Kojic acid–amino acid conjugates as tyrosinase inhibitors

Kojic acid (KA), a well known tyrosinase inhibitor, has insufficient inhibitory activity and stability. We modified KA with amino acids and screened their tyrosinase inhibitory activity. Among them, kojic acid–phenylalanine amide (KA-F-NH₂) showed the strongest inhibitory activity, which was maintained for over 3 months at 50 °C, and acted as a noncompetitive inhibitor as determined by kinetic analysis. It also exhibited dopachrome reducing activity. We also propose a new tyrosinase inhibition mechanism based on the docking simulation data.     

Distinct germline progenitor subsets defined through Tsc2–mTORC1 signaling

Adult tissue maintenance is often dependent on resident stem cells; however, the phenotypic and functional heterogeneity existing within this self-renewing population is poorly understood. Here, we define distinct subsets of undifferentiated spermatogonia (spermatogonial progenitor cells; SPCs) by differential response to hyperactivation of mTORC1, a key growth-promoting pathway. We find that conditional deletion of the mTORC1 inhibitor Tsc2 throughout the SPC pool using Vasa-Cre promotes differentiation at the expense of self-renewal and leads to germline degeneration. Surprisingly, Tsc2 ablation within a subset of SPCs using Stra8-Cre did not compromise SPC function. SPC activity also appeared unaffected by Amh-Cre-mediated Tsc2 deletion within somatic cells of the niche. Importantly, we find that differentiation-prone SPCs have elevated mTORC1 activity when compared to SPCs with high self-renewal potential. Moreover, SPCs insensitive to Tsc2 deletion are preferentially associated with mTORC1-active committed progenitor fractions. We therefore delineate SPC subsets based on differential mTORC1 activity and correlated sensitivity to Tsc2 deletion. We propose that mTORC1 is a key regulator of SPC fate and defines phenotypically distinct SPC subpopulations with varying propensities for self-renewal and differentiation.     

mTORC1 signaling under hypoxic conditions is controlled by ATM-dependent phosphorylation of HIF-1α

The mTOR complex-1 (mTORC1) coordinates cell growth and metabolism, acting as a restriction point under stress conditions such as low oxygen tension (hypoxia). Hypoxia suppresses mTORC1 signaling. However, the signals by which hypoxia suppresses mTORC1 are only partially understood, and a direct link between hypoxia-driven physiological stress and the regulation of mTORC1 signaling is unknown. Here we show that hypoxia results in ataxia telangiectasia mutated (ATM)-dependent phosphorylation of hypoxia-inducible factor 1-alpha (HIF-1α) on serine(696) and mediates downregulation of mTORC1 signaling. Deregulation of these pathways in pediatric solid tumor xenografts suggests a link between mTORC1 dysregulation and solid tumor development and points to an important role for hypoxic regulation of mTORC1 activity in tumor development.     

Synthesis of camptothecin–amino acid carbamate linkers

A more convenient and facile approach for the synthesis and production of camptothecin–amino acids carbamate linkers, that can be used in the synthesis of bioconjugate peptides JF-10-81, JF-10-71, and other peptide analogs designed to target somatostatin receptors has been described.     

Structure-based non-canonical amino acid design to covalently crosslink an antibody–antigen complex

Engineering antibodies to utilize non-canonical amino acids (NCAA) should greatly expand the utility of an already important biological reagent. In particular, introducing crosslinking reagents into antibody complementarity determining regions (CDRs) should provide a means to covalently crosslink residues at the antibody–antigen interface. Unfortunately, finding the optimum position for crosslinking two proteins is often a matter of iterative guessing, even when the interface is known in atomic detail. Computer-aided antibody design can potentially greatly restrict the number of variants that must be explored in order to identify successful crosslinking sites. We have therefore used Rosetta to guide the introduction of an oxidizable crosslinking NCAA, l-3,4-dihydroxyphenylalanine (l-DOPA), into the CDRs of the anti-protective antigen scFv antibody M18, and have measured crosslinking to its cognate antigen, domain 4 of the anthrax protective antigen. Computed crosslinking distance, solvent accessibility, and interface energetics were three factors considered that could impact the efficiency of l-DOPA-mediated crosslinking. In the end, 10 variants were synthesized, and crosslinking efficiencies were generally 10% or higher, with the best variant crosslinking to 52% of the available antigen. The results suggest that computational analysis can be used in a pipeline for engineering crosslinking antibodies. The rules learned from l-DOPA crosslinking of antibodies may also be generalizable to the formation of other crosslinked interfaces and complexes.     

