Effect of soybean oil on the enhanced expression of hirudin gene in Hansenula polymorpha

Summary A heterologous gene from bloodsucking leech for anticoagulant, hirudin, has been expressed in the methylotrophic yeast Hansenula polymorpha. The addition of 1%(v/v) soybean oil to the medium as a stabilizer enhanced the expression of the hirudin gene in H. polymorpha with MOX promoter. The production of hirudin in the medium with soybean oil was 320mg/L in a 5L fermenter.     

Tegumental ultrastructure of juvenile and adult Echinostoma cinetorchis]

The tegumental ultrastructure of juvenile and adult Echinostoma cinetorchis (Trematoda: Echinostomatidae) was observed by scanning electron microscopy. Three-day (juvenile) and 16-day (adult) worms were harvested from rats (Sprague-Dawley) experimentally fed the metacercariae from the laboratory-infected fresh water snail, Hippeutis cantori. The worms were fixed with 2.5% glutaraldehyde, processed routinely, and observed by an ISI Korea DS-130 scanning electron microscope. The 3-day old juvenile worms were elongated and ventrally curved, with their ventral sucker near the anterior two-fifths of the body. The head crown was bearing 37-38 collar spines arranged in a zigzag pattern. The lips of the oral and ventral suckers had 8 and 5 type II sensory papillae respectively, and between the spines, a few type III papillae were observed. Tongue or spade-shape spines were distributed anteriorly to the ventral sucker, whereas peg-like spines were distributed posteriorly and became sparse toward the posterior body. The spines of the dorsal surface were similar to those of the ventral surface. The 16-day old adults were leaf-like, and their oral and ventral suckers were located very closely. Aspinous head crown, oral and ventral suckers had type II and type III sensory papillae, and numerous type I papillae were distributed on the tegument anterior to the ventral sucker. Scale-like spines, with broad base and round tip, were distributed densely on the tegument anterior to the ventral sucker but they became sparse posteriorly. At the dorsal surface, spines were observed at times only at the anterior body. The results showed that the tegument of E. cinetorchis is similar to that of other echinostomes, but differs in the number and arrangement of collar spines, shape and distribution of tegumenal spines, and type and distribution of sensory papillae.     

Mapping of Altromycin B-DNA Adduct at Nucleotide Resolution in the Human Genomic DNA by Ligation-mediated PCR

The ligation-mediated PCR was used to map DNA alkylation sites induced by altromycin B at nucleotide resolution in genomic DNA purified from cultured human colon carcinoma. Altromycin B, one of the pluramycin group of antitumor antibiotics, is characterized as intercalator with the added ability to alkylate N7 guanine. DNA adducts formed in genomic DNA were cleaved into DNA strand breaks by hot piperidine treatment, and fragments containing ligatable breaks were then amplified in a single-sided, ligation-mediated PCR. The alkylation sites observed in exon 9 of the p53 gene revealed that the most high reactivity sites for altromycin B were found to be N7 of guanine in a 5-AG* sequence. Determination of the DNA alkylation sites in naked radiolabeled plasmid DNA also showed that altromycin B preferred N7 of guanine in a 5-AG* sequence. Thus, it can be concluded that the sequence selective DNA adduct formation induced by the intercalating alkylator, altromycin B, in genomic DNA is similar to that observed in naked plasmid DNA.     

Design and application of highly responsive fluorescence resonance energy transfer biosensors for detection of sugar in living Saccharomyces cerevisiae cells

A protein sensor with a highly responsive fluorescence resonance energy transfer (FRET) signal for sensing sugars in living Saccharomyces cerevisiae cells was developed by combinatorial engineering of the domain linker and the binding protein moiety. Although FRET sensors based on microbial binding proteins have previously been created for visualizing various sugars in vivo, such sensors are limited due to a weak signal intensity and a narrow dynamic range. In the present study, the length and composition of the linker moiety of a FRET-based sensor consisting of CFP-linker(1)-maltose-binding protein-linker(2)-YFP were redesigned, which resulted in a 10-fold-higher signal intensity. Molecular modeling of the composite linker moieties, including the connecting peptide and terminal regions of the flanking proteins, suggested that an ordered helical structure was preferable for tighter coupling of the conformational change of the binding proteins to the FRET response. When the binding site residue Trp62 of the maltose-binding protein was diversified by saturation mutagenesis, the Leu mutant exhibited an increased binding constant (82 microM) accompanied by further improvement in the signal intensity. Finally, the maltose sensor with optimized linkers was redesigned to create a sugar sensor with a new specificity and a wide dynamic range. When the optimized maltose sensors were employed as in vivo sensors, highly responsive FRET images were generated from real-time analysis of maltose uptake of Saccharomyces cerevisiae (baker's yeast).     

