Binding of Phylogenetically Distant Bacillus thuringiensis Cry Toxins to a Bombyx mori Aminopeptidase N Suggests Importance of Cry Toxin's Conserved Structure in Receptor Binding

We investigated the binding proteins for three Cry toxins, Cry1Aa, Cry1Ac, and the phylogenetically distant Cry9Da, in the midgut cell membrane of the silkworm. In a ligand blot experiment, Cry1Ac and Cry9Da bound to the same 120-kDa aminopeptidase N (APN) as Cry1Aa. A competition experiment with the ligand blot indicated that the three toxins share the same binding site on several proteins. The values of the dissociation constants of the three Cry toxins and 120-kDa APN are as low as the case of other Cry toxins and receptors. These results suggest that distantly related Cry toxins bind to the same site on the same proteins, especially with APN. We propose that the conserved structure in these three toxins includes the receptor-binding site. Received: 12 January 1998 / Accepted: 17 February 1999     

Ultrasound-mediated interferon β gene transfection inhibits growth of malignant melanoma

We investigated the effects of ultrasound-mediated transfection (sonotransfection) of interferon β (IFN-β) gene on melanoma (C32) both in vitro and in vivo. C32 cells were sonotransfected with IFN-β in vitro. Subcutaneous C32 tumors in mice were sonicated weekly immediately after intra-tumor injection with IFN-β genes mixed with microbubbles. Successful sonotransfection with IFN-β gene in vitro was confirmed by ELISA, which resulted in C32 growth inhibition. In vivo, the growth ratio of tumors transfected with IFN-β gene was significantly lower than the other experimental groups. These results may lead to a new method of treatment against melanoma and other hard-to-treat cancers.     

Emerging technologies in therapeutic ultrasound: Thermal ablation to gene delivery

Ultrasound is used today in medicine as a modality for diagnostic imaging. Recently, there have been numerous reports on the application of thermal and nonthermal ultrasound energy for treating various diseases. In addition to thermal ablation of tumors, non-thermal ultrasound combined with drugs and genes have led to much excitement especially for cancer treatment, vascular diseases, and regenerative medicine. Ultrasound energy can enhance the effects of thrombolytic agents such as urokinase for treatment of stroke and acute myocardial infarction. New ultrasound technologies have resulted in advanced devices such as a) ultrasound catheters, b) Non-invasive methods as high intensity focused ultrasound (HIFU) in conjunction with MRI and CT is already being applied in the clinical field, c) Chemical activation of drugs by ultrasound energy for treatment of tumors is another new field recently termed "Sonodynamic Therapy", and d) Combination of genes and microbubble have induced great hopes for ideal gene therapy (sonoporation). Various examples of ultrasound combined modalities are under investigation which could lead to revolutionary therapy.     

Immunohistochemical Localization of Arginase II and Other Enzymes of Arginine Metabolism in Rat Kidney and Liver

Arginine is a precursor for the synthesis of urea, polyamines, creatine phosphate, nitric oxide and proteins. It is synthesized from ornithine by argininosuccinate synthetase and argininosuccinate lyase and is degraded by arginase, which consists of a liver-type (arginase I) and a non-hepatic type (arginase II). Recently, cDNAs for human and rat arginase II have been isolated. In this study, immunocytochemical analysis showed that human arginase II expressed in COS-7 cells was localized in the mitochondria. Arginase II mRNA was abundant in the rat small intestine and kidney. In the kidney, argininosuccinate synthetase and lyase were immunostained in the cortex, intensely in proximal tubules and much less intensely in distal tubules. In contrast, arginase II was stained intensely in the outer stripes of the outer medulla, presumably in the proximal straight tubules, and in a subpopulation of the proximal tubules in the cortex. Immunostaining of serial sections of the kidney showed that argininosuccinate synthetase and arginase II were collocalized in a subpopulation of proximal tubules in the cortex, whereas only the synthetase, but not arginase II, was present in another subpopulation of proximal tubules. In the liver, all the enzymes of the urea cycle, i.e. carbamylphosphate synthetase I, ornithine transcarbamylase, argininosuccinate synthetase and lyase and arginase I, showed similar zonation patterns with staining more intense in periportal hepatocytes than in pericentral hepatocytes, although zonation of ornithine transcarbamylase was much less prominent. The implications of these results are discussed.     

Rational protein design for thermostabilization of glycoside hydrolases based on structural analysis

Glycosidases are used in the food, chemical, and energy industries. These proteins are some of the most frequently used such enzymes, and their thermostability is essential for long-term and/or repeated use. In addition to thermostability, modification of the substrate selectivity and improvement of the glycosidase activities are also important. Thermostabilization of enzymes can be performed by directed evolution via random mutagenesis or by rational design via site-directed mutagenesis; each approach has advantages and disadvantages. In this paper, we introduce thermostabilization of glycoside hydrolases by rational protein design using site-directed mutagenesis along with X-ray crystallography and simulation modeling. We focus on the methods of thermostabilization of glycoside hydrolases by linking the N- and C-terminal ends, introducing disulfide bridges, and optimizing β-turn structures to promote hydrophobic interactions.

