Lee, Hyuk;Park, Jung-In;Kim, Sun Young;Moon, Kyeung Hee;Yi, Ho Keun;Hwang, Pyeong Han
Clinical and Experimental Pediatrics
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v.48
no.5
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pp.551-556
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2005
Wiskott-Aldrich syndrome(WAS) is an X-linked recessive immunodeficiency characterized by thrombocytopenia with small platelet volume, eczema, and recurrent infections, and is also characterized by increased incidence of auto immune diseases and malignancies. The phenotype observed in this syndrome is caused by mutation in the Wiskott-Aldrich syndrome protein(WASP) gene localized to the proximal short arm of the X chromosome and recently isolated through positional cloning. The gene encodes a 502 amino acid protein, which contains 12 exons and spans 9 kb of genomic DNA. The function of the encoded protein is not well understood. The clinical diagnosis of WAS can be difficult and is usually confirmed by the detection of WASP gene mutations and the expression of WSAP in patient blood sample using genetic analysis. We reported a case of a 13-month old boy with WAS who was identified with the novel mutation in exon 2 of WASP gene by direct sequencing and the complete absence of WASP expression by immunoblotting.
An iron-fortified whey protein concentrate (Fe-WPC) was prepared by addition of ferric chloride to concentrated whey. A large part of the iron in the Fe-WPC existed as complexes with proteins such as ${\beta}$-lactoglobulin. The bioavailability of iron from Fe-WPC was evaluated using iron-deficient rats, in comparison with heme iron. Rats were separated into a control group and an iron-deficiency group. Rats in the control group were given the standard diet containing ferrous sulfate as the source of iron throughout the experimental feeding period. Rats in the iron-deficiency group were made anemic by feeding on an Fe-deficient diet without any added iron for 3 wk. After the iron-deficiency period, the iron-deficiency group was separated into an Fe-WPC group and a heme iron group fed Fe-WPC and hemin as the sole source of iron, respectively. The hemoglobin content, iron content in liver, hemoglobin regeneration efficiency (HRE) and apparent iron absorption rate were examined when iron-deficient rats were fed either Fe-WPC or hemin as the sole source of iron for 20 d. Hemoglobin content was significantly higher in the rats fed the Fe-WPC diet than in rats fed the hemin diet. HRE in rats fed the Fe-WPC diet was significantly higher than in rats fed the hemin diet. The apparent iron absorption rate in rats fed the Fe-WPC diet tended to be higher than in rats fed the hemin diet (p = 0.054). The solubility of iron in the small intestine of rats at 2.5 h after ingestion of the Fe-WPC diet was approximately twice that of rats fed the hemin diet. These results indicated that the iron bioavailability of Fe-WPC was higher than that of hemin, which seemed due, in part, to the different iron solubility in the intestine.
Phosducin (PDC) is a photoreceptor cell-specific protein that is phosphorylated by cyclic nucleotide-dependent protein kinase. PDC and PDC-like proteins (PDCL, PDCL2, and PDCL3) are members of a conserved family of small thioredoxin-like proteins that modulate the ${\beta}$- and ${\gamma}$-subunits of G-proteins. In mammals, Pdc, Pdcl, and Pdcl3 genes show ubiquitous expression; however, Pdcl2 gene expression is limited to the testis and ovary. The aim of the present study was to examine the expression patterns of chicken Pdcl2 (cPdcl2) during testicular and ovarian development. Protein sequence comparisons performed using the CLUSTAL X program revealed that the amino acid sequences and potential phosphorylation sites of cPDCL2 and mammalian PDCL2 proteins were highly conserved. Quantitative real-time PCR analysis revealed that cPdcl2 was differentially expressed in the testis and ovary. Specifically, cPdcl2 expression was detected at low levels in the ovary at all time points. In the testis, cPdcl2 expression was detected at low levels until 5 weeks of age. At 8 weeks of age, however, cPdcl2 showed increased expression levels in the testis. Using in situ hybridization, we detected high levels of cPdcl2 expression in the testis, particularly in the spermatocytes and round spermatids. In summary, our data describe expression patterns of germ cell-specific Pdcl2 during testicular and ovarian development in chickens.
Clinical resistance to chemotherapeutic agents is one of the major hindrances in the treatment of human cancers. EHZ2 is involved in drug resistance and is overexpressed in drug-resistant cancer cell lines. In this study, we investigated the effects of EHZ2 on cisplatin -resistance in A549/DDP and AGS/DDP cells. EHZ2 mRNA and protein were found to be significantly overexpressed in A549/DDP and AGS/DDP cells, compared to parental cells. EHZ2 siRNA successfully silenced EHZ2 mRNA and protein expression. Proliferation was inhibited and drug resistance to cisplatin was improved. Flow cytometry showed that silencing of EHZ2 arrested A549/DDP and AGS/DDP cells in the G0/G1 phase, increasing apoptosis, rh-123 fluorescence intensity and caspase-3/8 activities. Silencing of EHZ2 also significantly reduced the mRNA and protein expression levels of cyclin D1 and MDR1,while up-regulating p15, p21, p27 and miR-218 in A549/DPP cells. Furthermore, silencing of EHZ2 also significantly increased the expression level of tumor suppressor factor miR-218. We also found down-regulating EHZ2 expression increased methylation in A549/DDP and AGS/DDP cells. This study demonstrates that drug resistance can be effectively reversed in human cisplatin-resistant lung and gastric cancer cells through delivery of siRNAs targeting EHZ2.