Substrate Binding Mechanism of a Type I Extradiol Dioxygenase

A meta-cleavage pathway for the aerobic degradation of aromatic hydrocarbons is catalyzed by extradiol dioxygenases via a two-step mechanism: catechol substrate binding and dioxygen incorporation. The binding of substrate triggers the release of water, thereby opening a coordination site for molecular oxygen. The crystal structures of AkbC, a type I extradiol dioxygenase, and the enzyme substrate (3-methylcatechol) complex revealed the substrate binding process of extradiol dioxygenase. AkbC is composed of an N-domain and an active C-domain, which contains iron coordinated by a 2-His-1-carboxylate facial triad motif. The C-domain includes a β-hairpin structure and a C-terminal tail. In substrate-bound AkbC, 3-methylcatechol interacts with the iron via a single hydroxyl group, which represents an intermediate stage in the substrate binding process. Structure-based mutagenesis revealed that the C-terminal tail and β-hairpin form part of the substrate binding pocket that is responsible for substrate specificity by blocking substrate entry. Once a substrate enters the active site, these structural elements also play a role in the correct positioning of the substrate. Based on the results presented here, a putative substrate binding mechanism is proposed.     

Pitfalls of using lucigenin in endothelial cells: Implications for NAD(P)H dependent superoxide formation

Since an increased endothelial superoxide formation plays an important role in the pathogenesis of endothelial dysfunction its specific detection is of particular interest. The widely used superoxide probe lucigenin, however, has been reported to induce superoxide under certain conditions, especially in the presence of NADH. This raises questions as to the conclusion of a NAD(P)H oxidase as the major source of endothelial superoxide. Using independent methods, we showed that lucigenin in the presence of NADH leads to the production of substantial amount of superoxide (～ 15-fold of control) in endothelial cell homogenates. On the other hand, these independent methods revealed that endothelial cells without lucigenin still produce superoxide in a NAD(P)H-dependent manner. This was blocked by inhibitors of the neutrophil NADPH oxidase diphenyleniodonium and phenylarsine oxide. Our results demonstrate that a NAD(P)H-dependent oxidase is an important source for endothelial superoxide but the latter, however, cannot be measured reliably by lucigenin.     

Enzyme-linked assay of cellulose-binding domain functions from Cellulomonas fimi on multi-well microtiter plate

Cellulose-binding domain (CBD) enriches cellulolytic enzymes on cellulosic surfaces and contributes to the catalytic efficiency by increasing enzyme-substrate complex formations. Thus, high affinity CBDs are essential for the development of efficient cellulose-degrading enzymes. Here, we present a microtiter plate-based assay system to measure the binding affinity of CBDs to cellulose. The assay uses a periplasmic alkaline phosphatase (AP) as a fusion reporter and its activity is detected using a fluorogenic substrate, 4-methylumbelliferyl phosphate. Lignocellulose discs of 6 mm in diameter were used as substrates in 96-well plate. As a result, the enzyme-linked assay detected the binding of CBDs on the cellulosic discs in a highly sensitive manner, detecting from 0.05 to 1.0 μg/mL of APCBD proteins, which is several hundred times more sensitive than conventional protein measurements. The proposed method was applied to compare the binding affinity of different CBDs from Cellulomonas fimi to lignocellulose discs.     