     

Biological and Biochemical Characterization of Macrophage Activating Factor (MAF) in Murine Lymphocytes: Physicochemical Similarity of MAF to Gamma Interferon (IFN-γ)

During the course of an investigation designed to separate macrophage activating factor (MAF) activity from interferon (IFN) antiviral activity in the lymphokine-rich fraction (LKF) produced by stimulation of murine splenic cells with concanavalin A (Con A), we found molecular evidence for the similarity of the two activities. MAF activity was expressed as the rate of inhibition of intracellular growth of Salmonella typhimurium in macrophages based on the linear correlation between relative MAF activity and LKF concentration. The antiviral substance in LKF was identified as IFN-γ based on the observation that its activity was inactivated at pH 2 and neutralized with anti-mouse IFN-γ serum but not with anti-mouse IFN-α/β serum. MAF and IFN antiviral activities displayed identical sensitivity to pH 2 and temperature. Further, neither activity was affected by β-mercapto-ethanol, but both were inactivated by guanidine hydrochloride and by sodium dodecyl sulfate, suggesting that the structures related to conformation of the protein of the two molecules may be similar. In affinity chromatography of the LKF on a Con A-Sepharose column, MAF and IFN activities were found in both the nonadsorbing (F I) and adsorbing (F II) fractions. However, the rates of F II of MAF and IFN activities increased proportionally when the sample was applied on a column of higher capacity, suggesting that the molecular structure of the mannose-containing glycosyl moiety of the two molecules may also be similar. Moreover, the intact or modified form of MAF and IFN activities of different LKF preparations showed a strong correlation, indicating that the production and denaturation of MAF activity were proportional to those of IFN antiviral activity. The results of this study provide strong evidence that MAF and IFN antiviral activities may reside in virtually the same molecular species.     

Cancer metastasis directly eradicated by targeted therapy with a modified Salmonella typhimurium

Cancer metastasis is the life‐threatening aspect of cancer and is usually resistant to standard treatment. We report here a targeted therapy strategy for cancer metastasis using a genetically‐modified strain of Salmonella typhimurium. The genetically‐modified strain of S. typhimurium is auxotrophic for the amino acids arginine and leucine. These mutations preclude growth in normal tissue but do not reduce bacterial virulence in cancer cells. The tumor‐targeting strain of S. typhimurium, termed A1‐R, and expressing green fluorescent protein (GFP), was administered to both axillary lymph and popliteal lymph node metastasis of human pancreatic cancer and fibrosarcoma, respectively, as well as lung metastasis of the fibrosarcoma in nude mice. The bacteria were delivered via a lymphatic channel to target the lymph node metastases and systemically via the tail vein to target the lung metastasis. The cancer cells expressed red fluorescent protein (RFP) in the cytoplasm and GFP in the nucleus linked to histone H2B, enabling color‐coded real‐time imaging of the bacteria targeting the metastatic tumors. After 7–21 days of treatment, the metastases were eradicated without the need of chemotherapy or any other treatment. No adverse effects were observed. This new strategy demonstrates the clinical potential of targeting and curing cancer metastasis with engineered bacteria without the need of toxic chemotherapy. J. Cell. Biochem. 106: 992–998, 2009. © 2009 Wiley‐Liss, Inc.     

Real‐time imaging of single cancer‐cell dynamics of lung metastasis

We have developed a new in vivo mouse model to image single cancer‐cell dynamics of metastasis to the lung in real‐time. Regulating airflow volume with a novel endotracheal intubation method enabled controlling lung expansion adequate for imaging of the exposed lung surface. Cancer cells expressing green fluorescent protein (GFP) in the nucleus and red fluorescent protein (RFP) in the cytoplasm were injected in the tail vein of the mouse. The right chest wall was then opened in order to image metastases on the lung surface directly. After each observation, the chest wall was sutured and the air was suctioned in order to re‐inflate the lung, in order to keep the mice alive. Observations have been carried out for up to 8 h per session and repeated up to six times per mouse thus far. The seeding and arresting of single cancer cells on the lung, accumulation of cancer‐cell emboli, cancer‐cell viability, and metastatic colony formation were imaged in real‐time. This new technology makes it possible to observe real‐time monitoring of cancer‐cell dynamics of metastasis in the lung and to identify potential metastatic stem cells. J. Cell. Biochem. 109: 58–64, 2010. © 2009 Wiley‐Liss, Inc.     

Infectious prion protein in the filtrate even after 15nm filtration

The evaluation of the removal efficacy during manufacturing is important for the risk assessment of plasma products with respect to possible contamination by infectious prions, as recently reported in several papers on the potential for prion transmission through plasma products. Here, we evaluated a virus removal filter which has 15 nm pores. An antithrombin sample immediately prior to nano-filtration was spiked with prion material prepared in two different ways. The removal (log reduction factor) of prion infectivity using animal bioassays was ≥4.72 and 4.00 in two independent filtrations. However, infectivity was detected in both the pellet and supernatant following ultracentrifugation of the 15 nm filtered samples, indicating difficulty in complete removal. The data supports the conclusion that a certain amount of infectious prion protein is present as a smaller and/or soluble form (less than ～15 nm in diameter).