Biomedical subjects
Publications and source records attributed to L Peng.
The energies of CrKbeta X-Rays in metel Cr, Cr2Os and K2CrO4, which are excited respectively by gamma-ray 59.6keV from radioactive source 241Am, have been measured by Si(Li) spectrometry. It is shown in the experiment that the peak of CrKbeta X-ray in K2CrO4 shifts 2.12 +/- 0.27eV compared to that in metel Cr.
The adoptive transfer of tumor-sensitized T cells can eradicate disseminated malignancy in murine animal models. T cells must be sensitized to tumor antigens in vivo to acquire antitumor reactivity. T-cell sensitization has been demonstrated to be dependent on host antigen-presenting cells. Tumor-associated macrophages are a heterogeneous population of cells that may have both inhibitory and stimulatory influences on the sensitization of naive T cells. Here we demonstrate that a weakly immunogenic tumor, the MCA 205 sarcoma, produces substantial amounts of murine monocyte chemoattractant protein 1 (MCP-1). Neutralization of MCP-1 during in vivo T-cell sensitization resulted in T cells that possessed enhanced therapeutic activity against established pulmonary metastases. These T cells sensitized during MCP-1 depletion also exhibited enhanced production of IFN-gamma upon recognition of tumor targets. These results demonstrate that MCP-1 can have a potent inhibitory influence on the development of tumor-reactive T cells.
The ligand for flt-3 (FLT3L) exhibits striking structural homology with stem cell factor (SCF) and monocyte colony-stimulating factor (M-CSF) and also acts in synergy with a range of other hematopoietic growth factors (HGF). In this study, we show that FLT3L responsive hematopoietic progenitor cells (HPC) are CD34+CD38-, rhodamine 123dull, and hydroperoxycyclophosphamide (4-HC) resistant. To investigate the basis for the capacity of FLT3L to augment the de novo generation of myeloid progenitors from CD34+CD38- cells, single bone marrow CD34+CD38- cells were sorted into Terasaki wells containing serum-free medium supplemented with interleukin-3 (IL-3), IL-6, granulocyte colony-stimulating factor (G-CSF), SCF (4 HGF) +/- FLT3L. Under these conditions, FLT3L recruited approximately twofold more CD34+CD38- cells into division than 4 HGF alone. The enhanced proliferative response to FLT3L was evident by day 3 and was maintained at all subsequent time points examined. In accord with these findings, we also show that transduction of CD34+CD38- cells with the LAPSN retrovirus is enhanced by FLT3L. The results of these experiments therefore indicate that increased recruitment of primitive HPC into cell cycle underlies the ex vivo expansion potential of FLT3L and also its ability to improve retroviral transduction of HPC.
The platelet activation induced by two anti-human platelet P24/CD9 McAbs was investigated. The results showed that: the pathway of platelet aggregation induced by the two McAbs (HI117 and SJ9A4) is not the same; HI117 and SJ9A4 induced the phosphorylation of platelet proteins (40KD and 20KD) when platelets were activated; but HI117 didn't cause a rise in intracellular Ca2+ concentration in activated platelets compared with SJ9A4; the epitope recognized by HI117 and SJ9A4 is different and this is probably the real reason why the two CD9 McAbs play different roles in platelet activation. Additionally McAbs HI117 and SJ9A4 could not promote associates of other proteins (e.g.: GPIIb/IIIa) with P24/CD9 on activated human platelets. All these results indicate that the mechanism of platelet activation induced by HI117 or SJ9A4 is different form each other. It suggests that CD9 antigen play an important and complex role in platelet activation.
Adoptive immunotherapy with T cells directed at tumor antigens has been demonstrated to result in the regression of malignant tumors in humans. These encouraging results have prompted the further exploration of parameters necessary to treat tumor in various locations in animal models. We have demonstrated that T cells that are sensitized to tumor antigens and then ex vivo cultured are capable of eradicating pulmonary metastases. In this report, we demonstrate that these T cells are capable of eliminating subcutaneous tumor deposits. Critical to the successful treatment of subcutaneous tumor was treatment with a large number of adoptively transferred T cells and pretreatment of the mice with irradiation. The transfer of T cells from tumor-bearing mice into irradiated mice failed to inhibit the therapeutic effect of ex vivo cultured T cells, suggesting that irradiation was not acting only as an immunosuppressant. Irradiation resulted in increased expression of the F4/80 and 33D1 epitopes on antigen-presenting cells within the tumor. The therapeutic effect of the adoptively transferred T cells was eliminated if either CD4 cells or CD8 cells were depleted. Naive T cells subjected to the same culture conditions were completely ineffective at eliminating tumor. These results demonstrate that adoptively transferred T cells derived from tumor-bearing hosts can treat subcutaneous tumor deposits, and they define the conditions necessary for the elimination of tumor in this location.
