Biomedical subjects

Qian Huang

Publications and source records attributed to Qian Huang.

52 records · Page 3Linked to original sources

PubMed2003

Characterization of RP1L1, a highly polymorphic paralog of the retinitis pigmentosa 1 (RP1) gene.

PURPOSE: To determine the full-length sequence of a gene with similarity to RP1 and to screen for mutations in this newly characterized gene, named retinitis pigmentosa 1-like 1(RP1L1). Since mutations in the RP1 gene cause autosomal dominant retinitis pigmentosa, it is possible that mutations in RP1's most sequence similar relative, RP1L1, may also be a cause of inherited retinal degeneration. METHODS: A combination of cDNA clone sequencing, RACE, and database analysis were used to determine the RP1L1 mRNA sequence and its genomic organization. PCR analysis, semi-quantitative RT PCR, and in situ hybridization were used to determine the expression pattern of RP1L1. Single-strand conformational analysis and automated sequencing were used to screen probands from 60 adRP families for potential disease-causing mutations in RP1L1. RESULTS: The human RP1L1 gene is encoded in 4 exons, which span 50 kb on chromosome 8p. The length of the RP1L1 mRNA is large, over 7 kb, but its exact length is variable between individuals due to the presence of several length polymorphisms, including a 48 bp repeat. RP1L1 encodes a protein with a minimal length of 2,400 amino acids and a predicted weight of 252 kDa. Expression of RP1L1 is limited to the retina and appears to be specific to photoreceptors. Mutational analysis of 60 autosomal dominant retinitis pigmentosa probands revealed the presence of 38 sequence substitutions in RP1L1. Over half of these substitutions result in alteration of the RP1L1 protein, but none of these substitutions appear to be pathogenic. CONCLUSIONS: The RP1L1 gene encodes a large, highly polymorphic, retinal-specific protein. No RP1L1 disease-causing mutations were identified in any of the samples tested, making it unlikely that mutations in RP1L1 are a frequent cause of autosomal dominant retinitis pigmentosa. Additional experiments will be needed to determine if mutations in RP1L1 cause other forms of inherited retinal degeneration.

Amino Acid Sequence↗

PubMed2003

Presenilin-1 and presenilin-2 exhibit distinct yet overlapping gamma-secretase activities.

Presenilin-1 (PS1) and presenilin 2 (PS2) are proposed to be transmembrane aspartyl proteases that cleave amyloid precursor protein and Notch. PS1- and PS2-mediated activities were individually characterized using blastocyst-derived (BD) cells and membranes from PS1+/--PS2-/- and PS1-/-PS2+/+ mice, respectively. The relative amounts of PS1 and PS2 in the various BD cells were determined from the intensities of the anti-PS1 and anti-PS2 immunoblot signals by comparison with standard curves using radiolabeled PS1 and PS2 standards produced by in vitro transcription and translation. Cellular membranes from wild type, PS1-/-PS2+/+, and PS1+/--PS2-/- but not PS1-/-PS2-/- BD cells generated the Abeta40 and Abeta42 products from the C100FLAG substrate. PS1-associated gamma-secretase displays considerably higher specific activity than PS2-associated gamma-secretase. Moreover, the PS1+/-PS2-/- BD cells and corresponding membranes exhibited much higher gamma-secretase activity as compared with other BD cells and membranes. The PS1-mediated gamma-secretase activity correlated better with the amount of PS1 that is modifiable by a photoactivated active site-directed gamma-secretase inhibitor rather than total PS1; hence, only a small portion (<14%) of the PS1 in wild-type membranes appears to be engaged in an active gamma-secretase complex. This finding suggests that PS1 may serve other biological functions in addition to that associated with its gamma-secretase activity. Furthermore, the PS1 gamma-secretase complex and the PS2 gamma-secretase complex activities can be discriminated on the basis of their susceptibility to inhibition by a potent gamma-secretase inhibitor. The distinct yet overlapping enzymatic properties of the PS1 gamma-secretase complex and the PS2 gamma-secretase complex imply that these two putative aspartyl class proteases may contribute to different biological processes.

