Biomedical subjects

Zhong Huang

Publications and source records attributed to Zhong Huang.

18 recordsLinked to original sources

PubMed2006

SPARX, a new environment for Cryo-EM image processing.

SPARX (single particle analysis for resolution extension) is a new image processing environment with a particular emphasis on transmission electron microscopy (TEM) structure determination. It includes a graphical user interface that provides a complete graphical programming environment with a novel data/process-flow infrastructure, an extensive library of Python scripts that perform specific TEM-related computational tasks, and a core library of fundamental C++ image processing functions. In addition, SPARX relies on the EMAN2 library and cctbx, the open-source computational crystallography library from PHENIX. The design of the system is such that future inclusion of other image processing libraries is a straightforward task. The SPARX infrastructure intelligently handles retention of intermediate values, even those inside programming structures such as loops and function calls. SPARX and all dependencies are free for academic use and available with complete source.

Computational Biology↗

PubMed2006

PLP1 alternative splicing in differentiating oligodendrocytes: characterization of an exonic splicing enhancer.

Proteolipid protein (PLP) and DM20 are generated by alternative splicing of exon 3B of PLP1 transcript in differentiating oligodendrocytes. We investigated the role of exonic splicing enhancers (ESE) in the selection of PLP 5' donor site, focusing on putative ASF/SF2, and SC35 binding motifs in exon 3B on the basis of mutations that cause disease in humans. Mutations in a putative ASF/SF2 binding motif (nucleotides 406-412) reduced PLP 5' donor site selection, whereas a mutation in a putative SC35 binding motif (nucleotides 382-389) had no effect. UV crosslinking and immunoprecipitation (IP) assays using an antibody to ASF/SF2 showed that the ASF/SF2 protein specifically binds to the ESE (nucleotides 406-412). The single nucleotide mutations that reduced PLP splice site selection greatly diminished ASF/SF2 protein binding to this motif. We next tested the effect of overexpressed ASF/SF2 on PLP 5'splice selection in differentiating oligodendrocytes. ASF/SF2 positively regulates PLP splice site selection in a concentration-dependent manner. Disruption of the putative ASF/SF2 binding site in exon 3B reduced the positive effect of ASF/SF2 on PLP splicing. We conclude that an ESE in exon3B regulates PLP 5' donor site selection and that ASF/SF2 protein participates in the regulation of PLP alternative splicing in oligodendrocytes.

Alternative Splicing↗

PubMed2006

[pcDPG parathyroid hormone gene therapy of hypoparathyroidism: an experimental study].

OBJECTIVE: To construct recombination eukaryote expression plasmid for human parathyroid hormone (PTH) gene, assay PTH expression and biological activity after transfection in vitro and evaluate gene therapy effect on hypoparathyroidism (HPT). METHODS: (1) PTH gene was amplified from human embryonic parathyroid gland tissue, and plasmid pcDNA3.1-PTH-GFP (pcDPG) was constructed by TOPO recombination technique. Digestion, PCR and sequencing were used to identify the positive vectors. (2) pcDPG was transformed into 293 cells by Lipofectamine 2000(TM), fluorescent inverted microscope was used to observe GFP expression, and PTH gene expression was assayed by RT-PCR technique. (3) PTH protein in supernatant was purified and evaluated biological activity. (4) HPT rabbit models were developed and plasmid pcDPG was injected in skeletal muscles, respectively. Serum calcium, phosphonium and PTH were assayed and pathological changes observed. RESULTS: (1) The findings in digestion and PCR were accorded to anticipation and sequences in report were identified to reference at 99.30%. (2) 24 h after transfection GFP expression could be detected and enhanced with time prolonged arriving to 38.91% and 62.45% at 48 h. PTH gene expression could be detected by RT-PCR. (3) Purified PTH protein made the signs of HPT disappear. (4) Serum calcium and PTH levels were lower than those of pre-operation (P < 0.05) and serum phosphonium enhanced to normal standard 48 h after treatment at the plasmid pcDPG doses of 300 microg/kg and 500 microg/kg. CONCLUSION: Recombination plasmid pcDPG was transformed effectively in vitro and the transfected cells produced PTH protein with biological activity. Besides, the satisfactory therapeutic effect of HPT rabbits was attained by pcDPG plasmid, which provided a foundation for further study of HPT gene therapy.

