DSPP mutation in dentinogenesis imperfecta Shields type II.

We identified a nonsense mutation (Gln45stop) in exon 3 of the dentin sialophosphoprotein (DSPP) gene in a Chinese family with dentinogenesis imperfecta Shields type II (DGI-II), in which the affected members showed discoloration and severe attrition of their teeth, with obliterated pulp chambers.

Estimating Concentration Response Function and Change-Point using Time-Course and Calibration Data.

In this paper the problem of determining the functional relationship between time and the concentration of a chemical substance is studied. An intervention drug is administered on the experimental unit from which the chemical substance (specimen) is measured. This drug is hypothesized to cause a change in the concentration level of the chemical substance a certain lag-time after the intervention. However, the concentration value could not be directly measured, but rather a surrogate response can be measured. In the time-course study, this surrogate response is measured using different electrodes which possess varied behaviors. To utilize these surrogate measurements arising from the different electrodes (sensors), a calibration study is undertaken which measures the surrogate response for the different electrodes at known concentration levels. Based on the time-course and calibration data sets, a statistical procedure to estimate the signal function and the lag-time is proposed. Simulation studies indicate that the proposed procedure is able to reasonably recover the signal function and the lag-time. The procedure is then applied to the real data sets obtained during an analytical chemistry experiment.

Roles of monocyte chemotactic protein 1 and nuclear factor-κB in immune response to spinal tuberculosis in a New Zealand white rabbit model.

This study aimed to explore the roles of monocyte chemotactic protein 1 (MCP-1) and nuclear factor kappa B (NF-κB) in immune response to spinal tuberculosis in a New Zealand white rabbit model. Forty-eight New Zealand white rabbits were collected and divided into four groups: experimental group (n=30, spinal tuberculosis model was established), the sham group (n=15, sham operation was performed) and the blank group (n=3). The qRT-PCR assay and western blotting were applied to detect the mRNA and protein expressions of MCP-1 and NF-κB in peripheral blood. ELISA was used to measure serum levels of MCP-1, NF-κB, IFN-γ, IL-2, IL-4, and IL-10. Flow cytometry was adopted to assess the distributions of CD4+, CD8+ lymphocytes and CD4+ CD25+ Foxp3 lymphocyte subsets. Compared with the sham and blank groups, the mRNA and protein expressions of MCP-1 and NF-κB in the experimental group were significantly increased. The experimental group had lower serum levels of IL-2 and IFN-γ and higher serum level of IL-10 than the sham and blank groups. In comparison to the sham and blank groups, CD4+ T lymphocyte subsets percentage, CD4+/CD8+ ratio and CD4+ CD25+ Foxp3+ Tregs subsets accounting for CD4+ lymphocyte in the experimental group were lower, while percentage of CD8+ T lymphocyte subsets was higher. Our study provided evidence that higher expression of MCP-1 and NF-κB may be associated with decreased immune function of spinal tuberculosis, which can provide a new treatment direction for spinal tuberculosis.

NSPc1 promotes cancer stem cell self-renewal by repressing the synthesis of all-trans retinoic acid via targeting RDH16 in malignant glioma.

Polycomb group (PcG) proteins play an important role in development and stem cell maintenance, and their dysregulation have been closely linked to oncogenesis and cancer stem cell phenotypes. Here, we found that nervous system polycomb 1 (NSPc1) was highly expressed in stem cell-like glioma cells (SLCs). Knockdown of NSPc1 in SLCs resulted in impaired neurosphere formation and self-renewal abilities, down-regulated expression of stemness markers such as NESTIN, CD133 and SOX2, and decreased capacity to propagate subcutaneous xenografts. In contrast, glioma cells overexpressing NSPc1 exhibited a stem cell-like phenotype, up-regulated expression of stemness markers NESTIN, CD133 and SOX2, and an enhanced capacity to propagate subcutaneous xenografts. Furthermore, we identified that NSPc1 epigenetically repressed the expression of retinol dehydrogenase 16 (RDH16) by directly binding to a region upstream (-1073 to -823) of the RDH16 promoter. Next, we confirmed that RDH16 is a stemness suppressor that partially rescues SLCs from the NSPc1-induced increase in neurosphere formation. Finally, we showed that ATRA partly reversed the NSPc1-induced stemness enhancement in SLCs, through mechanisms correlated with an ATRA-dependent decrease in the expression of NSPc1. Thus, our results demonstrate that NSPc1 promotes cancer stem cell self-renewal by repressing the synthesis of ATRA via targeting RDH16 and may provide novel targets for glioma treatment in the future.