Affinity Labels↗

PubMed2003

Beta-secretase cleavage at amino acid residue 34 in the amyloid beta peptide is dependent upon gamma-secretase activity.

The amyloid beta peptides (Abeta) are the major components of the senile plaques characteristic of Alzheimer's disease. Abeta peptides are generated from the cleavage of amyloid precursor protein (APP) by beta- and gamma-secretases. Beta-secretase (BACE), a type-I transmembrane aspartyl protease, cleaves APP first to generate a 99-amino acid membrane-associated fragment (CT99) containing the N terminus of Abeta peptides. Gamma-secretase, a multi-protein complex, then cleaves within the transmembrane region of CT99 to generate the C termini of Abeta peptides. The production of Abeta peptides is, therefore, dependent on the activities of both BACE and gamma-secretase. The cleavage of APP by BACE is believed to be a prerequisite for gamma-secretase-mediated processing. In the present study, we provide evidence both in vitro and in cells that BACE-mediated cleavage between amino acid residues 34 and 35 (Abeta-34 site) in the Abeta region is dependent on gamma-secretase activity. In vitro, the Abeta-34 site is processed specifically by BACE1 and BACE2, but not by cathepsin D, a closely related aspartyl protease. Moreover, the cleavage of the Abeta-34 site by BACE1 or BACE2 occurred only when Abeta 1- 40 peptide, a gamma-secretase cleavage product, was used as substrate, not the non-cleaved CT99. In cells, overexpression of BACE1 or BACE2 dramatically increased the production of the Abeta 1-34 species. More importantly, the cellular production of Abeta 1-34 species induced by overexpression of BACE1 or BACE2 was blocked by a number of known gamma-secretase inhibitors in a concentration-dependent manner. These gamma-secretase inhibitors had no effect on enzymatic activity of BACE1 or BACE2 in vitro. Our data thus suggest that gamma-secretase cleavage of CT99 is a prerequisite for BACE-mediated processing at Abeta-34 site. Therefore, BACE and gamma-secretase activity can be mutually dependent.

Amino Acid Sequence↗

PubMed2003

HIV-1 Tat reprograms immature dendritic cells to express chemoattractants for activated T cells and macrophages.

Immature dendritic cells are among the first cells infected by retroviruses after mucosal exposure. We explored the effects of human immunodeficiency virus-1 (HIV-1) and its Tat transactivator on these primary antigen-presenting cells using DNA microarray analysis and functional assays. We found that HIV-1 infection or Tat expression induces interferon (IFN)-responsive gene expression in immature human dendritic cells without inducing maturation. Among the induced gene products are chemokines that recruit activated T cells and macrophages, the ultimate target cells for the virus. Dendritic cells in the lymph nodes of macaques infected with simian immunodeficiency virus (SIV) have elevated levels of monocyte chemoattractant protein 2 (MCP-2), demonstrating that chemokine induction also occurs during retroviral infection in vivo. These results show that HIV-1 Tat reprograms host dendritic cell gene expression to facilitate expansion of HIV-1 infection.

Animals↗

PubMed2003

Increased resistance of tumor cells to hyperthermia mediated by integrin-linked kinase.

PURPOSE: Integrin-linked kinase (ILK) is a serine-threonine kinase associated with anchorage-independent growth and tumorigenic transformation. Previous studies indicate that overexpression of ILK is common among several types of tumors, and it is involved in the regulation of tumor cell survival under stress. In this study, we examined the effects of ILK expression on tumor cellular response to hyperthermia. EXPERIMENTAL DESIGN: We used an adenovirus-mediated approach to overexpress the ILK gene in a prostate cancer cell line and examine its effects on heat stress-induced cell death. Clonogenic survival, as well as apoptosis, was evaluated in cells that overexpress ILK. In addition, the ability to form tumors in vivo was examined in syngeneic hosts. Finally, potential molecular mechanisms of ILK-mediated resistance to heat were examined by determining the status of a variety of signal transduction pathways. RESULTS: ILK overexpression made tumor cells significantly more resistant to the cell-killing effects of hyperthermia. This was correlated at the molecular level with the down-regulation of hyperthermia-induced activation of stress-activated protein kinase/c-Jun-NH(2)-terminal kinase, p38 mitogen-activated protein kinase activities, and caspase 9. The overexpression of ILK was also shown to induce a more rapid tumor growth in a murine prostate cancer cell line CONCLUSION: ILK plays an important role in tumor growth and tumor response to hyperthermia treatment.

