Guidelines for Manufacturing and Application of Organoids: Kidney

Recent advancements in organoid technology have led to a vigorous movement towards utilizing it as a substitute for animal experiments. Organoid technology offers versatile applications, particularly in toxicity testing of pharmaceuticals or chemical substances. However, for the practical use in toxicity testing, minimal guidance is required to ensure reliability and relevance. This paper aims to provide minimal guidelines for practical uses of kidney organoids derived from human pluripotent stem cells as a toxicity evaluation model in vitro.

Adenovirus-mediated transfer of siRNA against PTTG1 inhibits liver cancer cell growth in vitro and in vivo.

The pituitary tumor transforming (PTTG) gene family comprises PTTG1, 2, and 3. Forced expression of PTTG1 (securin) induces cellular transformation and promotes tumor development in animal models. PTTG1 is overexpressed in various human cancers. However, the expression and pathogenic implications of the PTTG gene family in hepatocellular carcinoma are largely unknown. Gene silencing using short interfering RNA (siRNA) has become an efficient means to study the functions of genes and has been increasingly used for cancer gene therapy approaches. We report that PTTG1, but not PTTG2 and 3, was highly and frequently expressed in liver cancer tissues from patients and highly in SH-J1, SK-Hep1, and Huh-7 hepatoma cell lines. Adenoviral vector encoding siRNA against PTTG1 (Ad.PTTG1-siRNA) depleted PTTG1 specifically and efficiently in SH-J1 hepatoma cells, which resulted in activation of p53 that led to increased p21 expression and induction of apoptosis. The depletion of PTTG1 in HCT116 colorectal cancer cells exhibited a cytotoxic effect in a p53-dependent manner. Ad.PTTG1-siRNA-mediated cytotoxic effect was dependent on expression levels of PTTG1 and p53 in hepatoma cell lines. Huh-7 hepatoma cells, once transduced with Ad.PTTG1-siRNA, displayed markedly attenuated growth potential in nude mice. Intra-tumor delivery of Ad.PTTG1-siRNA led to significant inhibition of tumor growth in SH-J1 tumor xenograft established in nude mice. In conclusion, PTTG1 overexpressed in hepatoma cell lines negatively regulates the ability of p53 to induce apoptosis. PTIG1 gene silencing using siRNA may be an effective modality to treat liver cancer, in which PTTG1 is abundantly expressed. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270-9139/ suppmat/index.html).

mesenchymal stem cells and osteogenesis

No abstract available.

Isolation and identification of Vibrio vulnificus and Vibrio parahaemolyticus from coast of Pusan and Daechon

This study was focused on the isolation of pathogenic Vibrio species, V. vulnificus and V. parahaemolyticus from marine environment from May to July of 1999. Isolation sites were coast near by Pusan and Daechon. The results obtained were as follows: 1. Seventy strains of V. parahaemolyticus and 19 strains of V. vulnificus were isolated from a total of 120 specimens. 2. Nineteen strains of V. vulnificus did not fermented arabinose and salicin but fermented lactose and cellobiose. All of V. parahaemolyticus isolates did not fermented lactose and cellobiose. 47 strains of V. parahaemolyticus fermented arabinose but 53 strains did not fermented salicin. 3. V. vulnificus and V. parahaemolyticus isolates showed three different API index numbers with 5046105 and 4346107 dominant. 4. V. vulnificus did not grow on 0% and 8% NaCl containing medium. V. parahaemolyticus grew on 8% NaCl containing medium. 5. V. vulnificus isolates and V. parahaemolyticus revealed different outer membrane protein p rofiles on SDS-PAGE.

Characterization of a Vibrio parahaemolyticus Phage Isolated from Marine

A novel bacteriophage, designated as VPP97, that infects the strains of Vibiro parahaemolyticus (hallophilic, Gram-negative bacterium) isolated most commonly from marine environments, has been discovered, and several of its properties have been determined. The plaques were clear and sized 0.6-1.0 mm in diameter. The virion forms a single band on 70% sucrose gradient and p1.50 CsC1 gradient by sucrose gradient centrifugation and CsCI gradient centrifugation respectively. It has a hexagonal head and a relatively long tail, as shown by electron microscopy. Vibrio alginolyticus, Vibrio fluvialis and Vibrio furnissii were also sensitive to this phage It was almost totally inactivated at 70 degree C and at pH below 5 or over 10. The nucleic acid of VPP97 is composed of DNA. The VPP97 had 9 specific structural proteins sized between 21.5 kDa and 97.4 kDa on SDS-PAGE. When V. parahaemolyticus cultures were treated with either phage VPP97 or one of the several antibiotics for 2 hours, the viable number of V. parahaemolyticus treated with the phage VPP97 is lower than that treated with chloramphenicol, erythromycin or penicillin, but not lower than that treated with tetracycline. Mice that have responded to the phage treatment revealed the lower numbers of V. parahaemolyticus in small intestine and less damage on small intestine compared to the untreated mice. Therefore, we suggest that the phage treatment appears effective to the infection by V. parahaemolyticus.

