Characterizing the effects of the protein environment on the reduction potentials of metalloproteins

The reduction potentials of electron transfer proteins are critically determined by the degree of burial of the redox site within the protein and the degree of permanent polarization of the polypeptide around the redox site. Although continuum electrostatics calculations of protein structures can predict the net effect of these factors, quantifying each individual contribution is a difficult task. Here, the burial of the redox site is characterized by a dielectric radius R _p (a Born-type radius for the protein), the polarization of the polypeptide is characterized by an electret potential ? _p (the average electrostatic potential at the metal atoms), and an electret-dielectric spheres (EDS) model of the entire protein is then defined in terms of R _p and ? _p. The EDS model shows that for a protein with a redox site of charge Q, the dielectric response free energy is a function of Q ², while the electret energy is a function of Q. In addition, R _p and ? _p are shown to be characteristics of the fold of a protein and are predictive of the most likely redox couple for redox sites that undergo different redox couples.     

A Method for Fractional Determination of Soybean Sterols in Four Classes by Florisil Column Chromatography

A procedure for fractional determination of soybean sterols is presented. Sterols in lipid extracts were fractionated into four classes, fatty acid esters, the free form, acylated glucosides and non-acylated glucosides, by Florisil column chromatography. Sterol contents in the four classes were determined colorimetrically with ferric chloride-perchloric acid reagent. Before the colorimetry, the fatty acid ester fraction was hydrolyzed with ethanolic KOH, and the sterol was isolated as tomatinide. The free sterol fraction was directly treated with tomatine solution. The tomatinides were dissociated with dimethyl sulfoxide. To avoid the contamination of pigments from the acylated glucoside fraction, the second Florisil column was rinsed with diethyl ether between the elution with the first solvent (0 to 50% diethyl ether in n-heхane) and that with the second solvent (0 to 30% methanol in diethyl ether).     

Production of the Carotenoids Lycopene, β-Carotene,and Astaxanthin in the Food Yeast Candida utilis

The food-grade yeast Candida utilis has been engineered to confer a novel biosynthetic pathway for the production of carotenoids such as lycopene, β-carotene, and astaxanthin. The exogenous carotenoid biosynthesis genes were derived from the epiphytic bacterium Erwinia uredovora and the marine bacterium Agrobacterium aurantiacum. The carotenoid biosynthesis genes were individually modified based on the codon usage of the C. utilis glyceraldehyde 3-phosphate dehydrogenase gene and expressed in C. utilis under the control of the constitutive promoters and terminators derived from C. utilis. The resultant yeast strains accumulated lycopene, β-carotene, and astaxanthin in the cells at 1.1, 0.4, and 0.4 mg per g (dry weight) of cells, respectively. This was considered to be a result of the carbon flow into ergosterol biosynthesis being partially redirected to the nonendogenous pathway for carotenoid production.Carotenoids are yellow, orange, and red pigments which are widely distributed in nature (3). Industrially, carotenoid pigments such as β-carotene are utilized as food or feed supplements. β-Carotene is also a precursor of vitamin A in mammals (11). Recently, carotenoids have attracted greater attention, due to their beneficial effect on human health: e.g., the functions of lycopene and astaxanthin include strong quenching of singlet oxygen (12), involvement in cancer prevention (2), and enhancement of immune responses (6). Astaxanthin has also been exploited for industrial use, principally as an agent for pigmenting cultured fish and shellfish.The genes responsible for the synthesis of carotenoids such as lycopene, β-carotene, and astaxanthin have been isolated from the epiphytic Erwinia species or the marine bacteria Agrobacterium aurantiacum and Alcaligenes sp. strain PC-1, and their functions have been elucidated (13, 14). The first substrate of the encoded enzymes for carotenoid synthesis is farnesyl pyrophosphate (diphosphate) (FPP), which is the common precursor for the biosynthesis of numerous isoprenoid compounds such as sterols, hopanols, dolicols, and quinones. The ubiquitous nature of FPP among yeasts has been utilized in the microbial production of lycopene and β-carotene by the yeast Saccharomyces cerevisiae carrying the Erwinia uredovora carotenogenic genes (19). However, the amount of carotenoids produced in these hosts was only 0.1 mg of lycopene and 0.1 mg of β-carotene per g (dry weight) of cells, respectively.The edible yeast Candida utilis is generally recognized as a safe substance by the Food and Drug Administration. Large-scale production of the yeast cells has been developed with cheap biomass-derived sugars as the carbon source for the production of single-cell protein and several chemicals such as glutathione and RNA (1, 4). This yeast was also found to accumulate a large amount of ergosterol in the cell during stationary phase (6 to 13 mg/g [dry weight] of cells) (17). Thus, C. utilis has the potential to produce a large amount of carotenoids by redirecting the carbon flux for the ergosterol biosynthesis into the nonendogenous pathway for carotenoid synthesis via FPP. Previously, a C. utilis strain was made to produce lycopene (0.8 mg/g [dry weight]) by expressing the three nonmodified genes crtE, crtB, and crtI derived from E. uredovora (15).In this paper, the de novo biosynthesis of lycopene, β-carotene, and astaxanthin has been performed in C. utilis by using six carotenogenic genes, which were synthesized according to the codon usage of the C. utilis glyceraldehyde-3-phosphate dehydrogenase (GAP) gene, which is expressed at high levels. By this approach, increased carotenoid production in C. utilis was achieved.     

