Effective immunotherapy of cancer by DNA vaccination.

Direct injection of naked plasmid DNA either intramuscularly or intradermally induces strong, long-lived cell-mediated and humoral immune responses to the antigen encoded by the gene vaccine. In the present study, we used gene vaccination with naked plasmid DNA to induce prophylactic immune responses to tumor associated antigens. MAGE-1 (melanoma antigen 1) is an ideal candidate for cancer vaccines because it belongs to a family of genes that are expressed in a number of human tumors of various histological types but not in normal adult tissues except for the testis, and because both humoral and cell-mediated immune responses against MAGE-1 antigen were detected in tumor patients. Intradermal administration of plasmid DNA encoding MAGE-1 (pcMAGE1) induced anti-MAGE-1-specific antibody in BALB/c mice. In contrast, no detectable level of anti-MAGE-1 antibody was induced by intramuscular injection of pcMAGE1. Also, intradermal injection of pcMAGE1 was capable of generating CTLs reactive with MAGE-1-transfected murine tumor cells, M-MSV-MAGE1. Most of the mice (8 out of 10) immunized with pcMAGE1 rejected the challenge of M-MSV-MAGE1 tumor cells, compared with control animals most of which developed tumors. This suggests that intradermal DNA vaccination could provide a novel immunotherapy of cancer.     

Expression of a bifunctional fusion of the Escherichia coli genes for trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase in transgenic rice plants increases trehalose accumulation and abiotic stress tolerance without stunting growth

Trehalose plays an important role in stress tolerance in plants. Trehalose-producing, transgenic rice (Oryza sativa) plants were generated by the introduction of a gene encoding a bifunctional fusion (TPSP) of the trehalose-6-phosphate (T-6-P) synthase (TPS) and T-6-P phosphatase (TPP) of Escherichia coli, under the control of the maize (Zea mays) ubiquitin promoter (Ubi1). The high catalytic efficiency (Seo et al., 2000) of the fusion enzyme and the single-gene engineering strategy make this an attractive candidate for high-level production of trehalose; it has the added advantage of reducing the accumulation of potentially deleterious T-6-P. The trehalose levels in leaf and seed extracts from Ubi1::TPSP plants were increased up to 1.076 mg g fresh weight(-1). This level was 200-fold higher than that of transgenic tobacco (Nicotiana tabacum) plants transformed independently with either TPS or TPP expression cassettes. The carbohydrate profiles were significantly altered in the seeds, but not in the leaves, of Ubi1::TPSP plants. It has been reported that transgenic plants with E. coli TPS and/or TPP were severely stunted and root morphology was altered. Interestingly, our Ubi1::TPSP plants showed no growth inhibition or visible phenotypic alterations despite the high-level production of trehalose. Moreover, trehalose accumulation in Ubi1::TPSP plants resulted in increased tolerance to drought, salt, and cold, as shown by chlorophyll fluorescence and growth inhibition analyses. Thus, our results suggest that trehalose acts as a global protectant against abiotic stress, and that rice is more tolerant to trehalose synthesis than dicots.     

Microbial flocculant from Arcuadendron sp. TS-49

Summary A flocculant was purified from the culture broth of Archuadendron sp. TS-49 by a series of precipitations with acetone, 60% ammonium sulfate-butanol, salting-out by dialysis, and cetylpyridinium chloride. The flocculating activity was observed most highly at pH 3.0 and markedly enhanced by the addition of salts, especially in the case of FeCl₃ or FeSO₄. This bioflocculant efficiently flocculated all tested solids, including various microorganisms and organic/ inorganic materials. Qualitative analyses of the bioflocculant showed that it might contain hexosamine, uronic acid, neutral sugar, and protein.     

A role for cell cycle proteins in the serum-starvation resistance of Epstein-Barr virus immortalized B lymphocytes.

Epstein-Barr virus (EBV) is a B-lymphotropic human herpes virus that infects B lymphocytes and is associated with a broad spectrum of benign and malignant diseases. B cell infection by EBV causes indefinite cell proliferation that results in the development of immortalized lymphoblastoid cell lines (LCLs). We found that SNU-1103, a latency type III EBV-transformed LCL developed from a Korean cancer patient, resisted the G1 arrest that was normally caused by serum starvation. Western blot analyses revealed several alterations in the expression of key regulatory cell cycle proteins involved in the G1 phase. High expression of cyclin D2 and time-dependent increases in cyclin-dependent kinase 6 (CDK6) and cyclin D3 were observed in SNU-1103 during serum starvation. Very unexpectedly, in SNU-1103, the key G1 phase CDK inhibitor p21CiP1 was expressed at a consistently high level, while p27KiP1 expression was increased. Of three pRb family proteins, pRb expression was reduced and it became hypophosphorylated in SNU-1103 during serum starvation. Instead, p107 and p130 were expressed at consistently high levels in SNU-1103 during serum starvation. In conclusion, compared with an EBV-negative BJAB cell line, multiple cell cycle regulatory proteins were abnormally or inversely expressed in SNU-1103 during serum starvation.     

