Isolation and Characterization of Mutations in the β-Tubulin Gene of SACCHAROMYCES CEREVISIAE

Of 173 mutants of Saccharomyces cerevisiae resistant to the antimitotic drug benomyl (BenR), six also conferred cold-sensitivity for growth and three others conferred temperature-sensitivity for growth in the absence of benomyl. All of the benR mutations tested, including the nine conditional-lethal mutations, were shown to be in the same gene. This gene, TUB2, has previously been molecularly cloned and identified as the yeast structural gene encoding beta-tubulin. Four of the conditional-lethal alleles of TUB2 were mapped to particular restriction fragments within the gene. One of these mutations was cloned and sequenced, revealing a single amino acid change, from arginine to histidine at amino acid position 241, which is responsible for both the BenR and the cold-sensitive lethal phenotypes. The terminal arrest morphology of conditional-lethal alleles of TUB2 at their restrictive temperature showed a characteristic cell-division-cycle defect, suggesting a requirement for tubulin function primarily in mitosis during the vegetative growth cycle. The TUB2 gene was genetically mapped to the distal left arm of chromosome VI, very near the actin gene, ACT1; no CDC (cell-division-cycle) loci have been mapped previously to this location. TUB2 is thus the first cell-division-cycle gene known to encode a cytoskeletal protein that has been identified in S. cerevisiae.     

Supersensitivity of Escherichia coli Cells to Several β-Lactam Antibiotics Caused by Rod− Morphology Mutations at the mrd Gene Cluster

Two kinds of spherical mutants, mrdA and mrdB mutants, have been isolated from Escherichia coli strain K12. The mrdA mutants have thermosensitive penicillin-binding protein 2, while the mrdB mutants have normal penicillin-binding proteins. Both kinds of mutants form spherical cells at 42°C and are resistant to the amidinopenicillin, mecillinam, at the same temperature. The two mutations have been mapped very close to lip at 14.2 min (revised chromosome linkage map, 1980) on the E. coli chromosome. Both mutations cause supersensitivities of cell growth to various β-lactam antibiotics, such as ampicillin, cephalexin, cefoxitin and nocardicin A at 42°C.     

Leptin boosts cellular metabolism by activating AMPK and the sirtuins to reduce tau phosphorylation and β-amyloid in neurons

Leptin is a pleiotropic hormone primarily secreted by adipocytes. A high density of functional Leptin receptors has been reported to be expressed in the hippocampus and other cortical regions of the brain, the physiological significance of which has not been explored extensively. Alzheimer’s disease (AD) is marked by impaired brain metabolism with decreased glucose utilization in those regions which often precede pathological changes. Recent epidemiological studies suggest that plasma Leptin is protective against AD. Specifically, elderly with plasma Leptin levels in the lowest quartile were found to be four times more likely to develop AD than those in the highest quartile. We have previously reported that Leptin modulates AD pathological pathways in vitro through a mechanism involving the energy sensor, AMP-activated protein kinase (AMPK). To this end, we investigated the extent to which activation of AMPK as well as another class of sensors linking energy availability to cellular metabolism, the sirtuins (SIRT), mediate Leptin’s biological activity. Leptin directly activated neuronal AMPK and SIRT in cell lines. Additionally, the ability of Leptin to reduce tau phosphorylation and β-amyloid production was sensitive to the AMPK and sirtuin inhibitors, compound C and nicotinamide, respectively. These findings implicate that Leptin normally acts as a signal for energy homeostasis in neurons. Perhaps Leptin deficiency in AD contributes to a neuronal imbalance in handling energy requirements, leading to higher Aβ and phospho-tau, which can be restored by replenishing low Leptin levels. This may also be a legitimate strategy for therapy.     

