Biomedical subjects

Hua Pan

Publications and source records attributed to Hua Pan.

At least 19 recordsLinked to original sources

PubMed2007

Comparison of global gene expression between porcine testis tissue xenografts and porcine testis in situ.

Testis tissue from immature mammalian donor animals, grafted ectopically to immunodeficient mouse hosts, can undergo complete spermatogenesis with the production of fertilization-competent spermatozoa. To further characterize testis tissue xenografts as a model for testis function in situ, the objective of this study was to compare gene expression between porcine testis tissue xenografts and testis tissue in situ. Pieces of testis tissue from 1-week-old piglets were grafted onto immunodeficient male mice and a littermate piglet was raised for comparison as control. Complete spermatogenesis was present in the testis tissue xenografts at 8 months after transplantation into mouse hosts and in the 8-month-old control porcine testis tissue. Total RNA was isolated from xenografts and control tissue, and the RNA was labeled and hybridized to the porcine genome array. By analyzing the expression of 23,256 transcripts, we found that 71 genes were differentially expressed with at least a fourfold difference between xenografts and control tissue. Interestingly, none of the 56 transcripts present on the array that were annotated in porcine testis showed differential expression between xenografts and control testis. This analysis indicates that global gene expression in porcine testis xenografts appears comparable to testis tissue in situ. These findings support the hypothesis that testis tissue xenografts can provide a representative model to study mammalian spermatogenesis.

Animals↗

PubMed2006

Ca2+ oscillatory pattern in fertilized mouse eggs affects gene expression and development to term.

The Ca2+ oscillations initiated by the fertilizing sperm (but terminating concomitant with pronucleus formation) apparently ensure that the events constituting egg activation occur in the correct temporal order; early events (e.g., cortical granule exocytosis) require fewer oscillations than later events (e.g., recruitment of maternal mRNA). Whether the Ca2+ signaling events impact long-term development, in particular development to term, is unknown. Using fertilized eggs that have undergone the first few Ca2+ oscillations, we developed procedures that result either in inhibiting or stimulating the natural pattern of Ca2+ signaling of inseminated eggs. Although the incidence of development to the blastocyst stage is unaltered by these procedures, fewer offspring are born following embryo transfer, indicating that developmental competence of the blastocysts is reduced. Interestingly, embryo transfer experiments reveal that when the natural regime of Ca2+ oscillations is precociously interrupted, the incidence of implantation is compromised whereas hyper-stimulation of Ca2+ signaling events compromises post-implantation development. Moreover, although there was no major difference in the overall growth rates of the offspring, those obtained following hyper-stimulation exhibited a far greater variability in their weight. Analysis of global patterns of gene expression by microarray analysis revealed that approximately 20% of the transcripts are mis-regulated when too few oscillations are experienced by the embryo and EASE analysis indicates that genes preferentially involved in RNA processing and polymerase II transcription are differentially affected. In addition, a set of genes involved in cell adhesion is also mis-expressed and could thus be mechanistically linked to the observed reduced implantation. Only about 3% of the transcripts were mis-regulated following hyper-stimulation, and EASE analysis indicates that genes preferentially involved in metabolism are differentially affected. In toto, these results indicate that a range Ca2+ signaling events following fertilization (an excess or reduction) has long-term effects on both gene expression and development to term.

Animals↗

PubMed2006

Maternal BRG1 regulates zygotic genome activation in the mouse.

Zygotic genome activation (ZGA) is a nuclear reprogramming event that transforms the genome from transcriptional quiescence at fertilization to robust transcriptional activity shortly thereafter. The ensuing gene expression profile in the cleavage-stage embryo establishes totipotency and is required for further development. Although little is known about the molecular basis of ZGA, oocyte-derived mRNAs and proteins that alter chromatin structure are likely crucial. To test this hypothesis, we generated a maternal-effect mutation of Brg1, which encodes a catalytic subunit of SWI/SNF-related complexes, utilizing Cre-loxP gene targeting. In conditional-mutant females, BRG1-depleted oocytes completed meiosis and were fertilized. However, embryos conceived from BRG1-depleted eggs exhibited a ZGA phenotype including two-cell arrest and reduced transcription for approximately 30% of expressed genes. Genes involved in transcription, RNA processing, and cell cycle regulation were particularly affected. The early embryonic arrest is not a consequence of a defective oocyte because depleting maternal BRG1 after oocyte development is complete by RNA interference (RNAi) also resulted in two-cell arrest. To our knowledge, Brg1 is the first gene required for ZGA in mammals. Depletion of maternal BRG1 did not affect global levels of histone acetylation, whereas dimethyl-H3K4 levels were reduced. These data provide a framework for understanding the mechanism of ZGA.