Animals↗

PubMed2006

Splice-site contribution in alternative splicing of PLP1 and DM20: molecular studies in oligodendrocytes.

Mutations in the proteolipid protein 1 (PLP1) gene cause the X-linked dysmyelinating diseases Pelizaeus-Merzbacher disease (PMD) and spastic paraplegia 2 (SPG2). We examined the severity of the following mutations that were suspected of affecting levels of PLP1 and DM20 RNA, the alternatively spliced products of PLP1: c.453G>A, c.453G>T, c.453G>C, c.453+2T>C, c.453+4A>G, c.347C>A, and c.453+28_+46del (the old nomenclature did not include the methionine codon: G450A, G450T, G450C, IVS3+2T>C, IVS3+4A>G, C344A, and IVS3+28-+46del). These mutations were evaluated by information theory-based analysis and compared with mRNA expression of the alternatively spliced products. The results are discussed relative to the clinical severity of disease. We conclude that the observed PLP1 and DM20 splicing patterns correlated well with predictions of information theory-based analysis, and that the relative strength of the PLP1 and DM20 donor splice sites plays an important role in PLP1 alternative splicing.

Alternative Splicing↗

PubMed2006

The endosome-associated protein Hrs is hexameric and controls cargo sorting as a "master molecule".

The structure of the endosomal-associated protein, Hrs, has been determined with cryo-electron microscopy. Hrs interacts with a number of proteins, including SNAP-25 and STAM1, forming a complex that binds ubiquitin moieties. Analytical ultracentrifugation studies revealed that Hrs exists as a hexamer. The symmetry and the structure of the hexameric form of Hrs were determined with the single-particle reconstruction method. Hrs comprises three antiparallel dimers with a central core and distinct caps on either end. Crystal structures of VHS and FYVE domains fit into the Hrs end caps in the EM density map. Thus, the location of domains that interact with the endosomal membrane, the VHS, FYVE, and C-terminal domains, facilitates the anchorage of Hrs to the membrane, initiating the functional processes of Hrs on the endosome. Based on our model, the Hrs hexamer interacts with the membrane and acts as a "master molecule" that presents multiple sites for protein binding.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Virus-like particles production in green plants.

Viruses-like particles (VLPs), assembled from capsid structural subunits of several different viruses, have found a number of biomedical applications such as vaccines and novel delivery systems for nucleic acids and small molecules. Production of recombinant proteins in different plant systems has been intensely investigated and improved upon in the last two decades. Plant-derived antibodies, vaccines, and microbicides have received great attention and shown immense promise. In the case of mucosal vaccines, orally delivered plant-produced VLPs require minimal processing of the plant tissue, thus offering an inexpensive and safe alternative to more conventional live attenuated and killed virus vaccines. For other applications which require higher level of purification, recent progress in expression levels using plant viral vectors have shown that plants can compete with traditional fermentation systems. In this review, the different methods used in the production of VLPs in green plants are described. Specific examples of expression, assembly, and immunogenicity of several plant-derived VLPs are presented.

Biotechnology↗

PubMed2006

Plant-derived vaccines: a look back at the highlights and a view to the challenges on the road ahead.

The sobering reality is that each year, 33 million children remain unvaccinated for vaccine-preventable diseases. Universal childhood vaccination would have profound effects on leveling the health inequities in many parts of the world. As an alternative to administration of vaccines by needle and syringe, oral vaccines offer significant logistical advantages, as the polio eradication campaign has demonstrated. Over the past decade, the expression of subunit vaccine antigens in plants has emerged as a convenient, safe and potentially economical platform technology, with the potential to provide a novel biotechnological solution to vaccine production and delivery. As this technology has come of age, many improvements have been made on several fronts, as a growing number of research groups worldwide have extensively investigated plants as factories for vaccine production. This review attempts to highlight some of the achievements over the past 15 years, identify some of the potential problems and discuss the promises that this technology could fulfill.

