Biomedical subjects

H Fan

Publications and source records attributed to H Fan.

At least 109 records · Page 6Linked to original sources

PubMed1998

Myc-epitope tagged proteins detected with the 9E10 antibody in immunofluorescence and immunoprecipitation assays but not in western blot analysis.

A human Myc epitope is frequently used to tag proteins for expression experiments in nonhuman cells. We used the monoclonal 9E10 antibody specific for this epitope to analyse the expression of four proteins carrying the Myc tag in cells transfected with expression vectors. While all four proteins can be detected by immunofluorescence and immunoprecipitation assays, surprisingly, only two proteins could be detected in Western blot analysis, indicating that epitope recognition by the monoclonal antibody can be blocked in some membrane-retained ectopic proteins. Other techniques such as immunofluorescence and immunoprecipitation assays can be successfully used with the 9E10 antibody to determine potential expression of Myc-tagged proteins.

Animals↗

PubMed1997

The R1 component of mammalian ribonucleotide reductase has malignancy-suppressing activity as demonstrated by gene transfer experiments.

Our recent studies have shown that deregulated expression of R2, the rate-limiting component of ribonucleotide reductase, enhances transformation and malignant potential by cooperating with activated oncogenes. We now demonstrate that the R1 component of ribonucleotide reductase has tumor-suppressing activity. Stable expression of a biologically active ectopic R1 in ras-transformed mouse fibroblast 10T(1/2) cell lines, with or without R2 overexpression, led to significantly reduced colony-forming efficiency in soft agar. The decreased anchorage independence was accompanied by markedly suppressed malignant potential in vivo. In three ras-transformed cell lines, R1 overexpression resulted in abrogation or marked suppression of tumorigenicity. In addition, the ability to form lung metastases by cells overexpressing R1 was reduced by >85%. Metastasis suppressing activity also was observed in the highly malignant mouse 10T(1/2) derived RMP-6 cell line, which was transformed by a combination of oncogenic ras, myc, and mutant p53. Furthermore, in support of the above observations with the R1 overexpressing cells, NIH 3T3 cells cotransfected with an R1 antisense sequence and oncogenic ras showed significantly increased anchorage independence as compared with control ras-transfected cells. Finally, characteristics of reduced malignant potential also were demonstrated with R1 overexpressing human colon carcinoma cells. Taken together, these results indicate that the two components of ribonucleotide reductase both are unique malignancy determinants playing opposing roles in its regulation, that there is a novel control point important in mechanisms of malignancy, which involves a balance in the levels of R1 and R2 expression, and that alterations in this balance can significantly modify transformation, tumorigenicity, and metastatic potential.

Animals↗

PubMed1997

A new iterative procedure for combining direct methods with solvent flattening - dealing with the phase ambiguity in protein crystallography.

A new procedure for combining direct methods with the solvent-flattening technique is proposed for phasing single isomorphous replacement (SIR) or one-wavelength anomalous scattering (OAS) data of proteins. The new procedure differs from the previous one [Zheng, Zheng, Gu, Mo, Fan & Hao (1997). Acta Cryst. D53, 49-55] in that the direct method not only provides input phases to but also accepts feedback phases from solvent flattening, thus forming an iterative process for breaking the ambiguities and refining the values of phases. The new procedure was tested with the experimental SIR data of the known structure ribonuclease Sa. For the strongest 1000 of the total 7264 reflections, the mean F(obs)-weighted phase error is 7.5 and 9.4 degrees lower than that of the previous procedure and that of solvent flattening alone, respectively.

Journal Article↗

PubMed1997

Ribonucleotide reductase R2 gene expression and changes in drug sensitivity and genome stability.