Adenoviridae↗

PubMed2003

[Detection of point mutation in p53 gene by capillary electrophoresis-PCR-SSCP analysis].

OBJECTIVE: To screen the point mutation of p53 gene rapidly by capillary electrophoresis (CE). METHODS: A simple capillary electrophoresis system for the strand conformational polymorphism (SSCP) analysis of genomic DNA was developed by choosing commercially available capillary and gel buffer. This CE-SSCP system was applied to the analysis of twenty PCR products amplified from the exon 7 of the p53 gene of tissue specimens from patients with colon cancer. RESULTS: Five of them (Ca4, Ca6, Ca7, Ca8, Ca14) were found to have mutation within 25 minutes, while the results of the SSCP analysis by PAGE-silver staining techniques only detected mutation of four samples. And sequencing of these PCR products proved that the results of CE-SSCP were correct. CONCLUSION: This simple, accurate and less expensive method could be used in the field of medical research and in the clinical DNA diagnosis of human cancers and other diseases.

Base Sequence↗

PubMed2003

[Acute fulminant invasive fungal sinusitis].

OBJECTIVE: To discuss the clinical presentation, diagnostic criterion and treatment principle of acute fulminant invasive fungal rhinosinusitis (AFFS). METHODS: Six patients were diagnosed as AFFS based on history, nose, especially eye symptoms with or without fever, sinus CT and MRI, endoscopic and cytological findings in the nasal cavity. Surgical debridement was performed on 5 of 6 biopsy proven AFFS patients, one of them being amputated of the orbital content. With the original disease controlled simultaneously, 4 of 5 patients were prescribed with systemic amphotericin B or liposomal amphotericin B (1 case) and one with Itraconazole orally. One patient had not been involved with any of the anti-fungi measures. RESULT: Mucor (Zygomycetes) was identified on culture in 1 patient, Rhizopus species in 2, Aspergillus in 1, Alternaria in 1 and mixed Mucor and Rhizopus in 1. All patients were proved of tissue invasion histopathologically through biopsy. One patient died without any anti-fungi therapy on the 7th admission day, 3 patients survived for 88 days, 32 and 6 months respectively and died of original diseases (diabetes 1, leukaemia 2). One patient survived 9 months and lost for follow-up, 1 patient survived 11 months after treatment. CONCLUSION: A high index of suspicion and early endoscopic investigation through nasal cavity with fungal investigation should highly be strengthened for recognition of this disease. MRI findings should be considered as, or even more, important as that of CT scan on the early diagnoses. Extensive and aggressive surgical debridement, prompt and enough dosage of antifungal therapy intravenously, together with serious controlling of the underlying disease, all take important roles in the complete control of the disease.

Adolescent↗

PubMed2003

[Long-term outcomes and analysis of its relative factors of nasal endoscopic sinus surgery in children].

OBJECTIVE: To evaluate the results of endoscopic sinus surgery (ESS) in children with chronic sinusitis. The long-term outcomes and the relative factors were analyzed retrospectively. MATERIALS AND METHODS: A total of 268 children (432 sides), 186 males and 82 females, aged 3 to 17 years (average, 14.3) were included in this study (underwent ESS). The postoperative follow-up time of 188 cases (305 sides, 70.1%) was over one year. The management during follow-up was adopted under nasal endoscope and local anesthesia. The postoperative treatment included cleaning operative cavity, nasal irrigation, antibiotics, steroid, immune regulator, etc. RESULTS: Overall cases included 89 cases (147 sides) of chronic sinusitis, 86 cases (142 sides) of nasal polyps and 13 cases (16 sides) of mucocele respectively. The cure rate was 70.2% (132 cases). 43 cases (22.9%) were improved, while other 13 cases were of no effect. Overall symptoms, 93.1% children with headache were improved, 85.1% children were free of nasal obstruction and 60.1% children gained a relief of purulent discharge. 11 cases had no change and 2 cases were worse compared with before surgery. 34 cases with anosmia were improved or recovered after ESS. The total effective rate of ESS was 93.1%. 8 cases received a revision ESS. CONCLUSIONS: ESS in children with chronic sinusitis and polyps has a good long-term outcomes. The reasonable reservation of mucosa during ESS must be emphasized. The quality of follow-up is an important factor affecting the results of ESS. Whether the disciplinary preoperative management was made or not is the hypothesis of ESS indications.

