PURPOSE: When Managing metastatic bladder tumors, to overcome the resistance mechanism of cisplatin is a main problem to be solved. The objective is to confirm the changes of general and ultrastructural morph ology with the induction of cisplatin resistance from the bladder cell line. MATERIALS AND METHODS: The samples of this investigation are 2ng/ml-cisplatin resistant human bladder cell lines T24R2 established by SNUH Urology and the drug resistant bladder cell lines T24 was obtained from ATCC, as a control group. We cultured the resistant cell line on the slide and observed it using light microscopy to see the general morphology. For the ultrastructural morphology, we fixed cultured cells, made an epon block, sliced an ultrathin section and observed it using H-71000 EM. RESULTS: Under light microscopy, the cytoplasm of the resistant cell line shows a plumper pattern than that of the parent cell. Under electronmicroscopy, the chromatin of the resistant cell line has a relatively finely dispersed chromatin pattern when compared to the parent cell line, which shows a coarse and aggregated chromatin pattern. Within the cytoplasm, the mitochondrial volume, dilated rough endoplasmic reticulum, polyribosomes and ribosomes are moderately increased in the resistant cell line when compared to the parent cell line. In particular, we found a great amount of double membrane vesicle near the cell surface and pinocytic vesicles on the surface, which are seldom observed within the parent cells. CONCLUSIONS: We concluded that the cisplatin resistant human bladder cell lines (T24R2) underwent a morphological change with the induction of cisplatin resistance, and we hypothesize that the resistant cell's ultrastructure, which shows morphological change, will be involved in the drug resistance mechanism. Regarding this matter, further research will be needed.
Cell Line* ; Cells, Cultured ; Chromatin ; Cisplatin* ; Cytoplasm ; Drug Resistance ; Endoplasmic Reticulum, Rough ; Humans ; Membranes ; Microscopy ; Mitochondrial Size ; Parents ; Polyribosomes ; Ribosomes ; Urinary Bladder Neoplasms ; Urinary Bladder* ; Urology

The human Shwachman-Diamond syndrome (SDS) is an autosomal recessive disease caused by mutations in a highly conserved ribosome assembly factor SBDS. The functional role of SBDS is to cooperate with another assembly factor, elongation factor 1-like (Efl1), to promote the release of eukaryotic initiation factor 6 (eIF6) from the late-stage cytoplasmic 60S precursors. In the present work, we characterized, both biochemically and structurally, the interaction between the 60S subunit and SBDS protein (Sdo1p) from yeast. Our data show that Sdo1p interacts tightly with the mature 60S subunit in vitro through its domain I and II, and is capable of bridging two 60S subunits to form a stable 2:2 dimer. Structural analysis indicates that Sdo1p bind to the ribosomal P-site, in the proximity of uL16 and uL5, and with direct contact to H69 and H38. The dynamic nature of Sdo1p on the 60S subunit, together with its strategic binding position, suggests a surveillance role of Sdo1p in monitoring the conformational maturation of the ribosomal P-site. Altogether, our data support a conformational signal-relay cascade during late-stage 60S maturation, involving uL16, Sdo1p, and Efl1p, which interrogates the functional P-site to control the departure of the anti-association factor eIF6.
Crystallography, X-Ray ; GTP Phosphohydrolases ; chemistry ; metabolism ; Humans ; Protein Domains ; Ribosome Subunits, Large, Eukaryotic ; chemistry ; metabolism ; Saccharomyces cerevisiae ; chemistry ; metabolism ; Saccharomyces cerevisiae Proteins ; chemistry ; metabolism

Aminoglycosides are one of the clinically relevant antibiotics. They kill bacteria by binding to bacterial 30S subunit of ribosome. Resistance to aminoglycosides occurs by three different mechanisms: 1. Production of an enzyme that modifies aminoglycosides, 2. Impaired entry of aminoglycoside into the cell by altering the OMP permeability, decreasing inner membrane transport, or active efflux, 3. The receptor protein on the 30S ribosomal subunit may be deleted or altered as a result of a mutation. By far, enzymatic modification has been the most important mechanism. In this review, the mechanisms of action and resistance, and the prevalence of resistance due to acquisition of enzymes are briefly described.
Aminoglycosides ; Anti-Bacterial Agents ; Bacteria ; Membranes ; Permeability ; Prevalence ; Ribosome Subunits ; Ribosomes