The functional expression of potassium $(K^+)$ channels has electrophysiologically been studied in bone cells from several species, however, their identity and regulation of gene expressions in bone cells are not well known. In the present study, to investigate how $K^+$ channel expressions are regulated by estrogen, we measured changes of transcript levels of various $Ca^{2+}$-activated ($K_{Ca}$) and ATP-sensitive $K^+$ channels in rat osteoblastic ROS 17/2.8 cells after treatment with estrogen. Application of 17${\beta}$-estradiol $(E_2)$ for 24 h and 48 h increased mRNA and protein expressions of inwardly rectifying $K^+$ channel (Kir) 6.2 and type 2 small conductance $K_{Ca}$ channel (SK2), respectively. Combined treatment of cells with 17${\beta}-E_2$ and ICI 182,780, a pure antiestrogen, suppressed 17${\beta}-E_2$-induced alterations of SK2 and Kir6.2 mRNA levels. In addition, treatment of cells with U0126, a specific inhibitor of extracellular receptor kinases (ERK)1/2, and SP600125, a specific inhibitor of c-jun N-terminal kinase (JNK) blocked the enhancing effects of 17${\beta}-E_2$ on SK2 and Kir6.2 protein expressions. On the other hand, blocking of p38 mitogen-activated protein kinase had no effect. Taken together, these results indicate that 17${\beta}-E_2$ modulates SK2 and Kir6.2 expressions through the estrogen receptor, involving ERK1/2 and JNK activations.
Agarase genes of non-marine agarolytic bacterium Cellvibrio sp. were cloned into Escherichia coli and one of the genes obtained using HindIII was sequenced. From nucleotide and putative amino acid sequences (713 aa, molecular mass; 78,771 Da) of the gene, designated as agarase AgaA, the gene was found to have closest homology to the Saccharophagus degradans (formerly, Microbulbifer degradans) 2-40 aga86 gene, belonging to glycoside hydrolase family 86 (GH86). The putative protein appears to be a non-secreted protein because of the absence of a signal sequence. The recombinant protein was purified with anion exchange and gel filtration columns after ammonium sulfate precipitation and the molecular mass (79 kDa) determined by SDS-PAGE and subsequent enzymography agreed with the estimated value, suggesting that the enzyme is monomeric. The optimal pH and temperature for enzymatic hydrolysis of agarose were 6.5 and $42.5^{\circ}C$, and the enzyme was stable under $40^{\circ}C$. LC-MS and NMR analyses revealed production of a neoagarobiose and a neoagarotetraose with a small amount of a neoagarohexaose during hydrolysis of agarose, indicating that the enzyme is a ${\beta}$-agarase.
A dog which was hospitalized to Veterinary Teaching Hospital, College of Veterinary Medicine, Jeonbug National University on December 28, 1988 was revealed severe anemia: hemoglobinuria and weakness. In the inspections, abdominal pain and spleno megaly at the ventral abdomen were detected by palpations. In the examinations of blood, the obtained results were summarized as follows: Babesla spp. was identified on the blood smear stained with Giemsa. The Babesia spp. was assumed to the Babesia gibsoni for the their small size and pleomorphism such as comma form, ring form and dot form. In the blood examinations of the patient, Ht: 22.5%, RBC:354${\times}$10$^4$/${\mu}\ell$, Hb: 8.8g/dl, serum protein: 8g/dl, and WBC count was 21, 425/${\mu}\ell$. In the chemical examinations of serum, the value of AST(GOT) was 30iu and ALT(GPT) was 20iu, respectively. The blood sugar was 60mg/d1. In the urine test, urine protein was 30mg/d1 and the hemoglobin In the urine was the +++ and occult blood reaction(Benzidine test) in the feces was +++. Splenomegaly was confirmed by X-ray examination. To confirm for the Babesia spp. infection, 5ml of the whole blood of the patient(3% of Parasitized erythrocytes) were inoculated into the cephalic vein of the two normal dogs. In the blood of experimental dogs which were inoculated parasitized blood, Babesia spp. was detected in the two doss and pleomorphic parasites were observed, too. In the blood examinations of No. 1 the Ht and RBC were decreased to 6.8% and 52${\times}$10$^4$/${\mu}\ell$, respectively. WBC count was 10.600/${\mu}\ell$ and serum protein was 6.8g/dl. The rates of parasitized erythrocytes were 15% in the experimental dog. Also +++ of the hemoglobin was detected in the urine. In the X-ray examination, splenomegaly was comfirmed and it was confirmed by autopsy of the experimental dog(No. 1).