There are multiple isoforms of the Na,K-ATPase in the nervous system, three isoforms of the alpha subunit, and at least two of the beta subunit. The alpha subunit is the catalytic subunit. The beta subunit has several roles. It is required for enzyme assembly, it has been implicated in neuron-glia adhesion, and the experimental exchange of beta subunit isoforms modifies enzyme kinetics, implying that it affects functional properties. Here we describe the specificities of antibodies against the Na,K-ATPase beta subunit isoforms beta1 and beta2. These antibodies, along with antibodies against the alpha subunit isoforms, were used to stain sections of the rat cerebellum and cultures of cerebellar granule cells to ascertain expression and subcellular distribution in identifiable cells. Comparison of alpha and beta isoform distribution with double-label staining demonstrated that there was no preferential association of particular alpha subunits with particular beta subunits, nor was there an association with excitatory or inhibitory neurotransmission modes. Isoform composition differences were seen when Purkinje, basket, and granule cells were compared. Whether beta1 and beta2 are specific for neurons and glia, respectively, has been controversial, but expression of both beta subunit types was seen here in granule cells. In rat cerebellar astrocytes, in sections and in culture, alpha2 expression was prominent, yet the expression of either beta subunit was low in comparison. The complexity of Na,K-ATPase isoform distribution underscores the subtlety of its regulation and physiological role in excitable cells.
Mouse astrocytes (glial cells) in primary cultures were exposed to a low-voltage static DC electric field with no current flow and thus with no generation of magnetic fields. The electric field altered the rate of glycolysis, measured by 2-deoxyglucose accumulation. The magnitude and direction of this effect depended on the polarization of the field and the applied voltage. The maximum effect was an increase of approximately 30%, which occurred with field across the cells at an intensity that can be calculated to be 0.3 mV/cm or less. Reversal of the polarization converted the stimulation to a small but statistically significant inhibition.
We have investigated a possible delivery system for the rat preproinsulin II gene (rI2) utilising a recombinant adeno-associated virus (rAAV) vector system, with the long-term goal of engineering stably infected insulin-producing cell lines. The rAAV vector was chosen because it is a safe and nonpathogenic method for gene transfer. The plasmid pBC12BI (ATCC) was purified and digested with restriction enzymes SepI and StuI to release a fragment containing the Rous sarcoma virus long terminal repeat (RSV-LTR) promoter-driven rat preproinsulin II gene (rI2). Subsequently, the RSV-rI2 gene fragment was cloned into the BamHI site of rAAV vector plasmid pWP-19 to produce the rI2 recombinant plasmid designated pLP-1. The pWP-19 also encodes the AAV inverted terminal repeats for integration and replication and the herpes virus thymidine kinase promoter-driven gene for neomycin resistance (neoR). The cell line 293 (ATCC) was then cotransfected with pLP-1 and helper plasmid pAAV/AD, which is required for viral replication. The rAAV genome, now containing rI2, was rescued using adenovirus and packaged into mature AAV virions termed vLP-1. Finally, human pancreatic adenocarcinoma cells (HPAC; ATCC) were exposed to vLP-1, selected for G418 resistance, and screened for insulin production. Successful rescue was confirmed by Southern blot analysis using the rI2 gene probe derived from the original plasmid. The final titer of 1.25 x 10(9) particles/ ml was determined by DNA slot blots using pLP-1 as the standard, HPAC cells were infected with vLP-1 (termed HPAC/rI2). Integration of the rI2 genome in G418-resistant clones was confirmed by Southern blot analysis and again after 6 months in culture by amplification of the rI2 gene by PCR. Insulin gene transcription was confirmed by RT-PCR. We have developed a rAAV-mediated gene transfer system for the rat preproinsulin II gene. Successful transduction and stable integration of rI2 into HPAC was achieved. Production of insulin by HPAC/rI2 was confirmed by RIA and RT-PCR, validating this system as an effective approach to experimental gene therapy.