Antigens, Plant↗

PubMed2005

Rapid, high-level production of hepatitis B core antigen in plant leaf and its immunogenicity in mice.

Hepatitis B core antigen (HBc or HBcAg) self-assembles into capsid particles and is extremely immunogenic. HBc has been extensively studied for its production in various expression systems and for the use of HBc particles for high-density, immunogenic presentation of foreign epitopes. Here we reported the high-level transient expression of HBc in plant leaf and its immunogenicity in mice. By using a novel plant viral expression system, HBc was produced in Nicotiana benthamiana leaves at levels up to 7.14% of total soluble protein (TSP) or 2.38 milligrams HBc per gram of fresh weight at 7 days post-infection (dpi). Plant-derived HBc (p-HBc) assembled into virus-like particles (VLPs) as revealed by sucrose gradients and electron microscopy. Partially purified p-HBc stimulated strong serum antibody responses in mice as Escherichia coli-derived HBc upon intraperitoneal (i.p.) injection. Furthermore, mice immunized mucosally (orally and intranasally) with p-HBc in the absence of adjuvants also developed HBc-specific serum IgG as well as intestinal IgA. Taken together, our results indicate the potential usefulness of p-HBc-VLP as a carrier for immunogenic presentation and mucosal delivery of foreign epitopes.

Animals↗

PubMed2005

Virus-like particle expression and assembly in plants: hepatitis B and Norwalk viruses.

Expression of vaccine antigens in plants and delivery via ingestion of transgenic plant material has shown promise in numerous pre-clinical animal studies and in a few clinical trials. A number of different viral antigens have been tested, and among the most promising are those that can assemble virus-like particles (VLP), which mimic the form of authentic virions and display neutralizing antibody epitopes. We have extensively studied plant expression, VLP assembly, and immunogenicity of hepatitis B surface antigen (HBsAg) and Norwalk virus capsid protein (NVCP). The HBsAg small protein (S protein) was found by TEM to assemble tubular membrane complexes derived from endoplasmic reticulum in suspension cultured cells of tobacco and soybean, and in potato leaf and tuber tissues. The potato material was immunogenic in mice upon delivery by ingestion. Here we describe the plant expression and immunogenicity of HBsAg middle protein (M protein or pre-S2 + S) which contains additional 55 amino acid pre-S2 region at N-terminus of the S protein. Plant-derived recombinant M protein provoked stronger serum antibody responses against HBsAg than did S protein when injected systemically in mice. We discuss implications for use of fusion proteins for enhanced immunogenicity and mucosal targeting of HBsAg, as well as delivery of heterologous fused antigens. NVCP expressed in plants assembled 38 nm virion-size icosahedral (T = 3) VLP, similar to those produced in insect cells. The VLP stimulated serum IgG and IgA responses in mice and humans when they were delivered by ingestion of fresh potato tuber. Here we show that freeze-drying of transgenic NVCP tomato fruit yielded stable preparations that stimulated excellent IgG and IgA responses against NVCP when fed to mice. However, the predominant VLP form in tomato fruit was the small 23 nm particle also observed in insect cell-derived NVCP.

Administration, Oral↗

PubMed2005

[Establishing the norm of cognitive adaptive test/clinical linguistic and auditory milestone scale in the children from 4 to 36 months of age in Shanghai, China].