Acetylation↗

PubMed2006

[A retrospective study of one case of human infection by the highly pathogenic avian influenza A (H5N1)].

OBJECTIVE: To describe the clinical features of the infection caused by the highly pathogenic avian influenza A (H(5)N(1)). METHODS: A previously healthy 24 year old woman presented to our hospital on November 7, 2005. She was confirmed to be an H(5)N(1) infected case after death. The clinical, radiological and epidemiological data were analyzed. RESULTS: The patient had a history of direct contact with diseased and dead poultry (chicken and duck). The disease course was 10 days from onset of illness to death, and fever preceded dyspnea by 5 days. On admission, the striking characteristics were acute community-acquired pneumonia (CAP) and acute respiratory distress syndrome (ARDS), and the major radiographic abnormalities included extensive infiltration bilaterally, focal consolidation and air bronchograms. The radiographic and clinical deterioration was rapid, and the patient died in less than 3 days after hospitalization. The diagnosis of influenza A (H(5)N(1)) was confirmed by means of reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time PCR on specimens of the lower respiratory tract, performed by Chinese Center for Disease Control. The postmortem examination showed bronchial hyperemia, extensive consolidation, serous cavity effusions, disseminated intravascular coagulation (DIC) and multiple organ failure (MOF). CONCLUSIONS: Human infection by the highly pathogenic avian influenza A (H(5)N(1)) is a fatal communicable disease. Information of avian influenza A (H(5)N(1)) virus, more attention to the epidemiologic data, and early intervention are critical in reducing the mortality.

Adult↗

PubMed2005

Transcript profiling during mouse oocyte development and the effect of gonadotropin priming and development in vitro.

The molecular basis for acquisition of meiotic and developmental competence, the two main outcomes of oocyte development and essential for producing an egg capable of being fertilized and supporting development to term, is largely unknown. Using microarrays, we characterized global changes in gene expression in oocytes derived from primordial, primary, secondary, small antral, and large antral follicles and used Expression Analysis Systematic Explorer (EASE) to identify biological and molecular processes that accompany these transitions and likely underpin acquisition of meiotic and developmental competence. The greatest degree of change in gene expression occurs during the primordial to primary follicle transition. Of particular interest is that specific chromosomes display significant changes in their overall transcriptional activity and that in some cases these changes are largely confined to specific regions on these chromosomes. We also examined the transcript profile of oocytes that developed in vitro, as well as following eCG priming. Remarkably, the expression profiles only differed by 4% and 2% from oocytes that developed in vivo when compared to oocytes that developed in vitro from either primordial or secondary follicles, respectively. About 1% of the genes were commonly mis-expressed, and EASE analysis revealed there is an over-representation of genes involved in transcription. Developmental competence of oocytes obtained from eCG-primed mice was substantially improved when compared to oocytes obtained from unprimed mice, and this correlated with decreased expression of genes implicated in basal transcription.

Animals↗

PubMed2005

Absence of non-specific effects of RNA interference triggered by long double-stranded RNA in mouse oocytes.

RNA interference (RNAi) is a conserved eukaryotic mechanism by which double-stranded RNA (dsRNA) triggers the sequence-specific degradation of homologous mRNAs. Recent concerns have arisen in mammalian systems about off-target effects of RNAi, as well as an interferon response. Most mammalian cells respond to long dsRNAs by inducing an antiviral response mediated by interferon that leads to general inhibition of protein synthesis and nonspecific degradation of mRNAs. Moreover, recent reports demonstrate that under certain conditions, short interfering RNAs (siRNAs, 21-25 bp) may activate the interferon system. Mouse oocytes and preimplantation embryos apparently lack this response, as potent and specific inhibition of gene expression triggered by long dsRNA is observed in these cells. In the present study, we analyzed the global pattern of gene expression by microarray analysis in transgenic mouse oocytes expressing long dsRNA and find no evidence of off-targeting. We also report that genes involved in the interferon response pathway are not expressed in mouse oocytes, even after exposure for an extended period of time to long dsRNA.