Adolescent↗

PubMed2003

[Effect of neurotrophic factors and growth factors on adult human retinal cells in vitro].

OBJECTIVE: To examine whether adult human retinal cells can be maintained in vitro over long time periods and to study the effects of brain-derived neurotrophic factor (BDNF),neurotrophin-4 (NT-4), epidermal growth factor (EGF), fibroblast growth factor (FGF), and retinoic acid (RA) on growth, proliferation, and apoptosis of the cultured adult human retinal cells. METHODS: Adult retinal cells were isolated from donor eyes and cultured. BDNF, NT-4, EGF, FGF, and RA were added individually to the cultures. The cells were identified by morphologic criteria, growth patterns, and immunocytochemical staining. The number of neurons in each group was compared. Expression levels of c-fos, c-jun, Bcl-2, and Bax were examined. RESULTS: Adult human retinal cells survived for 240 days in vitro. The addition of BDNF and FGF promoted survival of adult retinal neurons, increased the number of cells positive for neuron specific enzyme (NSE), Thy1.1, c-fos, c-jun, and Bcl-2. More c-fos and c-jun positive cells were observed in cell cultures containing medium with RA. However, NT-4 and EGF treatment groups showed no significant difference from control groups in that neuron cell survival was low and fewer cells were positive for NSE, Thy1.1, c-fos, c-jun, and Bcl-2. CONCLUSIONS: BDNF, FGF and RA improve survival of adult human retinal neurons in vitro via a mechanism that may involve c-fos, c-jun, and Bcl-2 expression, However, EGF and NT-4 do not improve survival of the adult human retinal neurons.

Adult↗

PubMed2003

[Surgery of ossifying fibroma of the sinuses].

OBJECTIVE: To explore the different surgical choices for treating the ossifying fibroma of the sinuses. To summarize the management and characteristics of each surgical operation. METHODS: A retrospective evaluation of thirty-five patients with ossifying fibroma of the sinuses from August 1994 to July 2001 was presented. RESULTS: Among 22 patients operated by nasal endoscopic management, complete ossifying fibroma removed was achieved in 8 cases, and the majority part of tumor removed in 14 cases. Six patients were operated through a lateral rhinotomy with radical operation in 4 cases. Five ossifying fibromas were removed with a coronal incision. Two cases underwent Caldwell-Luc' surgery. The clinical symptoms, location of ossifying fibroma, and surgical procedures were analyzed. All patients outcomes were successful, no serious complication from the surgical technique occurred. Thirty-three cases were followed-up for 1 to 8 years with an average of three and half years. Fourteen patients had no recurrence, fourteen cases lived with the remains of ossifying fibroma, and five cases recurred. CONCLUSIONS: The choice of surgical operations on ossifying fibroma of the sinuses was mainly decided by the location of ossifying fibroma, in the meanwhile, the organ function, the cosmetology, the surgical degree of difficulty, and the doctor's experience were taken into account.

Adolescent↗

PubMed2002

A 3'-terminal minihelix in the precursor of human spliceosomal U2 small nuclear RNA.