Ribonucleotide reductase is a highly regulated, cell cycle-controlled activity that plays an important role in DNA synthesis and repair. Recent studies have shown that elevated expression of the rate-limiting R2 component of ribonucleotide reductase increases Raf-1 protein activation and mitogen-activated protein kinase activity and acts as a novel malignancy determinant in cooperation with activated oncogenes like H-ras. We show that hydroxyurea-resistant mouse L cells with elevated R2 gene expression and increased ribonucleotide reductase activity exhibit significantly decreased sensitivities to the chemotherapeutic compounds N-(phosphonacetyl)-L-aspartate (PALA) and methotrexate (MTX). Furthermore, BALB/c 3T3 cells containing a retroviral expression vector encoding the R2 sequence also showed decreased sensitivity to PALA and MTX when compared to cells containing the same vector but without the R2 coding region. Colonies that developed in the presence of PALA or MTX contained amplifications of the CAD or dihydrofolate reductase genes and exhibited wild-type p53 function as determined in sequence-specific p53 binding activity assays. NIH-3T3 cells containing the R2 retroviral expression vector also showed significantly decreased sensitivity to hydroxyurea and MTX but not to PALA. Furthermore, NIH-3T3 cells transfected with a vector containing the R2 sequence in antisense orientation exhibited increased sensitivity to hydroxyurea, PALA, and MTX. Similarly, mouse 10T1/2 cells that are highly transformed and drug resistant due to alterations in H-ras and a mutant oncogenic form of p53 exhibited significant increases in sensitivity to hydroxyurea, PALA, and MTX when transfected with a vector containing the R2 sequence in antisense orientation and compared to cells containing the same vector without the antisense sequence. These results indicate that altered expression of the R2 component is capable of significantly modifying drug sensitivity properties of tumor cells. We hypothesize that this occurs, at least in part, through a mechanism of increased genetic instability that is independent of direct p53 mutation or loss and involves R2 stimulation of the mitogen-activated protein kinase signal pathway.

3T3 Cells↗

PubMed1997

Electrical stimulation induces the level of TGF-beta1 mRNA in osteoblastic cells by a mechanism involving calcium/calmodulin pathway.

It is well-known that electrical stimulation can prompt healing of bone fractures. However, the mechanism involved is less clear. In this study, we found that capacitively coupled electric field-induced proliferation of osteoblastic cells (MC3T3-E1) accompanied increased levels of transforming growth factor-beta 1 (TGF-beta1) mRNA determined by quantitative reverse transcription/polymerase chain reaction. Previous reports have shown that verapamil and W-7, both of which block voltage gated calcium channels and inhibit the activation of cytosolic calmodulin, respectively, blocked capacitively coupled electric field-induced proliferation of the osteoblast cells. Interestingly, we found that verapamil and W-7 can also block capacitively coupled electric field-induced elevation of TGF-beta1 mRNA. This result suggested that electrical stimulation induces the level of TGF-beta1 mRNA in osteoblastic cells by a mechanism involving calcium/calmodulin pathway. The potential roles of TGF-beta in the electrical signal-induced osteogenesis was discussed.

3T3 Cells↗

PubMed1997

Domain-specific N-glycosylation of the membrane glycoprotein dipeptidylpeptidase IV (CD26) influences its subcellular trafficking, biological stability, enzyme activity and protein folding.

Dipeptidyl peptidase IV (DPPIV, CD26) is an N-glycosylated type II plasma membrane protein. The primary structure of rat wild-type DPPIV contains eight potential N-glycosylation sites. To investigate the role of N-glycosylation in the function of DPPIV, three of its asparagine residues were separately converted to glutamine by site-directed mutagenesis. The resulting N-glycosylation mutants of rat DPPIV were studied in stable transfected Chinese hamster ovary cells. All three N-glycosylation mutants of DPPIV showed a reduced half-life, as well as differing degrees of inhibition of the processing of their N-glycans. Mutation of the first (Asn83-->Gln) or eighth (Asn686-->Gln) N-glycosylation site had only a small effect on its enzymatic activity, cell-surface expression and dimer formation, whereas the mutation of the sixth N-glycosylation site (Asn319-->Gln) abolished the enzymatic activity, eliminated cell-surface expression and prevented the dimerization of the DPPIV protein. The mutant [Gln319]DPPIV is retained in the cytoplasm and its degradation was drastically increased. Our data suggest that the N-glycosylation at Asn319 is involved in protein trafficking and correct protein folding.

Animals↗

PubMed1997

Phosphorylation of the human La antigen on serine 366 can regulate recycling of RNA polymerase III transcription complexes.