Animals↗

PubMed2005

Study of sonoporation dynamics affected by ultrasound duty cycle.

Sonoporation is the ultrasound-induced membrane porosity and has been investigated as a means for intracellular drug delivery and nonviral gene transfection. The dynamic characteristics of sonoporation, such as formation, duration and resealing of the pores in the cell membrane, determine the process of intracellular uptake of molecules or agents of interest that are otherwise obstructed by the cell membrane barrier. Sonoporation dynamics is also important for postultrasound cell survival. In this study, we investigated the effects of ultrasound duty cycle on sonoporation dynamics using Xenopus oocyte as a model system. Transducer with a center frequency of 0.96 MHz was used to generate pulsed ultrasound of desired duty cycle (5%, 10% and 15%) at a pulse repetition frequency of 1 Hz and an acoustic pressure of 0.4 MPa in our experiments. Employing voltage clamp techniques, we measured the transmembrane current as the direct result of decreased membrane resistance due to pore formation induced by ultrasound application. We characterized the sonoporation dynamics from these time-resolved recordings of transmembrane current to indicate cell membrane status, including pore formation, extension and resealing. We observed that the transmembrane current amplitude increased with increasing duty cycle, while the recovering process of membrane pores and cell survival rate decreased at higher duty cycles.

Animals↗

PubMed2005

Thirty-two cases of acne treated with blood-letting puncture, cupping and Chinese-drug facemask.

To observe the therapeutic effect on acne treated with blood-letting puncture, cupping and Chinese-drug facemask. Thirty-two patients with acne were treated with above-mentioned therapeutic method. Cure, improvement and ineffectiveness were respectively found in 24, 7 and 1 case, with a curative rate of 75.0% and a total effective rate of 96.8%. The combination of blood-letting puncture, cupping and external application of Chinese-drug facemask is effective in treating acne.

Acne Vulgaris↗

PubMed2004

Ultrasound-induced cell membrane porosity.

Recent studies of ultrasound (US) methods for targeted drug delivery and nonviral gene transfection revealed new, advantageous possibilities. These studies utilized US contrast agents, commonly stabilized microbubbles, to facilitate delivery and suggested that US delivery resulted from cell sonoporation, the formation of temporary pores in the cell membrane induced by US. Using voltage clamp techniques, we obtained real-time measurements of sonoporation of single Xenopus oocyte in the presence of Optison trade mark, an agent consisting of albumin-shelled C(3)F(8) gas bubbles (mean diameter 3.2 microm). Ultrasound increased the transmembrane current as a direct result of decreased membrane resistance due to pore formation. We observed a distinct delay of sonoporation following US activation and characteristic stepwise increases of transmembrane current throughout US duration. We discovered that the resealing of cell membrane following US exposure required Ca(2+) entering the cell through US-induced pores.

Albumins↗

PubMed2003

Ehrlichiae and ehrlichial diseases in china.

The various ticks collected from different areas of China were examined for the existence of ehrlichial agents by polymerase chain reaction (PCR) with genus- or species-specific primers designed on the basis of ehrlichial 16S rRNA genes and sequence analyses. In southern China, E. chaffeensis was detected in Amblyomma testudinarium ticks from infested cattle, Haemaphysalis yeni ticks from hare, and Ixodes ovatus ticks from Muntiacus reevesi. E. canis was identified in Rhipicephalus sanguineus ticks from dogs and Boophilus microplus ticks from goats. A new species of the genus Ehrlichia, closely related to E. chaffeensis, and Anaplasma marginale were found in B. microplus ticks from cattle in Tibet. In northern China, E. chaffeensis was detected in Dermacentor silvarum and I. persulcatus ticks; the granulocytic ehrlichial agents were detected in I. persulcatus ticks from an area where Lyme disease is endemic. Canine ehrlichiosis was found in southern China and E. canis and E. platys were identified in dogs; human ehrlichioses were demonstrated by amplifying the 16S rRNA genes of E. chaffeensis and granulocytic ehrlichial agents from patients' blood specimens. In comparison of 16S rRNA gene sequences, the sequences of E. chaffeensis, E. canis, and E. platys in China were found to be different from that in other countries at certain nucleotide positions. These results reveal that a variety of tick-borne ehrlichial agents and diseases exist in China, and the ehrlichial agents and their tick-vectors are same as or different from that in other countries at species or strain levels.

