Biomedical subjects

M R Huang

Publications and source records attributed to M R Huang.

6 recordsLinked to original sources

PubMed2000

[Insect-resistant transgenic poplar expressing AaIT gene].

Insect-specific scorpion neurotoxin AaIT gene inserted into a binary vector was transferred into a hybrid poplar clone N-106(P. deltoides x P. simonii) growing in the Southern of China. We obtained sixty-two regenerated plants by Agrobacterium tumefaciens transferring system. PCR and PCR-Southern analysis showed that AaIT gene was incorporated into the genome of some recovered poplar plants. One of the transformed plants named A5 was significantly resistant to feeding by first instar larvae of Lymantria dispar, compared with the untransformed control plant. It caused a decrease in leaf consumption by larvae, a lower larval weight gain and a higher larval motality rate of Lymantria dispar. ELISA analysis proved that AaIT gene was expressed in this transfomed poplar plant.

Animals↗

PubMed1999

[Introduction of rabbit defensin NP-1 gene into poplar (P. tomentosa) by Agrobacterium-mediated transformation].

Rabbit defensin NP-1 possesses a broad resistant spectrum to pathogens. In this work, we have transferred the NP-1 gene into poplar plants by Agrobacterium-mediated transformation. PCR amplification and Southern analysis showed that rabbit defensin NP-1 gene was integrated into the poplar genome. The transformation efficiency is about 15.6%. Antimicrobial activity test showed that the extract of transgenic plants inhibited the growth of the tested microbes.

Animals↗

PubMed1998

Characterization of mouse mast cell protease-8, the first member of a novel subfamily of mouse mast cell serine proteases, distinct from both the classical chymases and tryptases.

Using a recently developed PCR-based strategy, a cDNA encoding a novel mouse mast cell (MC) serine protease (MMCP-8) was isolated and characterized. The MMCP-8 mRNA contains an open reading frame of 247 amino acids (aa), divided into a signal sequence of 18 aa followed by a 2-aa activation peptide (Gly-Glu) and a mature protease of 227 aa. The mature protease has an M(r) of 25072, excluding post-translational modifications, a net positive charge of +12 and six potential N-glycosylation sites. MMCP-8 showed a high degree of homology with mouse granzyme B in the critical regions for determining substrate cleavage specificity, indicating that MMCP-8, similar to granzyme B, preferentially cleaves after Asp residues. A comparative analysis of the aa sequence of MMCP-8 with other hematopoietic serine proteases shows that it is more closely related to cathepsin G and T cell granzymes than to the MC chymases. We therefore conclude that MMCP-8 belongs to a novel subfamily of mouse MC proteases distinct from both the classical chymases and tryptases. Southern blot analysis of BALB/c genomic DNA indicated that only one MMCP-8 gene (or MMCP-8 like gene) is present in the mouse genome. Northern blot analysis of rodent hematopoietic cell lines revealed high levels of MMCP-8 mRNA in a mouse connective tissue MC-like tumor line. However, MMCP-8 mRNA could not be detected in mouse liver, intestine, lung or ears, indicating very low expression in normal tissues. Analysis of the expression of different MMCP in the tissues of Schistosoma mansoni-infected BALB/c mice showed a strong increase in MMCP-8 levels in the lungs but not in the intestines of infected animals, suggesting the presence of a novel subpopulation of MC in the lungs that expressed MMCP-8, either alone or in combination with MMCP-5 and carboxypeptidase A. The dramatic increase in MMCP-1 and MMCP-2 levels but not of MMCP-8 in the intestines of parasitized animals also shows that MMCP-8 is not expressed in mucosal MC in the mouse. This latter is in clear contrast to what has been observed in the rat where the MMCP-8 homologues, RMCP-8, -9 and -10, can be considered as true mucosal MC proteases.

Amino Acid Sequence↗

PubMed1997

Molecular cloning and characterization of a cDNA encoding the rat interleukin-8 receptor.