The mash of Takjoo, Korean flour wine, is fermented through two brewing processes ; the primary brewing process to saccharify and the main one to produce ethyl alcohol. The activities of acid proteinase (pH3), weak acid proteinase (pH 6), and alkaline proteinase (Ph 80 on the processes are determined with time by the Folin phenol method as a strength of casein digestion. Hydrogen ion concentration, the content of total organic acids, protein, free amino acids and oligopeptides, which effect the activities of proteinase, are also measured. The results are briefly summarized as follows : 1. In general, the activities of acid proteinase and weak acid proteinase in the mesh of primary brewing process are stronger than those in main brewing process. 2. The activities of acid proteinase are remarkably stronger than those of weak acid proteinase in both processes. It reveals that they decrease slowly through the fermentation. Activities of alkaline proteinase are weaker than others. 3. As the raw materials are mixtured, the total amount of organic acids is equivalent to 0.150 mg/ml acetic acid in the mesh of primary brewing process and 0.02 mg/ml acetic acid in the main one. They increase gradually with time. 4. Hydrogen ion concnetration shows 3.9 in the mesh of main brewing process and 3.28 in the primary one. They increase to the maximum in 60-72 hrs., and decrease since 108 hrs. 5. The content of crude protein shows 66.90mg/ml in the mesh of main brewing process, while shows 64.29mg/ml in the mesh of primary one. they decrease slowly with time. it seems that a small content of crude protein, as a substrate, converts into amino acids and soluble nitrogen compounds by proteinase. 6. The content of free amino acids and oligopeptides shows 0.36 mg/ml in the mesh of primary brewing process and 0.24mg/ml in the main brewing process. It is evident that the reason they increase continuously through the fermentation is the effect of proteinase. 7. According to the results, the strong activities of proteinase in primary brewing process has been derived from the decrease of hydrogen ion concentration due to the production of organic acids.
Background: The human protein methyl-transferase DOT1L catalyzes the methylation of histone H3 on lysine 79 (H3K79) at homeobox genes and is also involved in a number of significant processes ranging from gene expression to DNA-damage response and cell cycle progression. Inhibition of DOT1L activity by shRNA or small-molecule inhibitors has been established to prevent proliferation of various MLL-rearranged leukemia cells in vitro, establishing DOT1L an attractive therapeutic target for mixed lineage leukemia (MLL). Most of the drugs currently in use for the MLL treatment are reported to have low efficacy, hence this study focused on various natural compounds which exhibit minimal toxic effects and high efficacy for the target receptor. Materials and Methods: Structures of human protein methyl-transferase DOT1L and natural compound databases were downloaded from various sources. Virtual screening, molecular docking, dynamics simulation and drug likeness studies were performed for those natural compounds to evaluate and analyze their anti-cancer activity. Results: The top five screened compounds possessing good binding affinity were identified as potential high affinity inhibitors against DOT1L's active site. The top ranking molecule amongst the screened ligands had a Glide g-score of -10.940 kcal/mol and Glide e-model score of -86.011 with 5 hydrogen bonds and 12 hydrophobic contacts. This ligand's behaviour also showed consistency during the simulation of protein-ligand complex for 20000 ps, which is indicative of its stability in the receptor pocket. Conclusions: The ligand obtained out of this screening study can be considered as a potential inhibitor for DOT1L and further can be treated as a lead for the drug designing pipeline.
Lee, Sang-Hoon;Bidle, Kay;Falkowski, Paul;Marchant, David
Ocean and Polar Research
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v.27
no.2
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pp.205-214
/
2005
From ancient Antarctic glacier ice, we extracted total genomic DNA that was suitable for prokaryotic 16S rDNA gene cloning and sequencing, and bacterial artificial chromosome (BAC) library and end-sequencing. The ice samples were from the Dry Valley region. Age dating by $^{40}Ar/^{39}Ar$ analysis on the volcanic ashes deposited in situ indicated the ice samples are minimum 100,000-300,000 yr (sample DLE) and 8 million years (sample EME) old. Further assay proved the ice survived freeze-thaw cycles or other re-working processes. EME, which was from a small lobe of the basal Taylor glacier, is the oldest known ice on Earth. Microorganisms, preserved frozen in glacier ice and isolated from the rest of the world over a geological time scale, can provide valuable data or insight for the diversity, distribution, survival strategy, and evolutionary relationships to the extant relatives. From the 16S gene cloning study, we detected no PCR amplicons with Archaea-specific primers, however we found many phylotypes belonging to Bacteria divisions, such as Actinobacteria, Acidobacteria, Proteobacteria $({\alpha},\;{\beta},\;and\;{\gamma})$, Firmicutes, and Cytophaga-Flavobacterium-Bacteroid$. BAC cloning and sequencing revealed protein codings highly identical to phenylacetic acid degradation protein paaA, chromosome segregation ATPases, or cold shock protein B of present day bacteria. Throughput sequencing of the BAC clones is underway. Viable and culturable cells were recovered from the DLE sample, and characterized by their 16S rDNA sequences. Further investigation on the survivorship and functional genes from the past should help unveil the evolution of life on Earth, or elsewhere, if any.
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