• Title/Summary/Keyword: Alcian blue

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Keratanase II Digestion Accompanied with a Liquid Chromatography/Tandem Mass Spectrometry for Urinary Keratan Sulfate Quantitative Analysis

  • Chuang, Chih-Kuang;Lin, Hsiang-Yu;Wang, Tuen-Jen;Huang, Sung-Fa;Lin, Shuan-Pei
    • Journal of mucopolysaccharidosis and rare diseases
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    • v.3 no.1
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    • pp.20-27
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    • 2017
  • Purpose: Mucopolysaccharidosis IV (MPS IV) is a disease characterized by deficient activity of N-acetylgalactosamine-6-sulfatase (GALNS) causing excessive lysosomal storage of keratan sulfate (KS). The identification of the relevant disaccharide units of KS after keratanase II digestion followed by liquid chromatography/tandem mass spectrometry detection (LC-MS/MS) is validated and applicable for the preliminary diagnosis of MPS IV. Methods: A total of 67 urine samples were collected and analyzed from 11 MPS IV patients comprising 10 MPS IVA and one MPS IVB patients, and 56 normal controls. Urinary glycosaminoglycan was first precipitated by the Alcian blue method followed by a digestion of keratanase II. The protonated species of the digested disaccharide products were detected by using multiple reaction monitoring experiment. Results: One particular disaccharide of KS was selected. The transition mass-to-charge (m/z) of the parent ion and its daughter ion after collision was $462.0{\rightarrow}97.0$, whereas the chondrosine used as an internal standard in this assay was m/z $353.9{\rightarrow}73.0$. The results corresponded well with the two-dimensional electrophoresis method. The quantities of urinary KS were significantly raised in confirmed MPS IV patients when comparing with those of normal controls ($170.2{\pm}81.1$ vs. $4.06{\pm}1.92{\mu}g/mL$). Conclusion: The LC-MS/MS method for MPS IVA determination is specific, sensitive, validated, and applicable for urinary KS quantification. This method can be used not only as a first-line biochemistry examination of MPS IVA, but also as an outcome survey after enzyme replacement therapy.

Effects of Morphological Changes and Intestinal Transit time After Administration of Daesenggi-Tang in Rats

  • Yang Jun Seok;Shin Hyun Jong;Lee Chang Hyun;Lee Kwang Gyu;Kwon Gang Joo;Woo Won Hong;Kim Young Soo
    • Journal of Physiology & Pathology in Korean Medicine
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    • v.18 no.4
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    • pp.1199-1206
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    • 2004
  • This study was examined the effects of Daesenggi-Tang on intestinal mucosa and gastrointestinal transit time and plasma lipids in rats. Adult male rats were fed four weeks on diets containing no addition(basal diet group), 5% cellulose(cellulose group) and Daesenggi-Tang group(Daesenggi-Tang group). The results were as follows; 1. The gastrointestinal transit times was significantly decreased in Daesenggi-Tang administered group compare to basal diet and cellulose groups. Carmine red mixed with Daesenggi-Tang, as a marker, was administered through a gastric tube for stomach or intracecally by a chronically implanted catheter for colon transit. Small intestinal transit and large intestinal transit time were significantly decreased in Daesenggi-Tang administered group compare to basal diet. 3. The height of jejunal villi was developed in Daesenggi-Tang administered group compare to basal diet. The thickness of mucosa and muscle layer of colonic mucosa were significantly developed in Daesenggi-Tang administered group compare to basal diet group. 4. The numerical change of goblet cell in colonic mucosa was increased acid mucin stained alcian blue in Daesenggi-Tang administered group compare to basal diet and cellulose group. 6. HDL-cholesterol of plasma lipid was increased in Daesenggi-Tang administered group compare to basal diet and cellulose groups. Theses results suggests that Daesenggi-Tang may be used in prevention and treatment of constipation resulting in increase of fecal weight, decrease of gastrointestinal transit time, development of intestinal villi, intensify of stainability of acid mucin in colon.