Involvement of tumor necrosis factor receptor-associated protein 1 (TRAP1) in apoptosis induced by beta-hydroxyisovalerylshikonin

beta-Hydroxyisovalerylshikonin (beta-HIVS), a compound isolated from the traditional oriental medicinal herb Lithospermum radix, is an ATP non-competitive inhibitor of protein-tyrosine kinases, such as v-Src and EGFR, and it induces apoptosis in various lines of human tumor cells. However, the way in which beta-HIVS induces apoptosis remains to be clarified. In this study, we performed cDNA array analysis and found that beta-HIVS suppressed the expression of the gene for tumor necrosis factor receptor-associated protein 1 (TRAP1), which is a member of the heat-shock family of proteins. When human leukemia HL60 cells and human lung cancer DMS114 cells were treated with beta-HIVS, the amount of TRAP1 in mitochondria decreased in a time-dependent manner during apoptosis. A similar reduction in the level of TRAP1 was also observed upon exposure of cells to VP16. Treatment of DMS114 cells with TRAP1-specific siRNA sensitized the cells to beta-HIVS-induced apoptosis. Moreover, the reduction in the level of expression of TRAP1 by TRAP1-specific siRNA enhanced the release of cytochrome c from mitochondria when DMS114 cells were treated with either beta-HIVS or VP16. The suppression of the level of TRAP1 by either beta-HIVS or VP16 was blocked by N-acetyl-cysteine, indicating the involvement of reactive oxygen species (ROS) in the regulation of the expression of TRAP1. These results suggest that suppression of the expression of TRAP1 in mitochondria might play an important role in the induction of apoptosis caused via formation of ROS.     

Effects of Cytokinins on the Growth of Primary Roots of Zea mays

Intact primary roots of Zea mays seedlings, apical 6-mm segmentsisolated from the intact primary roots and 5-mm detipped segments,prepared from the 6-mm apical segments by removal of the apical1-mm meristematic region, were incubated in potassium-phosphatebuffer that contained various concentrations of kinetin, 6-benzylaminopurine(BAP) or zeatin. These cytokinins inhibited the growth of intactprimary roots but they promoted the growth of both tipped anddetipped apical segments. In other words, they promoted thegrowth of root segments irrespective of the presence or absenceof apical meristematic regions. Detipped segments were stoodvertically, with their apical or basal cutends in contact withan agar plate that contained the abovementioned buffer and variousconcentrations of kinetin, BAP or zeatin so that cytokininswere supplied either from the apical or basal cut-ends. Cytokininssupplied from the top promoted the growth of the segments, whilethose supplied from the base did not. These results indicate that the response of roots to the exogenouslyapplied cytokinins is not influenced by the presence of theroot meristem but is significantly affected by the way in whichcytokinins are supplied. (Received October 17, 1995; Accepted August 28, 1995)     

Cystic echinococcosis in immigrant from Peru: first case treated with percutaneous treatment in Japan

We report a case of cystic echinococcosis (CE) caused by Echinococcusgranulosus, for which a modified percutaneous evacuation (PEVAC) treatment was applied. The patient had immigrated from Peru to Japan and had 2 hydatid cystic masses, 1 located in segment (S)5 of the liver and the other in S3 (5.3 and 3.5 cm in diameter, respectively), both of which were visualized as pseudotumors by ultrasound (US) examinations. Albendazole treatment showed no effects and surgical treatment was refused. After punctuation of the S5 cyst under US guidance and S3 with CT guidance, 10- and 12-French gauge catheters, respectively, with multiple side holes were inserted. About 60 ml of the cyst contents was drawn out from the S5 lesion and 2 ml from the S3 lesion. Using repetitive manual injections and aspiration of small amounts of hypertonic saline, the remaining cyst content was removed as much as possible, after which 20 and 10 ml of 98% ethanol was injected into the S5 and S3 lesions, respectively. A short-term evaluation during the 4 month-period following the procedure using US revealed nearly complete evacuation of the S5 lesion, whereas that at S3 remained as a pseudo-solid mass. We consider that percutaneous treatment is a safe therapeutic modality for hydatid cysts. This is the first case report of CE treated percutaneously in Japan.     