The human La antigen is an RNA-binding protein that facilitates transcriptional termination and reinitiation by RNA polymerase III. Native La protein fractionates into transcriptionally active and inactive forms that are unphosphorylated and phosphorylated at serine 366, respectively, as determined by enzymatic and mass spectrometric analyses. Serine 366 comprises a casein kinase II phosphorylation site that resides within a conserved region in the La proteins from several species. RNA synthesis from isolated transcription complexes is inhibited by casein kinase II-mediated phosphorylation of La serine 366 and is reversible by dephosphorylation. This work demonstrates a novel mechanism of transcriptional control at the level of recycling of stable transcription complexes.

Autoantigens↗

PubMed1997

Intrathymically injected hemopoietic stem cells can differentiate into all lineage cells in the thymus: differences between c-kit+ cells and c-kit < low cells.

To investigate whether hemopoietic stem cells (HSCs) can differentiate into all lineage cells even in the thymus, we injected two types of HSCs (c-kit+ and c-kit < low cells) obtained from C57BL/6 Ly5.1 mice directly into the thymus of 7.5 Gy-irradiated C57BL/6 Ly5.2 mice. When c-kit < low cells (low density/lineage-/CD71-/major histocompatibility complex class I high/Sca-1+/Thy-1low/ c-kit < low) were injected, donor-derived (Ly5.1) cells were detected on day 8 after intrathymic (i.t.) injection, and the number reached a maximum on day 24 after injection. Granulocytes and macrophages were also detected on day 8 after injection. However, B220+ B cells were observed on day 13. Eighteen days after i.t. injection, the injected lobes showed red color due to the synchronous development of erythroid cells. Histological studies revealed the development not only of erythroid lineage cells but also of megakaryocytes in the thymus. In contrast, when c-kit+ cells were injected, a significant number of donor-derived cells were detected on day 5 after i.t. injection (three days earlier than in the case of c-kit < low cell injection). The differentiation into erythroid lineage cells was also observed six days earlier than when c-kit < low HSCs were injected. These findings suggest that c-kit < low HSCs are more primitive than c-kit+ HSCs, although both can differentiate into all lineage cells after i.t. injection.

Animals↗

PubMed1997

Leukemogenesis by Moloney murine leukemia virus: a multistep process.

Moloney murine leukemia virus is a prototypical simple retrovirus that has been an extremely useful model for leukemogenesis. Important steps in leukemogenesis include proviral activation of cellular proto-oncogenes, generation of mink cell focus-inducing recombinants, and early (preleukemic) virus-induced changes in hematopoiesis.

Animals↗

PubMed1997

Sheep pulmonary adenomatosis: a unique model of retrovirus-associated lung cancer.

Sheep pulmonary adenomatosis (SPA) is a contagious bronchiolo-alveolar carcinoma of sheep associated with an exogenous type D/B retrovirus known as jaagsiekte sheep retrovirus (JSRV). SPA represents a unique model for lung cancer, and studies on its aetiopathogenesis can provide further insight into the mechanisms of epithelial neoplasms.

Animals↗

PubMed1997

Induction of basal cell carcinoma features in transgenic human skin expressing Sonic Hedgehog.

Hedgehog (HH) signaling proteins mediate inductive events during animal development. Mutation of the only known HH receptor gene, Patched (PTC), has recently been implicated in inherited and sporadic forms of the most common human cancer, basal cell carcinoma (BCC). In Drosophila, HH acts by inactivating PTC function, raising the possibility that overexpression of Sonic Hedgehog (SHH) in human epidermis might have a tumorigenic effect equivalent to loss of PTC function. We used retroviral transduction of normal human keratinocytes to constitutively express SHH. SHH-expressing cells demonstrated increased expression of both the known HH target, BMP-2B, as well as bcl-2, a protein prominently expressed by keratinocytes in BCCs. These keratinocytes were then used to regenerate human skin transgenic for long terminal repeat-driven SHH (LTR-SHH) on immune-deficient mice. LTR-SHH human skin consistently displays the abnormal specific histologic features seen in BCCs, including downgrowth of epithelial buds into the dermis, basal cell palisading and separation of epidermis from the underlying dermis. In addition, LTR-SHH skin displays the gene expression abnormalities previously described for human BCCs, including decreased BP180/BPAG2 and laminin 5 adhesion proteins and expression of basal epidermal keratins. These data indicate that expression of SHH in human skin recapitulates features of human BCC in vivo, suggest that activation of this conserved signaling pathway contributes to the development of epithelial neoplasia and describe a new transgenic human tissue model of neoplasia.

