized that changes of protein structure in lung tissue induced by the isomerization of amino acids could result in decreased lung function. Therefore, we examined whether isomerization of amino acids takes place in the lungs of rats as they age. We measured the content of L- and D-amino acids in collagen 1 by HPLC using a chiral column. We found that collagen 1 was increasingly racemized with age, so that significantly higher proportions of D-Ser were present in 12- and 24-month-old rats than in 8-week-old rats. D-Asp increased slightly but not significantly. We also investigated the localization of collagen 1 in lung tissue. Stacks of collagen 1 were observed in the parenchyma and airway wall, and age-dependent changes were especially prominent in the airway wall. Racemization of collagen 1 could therefore influence lung function and contribute to pulmonary diseases.

In aquatic environments, genotoxicity results from the effects of pollution combined with the inflammatory response triggered by the immune system. Indeed, the production of nitrosylated DNA and proteins are though to arise from the production of peroxinitrite during phagocytosis and inflammation. The purpose of this study was to examine new DNA biomarkers that differentiate between immune- and pollution-mediated genotoxicity in wild clam populations. Intertidal clam populations were sampled and analyzed for gonadal DNA strand breaks, DNA nitrosylation and xanthine oxidoreductase (XOR) activity (purine salvage pathway). The clam weight-to-shell-length ratio, the gonado-somatic index (GSI), age status, lipid peroxidation, xenobiotic conjugation activity (glutathione S-transferase (GST) and phagocytic activity were examined to shed light on their relationships with the observed genotoxic endpoints. XOR activity and DNA strand breaks were generally elevated at polluted sites and correlated significantly with clam weight-to-shell-length ratios and DNA nitrosylation. DNA nitrosylation was also higher at some sites and correlated significantly with phagocytic activity and with DNA strand breaks. This study showed that DNA strand breaks were associated with both immune-and pollution-mediated effects. This suggests that there is a loss of DNA repair capacity due to the combined effects of aging, pollution and immune response in wild clam populations that are impacted by anthropogenic activity.

The enzymatic synthesis of capsaicin glycosides and 8-nordihydrocapsaicin glycosides was investigated using almond β-glucosidase and cyclodextrin glucanotransferase (CGTase). Capsaicin and 8-nordihydrocapsaicin were converted into their β-glucoside and β-maltooligosaccharide (amylose conjugate), i.e. β-maltoside and β-maltotrioside, by sequencial glycosylation with almond β-glucosidase and CGTase. The β-glucoside and β-maltoside of capsaicin and β-glucoside of 8-nordihydrocapsaicin showed inhibitory effects on high-fat-diet-induced elevations in body weight of mice.

Chemo-enzymatic synthesis of glycolyl-ester-linked taxol-glucose conjugate, ie, 7-glycolyltaxol 2′-O-α-D-glucoside, was achieved by using α-glucosidase as a biocatalyst. The water-solubility of 7-glycolyltaxol 2′-O-α-D-glucoside (21 μM) was 53 fold higher than that of taxol. The hepatitis B virus envelope L particles (bio-nanocapsules) are effective for delivering 7-glycolyltaxol 2′-O-α-D-glucoside to human hepatocellular carcinoma NuE cells.

To clarify the potential ability of marine actinomycetes as biocatalysts, the stereoselective reduction of α-keto esters and α-keto amide using Salinispora arenicola and Salinispora tropica was tested. The reduction of ethyl pyruvate and ethyl 2-oxobutanoate by S. tropica gave corresponding alcohol with high conversion ratio and in high e.e. (96% e.e. (S) and 99% e.e. (S), respectively). In the presence of l-glutamate as an additive, the reduction of ethyl pyruvate by S. tropica afforded the corresponding (S)-ethyl lactate with >99% e.e. Furthermore, 2-chlorobenzoylformamide was reduced by S. tropica to the corresponding (R)-2-chloromandelamide with high conversion ratio and excellent enantioselectivity (>99% e.e.). Thus, it was found that marine actinomycetes, Salinispora strains, had high ability for the stereoselective reduction of carbonyl compounds as useful biocatalysts.