U2 RNA is one of five small nuclear RNAs that participate in the majority of mRNA splicing. In addition to its role in mRNA splicing, the biosynthesis of U2 RNA and three of the other spliceosomal RNAs is itself an intriguing process involving nuclear export followed by 5'-cap hypermethylation, assembly with specific proteins, 3' end processing, and then nuclear import. Previous work has identified sequences near the 3' end of pre-U2 RNA that are required for accurate and efficient processing. In this study, we have investigated the structural basis of U2 RNA 3' end processing by chemical and enzymatic probing methods. Our results demonstrate that the 3' end of pre-U2 RNA is a minihelix with an estimated stabilization free energy of -6.9 kcal/mol. Parallel RNA structure mapping experiments with mutant pre-U2 RNAs revealed that the presence of this 3' minihelix is itself not required for in vitro 3'-processing of pre-U2 RNA, in support of earlier studies implicating internal regions of pre-U2 RNA. Other considerations raise the possibility that this distinctive structural motif at the 3' end of pre-U2 RNA plays a role in the cleavage of the precursor from its longer primary transcript or in its nucleocytoplasmic traffic.

Base Sequence↗

PubMed2002

[A novel model for visualization of tumor cell-induced angiogenesis in vivo].

The bladder transitional cell carcinoma cell line, BTT739 from the T739 mouse, was transfected with a plasmid that encoded an enhanced green fluorescence protein (GFP) and the cells stably expressing GFP were selected and subcloned. 1 x 10(3)-1 x 10(4) GFP-labeled BTT739 cells were injected under the skin of ear of T739 mice. On day 2-5 post injection, the most interesting manifestations observed were the chemotaxis-like movement of the tumor cells toward the pre-existing host vasculature, host vessel dilation and tortuosity and increased extravasation. On day 10 or later, the sprout from pre-existing host vasculature was observed. Once angiogenesis was triggered on, the tumor cells grew more rapidly and exhibited a specific growth pattern where tumor cells always associated with or surrounded the vessels. The newly formed microvessels always showed heavy extravasation. Immunohistochemistry staining revealed strong VEGF and VEGFR2 (Flk-1) expression in tumor cells. Angiography using Rhodamin-labeled dextran showed neovascularization with unprecedented clarity. However, the tumor mass, even bigger than 2 mm and being neovascularized, shrunk and then disappear in 3-5 days and left only delicated host vessels and recovered extravasation. The evidence from this observation indicated that angiogenesis induced by tumor cells after implantation into the host begins at very early stage. The micrometastases foci could not form or survive without vigorous and continuous angiogenesis. Furthermore, there was active VEGF paracrine and autocrine expression in tumor and high level VEGF secretion by tumor cells plays an important role in initiating angiogenesis and supporting micrometastases.

Angiography↗

PubMed2002

[Inhibitation of tumor angiogenesis, growth and metastasis by blocking VEGF paracrine pathway].

Solid tumors require an adequate vascular supply to grow beyond a certain dimension. It is known that formation of new blood vessels in tumor is mediated by unbalanced expression of angiogenic factors and their inhibitors. Among the former, the vascular endothelial growth factor (VEGF) has been assumed prime candidacy as a major positive physiological effector. To investigate the role of VEGF in angiogenesis associated with development of breast cancer, a sense VEGF and an anti-sense VEGF expression plasmids were constructed, and then were introduced into a human breast carcinoma cell line, MCF-7, expressing middle level of endogenous VEGF. Anti-sense VEGF(121) transfected MCF-7 cells that expressed reduced constitutive levels of VEGF and showed the same growing potential as untransfected MCF-7 cells in vitro, but it showed longer latency, smaller tumor, slower growth and prolonged survival time compared to parental or sense VEGF(165) transfected MCF-7 cells in vivo. Moreover, the tumors derived from anti-sense VEGF(121) transfected MCF-7 cells characterized by minimal vascularization and extensive necrosis. Finally, mice with primary subcutaneous tumors treated with intratumoral administration of anti-sense VEGF, or the plasmid expressing extracellular domain of the Flk-1 VEGF receptor (sFlk-1) followed by electroporation, showed significant tumor suppression. These results suggest that VEGF plays a major angiogenic role in breast cancer and a strategy, which blocks the VEGF paracrine pathway, may provide a means to control tumor growth topically without the risk of systemic antiangiogenesis.