Animals↗

PubMed1997

A 7-day activity diary for assessment of daily energy expenditure validated by the doubly labelled water method in adolescents.

OBJECTIVES: To validate the use of an activity diary and predicted BMR for assessment of daily total energy expenditure (TEE) and physical activity level (PAL = TEE/BMR) in adolescents. DESIGN: TEE and PAL estimated from activity diary records kept for seven days and BMR predicted from age, gender and body weight were compared with the results of doubly labelled water (DLW) measurements and indirect calorimetry performed during the same time period. SETTING: The Unit of paediatric Physiology of the Department of Clinical Physiology, University Hospital, Uppsala, Sweden. SUBJECTS: Fifty randomly selected 15 y old adolescents (25 boys and 25 girls). RESULTS: The mean difference between TEE estimated in all adolescents by the activity diary and by DLW methods was 1.2%. The limits of agreement (mean difference 2 s.d.) were -3.47 and 3.77 MD/d, equivalent to a coefficient of variation of 15%. The mean difference between PAL assessed by activity diary records and by DLW measurements was 0.001, and the limits of agreement between the two methods were 0.54. CONCLUSIONS: The results imply that the activity diary method provides a close estimate of TEE and PAL in population groups.

Activities of Daily Living↗

PubMed1997

The increased level of PDGF-A contributes to the increased proliferation induced by mechanical stimulation in osteoblastic cells.

Mechanical stimulation can prompt healing of bone fractures. However, it is largely unknown how osteogenesis is promoted by mechanical stimulation. In this study, we found that mechanical strain-induced proliferation of osteoblastic cells (MC3T3-E1) accompanied increased levels of platelet-derived growth factor-A (PDGF-A) mRNA, determined by quantitative reverse transcription/polymerase chain reaction. In addition, neomycin and W-7, which blocked mechanical strain-induced proliferation of the osteoblast cells, also blocked mechanical stimulation-induced elevation of PDGF-A mRNA. Finally, an antibody against PDGF can inhibit physical stimulation-induced proliferation of MC3T3-E1 cells, suggesting that the increased MC3T3-E1 cells produced by mechanical stimulation at least partially depends on the increased activity of PDGF.

Calcium↗

PubMed1997

Low-frequency loss of heterozygosity in Moloney murine leukemia virus-induced tumors in BRAKF1/J mice.

To identify potential involvement of tumor suppressor gene inactivation during leukemogenesis by Moloney murine leukemia virus (M-MuLV), a genome-wide scan for loss of heterozygosity (LOH) in tumor DNAs was made. To assess LOH, it is best to study mice that are heterozygous at many loci across the genome. Accordingly, we generated a collection of 52 M-MULV-induced tumor DNAs from C57BR/cdJ x AKR/J F1 (BRAKF1) hybrid mice. By using direct hybridization with oligonucleotides specific for three different classes of endogenous MuLV-related proviruses, 48 markers on 16 of 19 autosomes were simultaneously examined for allelic loss. No allelic losses were detected, with the exception of a common loss of markers on chromosome 4 in two tumors. The three autosomes that lacked informative endogenous proviral markers were also analyzed for LOH by PCR with simple-sequence length polymorphisms (SSLPs); one additional tumor showed LOH on chromosome 15. Further screening with chromosome 4 SSLPs identified one additional tumor with LOH on chromosome 4. Therefore, in total, the average fractional allelic loss was quite low (0.002), but the LOH frequency of 6% on chromosome 4 was highly statistically significant (P < 0.0005). Detailed SSLP mapping of the three tumors with LOH on chromosome 4 localized the region of common LOH to the distal 45 centimorgans, a region syntenic with human chromosomes 1 and 9. Candidate tumor suppressor genes, Mts1 (p16INK4a) and Mts2 (p15INK4b), have been mapped to this region, but by Southern blot analysis, no homozygous deletions were detected in either gene. One of three tumors with LOH on chromosome 4 also showed a proviral insertion near the c-myc proto-oncogene. These results suggested that tumor suppressor inactivation is generally infrequent in M-MuLV-induced tumors but that a subset of these tumors may have lost a tumor suppressor gene on chromosome 4.