Gallic acid is an organic acid known for its antioxidant and anticancer properties. The present study is focused on evaluating the role of gallic acid in providing better therapeutic outcomes against arsenic-induced toxicity. Animals pre-exposed to arsenic were treated with monoisoamyl meso-2,3-dimercaptosuccinic acid (MiADMSA), a new chelating drug, alone and in combination with gallic acid, consecutively for 10 days. The study suggests that (1) gallic acid in presence of MiADMSA is only moderately beneficial against arsenic, (2) monotherapy with gallic acid is more effective than in combination with MiADMSA after arsenic exposure in reducing oxidative injury, and (3) MiADMSA monotherapy as reported previously provides significant therapeutic efficacy against arsenic. Thus, based on the present results, we conclude that gallic acid is effective against arsenic-induced oxidative stress but provides limited additional beneficial effects when administered in combination with MiADMSA. We still recommend that lower doses of gallic acid be evaluated both individually and in combination with MiADMSA, as it might not exhibit the shortcomings we observed with higher doses in this study.

Stevioside is a natural sweetener from Stevia leaf, which is 300 times sweeter than sugar. It helps to reduce blood sugar levels dramatically and thus can be of benefit to diabetic people. Tissue culture is a very potential modern technology that can be used in large-scale disease-free stevia production throughout the year. We successfully produced stevia plant through in vitro culture for identification of stevioside in this experiment. The present study describes a potential method for identification of stevioside from tissue culture-derived stevia leaf. Stevioside in the sample was identified using HPLC by measuring the retention time. The percentage of stevioside content in the leaf samples was found to be 9.6%. This identification method can be used for commercial production and industrialization of stevia through in vitro culture across the world.

A plant-based paste fermented by lactic acid bacteria and yeast (fermented paste) was made from various plant materials. The paste was made of fermented food by applying traditional food-preservation techniques, that is, fermentation and sugaring. The fermented paste contained major nutrients (carbohydrates, proteins, and lipids), 18 kinds of amino acids, and vitamins (vitamin A, B₁ B₂, B₆, B₁₂, E, K, niacin, biotin, pantothenic acid, and folic acid). It contained five kinds of organic acids, and a large amount of dietary fiber and plant phytochemicals. Sucrose from brown sugar, used as a material, was completely resolved into glucose and fructose. Some physiological functions of the fermented paste were examined in vitro. It was demonstrated that the paste possessed antioxidant, antihypertensive, antibacterial, anti-inflammatory, anti-allergy and anti-tyrosinase activities in vitro. It was thought that the fermented paste would be a helpful functional food with various nutrients to help prevent lifestyle diseases.

Embryonic stem cells (ESCs) are pluripotent cells with great therapeutic potentials. The in vitro differentiation of ESC was designed by recapitulating embryogenesis. Significant progress has been made to improve the in vitro differentiation protocols by toning soluble maintenance factors. However, more robust methods for lineage-specific differentiation and maturation are still under development. Considering the complexity of in vivo embryogenesis environment, extracellular matrix (ECM) cues should be considered besides growth factor cues. ECM proteins bind to cells and act as ligands of integrin receptors on cell surfaces. Here, we summarize the role of the ECM and integrins in the formation of three germ layer progenies. Various ECM–integrin interactions were found, facilitating differentiation toward definitive endoderm, hepatocyte-like cells, pancreatic beta cells, early mesodermal progenitors, cardiomyocytes, neuroectoderm lineages, and epidermal cells, such as keratinocytes and melanocytes. In the future, ECM combinations for the optimal ESC differentiation environment will require substantial study.

There is substantial evidence that the growth hormone (GH)/insulin-like growth factor (IGF) system is involved in the pathophysiology of obesity. Both GH and IGF-I have direct effects on adipocyte proliferation and differentiation, and this system is involved in the cross-talk between adipose tissue, liver, and pituitary. Transgenic animal models have been of importance in identifying mechanisms underlying these interactions. It emerges that this system has key roles in visceral adiposity, and there is a rationale for targeting this system in the treatment of visceral obesity associated with GH deficiency, metabolic syndrome, and lipodystrophies. This evidence is reviewed, gaps in knowledge are highlighted, and recommendations are made for future research.