Facilitation of Expression and Purification of an Antimicrobial Peptide by Fusion with Baculoviral Polyhedrin in Escherichia coli

Several fusion strategies have been developed for the expression and purification of small antimicrobial peptides (AMPs) in recombinant bacterial expression systems. However, some of these efforts have been limited by product toxicity to host cells, product proteolysis, low expression levels, poor recovery yields, and sometimes an absence of posttranslational modifications required for biological activity. For the present work, we investigated the use of the baculoviral polyhedrin (Polh) protein as a novel fusion partner for the production of a model AMP (halocidin 18-amino-acid subunit; Hal18) in Escherichia coli. The useful solubility properties of Polh as a fusion partner facilitated the expression of the Polh-Hal18 fusion protein (~33.6 kDa) by forming insoluble inclusion bodies in E. coli which could easily be purified by inclusion body isolation and affinity purification using the fused hexahistidine tag. The recombinant Hal18 AMP (~2 kDa) could then be cleaved with hydroxylamine from the fusion protein and easily recovered by simple dialysis and centrifugation. This was facilitated by the fact that Polh was soluble during the alkaline cleavage reaction but became insoluble during dialysis at a neutral pH. Reverse-phase high-performance liquid chromatography was used to further purify the separated recombinant Hal18, giving a final yield of 30% with >90% purity. Importantly, recombinant and synthetic Hal18 peptides showed nearly identical antimicrobial activities against E. coli and Staphylococcus aureus, which were used as representative gram-negative and gram-positive bacteria, respectively. These results demonstrate that baculoviral Polh can provide an efficient and facile platform for the production or functional study of target AMPs.     

10 mAh cm−2 Cathode by Roll-to-Roll Process for Low Cost and High Energy Density Li-Ion Batteries

Roll-to-roll dry processing enables the manufacture of high energy density and low cost Li-ion batteries (LIBs). However, as the thickness of the electrode fabricated by dry processing becomes greater (≥10 mAh cm⁻²), Li-ion migration resistance (R_ion) and charge-transfer resistance (R_ct) in the electrode dramatically increase due to long diffusion lengths for Li-ion and electron. Therefore, it is important to reduce diffusion lengths in the electrode to achieve high energy density LIBs. The dry electrode with a high areal capacity of 10 mAh cm⁻² and low resistance can be achieved by following three characteristics. First, the fibrillization behavior of polytetrafluoroethylene (PTFE) binder is controlled by adjusting the processing temperature during the fibrillization process, which enables uniform distribution of PTFE binder and carbon black (CB). Second, pore size/distribution and conducting network are engineered by multi-dimensional conducting agents, enhancing Li-ions and electrons transport in the electrode. Finally, the structural integrity of LiNi_0.80Co_0.15Al_0.05O₂ (NCA) particles is improved without fractures, which enables uniform pore distribution in the electrode by controlling the calendering step. The prepared 10 mAh cm⁻² dry electrode with homogeneous microstructure shows reduced R_ion and R_ct due to short diffusion lengths, which improves electrochemical performances in LIBs with a high volumetric energy density of ≈710 Wh L⁻¹.     

Insight into the bovine milk peptide LPcin‐YK3 selection in the proteolytic system of Lactobacillus species

Antimicrobial peptides are class of small, positively charged peptides known for their broad‐spectrum antimicrobial activity. Antimicrobial activities for most antimicrobial peptides have largely remained elusive, particularly in the lactic acid bacteria. However, recently our investigation using LPcin‐YK3, an antimicrobial peptide from bovine milk, suggests that in vitro antimicrobial activity was reduced over 100‐fold compared with pathogenic bacteria. Additionally, for the structural study of how antimicrobial peptide undergoes its reaction at the proteolytic pathway of lactic acid bacteria based on degradation assay and propidium iodide staining, we performed molecular docking for interaction between oligopeptide‐binding protein A and LPcin‐YK3 peptide. Given that degradation related to the LPcin‐YK3 peptide in lactic acid bacteria proteolytic system, the inhibitory inactivity of LPcin‐YK3 against beneficial lactic acid bacteria strains may be one of the primary pharmacological properties of recombinant peptide discovered in bovine milk. These results provide structural and functional insights into the proteolytic mechanism and possibility as a putative substrate of oligopeptide‐binding protein A in respect of LPcin‐YK3 peptide.     

Treatment of growth hormone attenuates hepatic steatosis in hyperlipidemic mice via downregulation of hepatic CD36 expression

ABSTRACT

The recombinant human growth hormone (GH) has been used for the treatment of growth hormone deficiency (GHD) and diverse short stature state, and its physiological and therapeutic effects are well documented. However, since the effect of GH treatment on metabolic disorders has not been well characterized, we injected GH to Western diet-fed low-density lipoprotein receptor-deficient (Ldlr ^?/?) mice to understand the exact effect of GH on metabolic diseases including atherosclerosis, hepatic steatosis, and obesity. Exogenous GH treatment increased plasma IGF-1 concentration and decreased body weight without affecting serum lipid profiles. GH treatment changed neither atherosclerotic lesion size nor collagen and smooth muscle cells accumulation in the lesion. GH treatment reduced macrophage accumulation in adipose tissue. Importantly, GH treatment attenuated hepatic steatosis and inflammation. The hepatic expression IL-1β mRNA were decreased by GH treatment. The mRNA and protein levels of CD36 were markedly decreased in GH treated mice without significant changes in other molecules related to lipid metabolism. Therefore, the treatment of GH treatment could attenuate hepatic steatosis and inflammation with downregulation of CD36 expression in hyperlipidemic condition.