The sequence patterns of 11 types of frequently occurring connecting peptides, which lead to a classification of supersecondary motifs, were studied. A database of protein supersecondary motifs was set up. An artificial neural network method, i.e. the back propagation neural network, was applied to the predictions of the supersecondary motifs from protein sequences. The prediction correctness ratios are higher than 70%, and many of them vary from 75 to 82%. These results are useful for the further study of the relationship between the structure and function of proteins. It may also provide some important information about protein design and the prediction of protein tertiary structure.
A chemical mutagenesis protocol was used with the ruminal bacterium Prevotella ruminicola strain B(1)4 to generate mutant strains defective in peptidase activity. Compared with the wild-type parent strain, the isolated mutants possessed 1/10 of the enzyme activity responsible for cleavage of glycine-arginine-4-methoxy-beta-naphthylamide (Gly-Arg-MNA). A concomitant loss in activity against arginine-arginine-4-methoxy-beta-naphthylamide (Arg-Arg-MNA) was also observed. Both activities were similarly affected by various proteinase inhibitors, suggesting that the same enzyme is responsible for the Arg-Arg-MNA peptidase and Gly-Arg-MNA peptidase activities. Growth rates of wild-type and mutant strains grown in batch culture with various nitrogen sources did not differ. However, a role for the Gly-Arg-MNA peptidase activity was demonstrated in coculture experiments with gram-positive, ammonia-producing ruminal bacteria. The rate and extent of ammonia production were reduced by approximately 25% in cocultures containing the mutants when compared with that of wild-type-containing cultures. These reductions could not be accounted for simply by the decrease in ammonia production by the mutant strain alone. To our knowledge, this paper reports the first successful use of chemical mutagenesis with ruminal microorganisms.
The cellular receptors that mediate binding and internalization of retroviruses have recently been identified. The concentration and accessibility of these receptors are critical determinants in accomplishing successful gene transfer with retrovirus-based vectors. Murine retroviruses containing ecotropic glycoproteins do not infect human cells since human cells do not express the receptor that binds the ecotropic glycoproteins. To enable human cells to become permissive for ecotropic retrovirus-mediated gene transfer, we have developed a recombinant adeno-associated virus type 2 (AAV) vector containing ecotropic retroviral receptor (ecoR) cDNA under the control of the Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter (vRSVp-ecoR). Established human cell lines, such as HeLa and KB, known to be nonpermissive for murine ecotropic retroviruses, became permissive for infection by a retroviral vector containing a bacterial gene for resistance to neomycin (RV-Neo(r)), with a transduction efficiency of up to 47%, following transduction with vRSVp-ecoR, as determined by the development of colonies that were resistant to the drug G418, a neomycin analog. No G418-resistant colonies were present in cultures infected with either vRSVp-ecoR or RV-Neo(r) alone. Southern and Northern blot analyses revealed stable integration and long-term expression, respectively, of the transduced murine ecoR gene in clonal isolates of HeLa and KB cells. Similarly, ecotropic retrovirus-mediated Neo(r) transduction of primary human CD34+ hematopoietic progenitor cells from normal bone marrow was also documented, but only following infection with vRSVp-ecoR. The retroviral transduction efficiency was approximately 7% without prestimulation and approximately 14% with prestimulation of CD34+ cells with cytokines, as determined by hematopoietic clonogenic assays. No G418-resistant progenitor cell colonies were present in cultures infected with either vRSVp-ecoR or RV-Neo(r) alone. These results suggest that sequential transduction of primary human cells with two different viral vectors may overcome limitations encountered with a single vector. Thus, the combined use of AAV- and retrovirus-based vectors may have important clinical implications for ex vivo and in vivo human gene therapy.
