Biomedical subjects

P Wu

Publications and source records attributed to P Wu.

At least 127 records · Page 7Linked to original sources

PubMed1999

[Determination of triptolide in tripterygium preparations by gradient high performance liquid chromatography].

A gradient elution method was developed for the separation and determination of triptolide in Tripterygium wilfordii Hook preparations by high performance liquid chromatography (HPLC) using YWG column made in China. Methanol with 0.05 mol/L KH2PO4 was used as mobile phase, and the photodiode array detector was used at lambda = 218 nm. The sample of tripterygium oral preparation was extracted with ethanol and then by chloroform. The extract solution was evaporated and the residue was dissolved in 10 mL of ethanol as a sample solution. The spectrum was obtained by the subtraction of spectrum of ethanol from that of sample solution. In comparing with isocratic elution, the gradient operation used in this paper had higher resolution and sensitivity. The calibration curve was linear over the range of 1.32-21.10 mg/L (r = 0.9999). The average recovery of the method was 97.59%. The method was applied to separate and determine the triptolide in tripterygium tablets and the results indicated that the triptolide content by this method was less than that by isocratic and label-claimed one.

Chromatography, High Pressure Liquid↗

PubMed1998

Adeno-associated virus-mediated delivery of antiangiogenic factors as an antitumor strategy.

Antiangiogenic tumor therapies have recently attracted intense interest for their broad-spectrum action, low toxicity, and, in the case of direct endothelial targeting, an absence of drug resistance. To promote tumor regression and to maintain dormancy, antiangiogenic agents need to be chronically administered. Gene therapy offers a potential way to achieve sustained therapeutic release of potent antiangiogenic substances. As a step toward this goal, we have generated recombinant adeno-associated virus (rAAV) vectors that carry genes coding for angiostatin, endostatin, and an antisense mRNA species against vascular endothelial growth factor (VEGF). These rAAVs efficiently transduced three human tumor cell lines tested. Transduction with an rAAV-encoding antisense VEGF mRNA inhibited the production of endogenous tumor cell VEGF. Conditioned media from cells transduced with this rAAV or with rAAV-expressing endostatin or angiostatin inhibited capillary endothelial cell proliferation in vitro. Antiangiogenic rAAVs may offer a novel gene therapy approach to undermining tumor neovascularization and cancer progression.

Angiostatins↗

PubMed1998

Kinetic characterization of a bacteriophage T4 antimutator DNA polymerase.

Fidelity of DNA replication by bacteriophage T4 DNA polymerase is achieved in a multiplicative process: base selection by its polymerase activity and removal of misincorporated nucleotides by its exonuclease activity. The wild-type polymerase is capable of maintaining a balance between the two activities so that DNA replication fidelity is maximized without excessive waste of nucleotides. Antimutator enzymes exhibit a higher DNA replication fidelity than the wild-type enzyme, at the cost of increased nucleotide turnover. The antimutator A737V polymerase has been characterized kinetically using pre-steady-state and steady-state methods to provide a kinetic sequence which defines the effect of the mutation on the discrete steps controlling DNA replication fidelity. Comparison of this sequence to that of the wild type [Capson, L. T., Peliska, J. A., Kaboord, B. F., Frey, M. W., Lively, C., Dahlberg, M., and Benkovic, S. J. (1992) Biochemistry 31, 10984-10994] revealed that A737V polymerase differs in two ways. The rates at which DNA is transferred between the exonuclease and polymerase sites are reduced approximately 7-fold for a duplex DNA containing a mismatched 3'-terminus, and the partitioning of the mismatched duplex between the polymerase and exonuclease sites is 1:2 versus 4:1 for the wild-type enzyme. The exonuclease activity of A737V relative to the wild-type enzyme is unchanged on single-stranded DNA. However, the difference in partitioning the duplex DNA between the exonuclease and polymerase active sites results in an enhanced exonuclease activity for the antimutator enzyme.

Bacteriophage T4↗

PubMed1998

NCL1, a novel gene for a non-essential nuclear protein in Saccharomyces cerevisiae.

The nucleolar protein Nop2p is an essential gene product that is required for pre-rRNA processing and ribosome biogenesis in Saccharomyces cerevisiae (Hong, B. et al., 1997, Mol. Cell. Biol., 17, 378-388). A search for proteins similar to Nop2p identified a novel yeast gene product that also shares significant homology with the human proliferation associated nucleolar protein p120. The gene encoding this 78kDa protein was termed NCL1 (for nuclear protein 1; corresponding to YBL024w). Ncl1p and Nop2p contain an evolutionarily conserved motif that has been termed the 'NOL1/NOP2/fmu family signature' (NOL1 encodes p120). Epitope tagged Ncl1p was found to be localized to the nucleus, including the nucleolus, and was concentrated at the nuclear periphery. NCL1 is not essential. Strains containing a disruption of NCL1, or strains overexpressing NCL1, grow essentially identically to wildtype NCL1 strains on a number of different media and at different temperatures. Disruption of NCL1 does not affect steady-state levels of large and small ribosome subunits, monoribosomes, and polyribosomes. However, disruption of NCL1 leads to increased sensitivity to the antibiotic paromomycin.

Amino Acid Sequence↗

PubMed1998

Mutational analysis of the STAT6 SH2 domain.