3T3 Cells↗

PubMed2002

[Long-term outcome of gfp gene modified human RPE xenografts into the subretinal space of rabbits].

The possibility of delivery genes to retina by using retinal pigment epithelium (RPE) transplantation was investigated. Cultured human adult RPE cells were transfected with retrovirus encoding green fluorescent protein (GFP), then GFP-expressing RPE cells were transplanted into the subretinal space of rabbits through pars plana vitrectomy under microscopy. The transplant sites were examined in vivo by ophthalmoscope for fluorescence. The animals were euthanatized at 1, 2, 3, 4, 6, 10, 11, 14, 18, 20, 23, 24, 25, 33, 54 weeks, respectively, and the retinas were studied histologically, including epi-fluorescent microscopy, confocal microscopy and transmission electron microscopy. The GFP-expressing transplant could be followed in the living retina up to 21 days by ophthalmoscope. The epi-fluorescent microscopy examination disclosed survival of the transplanted cells in 1 year with continual and significant GFP expression. It was observed the transplanted hRPE-gfp cells were not only present at transplant sites but spread over two to three quarters of retina. Generally, RPE xenografts integrated individually into host RPE in a regular cobblestones arrangement or formed a monolayer sheet between host RPE and neural retina. It was observed that intravitreal injection of FK-506 weekly improved the survival of RPE xenografts and reduced the infiltration of mononuclear microphages and lymphocytes in choroids from 1 through 14 weeks after transplantation. The results demonstrate a long-term survival of gene modified human RPE xenografts into the subretinal space of rabbits and support the feasibility of this approach for delivery genes to retina.

Adult↗

PubMed2002

[Liposome-mediated gene transfer into retina].

OBJECTIVE: To evaluate the efficiency of liposome mediated gene transfer to retina, the time of gene expression in retinal cells, retinal damage caused by liposome and the possibility of using liposome mediated gene transfer to retina for treating fundus diseases. METHODS: The ratio of cationic liposome to green fluorescein protein (GFP) expression plasmid for high efficacy of transfection was tested in vitro. Then optimal liposome-plasmid mixture was injected into the subretinal space of Sprague-Dawley (SD) or Royal college of surgeon (RCS) rats. The GFP expression was observed by using fluorescence microscopy, and morphological changes were examined by using paraffin embedded sections, HE staining and transmission electron microscopy. RESULTS: The GFP expression in retinal cells was observed lasting more than 4 weeks. However, upon 4 weeks of transfection the swelling and degeneration in outer segment of photoreceptors could be observed. The aggravated degeneration with disappearance of outer segments, only one layer of photoreceptor nucleus surviving, apoptotic inner granular layer cells and neovascularization were observed 8 weeks after transfection. But no inflammatory cells were observed. Three of 5 RCS rats that received plasmid pCNTF (ciliary neurotrophic factor) delivery at 4 weeks old showed more surviving nuclei of photoreceptors after 3 to 5 weeks of transfection. CONCLUSION: Liposome is able to deliver genes to retinal cells with efficacy, and the expression of therapeutic gene such as CNTF in retinal cells can rescue photoreceptors from degeneration. But liposome used here is to some extent harmful to photoreceptors.

Animals↗

PubMedDate not supplied

gamma-Secretase: characterization and implication for Alzheimer disease therapy.

gamma-Secretase is a membrane-bound protease that cleaves within the transmembrane region of amyloid precursor protein to generate the C-termini of the Abeta peptides which are believed to play a central role in the neuropathology of Alzheimer's disease. An in vitro gamma-secretase assay using a recombinant substrate C100Flag has been developed to facilitate the characterization and identification of this enigmatic protease. Biochemical studies establish that gamma-secretase activity is catalyzed by a PS1-containing macromolecular complex. Moreover, the fact that the photoreactive active gamma-secretase inhibitor directed to the active site labels PS1 suggests that PS1 contains the active site of the protease. Presenilin/gamma-secretase as a potential target for AD therapy and its role in regulated intramembrane proteolysis are discussed.

Alzheimer Disease↗