Crystal structure of uridine-diphospho-N-acetylglucosamine pyrophosphorylase from Candida albicans and catalytic reaction mechanism

Uridine-diphospho-N-acetylglucosamine (UDP-GlcNAc) is a precursor of the bacterial and fungal cell wall. It is also used in a component of N-linked glycosylation and the glycosylphosphoinositol anchor of eukaryotic proteins. It is synthesized from N-acetylglucosamine-1-phosphate (GlcNAc-1-P) and uridine-5'-triphosphate (UTP) by UDP-GlcNAc pyrophosphorylase (UAP). This is an S(N)2 reaction; the non-esterified oxygen atom of the GlcNAc-1-P phosphate group attacks the alpha-phosphate group of UTP. We determined crystal structures of UAP from Candida albicans (CaUAP1) without any ligands and also complexed with its substrate or with its product. The series of structures in different forms shows the induced fit movements of CaUAP1. Three loops approaching the ligand molecule close the active site when ligand is bound. In addition, Lys-421, instead of the metal ion in prokaryotic UAPs, is coordinated by both phosphate groups of UDP-Glc-NAc and acts as a cofactor. However, a magnesium ion enhances the enzymatic activity of CaUAP1, and thus we propose that the magnesium ion increases the affinity between UTP and the enzyme by coordinating to the alpha- and gamma-phosphate group of UTP.     

Near Infrared Photoimmunotherapy Targeting EGFR Positive Triple Negative Breast Cancer: Optimizing the Conjugate-Light Regimen

Aim

Triple-negative breast cancer (TNBC) is considered one of the most aggressive subtypes of breast cancer. Near infrared photoimmunotherapy (NIR-PIT) is a cancer treatment that employs an antibody-photosensitizer conjugate (APC) followed by exposure of NIR light for activating selective cytotoxicity on targeted cancer cells and may have application to TNBC. In order to minimize the dose of APC while maximizing the therapeutic effects, dosing of the APC and NIR light need to be optimized. In this study, we investigate in vitro and in vivo efficacy of cetuximab (cet)-IR700 NIR-PIT on two breast cancer models MDAMB231 (TNBC, EGFR moderate) and MDAMB468 (TNBC, EGFR high) cell lines, and demonstrate a method to optimize the dosing APC and NIR light.

Method

After validating in vitro cell-specific cytotoxicity, NIR-PIT therapeutic effects were investigated in mouse models using cell lines derived from TNBC tumors. Tumor-bearing mice were separated into 4 groups for the following treatments: (1) no treatment (control); (2) 300 μg of cet-IR700 i.v., (APC i.v. only); (3) NIR light exposure only, NIR light was administered at 50 J/cm² on day 1 and 100 J/cm² on day 2 (NIR light only); (4) 300 μg of cet-IR700 i.v., NIR light was administered at 50 J/cm² on day 1 after injection and 100 J/cm² of light on day 2 after injection (one shot NIR-PIT). To compare different treatment regimens with a fixed dose of APC, we added the following treatments (5) 100 μg of cet-IR700 i.v., NIR light administered at 50 J/cm² on day 1 and 50 μg of cet-IR700 i.v. immediately after NIR-PIT, then NIR light was administered at 100 J/cm² on day 2, which were performed two times every week (“two split” NIR-PIT) and (6) 100 μg of cet-IR700 i.v., NIR light was administered at 50 J/cm² on day 1 and 100 J/cm² on day 2, which were performed three times per week (“three split” NIR-PIT).

Result

Both specific binding and NIR-PIT effects were greater with MDAMB468 than MDAMB231 cells in vitro. Tumor accumulation of cet-IR700 in MDAMB468 tumors was significantly higher (p < 0.05) than in MDAMB231 tumors in vivo. Tumor growth and survival of MDAMB231 tumor bearing mice was significantly lower in the NIR-PIT treatment group (p < 0.05). In MDAMB468 bearing mice, tumor growth and survival was significantly improved in the NIR-PIT treatment groups in all treatment regimens (one shot NIR-PIT; p < 0.05, “two split” NIR-PIT; p < 0.01, “three split” NIR-PIT; p < 0.001) compared with control groups.

Conclusion

NIR-PIT for TNBC was effective regardless of expression of EGFR, however, greater cell killing was shown with higher EGFR expression tumor in vitro. In all treatment regimens, NIR-PIT suppressed tumor growth, resulting in significantly prolonged survival that further improved by splitting the APC dose and using repeated light exposures.