The in vivo assembly of ribosomal subunits is a highly complex process, with a tight coordination between protein assembly and rRNA maturation events, such as folding and processing of rRNA precursors, as well as modifications of selected bases. In the cell, a large number of factors are required to ensure the efficiency and fidelity of subunit production. Here we characterize the immature 30S subunits accumulated in a factor-null Escherichia coli strain (∆rsgA∆rbfA). The immature 30S subunits isolated with varying salt concentrations in the buffer system show interesting differences on both protein composition and structure. Specifically, intermediates derived under the two contrasting salt conditions (high and low) likely reflect two distinctive assembly stages, the relatively early and late stages of the 3' domain assembly, respectively. Detailed structural analysis demonstrates a mechanistic coupling between the maturation of the 5' end of the 17S rRNA and the assembly of the 30S head domain, and attributes a unique role of S5 in coordinating these two events. Furthermore, our structural results likely reveal the location of the unprocessed terminal sequences of the 17S rRNA, and suggest that the maturation events of the 17S rRNA could be employed as quality control mechanisms on subunit production and protein translation.
Cryoelectron Microscopy ; Escherichia coli ; metabolism ; Escherichia coli Proteins ; genetics ; metabolism ; GTP Phosphohydrolases ; genetics ; metabolism ; Mass Spectrometry ; Protein Structure, Secondary ; Protein Structure, Tertiary ; RNA, Ribosomal ; analysis ; metabolism ; Ribosomal Proteins ; chemistry ; genetics ; metabolism ; Ribosome Subunits, Small, Bacterial ; chemistry ; metabolism ; ultrastructure ; Salts ; chemistry

A seed-borne fungus, Curvularia sp. EML-KWD01, was isolated from an indigenous wheat seed by standard blotter method. This fungus was characterized based on the morphological characteristics and molecular phylogenetic analysis. Phylogenetic status of the fungus was determined using sequences of three loci: rDNA internal transcribed spacer, large ribosomal subunit, and glyceraldehyde 3-phosphate dehydrogenase gene. Multi loci sequencing analysis revealed that this fungus was Curvularia spicifera within Curvularia group 2 of family Pleosporaceae.
Classification* ; DNA, Ribosomal ; Fungi ; Glyceraldehyde 3-Phosphate ; Humans ; Oxidoreductases ; Ribosome Subunits, Large ; Triticum

BACKGROUND: Trichomonas vaginalis is a pathogenic protozoa infecting human genitourinary tract. Metronidazole is currently the drug of choice to treat T. vaginalis infection. However, because of the side effects and the occurrence of resistant strains of metronidazole, it is needed to investigate alternatives. METHODS: The antiprotozoal effect of aquatic extract from Sophora flavescens on the growth and fine structure of T. vaginalis was examined by using trypan blue exclusion assay and electron microscopy. RESULTS: One hour after the addition of 4 mg/mL extract and half hour after the addition of 5 mg/mL showed antiprotozoal effect. One to two hours after the addition of 3 mg/mL extract, the movement of flagella and axostyle had disappeared, but death of the cells had not occurred until two hours after the addition. The fine structure of the cytoplasm was also changed half an hour to two hours after addition. The number of polyribosome decreased when that of single ribosomes in the cytoplasm increased. CONCLUSION: These results indicated that S. flavescens had the antiprotozoal effect on T. vaginalis by inhibition of cell multiplication as well as an impairment of protein synthesis.
Cell Proliferation ; Cytoplasm ; Flagella ; Humans ; Metronidazole ; Microscopy, Electron ; Polyribosomes ; Ribosomes ; Sophora* ; Trichomonas vaginalis* ; Trichomonas* ; Trypan Blue