Based on a previous study, glabridin displayed a dose-dependent increase in estrogenic activity and cell proliferative activity in Ishikawa cells. However, when treated in combination with 17β-E₂, synergistic estrogenic effect was observed but without the same synergistic increase in cell proliferative effect. This study aimed to identify the estrogen and nonestrogen-regulated activities induced by glabridin and in combination with 17β-E₂ in comparison with 17β-E₂. The results showed that 10 µM glabridin and the combination treatment of 100 nM glabridin with 1 nM 17β-E₂ regulated both the genomic and nongenomic estrogen pathways to possibly provide benefits of estrogens in cardiovascular, circulatory, and vasculature systems. Meanwhile, the combination of 100 nM glabridin with 1 nM 17β-E₂ seems to be more suitable to be used as an estrogen replacement. Finally, the results of this study have added on to the present knowledge of glabridin’s function as a phytoestrogen and suggested new ideas for the usage of glabridin.

Nanoporous gold (referred to as np-Au or NPG) has emerged over the past 10 years as a new support for enzyme immobilization. The material has appealing features of ease of preparation, tunability of pore size, high surface to volume ratio, and compatibility with multiple strategies for enzyme immobilization. The np-Au material is especially of interest for immobilization of redox enzymes for biosensor and biofuel cell applications given the ability to construct electrodes of high surface area and stability. Adjustment of the pore size of np-Au can yield enhancements in enzyme thermal stability. Glucose oxidase immobilization on np-Au has been a focus for development of glucose sensors. Immobilization of laccase and related enzymes has demonstrated the utility of np-Au for construction of biofuel cells. Np-Au has been used to immobilize other redox enzymes, enzyme conjugates for use in bioassays, and enzymes of interest for industrial processes.

Previous studies have identified genetic factors and Epstein-Barr virus underlying nasopharyngeal carcinoma. A hypothesis postulated that the local buildup of HCl, mediated by hydrogen bond donors and acceptors and basic amino acids, causes cancer. Nasopharyngeal carcinoma incidences are high in the humid southern coastal China, Southeast Asia, and Mediterranean regions, but not in the noncoastal and nonhumid southern Yunnan Province, China, and nonhumid Central China. The nearly saturated humidity in the Huinan period in Guangdong can trigger the expression of proteins with extensive hydrogen bonding to protons, augmenting the formation of HCl that is mutagenic. Given that the Epstein-Barr virus carries high content of hydrogen bond donors and acceptors, the moist environment in the nasal cavity may enable the virus to colonize the site, compounding pertinent investigations as both virus and high humidity are likely to trigger carcinogenesis. Therefore, the phenomena of exceptionally high humidity in regions with high nasopharyngeal cancer rates warrant further investigations.

Aerococcus urinae (Au) and Globicatella sanguinis (Gs) are gram-positive bacteria belonging to the family Aerococcaceae and colonize the human immunocompromised and catheterized urinary tract. We identified both pathogens in polymicrobial urethral catheter biofilms (CBs) with a combination of 16S rDNA sequencing, proteomic analyses, and microbial cultures. Longitudinal sampling of biofilms from serially replaced catheters revealed that each species persisted in the urinary tract of a patient in cohabitation with 1 or more gram-negative uropathogens. The Gs and Au proteomes revealed active glycolytic, heterolactic fermentation, and peptide catabolic energy metabolism pathways in an anaerobic milieu. A few phosphotransferase system (PTS)–based sugar uptake and oligopeptide ABC transport systems were highly expressed, indicating adaptations to the supply of nutrients in urine and from exfoliating squamous epithelial and urothelial cells. Differences in the Au vs Gs metabolisms pertained to citrate lyase and utilization and storage of glycogen (evident only in Gs proteomes) and to the enzyme Xfp that degrades d-xylulose-5′-phosphate and the biosynthetic pathways for 2 protein cofactors, pyridoxal 6′-phosphate and 4′-phosphopantothenate (expressed only in Au proteomes). A predicted ZnuA-like transition metal ion uptake system was identified for Gs while Au expressed 2 LPXTG-anchored surface proteins, one of which had a predicted pilin D adhesion motif. While these proteins may contribute to fitness and virulence in the human host, it cannot be ruled out that Au and Gs fill a niche in polymicrobial biofilms without being the direct cause of injury in urothelial tissues.

