Springer Science and Business Media LLC

Alpha-amylase inhibitors are widely used by the pharmaceutical and agricultural industries, such as the treatment of diabetes and obesity and insect controller. Here, we developed a colorimetric method to screen for α-amylase inhibitor producing strains or mutants with higher α-amylase inhibitor productivity. This method relies on absorbance changes at 402 nm that are due to the inhibition of α-amylase catalyzed hydrolysis of 2-Chloro-4-nitrophenyl-4-O-β-D-galactopyranosyl-maltoside by α-amylase inhibitors. The assay can be performed on a microtiter plate, making it simple and convenient. Using this method, α-amylase inhibitor producing strains and mutants with higher α-amylase inhibitor productivity can be rapidly screened. One strain, ZJB-08196, with the highest α-amylase inhibition was isolated and identified as Actinoplanes utahensis, and one mutant with higher acarbose production was obtained by screening 3,000 variants using this method.

Interferon-induced transmembrane protein 1 (IFITM1) is a transmembrane protein that is an essential mediator of interferon-induced anti-proliferation. Identifying the functional role of IFITM1 has been difficult because of high expression of the protein induces cell death. In this study, we showed that IFITM1 is localized to the mitochondria and that its expression is related to mitochondrial reactive oxygen species (mROS). Overexpression of IFITM1 resulted in a wrinkled and ruptured cell morphology, and subcellular fractionation revealed IFITM1 expression in the mitochondria and the plasma membrane. Interestingly, IFITM1 was expressed at high levels in the mitochondria of MPRO cells, but only at low levels in the plasma membrane. A mitochondrial staining assay confirmed that IFITM1 was localized to the mitochondria in MPRO cells. In addition, IFITM1 expression was significantly increased by treatment with H2O2, arachidonic acid, and dithiothreitol. These findings suggest that IFITM1 expression is not restricted to the membrane, but is present in the mitochondria as well. Furthermore, mitochondrial IFITM1 expression is regulated by mROS.

The ErbB signaling pathway plays important roles in normal physiology and cancer, which consists of four proto-oncogene receptor tyrosine kinases ErbB1/EGFR ErbB2/Her2, ErbB3/Her3, and ErbB4/Her4. Selective targeting of different ErbB kinases would result in distinct therapeutic effects, but traditional small-molecule inhibitors generally exhibit a strong cross-reactivity over these kinases due to the very high conservation in kinase’s active site. Instead of developing small-molecule drugs to selectively target the conserved active site of ErbB kinases, we herein attempt to design peptide agents for selectively disrupting the dimerization event of these kinases at their asymmetric dimer interfaces that have a relatively low homology. Three hotspot peptides S1P1, S1P2, and S1P3 are split from the functional segment 1 (Seg1) of mitogen-inducible gene 6 (Mig6), a natural EGFR-inhibitory protein that has been observed to inactivate the kinase by disrupting its dimerization. We demonstrate that the Mig6 peptides not only inhibit EGFR but also bind Her2, Her3, and Her4, although the peptide affinities to the four ErbB kinases are different considerably, exhibiting a typical selectivity. The S1P2 peptide locates in the core binding region of Mig6 Seg1 and contributes significantly to the segment interaction with kinases. An iteration algorithm is employed to guide the directed molecular engineering of S1P2 peptide selectivity towards each of the four kinases. Hundreds of parallel evolution running yield a series of peptide candidates with potential selectivity, which are then substantiated by fluorescence-based assays. The designed EGFR-selective peptide S1P2-p1EGFR is determined to have a moderate affinity to EGFR (Kd = 56 µM) and a high selectivity for EGFR over Her2, Her3, and H4 (FEGFR = 10.1-fold), which is improved considerably relative to wild-type S1P2 peptide (FEGFR = 2.7-fold). Structural examination observes different noncovalent interaction modes at the complex interfaces of S1P2-p1EGFR with EGFR and other three kinases, revealing a molecular origin of the peptide selectivity.

Baculovirus expression vector insect cell system (BEVIS) is useful for high level production of human therapeutic proteins. However, it has been reported that recombinant glycoproteins produced from this system are, in many cases, biologically inactive or less active than authentic counterparts, due to incomplete glycosylation potential of insect cells used so far, producing recombinant proteins with only high-or paucimannosidic oligosaccharides without sialylation. The presence of sialic acids in insects is still controversial. Egg proteins ofTrichoplusia ni andDanaus plexippus were isolated, and the presence of sialic acids was examined using reverse-phase fluorescent HPLC after derivatization of samples with 1,2-diamino-4,5-methylenedioxybenzene (DMB). The proteins of both eggs were shown to contain 5-N-acetylneuraminic acid. The results suggest that both insects may be able to produce proteins with sialylated complex-type oligosaccharide chains.

Adeno-associated virus (AAV)-mediated gene therapy holds significant promises to treat or potentially cure various human diseases. Although AAV holds promise for their significant therapeutic potential, batch-to-batch differences can exist from manufacturing; and therefore, a potency assay is required for clinical development of AAV. Among different serotypes, due to its ability to cross blood–brain barrier and wide-spread transduction capability in vivo upon systemic administration, AAV9 has been widely utilized for the development of treatment of neurological disorders. However, as AAV9 is known to show poor transduction in vitro, establishing a robust in vitro potency assay have been difficult. To this end, we engineered HEK293T and Schwann-like cell lines to express previously identified common AAV receptor, AAVR or endogenous host factor involved in AAV endosomal escapes, GPR108 that can increase infectivity of AAVs in an attempt to increase transduction capability of AAV9. We found that AAVR overexpressed Schwann-like cell line showed significant increase in AAV9 transduction; whereas, GPR108 overexpression showed no effect on AAV9 transduction. On the other hand, GPR108 engineered HEK293T showed increase in AAV9 transduction; whereas, AAVR overexpressed HEK293T cell line showed modest increase in AAV9 transduction. Gene expression analysis showed that AAVR is highly expressed in HEK293T compared to Schwann-like cell line; whereas, GPR108 is highly expressed in Schwann-like cell line when compared to HEK293T. These results indicate that different cell lines may require different gene engineering to increase AAV9 infectivity and analysis of endogenous expression of AAV entry factors for cell line to be engineered can improve efficiency of cell line engineering for AAV transduction.