OBJECTIVE: To meet the need for instrument assessing the cognitive abilities of infants and young children as well as discriminating between global developmental delay and particular deficits in either language or problem-solving skills, we intended to introduce Cognitive Adaptive Test/Clinical Linguistic and Auditory Milestone Scale (CAT/CLAMS) into China. METHODS: CAT/CLAMS were administered to 1604 normative children aged 4-36 months (in 16 age groups, about 100 children per age group) in Shanghai during the period from December 2003 to June 2004. In the meantime, Gesell Developmental Diagnosis was applied for 100 of these children, respectively aged 4, 6, 12, 18 and 30 months (20 children per age group). Interclass correlation coefficients (ICC) were adopted to analyze data in terms of inter-rater reliability and re-test reliability of the scales of CAT/CLAMS. Cronbach alpha coefficients were calculated to assess the inter consistency of the scales. Pearson correlation coefficients(r) were adopted to analyze the concurrent validity of the scales. The normative percentile graphs of CAT/CLAMS in the children from 4 to 36 Months of age in Shanghai, China were adopted. RESULTS: Administrations of the CAT/CLAMS for each subject usually took 10-20 minutes. Individual scores (CLAMS, CAT, and CAT/CLAMS) increased with ages (Pearson correlation coefficients were 0.96, 0.98 and 0.98, respectively, P < 0.01 for all). ICCs (intraclass correlation coefficient) in terms of individual scores for the inter-rater reliability test and the re-test reliability test were respectively > or = 0.96 (P < 0.01) and > or = 0.95 (P < 0.01), all the Cronbach alpha coefficients were > or = 0.98; in 100 children of the 5 age groups, there was significantly positive correlation between CAT/CLAMS and Gesell Developmental Diagnosis in terms of language skill DQ and adaptive skill DQ, and Full Scale DQ (r = 0.517, 0.703, 0.613, respectively, P < 0.01 for all). Moreover, this significant positive correlation was observed in each of the 5 age groups (r = 0.455-0.827, P < 0.05). CONCLUSION: CAT/CLAMS is suitable for discriminating between global developmental delay and particular deficits in either language or problem-solving skills. It is a quick, reliable, and valid instrument, with refined and quantified results. It is a good tool for developmental surveillance and screening of infants and young children.

Child Development↗

PubMed2004

Conformational analysis of hepatitis B surface antigen fusions in an Agrobacterium-mediated transient expression system.

Vaccine antigens have been successfully produced in transgenic plants for oral immunization. Recently, a fusion strategy has been adopted to produce multicomponent vaccines and to target antigens to mucosal sites for enhanced oral immunogenicity. However, antigen fusions may not be folded correctly due to steric hindrance and may thus lose their potency. Here, we describe an Agrobacterium-mediated transient assay that provides enough antigen-expressing material at 2 days post-transfection to evaluate antigen conformation. Using the hepatitis B surface antigen (HBsAg) as a model antigen and the green fluorescent protein (GFP) as a model fusion partner, we showed that transiently expressed HBsAg and an HBsAg fusion with GFP at the N-terminus (GFP:HBsAg), but not the HBsAg fusion with GFP at the C-terminus (HBsAg:GFP), formed the 'a' determinant and virus-like particles (VLPs), similar to yeast-derived vaccine HBsAg. Thus, it is feasible to modify the HBsAg with an N-terminal fusion of up to 239 amino acids without altering its major antigenic properties. Our results also demonstrate that the Agrobacterium-mediated transient expression system can be used to evaluate the conformation of plant-based vaccines or other pharmaceutical proteins in a high-throughput manner.

Journal Article↗

PubMed2004

Notch-induced E2A degradation requires CHIP and Hsc70 as novel facilitators of ubiquitination.