Animals↗

PubMed2003

Ehrlichia-like organism gene found in small mammals in the suburban district of Guangzhou of China.

The 16S rRNA gene of Ehrlichia genus was examined from the blood and spleen of 26 wild small mammals that were captured at a canine feed lot where the first case of canine ehrlichiosis was found in south of China in 1998. Samples (3 blood and 1 spleen) from Rattus norvegicus were positive by polymerase chain reaction. The amplimers were then cloned and sequenced. All of the 16S rRNA gene sequence of these samples were found to be same and unique when compared with the other members of genus EHRLICHIA: The most closely related species was Ehrlichia-like sp. Schotti variant (99.03% related), which was detected and identified in Dutch Ixodes ricinus ticks by Schouls in 1999. The organism found on these animals was obviously not the species of E. muris, which was isolated and identified from a wild mouse in Japan in 1995, since the relatedness of this gene between the former and the latter was 93.28%. It is unclear whether this organism can cause disease in other mammals, including human beings.

Animals↗

PubMed2002

Myf5 is a direct target of long-range Shh signaling and Gli regulation for muscle specification.

Sonic hedgehog (Shh) is a secreted signaling molecule for tissue patterning and stem cell specification in vertebrate embryos. Shh mediates both long-range and short-range signaling responses in embryonic tissues through the activation and repression of target genes by its Gli transcription factor effectors. Despite the well-established functions of Shh signaling in development and human disease, developmental target genes of Gli regulation are virtually unknown. In this study, we investigate the role of Shh signaling in the control of Myf5, a skeletal muscle regulatory gene for specification of muscle stem cells in vertebrate embryos. In previous genetic studies, we showed that Shh is required for Myf5 expression in the specification of dorsal somite, epaxial muscle progenitors. However, these studies did not distinguish whether Myf5 is a direct target of Gli regulation through long-range Shh signaling, or alternatively, whether Myf5 regulation is a secondary response to Shh signaling. To address this question, we have used transgenic analysis with lacZ reporter genes to characterize an Myf5 transcription enhancer that controls the activation of Myf5 expression in the somite epaxial muscle progenitors in mouse embryos. This Myf5 epaxial somite (ES) enhancer is Shh-dependent, as shown by its complete inactivity in somites of homozygous Shh mutant embryos, and by its reduced activity in heterozygous Shh mutant embryos. Furthermore, Shh and downstream Shh signal transducers specifically induce ES enhancer/luciferase reporters in Shh-responsive 3T3 cells. A Gli-binding site located within the ES enhancer is required for enhancer activation by Shh signaling in transfected 3T3 cells and in epaxial somite progenitors in transgenic embryos. These findings establish that Myf5 is a direct target of long-range Shh signaling through positive regulation by Gli transcription factors, providing evidence that Shh signaling has a direct inductive function in cell lineage specification.

3T3 Cells↗

PubMed2002

Sequence analysis of Orientia tsutsugamushi DNA from mites collected in the Xisa archipelago, China.

The genotype of Orientia tsutsugamushi DNA from mites in the Xisa archipelago of China were identified. A natural focus of tsutsugamushi disease in the archipelago was found. The DNA sequence that codes for the 56 kDa protein of O. tsutsugamushi was amplified by nested polymerase chain reaction (N-PCR). The purified positive products were cloned into a pGEM-T vector and sequenced. The DNA sequence was compared with various sequences on the internet for sequence homology. A 507 bp DNA fragment encoding the 56 kDa protein was amplified from the samples. The sequence homology was 85% (Karp strain), 68% (Gilliam strain), 65% (Kato strain), and 67% (Yonchon strain). Orientia tsutsugamushi is carried by the mites of the Xisa archipelago; the main genotype is the Karp strain.

Animals↗

PubMed2000

Cloning and Functional Expression of Neurotoxin cDNA from Naja naja atra.