Release of Mediator Enzyme β-Hexosaminidase and Modulated Gene Expression Accompany Hemocyte Degranulation in Response to Parasitism in the Silkworm Bombyx mori

In insects infections trigger hemocyte-mediated immune reactions including degranulation by exocytosis; however, involvement of mediator enzymes in degranulation process is unknown in insects. We report here that in silkworm Bombyx mori, infection by endoparasitoid Exorista bombycis and microsporidian Nosema bombycis activated granulation in granulocytes and promoted degranulation of accumulated structured granules. During degranulation the mediator lysosomal enzyme β-hexosaminidase showed increased activity and expression of β-hexosaminidase gene was enhanced. The events were confirmed in vitro after incubation of uninfected hemocytes with E. bombycis larval tissue protein. On infection, cytotoxicity marker enzyme lactate dehydrogenase (LDH) was released from the hemocytes illustrating cell toxicity. Strong positive correlation (R²?=?0.71) between LDH activity and β-hexosaminidase released after the infection showed parasitic–protein-induced hemocyte damage and accompanied release of the enzymes. Expression of β-hexosaminidase gene was enhanced in early stages after infection followed by down regulation. The expression showed positive correlation (R²?=?0.705) with hexosaminidase activity pattern. B. mori hexosaminidase showed 98% amino acid similarity with that of B. mandarina showing origin from same ancestral gene; however, 45–60% varied from other lepidopterans showing diversity. The observation signifies the less known association of hexosaminidase in degranulation of hemocytes induced by parasitic infection in B. mori and its divergence in different species.

     

Characterization of the β-Carotene Hydroxylase Gene DSM2 Conferring Drought and Oxidative Stress Resistance by Increasing Xanthophylls and Abscisic Acid Synthesis in Rice