Sibjeondaebotang and Yugmijihwangtang's Toxicological Effects on Pregnant Rats (십전대보탕과 육미지황탕이 실험동물의 모체에 미치는 생식독성학적 연구)

  • Han, Sang-Baek;Shin, Heon-Tae;Park, Hae-Mo;Lee, Sun-dong
    • Journal of Society of Preventive Korean Medicine
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    • v.11 no.2
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    • pp.159-174
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    • 2007
  • Introduction : The experiments was undertaken to evaluate the effects of herbal medicine, Sibjeondaebotang, Yugmijihwangtang, in pregnant rats and fetuses. Female Sprague-Dawley rats were orally administered with the Sibjeondaebotang, Yugmijihwangtang at dose of 5mg/kg/day for 20 days. Pregnant rats were sacrificed at 20th day of gestation, and observed internal and reproductive organs. Approximately live fetuses in the 20th day of gestation were randomly selected and fixed in 95% ethanol. To observe skeletal malformations, fetuses were stained with alcian blue and alizarin red S. Effect on pregnant rats : Maternal body weight of Sibjeondaebotang, Yugmijihwangtang treated group has a tendency to increase compared to that of control group. There were no significant difference in internal and reproductive organs. There were no significant changes between two groups in blood chemistry and hematological values. There were no significant changes in number of corpus luteum, implantation, live fetuses and implantation rate, delivery rate, and sex ratio. But Yugmijihwangtang administered group showed higher early resorption rate than the control group(p<0.05). Also Sibjeondaebotang administered group showed higher late resorption rate than the control group(p<0.05). From the sex ratio, number of females, bigger than number of males in the control group, and more males than females in Yugmijihwangtang administered group(p<0.05).

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Tissue-dependent variation of protease expression phenotype in mouse peritoneal mast cells (마우스복강비만세포에서 프로테아제 발현 표현형의 조직 의존적 변화)

  • Lee, Young-Mi
    • Korean Journal of Veterinary Research
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    • v.41 no.4
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    • pp.543-548
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    • 2001
  • To examine the fate of the injected peritoneal mast cells (PMCs), we injected PMCs (500 or $10^5$) derived from WBB6F1-green fluorescent protein(GFP) mice into stomach wall of $WBB6F1-W/W^v$ mice. When 500 PMCs were injected, the proportion of alcian blue $(AB)^+$ mast cells to $GFP^+$ mast cells in the muscle was 25.0% on day 1, but decreased to 0.9% on day 7. Then, it increased to 98.2% on day 35. In contrast,$GFP^+$ mast cells in the mucosa were not detectable on day 1, 3, and 7 after injection. On day 35, the proportion of $AB^+$ mast cells to $GFP^+$ mast cells in the mucosa was 97.0%. When $10^5$ PMCs were injected, the proportion of $AB^+$ mast cells to $GFP^+$ mast cells in the muscle was more than 88.2%, and that in the mucosa was more than 86.3% from day 1 through 35 after injection. These results indicated that percentage of degranulation on day 1, 3, 7, 14 after injection of 500 PMCs was significantly higher than that after injection of $10^5$ PMCs. Futhermore, when 500 PMCs were injected, protease expression phenotypes of PMCs changed from day 14 after injection. When $10^5$ PMCs were injected, protease expression phenotype of PMCs did not change after injection. Such degranulated PMCs may acquire the new phenotype and adapt the new tissue.

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Ultrastructural and Histochemical Study on the Epithelia of Digestive Tract of a Korean Slug, Incilaria fruhstorferi (한국산 산민달팽이 ( Incilaria fruhstorferi ) 소화관 상피조직의 미세구조 및 조직화학적 연구)