Three new cDNAs named NL1, NL2 and NL3 were cloned from the total RNA of Naja naja atra by RT-PCR. The protein sequences encoded by them showed 77%, 72% and 98% structure identity to cobrotoxin which is a postsynaptic neurotoxin from Taiwan cobra (Naja naja atra), respectively. The five conservative residues, Tyr(25), Lys(27), Trp(29), Arg(33) and Lys(47), essential for the function of cobrotoxin were also found in the three cDNAs. The NL3, the most homologous to cobrotoxin, was expressed in E.coli BL21(DE3) by cloning into pET28b+. The expressed product was insoluble inclusion bodies and could be purified up to 90% purity in the range of 6 mg per liter culture cells by a single affinity step. The purified protein was refolded in vitro and the toxicity was assessed to be less than the native cobrotoxin in mice.

Journal Article↗

PubMed1999

Cloning and Sequencing of Genes Encoding Phospholipase A(2) from Agkistrodon acutus.

Synthetic oligonucleotides were used to amplify phospholipase A(2) (PLA(2)) gene by RT-PCR from total RNA of snake Agkistrodon acutus venom gland. The PCR products were subcloned and positive clones were screened with acidic PLA(2) gene from Agkistrodon halys Pallas. Finally, four cDNAs of PLA(2) isoenzymes were isolated. Their complete sequences were determined by bidirectional sequencing and their amino acid sequences were deduced. They were designated as A.aAPLA(2)I A.aAPLA(2)II A.aBPLA(2) and A.aLys(49)-PLA(2) according to their isoelectric points calculated by computer and special structure characteristics respectively. The amino acid sequence of 1 10 residues of A.aAPLA(2)I deduced from the cDNA is identical to that of acidic PLA(2) which had been isolated from Agkistrodon acutus. A.aLys(49)-PLA(2) is unique because of the usual Asp(49) is replaced by Lys(49), which may lower its enzymatic activity. Their similarity scores were calculated and compared by computer. The successful cloning of these isoenzymes genes may provide more information for the study on structure-function relationship of PLA(2) family.

Journal Article↗

PubMed1999

Cloning and Expression of cDNA for Thrombin-like Enzyme from Agkistrodon halys Pallas Snake Venom.

Total RNAs were extracted from the venom gland of the Agkistrodon halys Pallas snake. The thrombin-like enzyme gene was amplified by RT-PCR, cloned, and its nucleotide sequences have been determined. The enzyme called pallase cDNA encodes 708 nucleotides, namely 236 amino acids. Based on the homology, the catalytic residues and disulfide bridges of pallase were deduced as follows catalytic residues, His(41), Asp(86) and Ser(182) and disulfide bridges, Cys(7)-Cys(139), Cys(26)-Cys(42), Cys(74)-Cys(234), Cys(118)-Cys(188), Cys(150)-Cys(167) and Cys(178)-Cys(203). The pallase expression plasmids under the control of the T7 promoter was constructed and the pallase was expressed in E. coli.

Journal Article↗

PubMed1999

CYP6B2 Expression in Insecticide-Resistant and -Suspcitible Strains of Helicoverpa armigera.

A fragment of CYP6B2 gene was obtained from the midgut cDNA library of the fifth instar larvae of the ballworm (Helicoverpa armigera) with RT-PCR. The product of 627 bp was cloned and sequenced. The results of dot blotting analysis of mRNA proved that CYP6B2 is expressed at higher level (mRNA) in an insecticide-resistant strain than in a susceptible strain, showing that the regulation was at transcription level. The mRNA is induced by pyrethroid in susceptible, but not in the insecticide-resistant strains. We guess that mutation in the 5' upstream regulator of the insecticide-resistant strain may lead to the difference in the expression level between resistant and susceptible strains.

Journal Article↗

PubMed1998

Expression of the APLA(2) Gene from Agkistrodon halys Pallas.

The APLA(2) gene from Agkistrodon halys Pallas has been cloned into the expression plasmid pBLMVL2 and expressed in E. coli RR1. The molecular weight of the expressed product is approximately 14 kD as shown by SDS-PAGE, its expression level is about 30% of the total cellular proteins. The protein was produced as insoluble inclusion bodies. After partially purified by washing the inclusion bodies, the product was denatured and refolded into active form. Then, the expressed APLA(2) was purified by FPLC Superose (TM) 12 and was a single band as shown by SDS-PAGE. The purified expressed protein had specific activity as the native enzyme and cross-reacted with antisera prepared against the native enzyme. The successful expression of the APLA(2) gene from Agkistrodon halys Pallas provides a good basis for further structure-function studies.

Journal Article↗