Advances in protein–amino acid nutrition of poultry

The ideal protein concept has allowed progress in defining requirements as well as the limiting order of amino acids in corn, soybean meal, and a corn–soybean meal mixture for growth of young chicks. Recent evidence suggests that glycine (or serine) is a key limiting amino acid in reduced protein [23% crude protein (CP) reduced to 16% CP] corn–soybean meal diets for broiler chicks. Research with sulfur amino acids has revealed that small excesses of cysteine are growth depressing in chicks fed methionine-deficient diets. Moreover, high ratios of cysteine:methionine impair utilization of the hydroxy analog of methionine, but not of methionine itself. A high level of dietary l-cysteine (2.5% or higher) is lethal for young chicks, but a similar level of dl-methionine, l-cystine or N-acetyl-l-cysteine causes no mortality. A supplemental dietary level of 3.0% l-cysteine (7× requirement) causes acute metabolic acidosis that is characterized by a striking increase in plasma sulfate and decrease in plasma bicarbonate. S-Methylmethionine, an analog of S-adenosylmethionine, has been shown to have choline-sparing activity, but it only spares methionine when diets are deficient in choline and(or) betaine. Creatine, or its precursor guanidinoacetic acid, can spare dietary arginine in chicks.     

mTORC1 signaling and regulation of pancreatic β-cell mass

The capacity of β cells to expand in response to insulin resistance is a critical factor in the development of type 2 diabetes. Proliferation of β cells is a major component for these adaptive responses in animal models. The extracellular signals responsible for β-cell expansion include growth factors, such as insulin, and nutrients, such as glucose and amino acids. AKT activation is one of the important components linking growth signals to the regulation of β-cell expansion. Downstream of AKT, tuberous sclerosis complex 1 and 2 (TSC1/2) and mechanistic target of rapamycin complex 1 (mTORC1) signaling have emerged as prime candidates in this process, because they integrate signals from growth factors and nutrients. Recent studies demonstrate the importance of mTORC1 signaling in β cells. This review will discuss recent advances in the understanding of how this pathway regulates β-cell mass and present data on the role of TSC1 in modulation of β-cell mass. Herein, we also demonstrate that deletion of Tsc1 in pancreatic β cells results in improved glucose tolerance, hyperinsulinemia and expansion of β-cell mass that persists with aging.     

mTORC1 signaling and regulation of pancreatic β-cell mass

The capacity of β cells to expand in response to insulin resistance is a critical factor in the development of type 2 diabetes. Proliferation of β cells is a major component for these adaptive responses in animal models. The extracellular signals responsible for β-cell expansion include growth factors, such as insulin, and nutrients, such as glucose and amino acids. AKT activation is one of the important components linking growth signals to the regulation of β-cell expansion. Downstream of AKT, tuberous sclerosis complex 1 and 2 (TSC1/2) and mechanistic target of rapamycin complex 1 (mTORC1) signaling have emerged as prime candidates in this process, because they integrate signals from growth factors and nutrients. Recent studies demonstrate the importance of mTORC1 signaling in β cells. This review will discuss recent advances in the understanding of how this pathway regulates β-cell mass and present data on the role of TSC1 in modulation of β-cell mass. Herein, we also demonstrate that deletion of Tsc1 in pancreatic β cells results in improved glucose tolerance, hyperinsulinemia and expansion of β-cell mass that persists with aging.     

Urinary amino acid analysis: A comparison of iTRAQ®–LC–MS/MS,GC–MS,and amino acid analyzer

Urinary amino acid analysis is typically done by cation-exchange chromatography followed by post-column derivatization with ninhydrin and UV detection. This method lacks throughput and specificity. Two recently introduced stable isotope ratio mass spectrometric methods promise to overcome those shortcomings. Using two blinded sets of urine replicates and a certified amino acid standard, we compared the precision and accuracy of gas chromatography/mass spectrometry (GC–MS) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) of propyl chloroformate and iTRAQ^® derivatized amino acids, respectively, to conventional amino acid analysis. The GC–MS method builds on the direct derivatization of amino acids in diluted urine with propyl chloroformate, GC separation and mass spectrometric quantification of derivatives using stable isotope labeled standards. The LC–MS/MS method requires prior urinary protein precipitation followed by labeling of urinary and standard amino acids with iTRAQ^® tags containing different cleavable reporter ions distinguishable by MS/MS fragmentation. Means and standard deviations of percent technical error (%TE) computed for 20 amino acids determined by amino acid analyzer, GC–MS, and iTRAQ^®–LC–MS/MS analyses of 33 duplicate and triplicate urine specimens were 7.27 ± 5.22, 21.18 ± 10.94, and 18.34 ± 14.67, respectively. Corresponding values for 13 amino acids determined in a second batch of 144 urine specimens measured in duplicate or triplicate were 8.39 ± 5.35, 6.23 ± 3.84, and 35.37 ± 29.42. Both GC–MS and iTRAQ^®–LC–MS/MS are suited for high-throughput amino acid analysis, with the former offering at present higher reproducibility and completely automated sample pretreatment, while the latter covers more amino acids and related amines.     