E2A transcription factors, E12 and E47, are important regulators of lymphocyte development. Notch signaling pathways have been shown to regulate E2A function by accelerating the degradation of E2A proteins through a mitogen-activated protein kinase-dependent and ubiquitin-mediated pathway. To further understand the mechanism underlying E2A ubiquitination and degradation, we conducted a yeast two-hybrid screen and identified the carboxyl terminus of Hsc70-interacting protein (CHIP) as an E47 binding protein. Here, we show that CHIP associates with E2A proteins in vivo and that overexpression of CHIP induces E47 degradation in a phosphorylation-dependent manner. Conversely, knocking down CHIP with small interfering RNA alleviates Notch-induced E47 degradation. CHIP binds E47 through the E protein homology domains 2 and 3 (EHD2 and EHD3). This interaction between CHIP and E47 is independent of the U-box domain with E3 ubiquitin ligase activity but requires the chaperone binding tetratricopeptide repeats domain. The ability of CHIP to induce E47 ubiquitination and degradation correlates with its ability to bind E47. We propose that CHIP, together with its partner Hsc70, forms a preubiquitination complex (PUC) with E47 and Skp2, thus facilitating the interaction between E47 and Skp2. CHIP also associates with Cul1, which introduces PUC to the SCF E3 ligase complex, responsible for E47 ubiquitination. Therefore, CHIP plays a crucial role in the ubiquitination and degradation of E2A proteins.

Amino Acid Sequence↗

PubMed2003

Sequence, distribution and quantification of the motilin precursor in the cat.

Due to motilin's relation to the migrating motor complex (MMC), the physiology of motilin has been mostly studied in man and dog. The cat does not have an MMC pattern, and little is known about cat motilin. Therefore we identified the cat motilin precursor (GenBank accession no. AF127917) and developed a quantitative polymerase chain reaction (PCR) to explore its distribution in the gastrointestinal tract and in the central nervous system (CNS). The precursor is closely related to the dog precursor and consists of an open reading frame of 348bp encoding the signal peptide (25 amino acids), the motilin sequence (22 amino acids) and the motilin associated peptide (69 amino acids). One amino acid of the signal peptide was subject to gene polymorphism. Quantification of motilin messenger RNA (mRNA) was for the first time achieved. It is most abundant in the gastrointestinal tract, with the highest concentration in the duodenum, the lowest in the colon and is not detectable in the corpus. However an important expression was also observed in several regions of the CNS, except the striatum and cerebral cortex. The highest level was in the hypothalamus (although 23-fold lower than in the duodenum), the lowest level in the pons. Moderate levels were found in the thyroid. These data suggest that the physiological role of motilin may extend beyond its effect on gastrointestinal motility.

Amino Acid Sequence↗

PubMed2002

How to describe protein motion without amino acid sequence and atomic coordinates.

This paper reports a computational method, the quantized elastic deformational model, that can reliably describe the conformational flexibility of a protein in the absence of the amino acid sequence and atomic coordinates. The essence of this method lies in the fact that, in modeling the functionally important conformational changes such as domain movements, it is possible to abandon the traditional concepts of protein structure (bonds, angles, dihedrals, etc.) and treat the protein as an elastic object. The shape and mass distribution of the object are described by the electron density maps, at various resolutions, from methods such as x-ray diffraction or cryo-electron microscopy. The amplitudes and directionality of the elastic deformational modes of a protein, whose patterns match the biologically relevant conformational changes, can then be derived solely based on the electron density map. The method yields an accurate description of protein dynamics over a wide range of resolutions even as low as 15-20 A at which there is nearly no visually distinguishable internal structures. Therefore, this method dramatically enhances the capability of studying protein motions in structural biology. It is also expected to have ample applications in related fields such as bioinformatics, structural genomics, and proteomics, in which one's ability to extract functional information from the not-so-well-defined structural models is vitally important.

Amino Acid Sequence↗

PubMed2002

A PLP splicing abnormality is associated with an unusual presentation of PMD.