BACKGROUND: T. vaginalis is a pathogenic protozoa infecting genitourinary tract. Metronidazole is a drug of choice to treat T. vaginalis infection. However, because of appearance of metronidazole- resistant T. vaginalis, it is needed to develop an alternative drug. So, we evaluated the effects of various concentration of kalopanaxsaponin A against T. vaginalis. METHODS: The antiprotozoal effect of kalopanaxsaponin A on the growth and fine structure of T. vaginalis was examined by using trypan blue exclusion assay and electron microscopy. RESULTS: The viability test showed markedly decreased number of T. vaginalis, treated with increasing concentration of kalopanaxsaponin A. We evaluated the electron microscopic findings for antiprotozoan effects against T. vaginalis. SEM showed that in T. vaginalis treated with 4 microgram/mL for 1 hour, axostyle was shrinked and flagella began to disappear. With gradual increase of the concentration of kalopanaxsaponin A, destruction of cell membrane was getting more severe, and degenerative change was observed in T. vaginalis treated with 8 microgram/mL for 2 hours. TEM showed that in T. vaginalis treated with 4 microgram/mL for 2 hours, the vacuoles in cytoplasm were larger and hydrogenosomes were smaller than those in control group. The number of vacuole was increased, the nucleus was destroyed, the number of polyribosome and free ribosome was also decreased in trichomonads treated with kalopanaxsaponin A (8 microgram/mL), which indicated the occurrence of degenerative changes. CONCLUSION: These results indicated that kalopanaxsaponin A had the antiprotozoal effect on T. vaginalis by inhibition of cell multiplication as well as an impairment of protein synthesis.
Cell Membrane ; Cell Proliferation ; Cytoplasm ; Flagella ; Metronidazole ; Microscopy, Electron ; Polyribosomes ; Ribosomes ; Trichomonas vaginalis* ; Trichomonas* ; Trypan Blue ; Vacuoles

BACKGROUND: T. vaginalis is a pathogenic protozoa infecting genitourinary tract. Metronidazole is a drug of choice to treat T. vaginalis infection. However, because of appearance of metronidazole- resistant T. vaginalis, it is needed to develop an alternative drug. So, we evaluated the effects of various concentration of kalopanaxsaponin A against T. vaginalis. METHODS: The antiprotozoal effect of kalopanaxsaponin A on the growth and fine structure of T. vaginalis was examined by using trypan blue exclusion assay and electron microscopy. RESULTS: The viability test showed markedly decreased number of T. vaginalis, treated with increasing concentration of kalopanaxsaponin A. We evaluated the electron microscopic findings for antiprotozoan effects against T. vaginalis. SEM showed that in T. vaginalis treated with 4 microgram/mL for 1 hour, axostyle was shrinked and flagella began to disappear. With gradual increase of the concentration of kalopanaxsaponin A, destruction of cell membrane was getting more severe, and degenerative change was observed in T. vaginalis treated with 8 microgram/mL for 2 hours. TEM showed that in T. vaginalis treated with 4 microgram/mL for 2 hours, the vacuoles in cytoplasm were larger and hydrogenosomes were smaller than those in control group. The number of vacuole was increased, the nucleus was destroyed, the number of polyribosome and free ribosome was also decreased in trichomonads treated with kalopanaxsaponin A (8 microgram/mL), which indicated the occurrence of degenerative changes. CONCLUSION: These results indicated that kalopanaxsaponin A had the antiprotozoal effect on T. vaginalis by inhibition of cell multiplication as well as an impairment of protein synthesis.
Cell Membrane ; Cell Proliferation ; Cytoplasm ; Flagella ; Metronidazole ; Microscopy, Electron ; Polyribosomes ; Ribosomes ; Trichomonas vaginalis* ; Trichomonas* ; Trypan Blue ; Vacuoles

