Inhibition of neuronal nitric oxide synthase activity promotes migration of human‐induced pluripotent stem cell‐derived neural stem cells toward cancer cells

The breakthrough in derivation of human‐induced pluripotent stem cells (hiPSCs) provides an approach that may help overcome ethical and allergenic challenges posed in numerous medical applications involving human cells, including neural stem/progenitor cells (NSCs). Considering the great potential of NSCs in targeted cancer gene therapy, we investigated in this study the tumor tropism of hiPSC‐derived NSCs and attempted to enhance the tropism by manipulation of biological activities of proteins that are involved in regulating the migration of NSCs toward cancer cells. We first demonstrated that hiPSC‐NSCs displayed tropism for both glioblastoma cells and breast cancer cells in vitro and in vivo. We then compared gene expression profiles between migratory and non‐migratory hiPSC‐NSCs toward these cancer cells and observed that the gene encoding neuronal nitric oxide synthase (nNOS) was down‐regulated in migratory hiPSC‐NSCs. Using nNOS inhibitors and nNOS siRNAs, we demonstrated that this protein is a relevant regulator in controlling migration of hiPSC‐NSCs toward cancer cells, and that inhibition of its activity or down‐regulation of its expression can sensitize poorly migratory NSCs and be used to improve their tumor tropism. These findings suggest a novel application of nNOS inhibitors in neural stem cell‐mediated cancer therapy.     

Synthesis and characterization of novel fluorogenic substrates of coagulation factor XIII-A

Further development of our recently published Glu(pNA)-containing peptides (Anal. Biochem. 428 (2012) 73–80) provided new fluorogenic substrates for the activated blood coagulation factor XIII. A first series was designed by incorporation of Glu(AMC) at the penultimate position from the N terminus. For the best derivative H-Tyr-Glu(AMC)-Val-Lys-Val-Ile-NH₂, a moderate k_cat/K_m value of 34 s⁻¹ M⁻¹ was determined, which is more than 100-fold reduced compared with the previously reported Glu(pNA) substrates. Furthermore, two fluorescence resonance energy transfer (FRET) substrates were prepared by incorporation of an N-methyl-anthraniloyl fluorophore and a 2,4-dinitrophenyl quencher. Both substrates were excellently cleaved by FXIII-A₂^∗, which is generated from its zymogen by activation of thrombin in the presence of calcium ions. In the absence and presence of H-Gly-ethyl ester, k_cat/K_m values of 8010 and 8660 s⁻¹ M⁻¹, respectively, were found for the conversion of H-Lys(N(Me)Abz)-Glu(NH-(CH₂)₄-NH-Dnp)-Val-Lys-Val-Ile-Gly-NH₂ (substrate 8). These values are more than 200-fold improved compared with the Glu(AMC) substrates. Substrate 8 is suitable for the measurement of FXIII-A₂^∗ activities in plasma samples as well as for in vitro measurements. Furthermore, it was used for the determination of the inhibitory potency of a newly synthesized chloromethyl ketone derivative, Cbz-Phe-Glu(CMK)-Val-Lys-Val-Ile-Gly-NH₂, which was found to be a potent irreversible inhibitor of FXIII-A₂^∗.     

CRISPR/Cas-induced double-strand breaks boost the frequency of gene replacements for humanizing the mouse Cnr2 gene

The CRISPR/Cas technology has been successfully used to stimulate the integration of small DNA sequences in a target locus to produce gene mutations. However, many applications require homologous recombination using large gene-targeting constructs. Here we address the potential of CRISPR/Cas-mediated double-strand breaks to enhance the genetic engineering of large target sequences using a construct for “humanizing” the mouse Cnr2 gene locus. We designed a small-guide RNA that directs the induction of double strand breaks by Cas9 in the Cnr2 coding exon. By co-transfection of the CRISPR/Cas system with the 10 kb targeting construct we were able to boost the recombination frequency more than 200-fold from 0.27% to 67%. This simple technology can thus be used for the homologous integration of large gene fragments and should greatly enhance our ability to generate any kind of genetically altered mouse models.     

A novel multifunctional cellulolytic enzyme screened from metagenomic resources representing ruminal bacteria

Metagenomic resources representing ruminal bacteria were screened for novel exocellulases using a robotic, high-throughput screening system, the novel CelEx-BR12 gene was identified and the predicted CelEx-BR12 protein was characterized. The CelEx-BR12 gene had an open reading frame (ORF) of 1140 base pairs that encoded a 380-amino-acid-protein with a predicted molecular mass of 41.8 kDa. The amino acid sequence was 83% identical to that of a family 5 glycosyl hydrolase from Prevotella ruminicola 23. Codon-optimized CelEx-BR12 was overexpressed in Escherichia coli and purified using Ni–NTA affinity chromatography. The Michaelis–Menten constant (K_m value) and maximal reaction velocity (V_max values) for exocellulase activity were 12.92 μM and 1.55 × 10⁻⁴ μmol min⁻¹, respectively, and the enzyme was optimally active at pH 5.0 and 37 °C. Multifunctional activities were observed against fluorogenic and natural glycosides, such as 4-methylumbelliferyl-β-d-cellobioside (0.3 U mg⁻¹), CMC (105.9 U mg⁻¹), birch wood xylan (132.3 U mg⁻¹), oat spelt xylan (67.9 U mg⁻¹), and 2-hydroxyethyl-cellulose (26.3 U mg⁻¹). Based on these findings, we believe that CelEx-BR12 is an efficient multifunctional enzyme as endocellulase/exocellulase/xylanase activities that may prove useful for biotechnological applications.     