Drought is a major limiting factor for crop production. To identify critical genes for drought resistance in rice (Oryza sativa), we screened T-DNA mutants and identified a drought-hypersensitive mutant, dsm2. The mutant phenotype was caused by a T-DNA insertion in a gene encoding a putative β-carotene hydroxylase (BCH). BCH is predicted for the biosynthesis of zeaxanthin, a carotenoid precursor of abscisic acid (ABA). The amounts of zeaxanthin and ABA were significantly reduced in two allelic dsm2 mutants after drought stress compared with the wild type. Under drought stress conditions, the mutant leaves lost water faster than the wild type and the photosynthesis rate, biomass, and grain yield were significantly reduced, whereas malondialdehyde level and stomata aperture were increased in the mutant. The mutant is also hypersensitive to oxidative stresses. The mutant had significantly lower maximal efficiency of photosystem II photochemistry and nonphotochemical quenching capacity than the wild type, indicating photoinhibition in photosystem II and decreased capacity for eliminating excess energy by thermal dissipation. Overexpression of DSM2 in rice resulted in significantly increased resistance to drought and oxidative stresses and increases of the xanthophylls and nonphotochemical quenching. Some stress-related ABA-responsive genes were up-regulated in the overexpression line. DSM2 is a chloroplast protein, and the response of DSM2 to environmental stimuli is distinctive from the other two BCH members in rice. We conclude that the DSM2 gene significantly contributes to control of the xanthophyll cycle and ABA synthesis, both of which play critical roles in the establishment of drought resistance in rice.Abiotic stresses such as drought, salinity, and adverse temperatures are major limiting factors for plant growth and reproduction. To respond to environmental cues, plants have evolved a variety of biochemical and physiological mechanisms to adapt to adverse conditions during their growth and development (Boyer, 1982). Abscisic acid (ABA) has been recognized as a stress hormone that coordinates the complex networks of stress responses. Under drought or salt stress conditions, plant endogenous ABA level can rise to about 40-fold, triggering the closure of stomata and accumulating reactive oxygen species (ROS), dehydrins, and late embryogenesis abundant proteins for osmotic adjustment (Verslues et al., 2006). The endogenous ABA level is determined by ABA biosynthesis, catabolism, and release of ABA from ABA-Glc conjugates (Nambara and Marion-Poll, 2005; Lee et al., 2006). Therefore, identification of all the components affecting active ABA content is essential for a complete understanding of the action of the hormone.Numerous ABA biosynthetic genes have been identified through mutant analysis, such as maize (Zea mays) viviparous mutants vp2, vp5, vp7, vp9, vp14, w3, y3, and y9 (Schwartz et al., 1997; Hable et al., 1998; Singh et al., 2003); rice (Oryza sativa) preharvest-sprouting mutants psh1, psh2, psh3, and psh4 (Fang et al., 2008); sunflower (Helianthus annuus) nondormant mutant nd-1 (Conti et al., 2004); Arabidopsis (Arabidopsis thaliana) ABA- and nonphotochemical quenching (NPQ)-deficient mutants aba1, aba2, aba3, aba4, npq1, npq2, b1, b2, and nced3 (Havaux et al., 2000; Xiong et al., 2001; Tian et al., 2003; Barrero et al., 2005; Kim and DellaPenna, 2006; North et al., 2007); and tomato (Solanum lycopersicum) white-flower mutant wf (Galpaz et al., 2006; Supplemental Fig. S1). The mutants unable to biosynthesize carotenoid precursors for endogenous ABA synthesis often produced preharvest-sprouting seeds and wilted or white leaves (Gubler et al., 2005; Nambara and Marion-Poll, 2005; Finch-Savage and Leubner-Metzger, 2006).ABA biosynthesis initiates with the synthesis of a C5 building block, isopentenyl pyrophosphate, and its isomer dimethylallyl pyrophosphate through a plastid methylerythritol phosphate pathway (Eisenreich et al., 2001; Hunter, 2007). The three isopentenyl pyrophosphate molecules are then added to dimethylallyl pyrophosphate by geranylgeranyl diphosphate synthase to produce C20 geranylgeranyl diphosphate. Two geranylgeranyl diphosphates are condensed by a committing enzyme, phytoene synthase, to produce colorless C40 carotenoid phytoene, which is then desaturated and isomerized into red-colored lycopene by phytoene desaturase (PDS), ζ-carotene desaturase (ZDS), and Z-ISO and CRTISO isomerases in plants (Isaacson et al., 2002; Park et al., 2002). Subsequently, several cyclization and hydroxylation reactions take place to yield α-carotene and β-carotene (Li et al., 1996; Hable et al., 1998; Park et al., 2002; Miki and Shimamoto, 2004; Fang et al., 2008). Heme-type cytochrome P450-type CYP97 and non-heme-type β-carotene hydroxylase (BCH) are primarily responsible for the hydroxylation of α-carotene and β-carotene to produce lutein and zeaxanthin, respectively. Zeaxanthin, an important component of the xanthophyll cycle, is epoxidated by zeaxanthin epoxidase to produce violaxanthin, and this reaction can be reversed by violaxanthin deepoxidase to increase the xanthophyll cycle for plants to adapt to high-light stress (Johnson et al., 2008). Neoxanthin synthase converts violaxanthin into neoxanthin (North et al., 2007). In chloroplast, 9-cis-epoxycarotenoid dioxygenase (NCED) cleaves violaxanthin and neoxanthin to produce xanthoxin, the direct substrate for ABA synthesis via ABA aldehyde (Schwartz et al., 1997, 2003; Xiong and Zhu, 2003). Increasing evidence suggest that the endogenous ABA level is fine-tuned by differential regulation of the multiple steps of ABA biosynthesis (Seo and Koshiba, 2002; Nambara and Marion-Poll, 2005; Destefano-Beltrán et al., 2006; Thompson et al., 2007; Rodríguez-Gacio et al., 2009; Supplemental Fig. S1).The xanthophyll cycle (light-dependent reversible conversion between violaxanthin and zeaxanthin) is involved in photoprotection in PSII by regulating the nonradiative dissipation of excess absorbed light energy as heat (Gilmore et al., 1994). Mutants with defects in the xanthophyll cycle exhibit a weak photoprotective ability and produce ROS such as hydrogen peroxide (H₂O₂) when the absorption of light energy exceeds that consumed for photosynthesis (Niyogi, 1999). Under dehydration stress, electrons at a high energy state can easily form ROS, which are toxic to proteins, DNA, and lipids (Mittler, 2002; Apel and Hirt, 2004). However, plants have evolved a variety of biochemical and physiological mechanisms to scavenge ROS, thus maintaining a balance between ROS production and scavenging (Mittler et al., 2004).An association between the xanthophyll cycle and stress tolerance has been reported in plants. In Arabidopsis, overexpression of a bacterial BCH gene caused a specific 2-fold increase in the size of the xanthophyll cycle and enhanced photooxidative tolerance (Davison et al., 2002). Constitutive overexpression of a bacterial BCH gene, crtZ, in tobacco (Nicotiana tabacum) led to increased zeaxanthin synthesis and enhanced UV light tolerance (Götz et al., 2002). In Arabidopsis, zeaxanthin synthesis can be catalyzed by both heme-type CYP97 hydroxylases LUT1 and LUT5 and non-heme-type hydroxylases BCH1 and BCH2, and these two types exhibit some overlapping activities (Tian et al., 2003, 2004; Kim and DellaPenna, 2006). In contrast to the intensive molecular and genetic studies of BCH in Arabidopsis, the counterpart in economically important crops such as rice has not been identified.In this study, we characterized the rice drought-sensitive mutant dsm2, impaired in the gene DSM2 encoding a BCH. Our results demonstrate that DSM2 acts as a putative enzyme catalyzing the biosynthesis of zeaxanthin, one of the precursors of ABA that participates in the process of NPQ. Decreases of NPQ, maximal efficiency of PSII photochemistry (F_v/F_m), xanthophylls, and ABA in the dsm2 mutant suggest that the drought hypersensitivity of dsm2 is due to the combination of impairments in the xanthophyll cycle and ABA synthesis under drought stress conditions. DSM2 overexpression lines, possessing high F_v/F_m and NPQ, showed significantly improved drought resistance at both seedling and reproductive stages. Furthermore, our results imply that DSM2 may be the major member of the BCH family in rice for controlling zeaxanthin synthesis in response to dehydration stresses.     