An autosomal genomic scan for loci linked to type 2 diabetes in northern Han Chinese.

We report the results of a genome-wide scan conducted in 219 individuals from 34 large multiplex nuclear pedigrees from the northern Han Chinese population at an average resolution of about 10 cM. Nonparametric two-point and multipoint linkage analyses were performed to detect evidence of linkage with type 2 diabetes in this study. On chromosome 1 four regions showed evidence of linkage with type 2 diabetes in northern Han Chinese. Of these regions a marker D1S193 (73 cM) showed evidence of linkage (two-point nonparametric linkage 2.409), and another region (around 190 cM) was a replication of several other studies performed in different ethnic populations. Evidences of linkage have been confirmed by typing additional markers (average distance 1-5 cM) flanking these two positive regions on chromosome 1. We also found indication of linkage with type 2 diabetes on chromosomes 2, 10, 12, 18, 20, and 22 by two-point linkage analyses.

ASH2L: alternative splicing and downregulation during induced megakaryocytic differentiation of multipotential leukemia cell lines.

Abstract Drosophila ash2 is a member of the trxG gene super family, some human homologues of which are involved in hematopoiesis and leukemia. We report here the identification of the human homologue of Drosophila ash2 and its alternative splicing isoform, ASH2L1 and ASH2L2. ASH2L proteins are 60% homologous to Drosophila ash2. ASH2L also has a zinc finger motif (C2C2) although it is not identical to that in ASH2. Expression profile analysis showed that the amount of ASH2L transcripts is extremely high in fetal liver, testis, and leukemia cell lines with erythroid and megakaryocytic potential such as K562, Hel, and Dami. We treated these cells with differentiation inducers phorbol ester and hemin. We found that ASH2L is downregulated rapidly and dramatically in K562, Hel, and Dami cells during phorbol ester induced differentiation with megakaryocytic features. However, its expression is maintained at a high level during erythroid differentiation of K562 cells induced with hemin. These results suggest that ASH2L plays a role in hematopoiesis and is associated with some special kinds of leukemia.

The apparent specificity of NotI (5'-GCGGCCGC-3') is enhanced by M.FnuDII or M.BepI methyltransferases (5'-mCGCG-3'): cutting bacterial chromosomes into a few large pieces.

The restriction endonuclease (ENase) NotI is blocked by methylation within its recognition sequence at 5'-GCGGCmCGC-3'. This sensitivity to methylation can be used to enhance the specificity of NotI in vivo and in vitro. Modification by M.FnuDII or M.BepI methyltransferases (MTase) (5'-mCGCG-3') will block NotI (5'-GCGGCCGC-3') cleavage at overlapping MTase/ENase sites 5'-CGCGGCCGC-3' (equivalent to 5'-GCGGCCGCG-3'), and increase the apparent cleavage specificity of NotI about twofold. This 'cross-protection' procedure reduces the number of NotI fragments in the genomes of Escherichia coli and Bacillus subtilis, as resolved by pulsed field electrophoresis. Application of this method to large DNAs in vitro requires the preparation of highly purified DNA MTases.

Nuclear and mitochondrial DNA of the primate malarial parasite Plasmodium knowlesi.

Restriction analyses and DNA/DNA hybridisation of parasite DNA isolated from monkeys infected with the malarial parasite Plasmodium knowlesi has permitted unambiguous identification of the nuclear DNA of this species. Its (G+C) content, as determined by estimations of buoyant density as well as by direct analysis, is about 38%, essentially indistinguishable from that of its primate laboratory host, and grossly different from that of the major human malaria parasite, P. falciparum, which has a (G+C) content of approx. 19%. In addition, gradient fractionation of total P. knowlesi DNA revealed a minor DNA component (approx. 1% of the total) with a (G+C) content of about 19%. This DNA comprises covalently closed circular molecules which have a contour length about 11.6 microns, carry a small cruciform structure, and are thought to originate in the parasite's mitochondria.

Cloning and characterization of an abundant Plasmodium knowlesi antigen which cross reacts with Gambian sera.