Immunostimulating Effects of Extract of Acanthopanax sessiliflorus

As malfunction/absence of immune cells causes a variety of immunosuppressive disorders and chemical synthetic drugs for curing these diseases have many adverse effects, vigorous studies are being conducted. The Acanthopanax family has been used as traditional medicines for gastric ulcer, diabetes, etc. and culinary materials in East-South Asia. In this study, the immunostimulating properties of A. sessiliflorus were evaluated. A. sessiliflorus increased not only the splenocyte number but also immune-related cytokines such as TNF-α. However, it could not upregulate the expressions of IFN-γ and IL-2. A. sessiliflorus increased the swimming time, and comparison of organ weights relative to body weights for immune-related organs such as the spleen and thymus after a forced swim test showed that it could recover the spleen and thymus weights. It also increased the expression of TNF-α and slightly increased the concentration of IFN-γ but not IL-2. From the results, we concluded that as A. sessiliflorus has not only a host defense effect but also a stress-ameliorating property, further study it will be a promising material of immunostimulating material.     

A transgene expression system for the marine microalgae Aurantiochytrium sp. KRS101 using a mutant allele of the gene encoding ribosomal protein L44 as a selectable transformation marker for cycloheximide resistance

In the present study, we established a genetic system for manipulating the oleaginous heterotrophic microalgae Aurantiochytrium sp. KRS101, using cycloheximide resistance as the selectable marker. The gene encoding ribosomal protein L44 (RPL44) of Aurantiochytrium sp. KRS101 was first identified and characterized. Proline 56 was replaced with glutamine, affording cycloheximide resistance to strains encoding the mutant protein. This resistance served as a novel selection marker. The gene encoding the Δ12-fatty acid desaturase of Mortierella alpina, used as a reporter, was successfully introduced into chromosomal DNA of Aurantiochytrium sp. KRS101 via 18S rDNA-targeted homologous recombination. Enzymatic conversion of oleic acid (C18:1) to linoleic acid (C18:2) was detected in transformants but not in the wild-type strain.     

Long-term survival after adoptive bone marrow T cell therapy of advanced metastasized breast cancer: follow-up analysis of a clinical pilot trial

Background

The bone marrow (BM) of breast cancer patients harbors tumor-reactive memory T cells (TCs) with therapeutic potential. We recently described the immunologic effects of adoptive transfer of ex vivo restimulated tumor-reactive memory TCs from the BM of 12 metastasized breast cancer patients in a clinical phase-I study. In this trial, adoptive T cell transfer resulted in the occurrence of circulating tumor antigen-reactive type-1 TCs. We here describe the long-term clinical outcome and its correlation with tumor-specific cellular immune response in 16 metastasized breast cancer patients, including 12 included in the original study.

Methods

Sixteen metastatic breast cancer patients with preexisting tumor-reactive BM memory TCs were included into the study. The study protocol involved one transfusion of TCs which were reactivated in vitro with autologous dendritic cells pulsed with lysates of MCF-7 breast cancer cells as source of tumor antigens. The presence of tumor-reactive memory TCs was analyzed by IFN-γ ELISpot assays.

Results

Tumor-reactive memory TCs in the peripheral blood were induced de novo in 7/16 patients (44 %) after adoptive TC transfer. These patients were considered immunologic responders to the therapy. Positive adoptive immunotherapy (ADI) response was observed significantly more often in patients without bone metastases (p = 0.0051), in patients with high levels of tumor-reactive BM TCs prior to therapy (p = 0.036) and correlated significantly with the estimated numbers of transferred tumor-reactive TCs (p = 0.0021). After the treatment, we observed an overall median survival of 33.8 months in the total cohort with three patients alive at last follow-up and more than 7 years after ADI. Numbers of transferred tumor-reactive TCs correlated significantly with the overall survival of patients (p = 0.017). Patients with an immunologic response to ADI in the peripheral blood had a significantly longer median survival than nonresponders (median survival 58.6 vs. 13.6 months; p = 0.009).

Conclusion

In metastasized breast cancer patients, adoptive transfer of BM TCs can induce the presence of tumor antigen-reactive type-1 TCs in the peripheral blood. Patients with immunologic response after ADI show a significantly longer overall survival. Patients with bone metastases significantly less frequently respond to the treatment and, therefore, might not be optimal candidates for ADI. Although the present study does not yet prove the therapeutic effect of ADI, these findings shed light on the relation between immune response and cancer prognosis and suggest that transfer of reactivated BM TCs might bear therapeutic potential.