Type 1 diabetes (the pancreas producing little or no insulin) is usually diagnosed in children and young adults and was previously known as juvenile diabetes. McArdle disease is a common metabolic defect caused by an inherited deficit of myophosphorylase. These 2 diseases might have some clinical heterogeneity. Here, we discuss a McArdle disease case where insulin-dependent diabetes overshadows its early diagnosis. In this case, an insulin-dependent 22-year-old female patient with diabetes mellitus had been on diabetes treatment for 15 years. Although her blood glucose was regulated, her anamnesis showed that muscle weakness, fatigue, cramps or myalgia never healed. Based on her anamnesis, the patient was asked to take a nonischemic forearm exercise test, which revealed significant elevation in levels of creatine kinase (5968-7906 U/L), but no increase was found in lactate concentration, but a slight increase in ammonia concentration (not statistically significant) at the end of the test made us consider McArdle disease. A genetic test was done to confirm this possibility. A homozygous c.2128_2130delTTC/p.Phe710del mutation was detected in the examination of exons of the PYGM gene, which confirmed the diagnosis of McArdle disease in our patient. According to the data, this is a rare case of McArdle disease with type 1 diabetes. During treatment for diabetes, if the above-mentioned symptoms are present in a patient, and especially if the patient’s creatine kinase concentration is high, muscle diseases should be suspected. Therefore, we suggest that this case report will provide new insight to clinicians on metabolic defects in this disease and increase the patient comfort. In such cases, an early diagnosis should reduce health costs.

Tumor necrosis factor-alpha (TNF-α), a member of the TNF superfamily, was the first cytokine to be evaluated for cancer biotherapy. However, the clinical use of TNF-α is severely limited by its toxicity. Currently, TNF-α is administered only through locoregional drug delivery systems such as isolated limb perfusion and isolated hepatic perfusion. To reduce the systemic toxicity of TNF-α, various strategies have been explored over the last several decades. This review summarizes current state-of-the-art targeted cancer therapy using TNF-α. Passive targeting, cell-based therapy, gene therapy with inducible or tissue-specific promoters, targeted polymer-DNA complexes, tumor pre-targeting, antibody-TNF-α conjugate, scFv/TNF-α fusion proteins, and peptide/TNF-α fusion proteins have all been investigated to combat cancer. Many of these agents are already in advanced clinical trials. Molecular imaging, which can significantly speed up the drug development process, and nanomedicine, which can integrate both imaging and therapeutic components, has the potential to revolutionize future cancer patient management. Cooperative efforts from scientists within multiple disciplines, as well as close partnerships among many organizations/entities, are needed to quickly translate novel TNF-α-based therapeutics into clinical investigation.

The insulin-like growth factors (IGF-I and IGF-II) and their receptors are widely expressed in nervous tissue from early embryonic life. They also cross the blood brain barriers by active transport, and their regulation as endocrine factors therefore differs from other tissues. In brain, IGFs have paracrine and autocrine actions that are modulated by IGF-binding proteins and interact with other growth factor signalling pathways. The IGF system has roles in nervous system development and maintenance. There is substantial evidence for a specific role for this system in some neurodegenerative diseases, and neuroprotective actions make this system an attractive target for new therapeutic approaches. In developing new therapies, interaction with IGF-binding proteins and other growth factor signalling pathways should be considered. This evidence is reviewed, gaps in knowledge are highlighted, and recommendations are made for future research.