Aversion to nicotine is regulated by the balanced activity of β4 and α5 nicotinic receptor subunits in the medial habenula

Nicotine dependence is linked to single nucleotide polymorphisms in the CHRNB4-CHRNA3-CHRNA5 gene cluster encoding the α3β4α5 nicotinic acetylcholine receptor (nAChR). Here we show that the β4 subunit is rate limiting for receptor activity, and that current increase by β4 is maximally competed by one of the most frequent variants associated with tobacco usage (D398N in α5). We identify a β4-specific residue (S435), mapping to the intracellular vestibule of the α3β4α5 receptor in close proximity to α5 D398N, that is essential for its ability to increase currents. Transgenic mice with targeted overexpression of Chrnb4 to endogenous sites display a strong aversion to nicotine that can be reversed by viral-mediated expression of the α5 D398N variant in the?medial habenula (MHb). Thus, this study both provides insights into α3β4α5 receptor-mediated mechanisms contributing to nicotine consumption, and identifies the MHb as a critical element in the circuitry controlling nicotine-dependent phenotypes.     

Directed modification of the Aspergillus usamii β-mannanase to improve its substrate affinity by in silico design and site-directed mutagenesis

β-Mannanases (EC 3.2.1.78) can catalyze the cleavage of internal β-1,4-d-mannosidic linkages of mannan backbones, and they have found applications in food, feed, pulp and paper, oil, pharmaceutical and textile industries. Suitable amino acid substitution can promote access to the substrate-binding groove and maintain the substrate therein, which probably improves the substrate affinity and, thus, increases catalytic efficiency of the enzyme. In this study, to improve the substrate affinity of AuMan5A, a glycoside hydrolase (GH) family 5 β-mannanase from Aspergillus usamii, had its directed modification conducted by in silico design, and followed by site-directed mutagenesis. The mutant genes, Auman5A ^Y111F and Auman5A ^Y115F, were constructed by megaprimer PCR, respectively. Then, Auman5A and its mutant genes were expressed in Pichia pastoris GS115 successfully. The specific activities of purified recombinant β-mannanases (reAuMan5A, reAuMan5A^Y111F and reAuMan5A^Y115F) towards locust bean gum were 152.5, 199.6 and 218.9 U mg^?1, respectively. The two mutants were found to be similar to reAuMan5A regarding temperature and pH characteristics. Nevertheless, the K _m values of reAuMan5A^Y111F and reAuMan5A^Y115F, towards guar gum, decreased to 2.95 ± 0.22 and 2.39 ± 0.33 mg ml^?1 from 4.49 ± 0.07 mg ml^?1 of reAuMan5A, which would make reAuMan5A^Y111F and reAuMan5A^Y115F promising candidates for industrial processes. Structural analysis showed that the two mutants increased their affinity by decreasing the steric conflicts with those more complicated substrates. The results suggested that subtle conformational modification in the substrate-binding groove could substantially alter the substrate affinity of AuMan5A. This study laid a solid foundation for the directed modification of substrate affinities of β-mannanases and other enzymes.     