Preparation,cellular uptake and angiogenic suppression of shikonin-containing liposomes in?vitro and in?vivo

Shikonin has anticancer activity, but it has not yet been applied into clinical use. In the present study, shikonin was prepared using liposomes. We aimed to examine several aspects of sh-L (shikonin-containing liposomes): preparation, angiogenic suppression and cellular uptake through self-fluorescence. Sh-L were prepared using soybean phospholipid and cholesterol to form the membrane and shikonin was encapsulated into the phospholipid membrane. Three liposomes were prepared with shikonin. They had red fluorescence and were analysed using a flow cytometer. Angiogenic suppression of sh-L was determined using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide], Transwell tests, chick CAM (chorioallantoic membrane) and Matrigel™ plug assay. MTT assay showed the median IC₅₀ (inhibitory concentrations) as follows: shikonin, sh-L₁ and sh-L₂ were 4.99±0.23, 5.81±0.57 and 7.17±0.69 μM, respectively. The inhibition rates of migration were 53.58±7.05, 46.56±4.36 and 41.19±3.59% for 3.15 μM shikonin, sh-L₁ and sh-L₂, respectively. The results of CAM and Matrigel plug assay demonstrated that shikonin and sh-L can decrease neovascularization. Effect of shikonin was more obvious than sh-L at the same concentration. The results showed that sh-L decreased the toxicity, the rate of inhibition of migration and angiogenic suppression. The cellular uptake of the sh-L could be pictured because of the self-fluorescence. The self-fluorescence will be useful for conducting further research. Sh-L might be an excellent preparation for future clinical application to cancer patients.     

Novel recombinant human lactoferrin: Differential activation of oxidative stress related gene expression

Lactoferrin, an iron-binding protein found in high concentrations in mammalian exocrine secretions, is an important component of the host defense system. It is also a major protein of the secondary granules of neutrophils from which is released upon activation. Due to its potential clinical utility, recombinant human lactoferrin (rhLF) has been produced in various eukaryotic expression systems; however, none of these are fully compatible with humans. Most of the biopharmaceuticals approved by the FDA for use in humans are produced in mammalian expression systems. The Chinese hamster ovary cells (CHO) have become the system of choice for proteins that require post-translational modifications, such as glycoproteins.     

Effect of Feeding Millet (Sorghum vulgarie) Protein at Various Protein Levels on Adrenal Lipids of Rats

A significant increase in adrenal weight, total lipids, cholesterol phospholipids and glycerides (mono-and triglycerides) was observed in rats fed millet at 5, 10 and 15 % protein levels respectively for a period of six weeks as compared to rats fed casein at 10 per cent level. Increases in cholesterol were in both its free and esterified fraction. Adrenal phosphatidyl etha-nolamine was increased in all millet fed rats whereas phosphatidyl choline increased in M–15 % and decreased in M–5 % groups. Other phospholipid fractions viz. monophosphatidyl inositol, lysophosphatidyl ethanolamine, sphingomyeline, phosphatidic acid and polyglycerophosphatide also showed significant alterations in rats fed millet protein as compared to control. Incorporation of acetate–l–¹⁴C into adrenal lipids was lower and that of glucose–U–¹⁴C, palmitate–l–¹⁴C and NaH₂³²PO₄ was higher than the control.     

Isolation and Characterization of a Free Gibberellin and Glucosyl Esters of Gibberellins in Mature Seeds of Phaseolus vulgaris

Treatment of N⁶,N⁶,5′-O-tribenzoyl-2′,3′-O-isopropylidenetubercidin (VI) with aqueous acetic acid afforded N⁶,5′-O-dibenzoyltubercidin (V), which was mesylated to yield the dimesylate X. On treatment of X with sodium iodide and zinc dust, the 2′,3′-unsaturated derivatives of tubercidin XI and XIII were obtained.

N⁶,5′-O-Dibenzoyltubercidin 2′,3′-thionocarbonate (XIV), prepared from V by treatment with Corey-Winter reagent, was converted to the 1-methyl-2′,3′-unsaturated derivative XV in refluxing trimethyl phosphite.     

Homogeneous Enzymatic Assay for L-Cysteine with βC-S Lyase

We have developed a new enzymatic assay for determining L-cysteine concentration. The method involves the use of βC-S lyase from Streptococcus anginosus, which catalyzes the α,β-elimination of L-cysteine to hydrogen sulfide, pyruvate, and ammonia. The production of pyruvate is measured by D-lactate dehydrogenase and NADH. The decrease in NADH was proportional to the L-cysteine concentration up to 1.0 mM. When serum samples were used, within-day and day-to-day coefficient variations were below 4%. This method is simple, and can easily and reliably be used for accurate determination of L-cysteine concentration in serum or other samples.