A 110 kDa Plasmodium knowlesi antigen, termed PK110, has been identified on the basis of messenger RNA abundance in late schizonts. Most Plasmodium genes previously cloned have been identified by immune sera, which have selected immunodominant antigens composed of repeating epitopes. Although PK110 was not selected by immune sera, it also contains amino acid repeats, indicating that this structure may be a common feature of malarial proteins. Determination of 296 codons in the PK110 gene revealed the presence of thirteen tandem repeats of twelve amino acids whose consensus sequence is E E T Q K T V E P E Q T. A termination codon interrupts the fourteenth repeat, indicating that these repeats are at the C-terminus of the protein. Indirect immunofluorescence experiments with sera raised against the lambda gt11 fusion protein indicate that PK110 is present in intra-erythrocytic late schizonts. Cloned PK110 is recognized by Gambian sera, and shares epitopes with Plasmodium ovale. PK110 does not cross react immunologically or by DNA hybridization with Plasmodium falciparum.

Overexpression of human reticulon 3 (hRTN3) in astrocytoma.

Neuroendocrine-specific protein (NSP) reticulons (RTN) are endoplasmic reticulum-associated protein complexes, which are localized in the endoplasmic reticulum (ER) and identified as markers for neuroendocrine differentiation. At least 4 different RTN genes have been identified in mammals, but in most cases, the functions of the encoded proteins except mammalian RTN4-A and RTN4-B are still elusive. In the present study, the expression of human reticulon 3 (hRTN3) in a variety of tissues was investigated. Northern blotting hybridization revealed higher expression of hRTN3 in normal brain and some endocrine-related organs such as thyroid relative to other non-endocrine organs. Twelve surgical specimens meeting the histological criteria for astrocytoma grade II, III and IV were investigated by in situ hybridization and immunohistochemical analysis, which demonstrated the overexpression of hRTN3 in astrocytoma tumor cells, while in non-cancerous brain tissues hRTN3 was mainly expressed in neurons and almost no signals in glia cells could be detected. The differential expression of hRTN3 between astrocytoma and non-cancerous tissue may provide insight into the progress of astrocytoma.

Confirmation of susceptibility gene loci on chromosome 1 in northern China Han families with type 2 diabetes.

OBJECTIVE: To confirm previous effort to identify type 2 diabetes susceptibility genes in a Northern Chinese population by conducting a new genome scan with both an increased number of type 2 diabetes families and a new set of microsatellite markers within the previously localized regions. METHODS: A genome scan method was applied. After multiplexed PCR, electrophoreses, genescan and genotyping analysis, we obtained size information for all loci, and then a further study was done by both parametric and non-parametric linkage analysis to investigate the P values and Z values of these loci. RESULTS: We surveyed 34 microsatellite markers which distributed within 5 regions along chromosome 1, and a total of 12,000 genotypes were screened. Evidence of linkage with diabetes was identified for 8 of the 34 loci. All P values of the 8 loci were lower than 0.05, and the highest Z value was 2.17. A very interesting finding is that all 5 markers at the p- terminal 1p36.3-1p36.23 region, spanning a long range of 16.9 cM, were identified to have a low P value of less than 0.05, which suggests that this region may contain multiple susceptibility genes. Regions 4 and 5 also confirmed the previous findings, and we narrowed these two regions to a 2.7 cM and 2.5 cM regions, respectively. CONCLUSIONS: We further confirmed the results gained in the previous genome-wide scan using an increased number of NIDDM families and a new set of microsatellite markers lying within the initially localized regions. The fact that all 5 loci at the p- terminal region displayed a low P value of less than 0.05 suggests that more than 1 susceptibility gene may reside in this region.

Isolation and identification of cDNA fragments and full-length cDNA differentially expressed in human glioblastoma cell line BT-325 versus all-trans retinoic acid induction .

OBJECTIVE: To investigate the differentiation process of the human glioblastoma cells. METHODS: Differential display reverse transcribed-PCR (DDRT-PCR) was used to isolate the genes differentially expressed in control and all-trans retinoic acid treated human glioblastoma cell line BT-325. Routine method of cDNA library screening was performed to clone full-length cDNA. RESULTS: Thirty-six RT-PCR reactions were performed and 64 differentially expressed fragments were recovered, amplified and cloned. Of them, 46 ESTs were sequenced and delivered into the GenBank. The homology comparison using BLAST algorithm revealed that 22ESTs are highly homologous with the known genes and many of them play important roles in the cell differentiation progress. A dot-blot hybridization was conducted to certify the differentiation expression. The result showed that 27 EST clones are expressed at different level in control and all-trans retinoic acid treated BT-325 cells. A full-length cDNA was cloned using the EST-HGBB098. CONCLUSION: DDRT-PCR was a simple and effective method to serially analyze the differentially expressed genes.