The development and differentiation of cells in the spinal ganglion were studied by electron microscopy in human fetuses ranging from 12 mm to 260 mm crown rump length. At 12 mm embryo the primitive neuroblasts which had a single process, contained a large numbers of free ribosome and mitochondria but very little rough endoplasmic reticulum. At 30 mm fetus, the primitive spinal ganglion consisted of bipolar neuroblasts, satellite cells and undifferentiated cells. Spindle-shaped bipolar neuroblasts formed spinal ganglion of loosely grouped cells at 50 mm fetus. Two neuroblast cell types, a small cell contained large clumps of rough endoplasmic reticulum at periphery, could be distinguished. At 80 mm fetus, the spinal ganglion constituted of bipolar neuroblast with apparently random distribution of small and large neurons with processes, together with satellite cells and blood vessels. The presences of a large numbers of neurotubules in the Golgi-central region were one of the first sign of further maturation of the neuroblast. During next prenatal stage from 120 mm on fetus, the ganglion cells were large and contained much rough endoplasmic reticulum, neurotubules and extensive Golgi complex. A large number of neuroblasts became transformed into unipolar cells from 180 mm to 260 mm feuts. Nissl bodies appeared during this stage. The ganglion-satellite cell boundary became complicated with increasing age, then enlarging in parallel with the increase in volume of the nerve cell. During next prenatal stage up to 180 mm fetus, the unipolar ganglion cell increased in number and size, and the cytoplsm contained all intracytoplasmic structures which were also found in mature spinal ganglion except for large pigment granules.
Blood Vessels ; Crown-Rump Length ; Embryonic Structures ; Endoplasmic Reticulum, Rough ; Fetus* ; Ganglia, Spinal* ; Ganglion Cysts ; Golgi Apparatus ; Humans* ; Microscopy, Electron ; Mitochondria ; Neurons ; Nissl Bodies ; Ribosomes

BACKGROUND: Onychomycosis has become one of the common fungal infection. However, highly reliable and sensitive methods of detecting and identifying causative fungi of onychomycosis are not established yet. Polymerase chain reaction (PCR) analysis of clinical specimens including blood, sputum, urine, and cerebrospinal fluid collected from patient systemically infected fungus is known as a sensitive diagnostic method. But it has been questionable whether PCR analysis is also applicable to onychomycosis. OBJECTIVE: The purpose of this study was to develop a DNA-based diagnostic method to improve the sensitivity and specificity of detection and identification of pathogenic fungi of onychomycosis. METHODS: To detect the fungi in the nail, PCR was performed by using 4 sets of primer (TR1 & TR2, NS5 & NS6, B2F & B4R and CA1 & CA2) designed in conserved sequences of the small ribosomal subunit (185-rRNA) genes and restriction enzyme analysis of amplified product by Hae III was done to identify species. Nail specimens were obtained from 19 cases of onychomycosis confirm by fungus culture. RESULTS: 1. Preparation of nail powder, which is necessary for removal of keratin, and composition of lysis buffer with guanidinium thiocyanate, Tris-HCl, and beta -mercaptoethanol are the most proper modalities for isolation of fungal DNA from fungus-infesting nails. 2. Specific fragments of the 18S-rRNA gene of fungi, 581 bp, 308 bp, 688 bp and 1106 bp were amplified respectively. From sequences of 18S-rRNA gene of fungi by universal primers, dermatophytes (Trichophyton rubrum, Trichophyton mentagrophytes) and yeast (Candida albicans) yielded identical products. 3. Using Hae III endonuclease, digested patterns of fragment of Trichophyton rubrum and Candida albicans resulted in different pattern. CONCLUSION: This method released enough DNA from fungus-infected nails to result in proper amplification and it can be possible to differentiate dermatophytes, yeasts, and molds using Hae III endonuclease. The present study is the first one to demonstrate the feasibility of this molecular biologic approach to identify fungi in the infected nail. Therefore, precise detection and identification of the causative fungi would be of help in investigating distribution of the causative fungi of onychomycosis as well as appropriate treatment of the disease.
Arthrodermataceae ; Candida albicans ; Cerebrospinal Fluid ; Conserved Sequence ; Diagnosis* ; DNA ; DNA, Fungal ; Fungi* ; Guanidine ; Humans ; Onychomycosis* ; Polymerase Chain Reaction* ; Restriction Mapping* ; Ribosome Subunits, Small ; Sensitivity and Specificity ; Sputum ; Trichophyton ; Yeasts