  • 이정찬;장남섭;한종민
    • The Korean Journal of Malacology
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    • v.13 no.2
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    • pp.143-160
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    • 1997
  • 한국산 산민달팽이 Incilariafruhstorferi의 소화관을 부위별로 관찰하고, 소화관 상피조직을 구성하는 세포의 종류와 분포수 그리고 분비되는 과립 등을 미세구조적, 조직화학적으로 관찰하고 그 결과를 보고하고자 한다. 산민달팽이의 소화관은 식도, 위, 장, 직장 등으로 구성되어있으며, 식도는 다시 전식도, 소낭, 후식도로 나누어지고, 장은 전장과 중장, 후장 등으로 구분되었다. 소화관을 부위별롸 관찰한 결과 섬모원주상피세포 2종, 녹색과립세포 3종, 청색과립세포, 점액세포, 투명세포, 망상형세포 그리고 괴사형세포 등 모두 10종류가 확인되었다. 섬모원주세포는 A형과 B형 등 두 종으로 나뉘며, A형은 세포의 상단 자유면에 섬모와 미세융모가 밀생된데 비해, B형은 전자밀도가 높아서 검게 보이고, 장과 직장에서만 관찰되는 특징을 보였다. 섬모의 구조는 9 x 2 + 2 axoneme이었다. 녹색과립세포는 미세구조에 의해 A형, B형 그리고 C형등 3종으로 나누어지며, 소낭과 후식도, 위, 직장에서 주로 관찰되었다. A형과 B형은 Sudan black에 양성인 지방과립(1.36 x 1.6$\mu\textrm{m}$)만을 포함하고 있는데비해 C형은 글리코겐 과립도 포함하고 있었다. 청색과립세포는 10종류의 세포 중 가장 키가 크고 (35$\mu\textrm{m}$), Millon반응에 양성을 보이는 둥근과립(직경, 1.3$\mu\textrm{m}$)을 포함하고 있는데 과립들은 단백질성으로 확인되었다. 이들은 중장에서만 관찰되었다. 장과 직장에서 주로 많이 관찰되는 점액세포는 세포질 속에 전자밀도가 낮은 투명한 과립과 전자밀도가 높아서 검게 보이는 둥근과립(크기, 1.33 x 0.89$\mu\textrm{m}$)들을 포함하고 있었는데, 이들은 미성숙 시기에는 투명과립(직경, 2.66$\mu\textrm{m}$)으로 관찰되었다. PAS-alcian blue(pH 2.5)반응에서 투명과립은 alcianophillia로 나타난 반면, 검은 과립은 PAS에 양성반을을 보이며, 각각 산성점액과 중성점액과립으로 확인되었다. 투명세포는 광학현미경 관찰에서 A형과 B형으로 구분되었으나, A형은 전자현미경 관찰에서 신경내분비세포로 확인된 반며, B형은 지방과립을 소지한 지방저장세포로 각각 확인되었다. 신경내분비세포가 소지한 과립의 크기는 0.16$\mu\textrm{m}$ 정도였다. 망상형세포는 주로 위에서 관찰되는 형태가 불규칙한 작은 세포로서 세포질에 비해 큰 핵을 소지하고 있었다. 소량의 세포질 돌기에는 사립체와 과립성소포체를 포함하고 있다. 괴사형세포는 후장과 직장에서 주로 많이 관찰되고, 점액세포의 점액과립이 분비된 후, 붕괴되는 과정에서 형성된 것으로 확인되었다.

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Subpopulations of miniature pig mesenchymal stromal cells with different differentiation potentials differ in the expression of octamer-binding transcription factor 4 and sex determining region Y-box 2

  • Jeon, Ryounghoon;Park, Sungjo;Lee, Sung-Lim;Rho, Gyu-Jin
    • Asian-Australasian Journal of Animal Sciences
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    • v.33 no.3
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    • pp.515-524
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    • 2020
  • Objective: Human mesenchymal stromal cells (MSCs) exhibit variable differentiation potential and can be divided accordingly into distinct subpopulations whose ratios vary with donor age. However, it is unknown whether the same is true in pigs. This study investigated MSC subpopulations in miniature pig and compared their characteristics in young (2 to 3 months) and adult (27 to 35 months) pigs. Methods: Osteogenic, chondrogenic, and adipogenic capacity of isolated MSCs was evaluated by von Kossa, Alcian blue, and oil red O staining, respectively. Cell surface antigen expression was determined by flow cytometry. Proliferative capacity was assessed with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Expression of marker genes was detected by quantitative real-time polymerase chain reaction. Results: Porcine MSCs comprised cells with trilineage and bilineage differentiation potential (tMSCs and bMSCs, respectively) and non-differentiating stromal cells (NDSCs). The tMSC and bMSC fractions were smaller in adult than in young pigs (63.0% vs 71.2% and 11.6% vs 24.0%, respectively, p<0.05); NDSCs showed the opposite trend (25.4% vs 4.8%; p<0.05). Subpopulations showed no differences in morphology, cell surface antigen expression, or proliferative capacity, but octamer-binding transcription factor 4 (OCT4) expression was higher in tMSCs than in bMSCs and NDSCs (p<0.05), whereas sex determining region Y-box 2 (SOX2) expression was higher in tMSCs and bMSCs than in NDSCs (p<0.05). Aging had no effect on these trends. Conclusion: Porcine MSCs comprise distinct subpopulations that differ in their differentiation potential and OCT4 and SOX2 expression. Aging does not affect the characteristics of each subpopulation but alters their ratios.