Animals↗

PubMed1997

A carboxy-terminal basic region controls RNA polymerase III transcription factor activity of human La protein.

Human La protein has been shown to serve as a transcription factor for RNA polymerase III (pol III) by facilitating transcription termination and recycling of transcription complexes. In addition, La binds to the 3' oligo(U) ends common to all nascent pol III transcripts, and in the case of B1-Alu RNA, protects it from 3'-end processing (R. J. Maraia, D. J. Kenan, and J. D. Keene, Mol. Cell. Biol. 14:2147-2158, 1994). Others have previously dissected the La protein into an N-terminal domain that binds RNA and a C-terminal domain that does not. Here, deletion and substitution mutants of La were examined for general RNA binding, RNA 3'-end protection, and transcription factor activity. Although some La mutants altered in a C-terminal basic region bind RNA in mobility shift assays, they are defective in RNA 3'-end protection and do not support transcription, while one C-terminal substitution mutant is defective only in transcription. Moreover, a C-terminal fragment lacking RNA binding activity appears able to support low levels of transcription by pol III. While efficient multiround transcription is supported only by mutants that bind RNA and contain a C-terminal basic region. These analyses indicate that RNA binding contributes to but is not sufficient for La transcription factor activity and that the C-terminal domain plays a role in transcription that is distinguishable from simple RNA binding. The transcription factor activity of La can be reversibly inhibited by RNA, suggesting the potential for feedback inhibition of pol III transcription.

Animals↗

PubMed1997

[Determination of trace silver in surface water samples by flame atomic absorption with continuous flow on-line absorption preconcentration].

In a continuous flow on-line preconcentration system, trace silver was adsorbed onto activated carbon particles packed in micro colum at pH 1, eluted with 0.3 ml of 10 g/L Na2S2O3 solution and determined by flame atomic absorption spectrometry. For surface water samples, 25 ml sufficed precise determination of silver at level of microgram/L. The relative standard deviation of 10 parallel sample determinations was 6.9%. The proposed method was successfully applied to water samples with recoveries ranging from 93.2% to 104.6% and a throughout of 7-8 sampling/h.

Silver↗

PubMed1997

[Application of serum bile acid chromatography to the diagnoses of liver diseases].

In order to explore the specificity of serum bile acid (SBA) chromatograph in the diagnoses of different kinds of hepatosis, we investigated by means of gas chromatography the changes of serum bile acids in workers who exposed to hexogen or chloroethylene and in patients who suffered from hepatosis such as acute jaundice hepatitis, chronic active hepatitis, cirrhosis and liver cancer. The results revealed different disturbances of SBA occurring in the liver injuries induced by the two kinds of hepatotoxicant. Serum lithocholic acid (LCA), deoxycholic acid (DCA) and chenodeoxycholic acid (CDCA) in workers exposed to hexogen and wre significantly different from those of the control group respectively (P < 0.01, P < 0.01, P < 0.05); on the other hand, only serum LCA and DCA went up in workers exposed to chloroethylene (P < 0.0005, P < 0.001). The main changes both concentrated on the secondary bile acids. In acute jaundice hepatitis, chronic active hepatitis, cirrhosis and liver cancer, serum LCA, DCA, CDCA and cholic acids (CA) all went up in different degrees compared with the control group respectively (P < 0.005, P < 0.025, P < 0.005, P < 0.005). But no difference was noted among the 4 kinds of bile acids (P > 0.5), except that between CA and CDCA. These provided the evidence of the diagnosis and identification of clinical hepato-biliary diseases and occupational liver injures.

Cholic Acids↗

PubMed1997

Detection of human herpesvirus 6(HHV-6) DNA in salivary glands by the polymerase chain reaction.

To assess the presence of HHV-6-Specific DNA in human salivary glands, eighteen specimens of salivary gland tissue were investigated using the polymerase chain reaction. Eight of nine parotid glands, five of seven submandibular glands and one of two sublingual glands were found to have amplification of the HHV-6-specific sequence. The findings suggest that salivary gland tissue is one of the potential sites for HHV-6 persistence following primary infection and that saliva is a vehicle for transmission of the virus.

DNA, Viral↗