A common polymorphism of the angiotensin-converting enzyme (ACE) gene (ACE in humans, Ace in mice) is associated with differences in circulating ACE levels that may confer a differential risk for cardiovascular diseases. To study the effects of genetically determined changes in Ace gene function within a defined genetic and environmental background, we have studied mice having one, two, or three functional copies of the Ace gene at its normal chromosomal location. ACE activities in the serum increased progressively from 62% of normal in the one-copy animals to 144% of normal in the three-copy animals (P < 10(-15), n = 132). The blood pressures of the mice having from one to three copies of the Ace gene did not differ significantly, but the heart rates, heart weights, and renal tubulointerstitial volumes decreased significantly with increasing Ace gene copy number. The level of kidney renin mRNA in the one-copy mice was increased to 129 +/- 9% relative to that of the normal two-copy mice (100 +/- 4%, P = .01, n = 16). We conclude that significant homeostatic adaptations successfully normalize the blood pressures of mice that have quantitative changes in Ace gene function. Our results suggest only that quantitative changes in expression of the Ace gene will observably affect blood pressures when accompanied by additional environmental or genetic factors that together with Ace exceed the capacity of the homeostatic mechanisms.
Regulation of galanin gene expression in the anterior pituitary (AP) is positively influenced by estrogen in rodents and undetermined in humans. The objective of this study was to investigate the mechanism behind estrogen induction of galanin by identifying any putative estrogen receptor (ER) binding sequences within the human galanin promoter that may function as estrogen response elements (ERE). Two regions, gERE1 and gERE2, were identified in the galanin 5'-flanking sequence with similarity to the full 13-base ERE consensus previously defined in the vitellogenin gene (vERE). Both sequences were tested in mobility shift assays for the ability to bind nuclear proteins isolated from rat AP tissue or MtTW-10 pituitary tumors. Only the distal sequence at -527 (gERE1) yielded an ERE-specific DNA/protein complex distinguished by mobility and cross-competition with vERE. The gel mobility pattern of the DNA/protein complex was comparable between the pituitary tissue and tumor extracts. However, DNA/protein affinity estimations demonstrated a greater affinity of pituitary proteins for gERE1 over the vERE sequence. Evidence that the human ER (hER) does recognize the gERE1 sequence in the human galanin gene was provided by electrophoretic mobility shift assays (EMSAs) with Sf9 extracts enriched in recombinant hER. In addition, antibodies specific for the hER recognized the gERE1/protein complex in supershift experiments. Enhancer activity by gERE1 was detected in transient transfections of the rat GH3 pituitary cell line, resulting in a 4-fold induction of expression driven by the heterologous thymidine kinase promoter in the presence of estrogen. Evidence for ER regulation of the gERE1 enhancer was demonstrated by: 1) inhibition of enhancement using the specific ER antagonist ICI 164,384; and 2) enhancement in HeLa cells that was dependent upon coexpression with hER. Enhancement by gERE1 was half the magnitude as that from the vERE element and may reflect a difference in affinity or composition of the ER complex between the two sequences. These data demonstrate the presence of a functional ERE sequence within the human galanin gene that could potentially function as a regulatory element for estrogen action in the AP.
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The clinical application of brain transplantation for treating Parkinson's disease (PD) is limited because it is difficult to obtain ideal donor cells. In this study, the tyrosine hydroxylase (TH) gene was introduced into NIH-3T3 cell line and the genetically modified cells reacted positively with TH antiserum and released L-dopa. These cells were implanted into the striatum of PD rats. 3,6,9,12 days after implantation, the average rate of the abnormal rotational behavior recovery was 52.6%, 68.4%, 63.2%, 44.7%, respectively. This result is considered to be potential for further clinical application.
Ventricular QRS classification is key technique of ventricular arrhythmias detection in single-chip-microcomputer based dynamic electrocardiogram real-time analyser. This paper adopts morphological feature vector including QRS amplitude, interval information to reveal QRS morphology. After studying the distribution of QRS morphology feature vector of MIT/BIH DB ventricular arrhythmia files, we use morphological feature vector cluster to classify multi-morphology QRS. Based on the method, morphological feature parameters changing method which is suitable to catch occasional ventricular arrhythmias is presented. Clinical experiments verify missed ventricular arrhythmia is less than 1% by this method.
A recombinant retroviral vector containing tissue-type plasminogen activator (t-PA) cDNA was constructed and transfected into PA317 viral packaging cells, forming intact virus particles. Under electron microscope the recombinant retroviral particles were composed of envelope, capsid and core. These viral particles were spherical with a diameter of 90-180nm, and spread dispersely in the cells. NIH3T3 cell infected by retrovirus particles were screened with G418. The virus titer of 6 x 10(8) CFU/L was verified by counting the positive clones two weeks after screening. The expression of t-PA was demonstrated in the NIH3T3 cells infected with the recombinant virus.