Growth Arrest by the Antitumor Steroidal Lactone Withaferin A in Human Breast Cancer Cells Is Associated with Down-regulation and Covalent Binding at Cysteine 303 of β-Tubulin

Withaferin A (WA), a C₅,C₆-epoxy steroidal lactone derived from a medicinal plant (Withania somnifera), inhibits growth of human breast cancer cells in vitro and in vivo and prevents mammary cancer development in a transgenic mouse model. However, the mechanisms underlying the anticancer effect of WA are not fully understood. Herein, we report that tubulin is a novel target of WA-mediated growth arrest in human breast cancer cells. The G₂ and mitotic arrest resulting from WA exposure in MCF-7, SUM159, and SK-BR-3 cells was associated with a marked decrease in protein levels of β-tubulin. These effects were not observed with the naturally occurring C₆,C₇-epoxy analogs of WA (withanone and withanolide A). A non-tumorigenic normal mammary epithelial cell line (MCF-10A) was markedly more resistant to mitotic arrest by WA compared with breast cancer cells. Vehicle-treated control cells exhibited a normal bipolar spindle with chromosomes aligned along the metaphase plate. In contrast, WA treatment led to a severe disruption of normal spindle morphology. NMR analyses revealed that the A-ring enone in WA, but not in withanone or withanolide A, was highly reactive with cysteamine and rapidly succumbed to irreversible nucleophilic addition. Mass spectrometry demonstrated direct covalent binding of WA to Cys³⁰³ of β-tubulin in MCF-7 cells. Molecular docking indicated that the WA-binding pocket is located on the surface of β-tubulin and characterized by a hydrophobic floor, a hydrophobic wall, and a charge-balanced hydrophilic entrance. These results provide novel insights into the mechanism of growth arrest by WA in breast cancer cells.     

TRAF4 participates in Wnt/β-catenin signaling in breast cancer by upregulating β-catenin and mediating its translocation to the nucleus

Tumor necrosis factor receptor-associated factor 4 (TRAF4) is upregulated in various subtypes of breast cancers and cell lines; however, the precise functions of TRAF4 are poorly understood. Our objective was to investigate its relationship with β-catenin. TRAF4 participates in several signaling pathways, such as NF-κB and JNK signaling pathways. In this study, we identified β-catenin as a TRAF4-binding protein, have shown that TRAF4 enhanced expression of β-catenin, and found that TRAF4 mediated the translocation of β-catenin from the cytoplasm to the nucleus, thereby facilitating activation of the Wnt signaling pathway in breast cancer.     

A Missense Mutation in CRYBB2 Leads to Progressive Congenital Membranous Cataract by Impacting the Solubility and Function of βB2-Crystallin

Congenital cataract is a major cause of visual impairment and childhood blindness. The solubility and stability of crystallin proteins play critical roles in maintaining the optical transparency of the lens during the life span. Previous studies have shown that approximately 8.3%∼25% of congenital cataracts are inherited, and mutations in crystallins are the most common. In this study, we attempted to identify the genetic defect in a four-generation family affected with congenital cataracts. The congenital cataract phenotype of this four-generation family was identified as membranous cataract by slit-lamp photography. Mutation screening of the candidate genes detected a heterozygous c.465G→C change in the exon6 of the βB2-crystallin gene (CRYBB2) in all family members affected with cataracts, resulting in the substitution of a highly conserved Tryptophan to Cystine (p.W151C). The mutation was confirmed by restriction fragment length polymorphism (RFLP) analysis and found that the transition resulted in the absence of a BslI restriction site in the affected members of the pedigree. The outcome of PolyPhen-2 and SIFT analysis predicted that this W151C mutation would probably damage to the structure and function of βB2-crystallin. Wild type (wt) and W151C mutant βB2-crystallin were expressed in human lens epithelial cells (HLECs), and the fluorescence results showed that Wt-βB2-crystallin was evenly distributed throughout the cells, whereas approximately 34.7% of cells transfected with the W151C mutant βB2-crystallin formed intracellular aggregates. Taken together, these data suggest that the missense mutation in CRYBB2 gene leads to progressive congenital membranous cataract by impacting the solubility and function of βB2-crystallin.     