Use of Neonatal Chondrocytes for Cartilage Tissue Engineering

  • KANG SUN WOONG;PARK JUNG HO;KIM BYUNG SOO
    • Journal of Microbiology and Biotechnology
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    • v.15 no.2
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    • pp.259-264
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    • 2005
  • Transplantation of cultured chondrocytes can regenerate cartilage tissues in cartilage defects in humans. However, this method requires a long culture period to expand chondrocytes to a large number of cells for transplantation. In addition, chondrocytes may dedifferentiate during long-term culture. These problems can potentially be overcome by the use of undifferentiated or partially developed cartilage precursor cells derived from neonatal cartilage, which, unlike chondrocytes from adult cartilage, have the capacity for rapid in vitro cell expansion and may retain their differentiated phenotype during long-term culture. The purpose of this study was to compare the cell growth rate and phenotypic modulation during in vitro culture between adult chondrocytes and neonatal chondrocytes, and to demonstrate the feasibility of regenerating cartilage tissues in vivo by transplantation of neonatal chondrocytes expanded in vitro and seeded onto polymer scaffolds. When cultured in vitro, chondrocytes isolated from neonatal (immediately postpartum, 2 h of age) rats exhibited much higher growth rate than chondrocytes isolated from adult rats. After 5 days of culture, more neonatal chondrocytes were in the differentiated state than adult chondrocytes. Cultured neonatal chondrocytes were seeded onto biodegradable polymer scaffolds and transplanted into athymic mice's subcutaneous sites. Four weeks after implantation, neonatal chondrocyte-seeded scaffolds formed white cartilaginous tissues. Histological analysis of the implants with hematoxylin and eosin showed mature and well-formed cartilage. Alcian blue/ safranin-O staining and Masson's trichrome staining indicated the presence of highly sulfated glycosarninoglycans and collagen, respectively, both of which are the major extracellular matrices of cartilage. Immunohistochemical analysis showed that the collagen was mainly type II, the major collagen type in cartilage. These results showed that neonatal chondrocytes have potential to be a cell source for cartilage tissue engineering.

AN IMMUNOHISTOCHEMICAL STUDY ON CALCIFYING TISSUES OF THE FLORID CEMENTO-OSSEOUS DYSPLASIA EASILY INVOLVING CHRONIC DIFFUSE SCLEROSING OSTEOMYELITIS (만성 미만성 경화성 골수염을 동반한 개화성 백악질-골 이형성증의 면역조직화학적 연구)

  • Kim, Ji-Hyuck;Jo, Joung-Ae;Kim, Soung-Min;Park, Young-Wook;Huh, Jin-Young;Lee, Suk-Keun
    • Journal of the Korean Association of Oral and Maxillofacial Surgeons
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    • v.29 no.5
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    • pp.293-297
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    • 2003
  • Florid cemento-osseous dysplasia (FCOD) is a benign, non-neoplastic lesion characterized by multiple sclerosing masses only within jawbones. It is frequently confused with chronic diffuse sclerosing osteomyelitis (CDSO) in previous literatures. In our study, two cases of FCOD were examined to know the characteristics of their calcifying tissues. The first case was non-infected, while the second case was severely infected, displaying the typical features of CDSO in clinico-radiologic findings. The infected FCOD case showed a lot of bacterial colonies in the main lesion with relatively rare inflammatory reaction. The globular cementum-like materials of FCOD showed woven bone pattern and was positive for Alcian blue stain, and also positive for the antibodies of ameloblastin, bone morphogenetic protein (BMP) -2 and -4. On the other hands, in the immunostains of matrix metalloproteinase (MMP) -3, -9, -10, and $TNF-{\alpha}$, macrophage infiltrated in the FCOD lesion was rarely observed. These data suggest that the cementum-like materials of FCOD contain various matrix proteins, and that the cementum-like materials are relevant to the overgrowth of the bacterial colonies by inhibition of the regional inflammatory reactions.