Alteration in enterocyte gene expression may explain structural and functional changes following glutamine supplemented parenteral nutrition.

Following extensive bowel resection, the intestinal tract undergoes a variety of adaptive responses to enhance bowel function. The purpose of this study was to determine the effect of glutamine-supplemented parenteral nutrition on mucosal cellularity and gut function. In addition, enterocyte gene expression of two relevant systems was also characterized and related to the structural and functional changes that occurred. Male Wistar rats underwent a 60% small bowel resection and jugular vein catheterization and were randomized into two groups. The control group (n = 10) received a standard intravenous nutritional solution and the study group (n = 10) received a similar solution but enriched with alanylglutamine dipeptide. After 7 days blood was taken for amino acid analysis, and bowel was harvested to determine mucosal morphology and expression of mucosal cell glutaminase and IGF-I mRNA. Mesentery lymphnodes were cultured to determine the presence of bacteria and thus access bacteria translocation. Serum glutamine concentration and mucosal architecture were maintained in the study group compared to the controls. Seventy percent of lymphnodes were cultured positive in control vs. only 20% in the study group (P < 0.05). Jejunal mucosal glutaminase and ileum mucosal IGF-I mRNA increased twofold and threefold respectively compared to control animals. Parenteral nutrition supplemented with alanyl-glutamine dipeptide supports mucosal cellularity and regional immune function in rodents following intestinal resection, These alterations are associated with enhanced enterocyte expression of glutaminase and IGF-I. These changes may facilitate the structural and functional alterations which were observed in the glutamine treated animals.

[Differential display analysis of human fetal brain mRNAs and isolation of brain-specific novel ESTs].

Using the differential display technique (DDRT-PCR) originally developed by Liang and Pardee, we tried to isolate the stage-specific or tissue-specific genes in the developing human neural system (brain). With about fifty sets of arbitrary primers and anchored primers, DDRT-PCR was carried out to analyze the differentially expressed mRNAs between human fetal brains of different developmental stage or different parts of human fetal brain at the same developmental stage. About one hundred bands containing cDNA fragments of differential interest were cut out from the polyacrylamide gel, thirty-four of which were cloned and sequenced. They were accepted as novel cDNA sequences by GeneBank. In order to check the feasibility of DDRT-PCR, three of the thirty-four cDNA sequences were randomly chosen as probes for further analysis by dot blot RNA hybridization, and two proved to be specifically expressed in human brain.

[Construction of poliovirus cDNA chimera with (NKNDD)n and (NANP)n].

Oligonucleotides encoding one copy of NKNDD, containing a P.f. B epitope NKND, and two copies of CSP repeated B epitope NANP were synthesized. After self-ligation, NKNDD and (NANP)2 were cloned into the intermediate vector pSKMM. Series of clones with different copies of NKNDD and NANP were identified. (NKNDD)n/MM with three, four, five, eight copies of NKNDD and (NANP)n/MM with four, six copies of NANP were chosen. Then (NKNDD)n and (NANP)n of these recombinants were recovered respectively and finally inserted into the N-Ag I site of poliovirus type I Mahoney strain full-length cDNA, which is contained in the expression plasmid pSV202H+. As using semi-quantitative hybridization in identifying tranformants, the procedure in our experiment for cloning different copies of NKNDD and NANP was greatly simplified.

[Differentiation of human glioma cells BT-325 induced by sodium butyrate].

OBJECTIVE: To study the differentiation of human glioma cells BT-325 induced by sodium butyrate in vitro. METHODS: BT-325 cells were treated with 1 mmol/L sodium butyrate. Flow cytometry was used to analyze the cell cycle. Cell differentiation was identified by flow cytometry and Western blot analysis. RESULTS: After 6-8 days of sodium butyrate treatment, the differentiation characters could be observed distinctively, such as the reduced cell density, the increased cell size and the marked increase in cell process formation and cell-to-cell connection. At the same time, Western blot showed that the amount of glial fibrillary acidic protein was elevated after the sodium butyrate treatment. CONCLUSION: Human glioma cells BT-325 could be induced to differentiation by sodium butyrate.