Assignment of the Gene for Porcine Insulin-Like Growth Factor Binding Protein 1 to Chromosome 18 and Detection of Polymorphisms in Intron 2 by PCR–RFLP

We have obtained a partial cDNA and three BAC clones for the porcine insulin-like growth factor binding protein 1 gene (IGFBP-1). Results of fluorescence in situ and radiation hybrid (RH) mapping assigned this gene to porcine chromosome (SSC) 18q24-qter. We found two types of polymerase chain reaction–restriction-fragment-length polymorphisms (PCR–RFLP) in intron 2 by using FokI and AluI.     

The adjuvant effect of Sophy β-glucan to the antibody response in poultry immunized by the avian influenza A H5N1 and H5N2 vaccines

Avian influenza virus vaccines produced in oil-emulsified inactivated form with antigen content of at least 160 hemagglutinin units (HAU) induced immunity in birds. However, in addition to enhancing the effect of the adjuvant(s), other additional supplemented biological compounds included in inactivated vaccines could produce higher levels of antibody. We examined in chickens, Vietnamese ducks, and muscovy ducks the adjuvant effect of Sophy β-glucan (SBG), a β-1,3-1,6 glucan produced by the black yeast Aureobasidium pollulans strain AF0-202, when administered with an avian influenza H5 subtype vaccine. In Experiment 1, 40 chickens (ISA Brown hybrid), allocated to four groups of ten each, were immunized with Oil-H5N1(VN), Oil-H5N1(CN), Oil-H5N2(CN), and saline (control group), respectively. In Experiment 2, chickens (ISA Brown hybrid), muscovy ducks (French hybrid), and Vietnamese ducks (indigenous Vietnamese) were used to further assess the effect of SBG on immunogenicity of the Oil-H5N1(VN) Vietnamese vaccine. ELISA and hemagglutination inhibition (HI) assays were used to assess the antibody response. The H5 subtype vaccines initiated significantly higher immune responses in the animals dosed with SBG, with 1.0-1.5 log2 higher HI titers and 10-20% ELISA seroconversion, compared with those not dosed with β-glucan. Notably, some of the animals dosed with SBG induced HI titers higher than 9.0 log2 following boosting immunization. Taken together, our serial studies indicated that SBG is a potential effector, such as enhancing the immune response to the H5 vaccines tested.     

One Hundred Seventy-Fold Increase in Excretion of an FV Fragment-Tumor Necrosis Factor Alpha Fusion Protein (sFV/TNF-α) from Escherichia coli Caused by the Synergistic Effects of Glycine and Triton X-100