Determining the Proportions of Bone and Cartilage Growth in the Crucian Carp (carassius auratus) Using the Modified Simultaneous Differential Staining Technique

  • Lee, Jin-Heon
    • Journal of Environmental Health Sciences
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    • v.36 no.4
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    • pp.337-341
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    • 2010
  • The modified simultaneous differential staining technique, which enables double staining of cartilage and bones, needs to be improved to prevent soft tissues from being damaged during the staining process. Key factors influencing the extent to which soft tissues are damaged include the fixative used, macerating time, potassium hydroxide concentration, incubation temperature and the removal of skin from specimens. Here we describe a protocol that enables the hardening of tissues during bleaching and maceration. We also describe a method for objectively measuring rates of cartilage and bone growth. The use of formalin as a fixative rendered soft tissues more rigid due to the resulting chemical bonds formed between proteins. Blotted specimens were immersed in 1% potassium hydroxide (KOH) and incubated at $37^{\circ}C$ for 1 day (smaller specimens) or 2-3 days (larger specimens). The 1% KOH solution was also used as the diluent solution for the subsequent immersion in a graded series of 30%, 50%, 70%, 90%, 100% glycerol solutions, a procedure that made soft tissues even more transparent and hardened. It was not necessary to remove the skin of specimens shorter than 2 cm, since the macerating solution could easily penetrate their thin skin layer and continuously remove those pigments hindering visibility. Since excessive osmosis is another factor that can damage soft tissues in the macerating process by causing the rupture of those cells not able to withstand the osmotic pressure, here it was minimized by balancing the salt concentration between the interior and exterior of cells with the addition of 0.9% sodium chloride (NaCl) in the macerating solution. Finally, to determine the proportions of cartilage and bone growth, photographs of the stained specimens were taken with a dissecting microscope and sections corresponding to the cartilage and bones were cut out from the printed pictures and weighed. Our results show that this method is suitable for the objective evaluation of bone and cartilage growth.

Phenotypic Characterization of MPS IIIA (Sgshmps3a/ Sgshmps3a) Mouse Model

  • Park, Sung Won;Ko, Ara;Jin, Dong-kyu
    • Journal of mucopolysaccharidosis and rare diseases
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    • v.4 no.1
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    • pp.26-36
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    • 2018
  • Mucopolysaccharidosis IIIA is a heritable neurodegenerative disorder resulting from the dysfunction of the lysosomal hydrolase sulphamidase. This leads to the primary accumulation of the complex carbohydrate heparan sulphate in a wide range of tissues and CNS degeneration. Characterization of animal model is the beginning point of the therapeutic clinical trial. Mouse model has a limitation in that it is not a human and does not have all of the disease phenotypes. Therefore, delineate of the phenotypic characteristics of MPS IIIA mouse model prerequisite for the enzyme replace treatment for the diseases. We designed 6-month duration of phenotypic characterization of MPS IIIA mouse biochemically, behaviorally and histologically. We compared height and weight of MPS IIIA mouse with wild type from 4 weeks to 6 months in both male and female. At 6 months, we measured GAG storage in urine kidney, heart, liver, lung and spleen. The brain GAG storage is presented with Alcian blue staining, immunohistochemistry, and electron-microscopy. The neurologic phenotype is evaluated by brain MRI and behavioral study including open field test, fear conditioning, T-maze test and Y-maze test. Especially behavioral tests were done serially at 4month and 6month. This study will show the result of the MPS IIIA mouse model phenotypic characterization. The MPS IIIA mouse provides an excellent model for evaluating pathogenic mechanisms of disease and for testing treatment strategies, including enzyme or cell replacement and gene therapy.