[The identification and cloning of human ubiquitin binding enzyme cDNA].

OBJECTIVE: To identify and clone the gene encoding human ubiquitin binding enzyme and study its expression spectrum. METHODS: According to the sequence of human EST, which is highly homologous to the mouse ubiquitin conjugating enzyme (E2), primers used for library screening were synthesized to screen the human fetal brain cDNA library. The gene was analyzed by making use of bioinformatics and its expression spectrum was studied by using multiple-tissue Northern blot. RESULTS: Two cDNA clones encoding human ubiquitin conjugating enzyme were isolated and identified. Both containing the ubiquitin conjugating domain, they were 88% identical in amino acid sequences and found to be isoforms of each other with only an exon excised from the short sequence. They belonged to a highly conserved, and widely expressed E2 enzyme family. Northern blot showed that they were expressed exclusively in heart, placenta, and pancreas while no transcripts could be detected in brain, lung, liver, skeletal muscle, or kidney. CONCLUSIONS: The gene encoding human ubiquitin binding enzyme is expressed under both temporal and spatial control. As a key enzyme in the degradation of proteins, ubiquitin conjugating enzymes play a central role in the expression regulation on the level of post-translation.

Roles of monocyte chemotactic protein 1 and nuclear factor-kappaB in immune response to spinal tuberculosis in a New Zealand white rabbit model

This study aimed to explore the roles of monocyte chemotactic protein 1 (MCP-1) and nuclear factor kappa B (NF-κB) in immune response to spinal tuberculosis in a New Zealand white rabbit model. Forty-eight New Zealand white rabbits were collected and divided into four groups: experimental group (n=30, spinal tuberculosis model was established), the sham group (n=15, sham operation was performed) and the blank group (n=3). The qRT-PCR assay and western blotting were applied to detect the mRNA and protein expressions of MCP-1 and NF-κB in peripheral blood. ELISA was used to measure serum levels of MCP-1, NF-κB, IFN-γ, IL-2, IL-4, and IL-10. Flow cytometry was adopted to assess the distributions of CD4+, CD8+ lymphocytes and CD4+ CD25+ Foxp3 lymphocyte subsets. Compared with the sham and blank groups, the mRNA and protein expressions of MCP-1 and NF-κB in the experimental group were significantly increased. The experimental group had lower serum levels of IL-2 and IFN-γ and higher serum level of IL-10 than the sham and blank groups. In comparison to the sham and blank groups, CD4+ T lymphocyte subsets percentage, CD4+/CD8+ ratio and CD4+ CD25+ Foxp3+ Tregs subsets accounting for CD4+ lymphocyte in the experimental group were lower, while percentage of CD8+ T lymphocyte subsets was higher. Our study provided evidence that higher expression of MCP-1 and NF-κB may be associated with decreased immune function of spinal tuberculosis, which can provide a new treatment direction for spinal tuberculosis.

Noninvasive ultrasound image guided surface wave method for measuring the wave speed and estimating the elasticity of lungs: A feasibility study.

Lung diseases, such as acute respiratory distress syndrome (ARDS) and idiopathic pulmonary fibrosis (IPF), are closely associated with altered lung elastic properties. Pulmonary function testing and imaging are routinely performed for evaluating lung diseases. However, lung compliance, a measure of lung elastic properties, is rarely used in clinic, because it is invasive and provides only a global and arguably biased estimate of lung elastic properties. Current ultrasound methods also cannot be used for imaging lungs because ultrasound cannot penetrate the lung tissue. In this paper, an ultrasound image guided and surface wave based method is proposed to measure regional lung surface wave speed and estimate lung elasticity noninvasively. The method described here was not explored before to the best knowledge of the authors. Experiments in an ex vivo pig lung and an in vivo human lung pilot study are reported. The surface wave speed is measured to be 1.83±0.02m/s at 100Hz by ultrasound for the ex vivo pig lung at 3mmHg pressure, which is validated by an optical measurement. An in vivo human lung pilot experiment measures the surface wave speed to be 2.41±0.33m/s for the 100Hz sinusoidal wave at total lung capacity (TLC) and 0.99±0.09m/s at functional residual capacity (FRC). These values of wave speed fall well within the range of available literature.