This study examined the role and physiological relevance of 3-hydroxyisobutyrate dehydrogenase-I (3HIBDHI) of Pseudomonas denitrificans ATCC 13867 in the degradation of 3-hydroxypropionic acid (3-HP) during 3-HP production. The gene encoding 3HIBDH-I of P. denitrificans ATCC 13867 was cloned and expressed in Escherichia coli BL21 (DE3). The recombinant 3HIBDH-I was then purified on a Ni-NTA-HP column and characterized for its choice of substrates, cofactors, metals, reductants, and the optimal temperature and pH. The recombinant 3HIBDH-I showed a high catalytic constant (k cat/K m) of 604.1 ± 71.1 mM/S on (S)-3-hydroxyisobutyrate, but no detectable activity on (R)-3-hydroxyisobutyrate. 3HIBDH-I preferred NAD+ over NADP+ as a cofactor for its catalytic activity. The k cat/K m determined for 3-HP was 15.40 ± 1.43 mM/S in the presence of NAD+ at 37°C and pH 9.0. In addition to (S)-3-hydroxyisobutyrate and 3-HP, 3HIBDH-I utilized l-serine, methyl-d,l-serine, and methyl-(S)-(+)-3-hydroxy-2-methylpropionate; on the other hand, the k cat/K m values determined for these substrates were less than 5.0mM/S. Ethylenediaminetetraacetic acid, 2-mercaptoethanol, dithiothreitol and Mn2+ increased the activity of 3HIBDHI significantly, whereas the presence of Fe2+, Hg2+ and Ag+ in the reaction mixture at 1.0 mM completely inhibited its activity. This study revealed the characteristics of 3HIBDH-I and its significance in 3-HP degradation.

Polymerase chain reaction (PCR) is one of the most widely used analytical tool and is an important module that would benefit from being miniaturized and integrated onto diagnostic or analytical chips. There are potentially two different approaches for the miniaturization of the PCR module: chamber-type and flow-type micro-PCR. These miniaturized PCRs have distinct characteristics and advantages. In this article, we review the necessity of micro-PCR, the materials for the chip fabrication, the surface modification, and characteristics of the two types of micro-PCR. The motivation underlying the development of micro-PCR, the advantages and disadvantages of the various materials used in fabrication and the surface modification methods will be discussed. And finally, the precise features of the two different types of micro-PCR will be compared.

Frataxin, a small nuclear-encoded protein targeted to mitochondria, is known to play an important role in both the mitochondrial respiratory chain and iron homeostasis. The protein is highly conserved in most eukaryotic organisms with no major structural changes, suggesting that it serves a crucial function in all organisms. Recently, purified frataxin was used as a therapeutic treatment of Friedreich’s ataxia, a common degenerative disorder that results from a frataxin protein deficiency, by directly applying the protein to the diseased cells. In this report, we describe a novel and rapid method of synthesizing genes encoding frataxin proteins for the purpose of efficient protein production. The artificial yeast and human frataxin genes were synthesized by direct assembly of serial deoxyoligonucleotide primers designed based on the optimal nucleotide sequences. When we tested the expression of these synthetic genes in two E. coli host strains, the yeast frataxin gene was expressed 20 folds higher in Rosetta (DE3) cells than in BL21 (DE3) cells, whereas the expression levels of human frataxin were similar in both E. coli strains. Attenuation of the Fenton reactions by the purified yeast and human frataxin proteins was observed under the defined conditions, which suggests that the recombinant frataxin proteins are active and functional. The procedure described here could be applied to many known genes or to generate novel synthetic genes that can be redesigned by arranging functional domains from previously identified genes and to study the structure and function of synthetic recombinant proteins and potential usage.

Exposure to high levels of atmospheric particulate matter (PM) with an aerodynamic diameter of less than 2.5 µm (PM2.5) causes respiratory injury mainly due to oxidative stress. Although the fisetin has biological activities such as the antiviral, neuroprotective, and anti-inflammatory activities, the effect of fisetin on PM-mediated oxidative damage has not been studied. In this study, we tested the protective effect of fisetin against PM2.5-induced toxicity in human pulmonary artery endothelial cells (HPAECs) and its molecular mechanism. Exposure to PM2.5 decreased cell viability in HPAECs in a time- and dose-dependent manner, possibly due to increased release of extracellular lactate dehydrogenase and generation of intracellular reactive oxygen species (ROS). Cell viability assay demonstrated that treatment of HPAECs with fisetin increased cell viability and reduced PM2.5-induced oxidative stress in a dose-dependent manner. Serum- and glucocorticoid-inducible kinase 1 (SGK1), a crucial cell survival factor, was downregulated by PM2.5 which was recovered by fisetin. Furthermore, fisetin treatment inhibited intracellular ROS in HPAECs generated by PM2.5. Moreover, decreased antioxidant enzymes activities of superoxide dismutase and catalase level in PM2.5-treated cells were reversed by fisetin treatment. Our results suggest that fisetin effectively protects human HPAECs from PM2.5-induced oxidative damage via antioxidant effects.