To target tumor necrosis factor alpha (TNF-α) to tumor cells, recombinant DNA techniques were used to construct and express the fused gene V_KLV_H–TNF-α, which encodes the secreted form of single-chain fusion protein sFV/TNF-α in Escherichia coli. sFV/TNF-α was secreted into the culture medium and purified by affinity chromatography. The production of the fusion protein in the culture medium under the optimal conditions of 30°C and 37 μmol of isopropyl-β-d-thiogalactopyranoside (IPTG) per liter was 16- and 5-fold higher than that under the standard conditions of 37°C and 1 mmol of IPTG per liter. Fusion protein excretion into culture medium with 2% glycine, 1% Triton X-100, or both of these two chemicals was either 14-, 38-, or 170-fold higher, respectively than that without the two chemicals. The final yield of sFV/TNF-α was estimated to be 50 mg/liter. The loss of integrity of the cellular membrane may be a potential mechanism for enhancement of fusion protein production and excretion by treatment with glycine and Triton X-100. This study thus provides a practical, large-scale method for more efficient production of the heterologous fusion protein sFV/TNF-α in E. coli by using glycine and Triton X-100.The bacterium Escherichia coli has become a commonly used system for expression of heterologous recombinant proteins of interest in both biological research and the biotechnology industry. A variety of properties make the E. coli expression system attractive, namely, ease of genetic manipulation, efficient transformation, fast growth, simple fermentation, and favorable economics. Many recombinant proteins, including antibodies (15, 24) and single-chain fusion proteins (5, 25), have been successfully expressed in E. coli. Two major forms of heterologous proteins are usually expressed in this bacterium, i.e., insoluble and soluble. The former do not contain a signal peptide and are expressed in the cytoplasm and subsequently packaged into highly condensed inclusion bodies (2), while the latter have a signal peptide that is expressed in the cytoplasm and subsequently secreted into the periplasmic compartment. Although a high level of heterologous protein expression in inclusion bodies can be attained, these proteins are insoluble and therefore nonfunctional. A process of protein denaturing followed by a complex renaturing procedure must be conducted to obtain properly refolded functional proteins (3). However, the final yield of these soluble refolded proteins is usually very low, due mainly to protein aggregation resulting from the exposure of hydrophobic peptide regions (10).An alternative is to express the secretory form of heterologous proteins from the E. coli periplasmic fractions (23). Sometimes, however, the secreted heterologous proteins can leak from the periplasm into the culture medium, possibly due to the increased permeability of cellular membranes during long incubation periods (24). Secretion of these proteins into the E. coli periplasm is a useful ploy that can lead to the rapid isolation of recombinant proteins for biological evaluation. Its application on an industrial scale is limited by the general unavailability of efficient large-scale methods for the selective release of periplasmic proteins from the cell. Since it is easier to process the heterologous proteins in the culture medium than in the periplasmic fraction, various approaches have been developed to enhance the secretion of heterologous proteins of E. coli into culture media. These include (i) optimizing culturing conditions by modifying the temperature (4) or the concentration of isopropyl-β-d-thiogalactopyranoside (IPTG) (3), (ii) coexpression of molecular chaperones (13, 26), (iii) genetic modification of expression vectors (6, 11) or recombinant genes (8), and (iv) addition of chemicals such as glycine (9, 14).Recombinant single-chain fusion proteins have increasingly attracted attention in both research and clinical use due to their novel bifunctional activity and small size (5, 25). We have previously reported the construction and expression of single-chain fusion protein FV/TNF-α in inclusion bodies of E. coli (29). The fusion protein contains a single-chain FV fragment consisting of an immunoglobulin variable region of the heavy (V_H) (12.5 kDa) and light (V_K) (12.5 kDa) chains of the B72.3 antibody recognizing the human tumor-associated TAG72 antigen (28) and the tumor necrosis factor alpha (TNF-α) moiety (18 kDa). Previous studies have demonstrated that small antibody fragments such as FV (25 kDa) showed deeper, as well as more homologous, penetration of tumors by the molecule (19) and a higher localization index of tumors versus normal tissues (7) than the large intact antibody molecule (150 kDa). Therefore, this fusion protein (43 kDa) has the potential to efficiently target TNF-α to tumors expressing the TAG72 antigen for induction of active antitumor immune responses. Although the fusion protein retained its bifunctional activity after the process of denaturing and refolding, it still tended to aggregate especially in concentrations used in experimental animal model studies. This greatly limited its potential use as an antitumor therapeutic reagent. In this report, we describe the construction and expression of a secreted form of single-chain fusion protein sFV/TNF-α in E. coli. We also demonstrate dramatic enhancement of the excretion of this heterologous fusion protein from E. coli into culture media by the synergistic effect of glycine and Triton X-100.     

Distinct signaling pathways leading to the induction of human β-defensin 2 by stimulating an electrolyticaly-generated acid functional water and double strand RNA in oral epithelial cells

Abstract

Defensins, a major family of cationic antimicrobial peptides, play important roles in innate immunity. In the present study, we investigated whether double-stranded RNA (dsRNA), a by-product of RNA virus replication, can induce human β-defensins-2 (hBD-2) expression in oral epithelial cells (OECs). We also examined the hBD-2-inducible activity of acid-electrolyzed functional water (FW). The results indicated that both dsRNA- and FW-induced hBD-2 expression in OECs. The induction efficiency was much higher for FW than for dsRNA. FW-induced production of hBD-2 was clearly observed by immunofluorescence staining. A luciferase assay was performed with 1.2?kb of the 5′-untranslated region (5′-UTR) of the hBD-2 gene. The results indicated that the nuclear factor-kappa B (NF-κB)-binding site proximal to the translation initiation site was indispensable for dsRNA-stimulated hBD-2 expression, but not in the case of FW. Moreover, FW-stimulated hBD-2 expression did not depend on NF-κB activity; instead, FW inhibited NF-κB activity. Pretreatment of the cells with specific inhibitors against NF-κB further confirmed NF-κB-independent hBD-2 induction by FW. In analogy to the results for intestinal epithelial cells (IECs), the dsRNA signal, but not FW, was sensed by toll-like receptor 3 (TLR3) in OECs. These results suggested that hBD-2 expression induced by dsRNA and FW is regulated by distinct mechanisms in OECs.