Transforming growth factor ß1 acid interaction

Chick embryo skin fibroblasts release transforming growth factor ₁ that is able to modulate glycosaminoglycan synthesis and secretion. When incubated with individual classes of glycosaminoglycans, the factor's modulatory activity was altered. To determine whether direct interactions between transforming growth factor ₁ and glycosaminoglycans occur, we have assessed the activity of the growth factor after pre-incubation with single classes of glycosaminoglycans by assaying its inhibitory effect upon the proliferative response of thymocytes stimulated with interleukin-1. Untreated transforming growth factor ₁ suppressed the proliferative response of thymocytes to interleukin-1, as did transforming growth factor ₁ pre-incubated with sulphated glycosaminoglycans. By contrast, transforming growth factor ₁ lost its inhibitory capacity when preincubated with high molecular weight hyaluronic acid. Digestion of transforming growth factor ₁-hyaluronic acid complex with hyaluronidase released active transforming growth factor ₁. Trypsin degraded transforming growth factor ₁ alone, but did not degrade the transforming growth factor ₁-hyaluronic acid complex. These results suggest that hyaluronic acid interacts with transforming growth factor ₁, thus protecting the factor from tryptic degradation and may be a means of concentrating growth factor activity.     

Distinct amino acid–sensing mTOR pathways regulate skeletal myogenesis

Signaling through the mammalian target of rapamycin (mTOR) in response to amino acid availability controls many cellular and developmental processes. mTOR is a master regulator of myogenic differentiation, but the pathways mediating amino acid signals in this process are not known. Here we examine the Rag GTPases and the class III phosphoinositide 3-kinase (PI3K) Vps34, two mediators of amino acid signals upstream of mTOR complex 1 (mTORC1) in cell growth regulation, for their potential involvement in myogenesis. We find that, although both Rag and Vps34 mediate amino acid activation of mTORC1 in C2C12 myoblasts, they have opposing functions in myogenic differentiation. Knockdown of RagA/B enhances, whereas overexpression of active RagB/C mutants impairs, differentiation, and this inhibitory function of Rag is mediated by mTORC1 suppression of the IRS1-PI3K-Akt pathway. On the other hand, Vps34 is required for myogenic differentiation. Amino acids activate a Vps34-phospholipase D1 (PLD1) pathway that controls the production of insulin-like growth factor II, an autocrine inducer of differentiation, through the Igf2 muscle enhancer. The product of PLD, phosphatidic acid, activates the enhancer in a rapamycin-sensitive but mTOR kinase–independent manner. Our results uncover amino acid–sensing mechanisms controlling the homeostasis of myogenesis and underline the versatility and context dependence of mTOR signaling.     

Synthesis and antibacterial evaluation of amino acid–antibiotic conjugates

Amino acid conjugates of quinolone, metronidazole and sulfadiazine antibiotics were synthesized in good yields using benzotriazole methodology. All the conjugates were screened for their antibacterial activity using methods adapted from the Clinical and Laboratory Standards Institute. Antibiotic conjugates were tested for activity in four medically relevant organisms; Staphylococcus aureus (RN4220), Escherichia coli (DH5α), Pseudomonas aeruginosa (PAO1), and Bacillus subtilis (168). Several antibiotic conjugates show promising results against several of the strains screened.     

Downregulation of mTORC1 and Mcl-1 by lipid-oversupply contributes to islet β-cell apoptosis and dysfunction

Pancreatic β-cell apoptosis is a key feature of diabetes and can be induced by chronic exposure to saturated fatty acids (FAs). However, the underlying mechanisms remain poorly understood. We presently evaluated the role of Mcl-1 and mTOR in mice fed with high-fat-diet (HFD) and β-cells exposed to the overloaded palmitic acid (PA). Compared with normal-chow-diet (NCD)-fed mice, HFD group showed impaired glucose tolerance after two months. Along with the diabetes progression, pancreatic islets first became hypertrophic and then atrophic, the ratio of β-cell:α-cell increased in the islets of four months HFD-fed mice while decreased after six months. This process was accompanied by significantly increased β-cell apoptosis and AMPK activity, and decreased Mcl-1 expression and mTOR activity. Consistently, glucose-induced insulin secretion dropped. In terms of mechanism, PA with lipotoxic dose could activate AMPK, which in turn inhibited ERK-stimulated Mcl-1^Thr163 phosphorylation. Meanwhile, AMPK blocked Akt activity to release Akt inhibition on GSK3β, followed by GSK3β-initiated Mcl-1^Ser159 phosphorylation. The context of Mcl-1 phosphorylation finally led to its degradation by ubiquitination. Also, AMPK inhibited the activity of mTORC1, resulting in a lower level of Mcl-1. Suppression of mTORC1 activity and Mcl-1 expression positively related to β-cell failure. Alteration of Mcl-1 or mTOR expression rendered different tolerance of β-cell to different dose of PA. In conclusion, lipid oversupply-induced dual modulation of mTORC1 and Mcl-1 finally led to β-cell apoptosis and impaired insulin secretion. The study may help further understand the pathogenesis of β-cell dysfunction in case of dyslipidemia, and provide promising therapeutic targets for diabetes.