The Effect of Iron Limmted Condition on Outer Membrane of Vibrio mimicus

Vibrio mimicus, marine bacteria pathogenic for fish, can causes acute gastroenteritis in human. Iron limmited condition like in human body, may change the surface structure of V. mimicus. In this study we obse'rved the effect of iron limmited condition on outer membrane protein of V. mimicus. Ethylenediamine-di (O-hydroxy-phenylacetic) acid (EDDA), an iron chelator, delayed the time to reach expotential growth of V. mimicus in brain heart infusion medium from 3 hours to 20 hours. Outer membrane protein of V. mimicus-CON (cultured in BHI) and V. mimicus-EDDA (cultured in BHI contain EDDA) were seperated by 1% sarcosine from total cell envelop. SDS-PAGE of V. mimicus-EDDA and V. mimicus-CON showed similar protein profiles contain 37 kDa major protein but 86 and 90 kDa protein were induced differently. Immunological properties of above protein were determined by ELISA and western blotting. 86 kDa EDDA- specific OMP was induced in V. mimicus (isolate 96-1), V. parahaemolyticus (serotype 09), V. alginolyticus (isolate 95-1), E. coli (human isolate) and V. vulnificus ATCC 27562 in iron limmited condition.

Identification of Vibrio vulnificus in Pusan and Southern Sea of Korea in 1996 using API 20E Kit

The halophilic bacterium, Vibrio vulnificus, causes acute fulminating wound infections and septicemia in human. Especially the septicemia shows high mortality above 50%. In Korea, septicemia by V. vulnificus was reported at westem and southern coast in every year. Here, we try to isolate this V. vulnipcus at Kyoung-nam area and coast of Pusan during 1996. Purposed sites were Dadaepo, Songjung, Chungsapo and Mipo of Pusan and Kijang, Ilkuang, Juksoung, Dongam, Waljun and Chilam of southern sea. Total 40 strains of V. vulnipcus were isolated from sea samples. Biochemical characteristics of isolated V. vulnificus were almost same with reference strain V. vulnificus ATCC 27562 on Farmer's tests and on API 20E kit test. V. vulnificus isolates in 1996, fermented cellobiose and salicin but arabinose. and had resistance to 7% sodium chloride.

Viral Disease of the Cultured Penaeus chinensis and Penaeus japonicus

From June to October in 1993~1995, cultured Penaeus chinesis and Penaeus japonicus occurred mass mortality at the farm in Western Sea of Korea. The disease was reproduced in healthy shrimp by injection of filtered (at 0.45 micromeger) homogenate of infected shrimp. So we concluded hat it was filterable agents like virus. Clinical symptoms were white spots on the carapace and reddish tail. Histopathological changes were characterized by hypertrophied nuclei at cuticular epidermis lymphoid organ, hematopoietic tissue. In negatively stained preparation, the virion revealed rod-shaped, enveloped, nonoccluded. Cytopathic effect (CPE) were not observed by virus in CHSE-214, RTG-2, EPC, FHM cell lines. Base on the above facts, the reason of mass mortality of penaied shrimp was baculovirus.

Isolation and Detection of Vibrio vulnificus from the Southern Sea of Korea for the Prevention of V. vulnificus Infection (The Characteristics of Vibrio vulnificus Isolates from the Southern Sea of Korea)

Vibrio vulnificus, which causes serious septicemia, has been isolated from the Southern Sea of Korea. Five strains were identified by Farmer's biochemical test and API 20E kit. V. vulnificus ATCC 27562 was used as the reference strain and V. parahaemolyticus was used as the comparative strain. Three of the five strains could grow at 37% and the others only at 30 degrees C. The proteins pattern of cell lysates from the isolates were analyzed by SDS-PAGE and densitometery. Distinct protein band pattern was observed with the reference strain and the isolates in comparison with V. parahaemolyticus. Antiserum made against V. vulnificus ATCC 27562, was used for ELISA and Western blotting analysis to test the isolates. In ELISA analysis, the three strains being able to grow at 37 degrees C showed significantly higher reactivity to the antiserum than that of V. parahaemolyticus, while the other two grown only at 30 degrees C showed no significant difference. By Western blotting analysis, distinctive 30 and 36 kDa bands were observed only in the reference strain and the isolates. Twenty six and 54 kDa bands were observed with only three of the five strains being able to grow at 37 degrees C. The SDS-PAGE profiles of the outer membrane proteins of the isolates shared common features with the reference strain but distinctive from V. parahaemolyticus.

Immunogenecity of Outer Membrane Proteins of vibro vulnificus

To provide basic information on the pathogengenesis of Vibrio vulnificus infection and for the manufacture of effective vaccine, outer membrane proteins (OMPs) were extracted from V. vulnificus ATCC 27562 strain and analysed by 12% of sodium dodecyl sulfate-polyarylamide gel(SDS-PAGE). Major 36 kDa and 48 kDa, 46 kDa, 66 kDa and 24 kDa protein bands appeared on gel by Coomassie stain and determined by densitometer. Immunogenecity of these proteins was examined by enzyme-linked immunosorbant assay(ELISA) and western blotting with rabbit anti-V. vulnificus serum. OMPs reacted with this antiserum, and the major 36 kDa protein appeared most immunogenic.