In this study we present the cloning, characterization and expression analysis of a cDNA encoding a rat interleukin-8 receptor (rIL-8R). A 1324 bp cDNA containing an open reading of 359 amino acids with an 86.1% overall identity with the previously characterized mouse IL-8R was isolated. Genomic DNA analysis using several restriction enzymes revealed a single band suggesting that the rIL-8R gene exists as a single-copy, which is in contrast to humans where there are two different IL-8Rs genes. Expression of rIL-8R mRNA was found in several tissues including spleen, heart, lung, liver, skeletal muscle and kidney. In brain and testis rIL-8R mRNA was not detectable. Rat IL-8R mRNA expression at the cellular level was studied in the spleen using RNA-RNA in situ hybridization and immunohistochemistry. IL-8R mRNA containing cells were predominately found in the mantle zone of the germinal center. These cells were identified as B lymphocytes using the OX-33 monoclonal antibody.

Amino Acid Sequence↗

PubMed1997

Characterization of cDNA clones encoding mouse proteinase 3 (myeloblastine) and cathepsin G.

Serine proteases are the most abundant granule constituents of several major hematopoietic cell lineages. Due to their high abundance and their strict tissue specificity they have become important phenotypic cell markers used for studies of various aspects of hematopietic cell development. Using a polymerase chain reaction (PCR)-based strategy for the isolation of trypsin-related serine proteases, we were able to isolate cDNAs for two of the major neutrophil and monocyte serine proteases in the mouse, cathepsin G and mouse protease 3 (myeloblastin). The internal PCR fragments were used as probes to screen a mouse mast cell cDNA library and a cDNA library originating from a mouse monocytic cell line (WEHI-274.1). Full-length cDNAs for mouse cathepsin G and proteinase 3 were isolated and their complete sequences were determined. Northern blot analysis revealed expression of cathepsin G in immature cells of the monocyte macrophage lineage but also in the connective tissue mast cell line MTC. Proteinase 3 was expressed in several cell lines of myelo-monocytic origin and in one B-cell line, but not in any of the other cell lines tested. The isolation of cDNAs for mouse cathepsin G and mouse proteinase 3, together with the previous characterization of the gene for mouse N-elastase, and the entire or partial amino acid sequences for porcine azurocidine, equine N-elastase and proteinase 3, rat, dog, and rabbit cathepsin Gs in evolutionary relatively distantly related mammalian species, indicates that these four members of the serine protease family have been maintained for more than 100 million years of mammalian evolution. This latter finding indicates a strong evolutionary pressure to maintain specific immune functions associated with these neutrophil and monocyte proteases. All amino acid positions of major importance for the cleavage site selection have also been fully conserved between mouse and human proteinase 3 and a few minor changes have occurred between mouse and human cathepsin G.

Amino Acid Sequence↗

PubMed1997

Efficient adenovirus-mediated gene transduction of normal and leukemic hematopoietic cells.

We evaluated the efficiency of adenovirus-mediated gene transfer into normal and malignant human hematopoietic cells. An E-1 and E-3 deleted, replication-defective recombinant Ad.RSV beta gal vector was used and the transduction efficiency was studied at a multiplicity of infection of 13 p.f.u. per cell. Approximately 40-50% of normal monocytes were transduced, whereas purified normal resting T cells and B cells were resistant to infection. We showed that 50-80% of primary chronic myeloid leukemia cells (CML, n = 12) were efficiently transduced in contrast to CML, successful transduction of resting primary chronic B lymphocytic leukemia cells required appropriate preactivation of targeted cells. A novel protocol for the efficient transduction of adenovirus into B-CLL cells was presented. We showed that anti-CD40 mAb or CD40 ligand acts in synergy with rhIL-4 to enable the transduction of approximately 50-75% of B-CLL cells (B-CLL, n = 6). Expression of beta-galactosidase in transduced CML cells and B-CLL cells was detected for at least 15 days after transduction. The present studies underline the utility of adenovirus vectors for the construction of cytokine gene-modified tumor vaccines for the treatment of hematopoietic malignancies such as CML and B-CLL.

Adenoviridae↗