We report that a deletion of 19 base pairs (bp) in intron 3 of the proteolipid protein (PLP/DM20) gene causes a neurological disease characterized by mild developmental delay, followed by progressive decline of acquired motor and cognitive milestones. The clinical features are associated with mild delay in myelination demonstrated by magnetic resonance imaging studies and with ongoing demyelination and axonal loss demonstrated by magnetic resonance spectroscopy. We demonstrate that the purine-rich 19bp element regulates PLP-specific splice site selection in transient transfections of chimeric constructs into cultured oligodendrocytes. Runs of 4 and 5 Gs centered in the 19bp element are critical for efficient PLP-specific splicing. The intronic element is sequence specific in oligodendrocytes and is not a repressor of PLP-specific splicing in nonglial cells. These data support the conclusion that deletion of the 19bp purine-rich region in PLP intron 3 causes a reduction in PLP message and protein, which affects myelin stability and axonal integrity.

Animals↗

PubMed2002

Down-regulation of the retinoblastoma protein (rb) is associated with rat oligodendrocyte differentiation.

Terminal differentiation of oligodendrocytes is associated with permanent withdrawal from the cell cycle. We studied the expression of the retinoblastoma protein, expression and activity of G1 cyclins and kinases in oligodendrocyte progenitor cells cultured in vitro. We found that Rb stopped to be expressed concomitantly with the activation of CNPase in oligodendrocytes differentiated with thyroid hormone. In contrast, Rb continued to be expressed at reduced levels in oligodendrocytes that were arrested in G1 by removal of mitogens. Cyclin D1, cdk2, and cdk4 kinase activities were decreased in G1-arrested and differentiated oligodendrocytes. Cyclin E, however, continued to be expressed in G1-arrested oligodendrocytes. Inhibition of differentiation induced by mitogens in oligodendrocytes arrested in G1 by Ad-p27 was accompanied by continued expression of Rb, D1, and E cyclins. After removal of mitogens and addition of thyroid hormone, Rb stopped being expressed and CNPase expression was activated with a temporal course similar to that of oligodendrocytes infected with a control adenovirus. Our results indicate that Rb may play an important function in differentiation of oligodendrocytes in response to external mitogens and differentiation factors.

Animals↗

PubMedDate not supplied

Automated determination of parameters describing power spectra of micrograph images in electron microscopy.

The current theory of image formation in electron microscopy has been semi-quantitatively successful in describing data. The theory involves parameters due to the transfer function of the microscope (defocus, spherical aberration constant, and amplitude constant ratio) as well as parameters used to describe the background and attenuation of the signal. We present empirical evidence that at least one of the features of this model has not been well characterized. Namely the spectrum of the noise background is not accurately described by a Gaussian and associated "B-factor;" this becomes apparent when one studies high-quality far-from focus data. In order to have both our analysis and conclusions free from any innate bias, we have approached the questions by developing an automated fitting algorithm. The most important features of this routine, not currently found in the literature, are (i). a process for determining the cutoff for those frequencies below which observations and the currently adopted model are not in accord, (ii). a method for determining the resolution at which no more signal is expected to exist, and (iii). a parameter-with units of spatial frequency-that characterizes which frequencies mainly contribute to the signal. Whereas no general relation is seen to exist between either of these two quantities and the defocus, a simple empirical relationship approximately relates all three.

Algorithms↗

PubMedDate not supplied

Application of template matching technique to particle detection in electron micrographs.

Template matching together with the comprehensive theory of image formation in electron microscope provides an optimal (in Bayesian sense) tool for solving one of the outstanding problems in single particle analysis, i.e., automatic selection of particle views from noisy micrograph fields. The method is based on the assumption that the reference three-dimensional structure is known and that the relevant parameters of the model of the image formation process can be estimated. In the first stage of the procedure, a set of possible particle views is generated using the available reference structure. The template images are constructed as linear combinations of available particle views using a clustering technique. Next, the micrograph noise characteristic is established using an automated contrast transfer function (CTF) estimation procedure. Finally, the CTF parameters calculated are used to construct a matched filter and correlation functions corresponding to the available template images are calculated. In order to alleviate the problem of the biased caused by varying image formation conditions, a decision making strategy based on the predicted distribution of correlation coefficients is proposed. It is demonstrated that due to the inclusion of CTF considerations, the template matching method performed very well in a broad range of microscopy conditions.

Algorithms↗