The SH2 domain of the STAT family of transcription factors is essential for STAT binding to phosphorylated cytoplasmic domains of activated cytokine receptors. Furthermore, the same domain mediates dimerization of activated STAT monomers, a prerequisite for DNA binding by this family of proteins. To identify amino acid residues within the STAT protein that mediate these various interactions, we have carried out an extensive mutational analysis of the Stat6 SH2 domain. Recombinant proteins carrying C-terminal deletions or double alanine substitutions were expressed in mammalian and insect cells and assayed for DNA binding, transcription activation, tyrosine phosphorylation, and the ability to interact with a tyrosine-phosphorylated peptide derived from the interleukin-4 receptor signaling chain. From these studies, we have identified amino acids that are required for both DNA binding and interleukin-4 receptor interaction, as well as residues that when mutated impair only one of the two functions. Our results suggest that the structural homology between the SH2 domain of Stat6 and that of the distantly related Src protein may be higher than predicted on the basis of primary amino acid sequence comparisons. However, the two types of SH2 domains may differ at their C-terminal ends.

Alanine↗

PubMed1998

Isoenzymes of pyruvate dehydrogenase phosphatase. DNA-derived amino acid sequences, expression, and regulation.

Pyruvate dehydrogenase phosphatase (PDP) is one of the few mammalian phosphatases residing within the mitochondrial matrix space. It is responsible for dephosphorylation and reactivation of the pyruvate dehydrogenase complex (PDC) and, by this means, is intimately involved in the regulation of utilization of carbohydrate fuels in mammals. PDP is a dimeric enzyme consisting of catalytic and regulatory subunits. The catalytic subunit of PDP is a Mg2+-dependent enzyme homologous to the cytosolic phosphatases of the 2C family. In the present study, we isolated two cDNAs encoding for mitochondrial phosphatases. The first cDNA is highly homologous to the previously identified cDNA encoding for the catalytic subunit of PDP (PDP1). The second cDNA encodes a previously unknown catalytic subunit of PDP (PDP2). The new phosphatase, expressed as the recombinant protein in Escherichia coli, shows strict substrate specificity toward PDC and does not use phosphorylated branched chain alpha-ketoacid dehydrogenase as substrate. Like PDP1, PDP2 is a Mg2+-dependent enzyme, but its sensitivity to Mg2+ ions is almost 10-fold lower than that of PDP1. In contrast to PDP1, PDP2 is not regulated by Ca2+ ions. Instead, it is sensitive to the biological polyamine spermine, which, in turn, has no effect on the enzymatic activity of PDP1. Western blot analysis of PDP extracted from mitochondria isolated from liver and skeletal muscle revealed that PDP1 is predominantly expressed in mitochondria from skeletal muscle, whereas PDP2 is much more abundant in the liver rather than muscle mitochondria. Both isoenzymes are expressed in mitochondria from 3T3-L1 adipocytes, but the level of expression of PDP2 is considerably higher. These observations are consistent with previous findings on the enzymatic parameters of PDP in adipose tissue. Thus, our results provide the first evidence that there are at least two isoenzymes of PDP in mammals that are different with respect to tissue distribution and kinetic parameters and, therefore, are likely to be different functionally.

3T3 Cells↗

PubMed1998

Nop5p is a small nucleolar ribonucleoprotein component required for pre-18 S rRNA processing in yeast.

We have identified a novel nucleolar protein, Nop5p, that is essential for growth in Saccharomyces cerevisiae. Monoclonal antibodies B47 and 37C12 recognize Nop5p, which has a predicted size of 57 kDa and possesses a KKX repeat motif at its carboxyl terminus. Truncations that removed the KKX motif were functional and localized to the nucleolus, but conferred slow growth at 37 degreesC. Nop5p shows significant sequence homology with yeast Sik1p/Nop56p, and putative homologues in archaebacteria, plants, and human. Depletion of Nop5p in a GAL-NOP5 strain lengthened the doubling time about 5-fold, and selectively reduced steady-state levels of 40 S ribosomal subunits and 18 S rRNA relative to levels of free 60 S subunits and 25 S rRNA. Northern blotting and primer extension analyses showed that Nop5p depletion impairs processing of 35 S pre-rRNA at the A0 and A2 cleavage sites. Nop5p is associated with the small nucleolar RNAs U3, snR13, U14, and U18. Depletion of Nop5p caused the nucleolar protein Nop1p (yeast fibrillarin) to be localized to the nucleus and cytosol. Also, 37C12 co-immunoprecipitated Nop1p. These results suggest that Nop5p functions with Nop1p in the execution of early pre-rRNA processing steps that lead to formation of 18 S rRNA.

Amino Acid Sequence↗

PubMed1998

Selective inhibition of IL-5 receptor alpha-chain gene transcription by IL-5, IL-3, and granulocyte-macrophage colony-stimulating factor in human blood eosinophils.

High affinity receptor for IL-5 (IL-5R), a predominant eosinophil maturation factor, is composed of an IL-5-binding alpha-chain (IL-5R alpha) and a signal-transducing beta-chain that is shared by IL-3 and granulocyte-macrophage CSF (GM-CSF) receptors (IL-3R and GM-CSFR). By Northern blot analysis of mRNAs obtained from normal human blood eosinophils, we show in this report that the hematopoietic cytokines IL-5, IL-3, and GM-CSF down-regulate IL-5R alpha mRNA while up-regulating alpha-chain mRNAs for both IL-3R and GM-CSFR as well as the beta-chain mRNA. More detailed characterization reveals that the down-regulation of IL-5R alpha mRNA is specific to IL-3, IL-5, and GM-CSF; occurs very rapidly (reaching maximum inhibition within 2 h); is cytokine dose dependent; and does not require protein synthesis. Nuclear run-on and mRNA stability experiments demonstrate that cytokine-induced inhibition of IL-5R alpha mRNA accumulation occurs at the level of IL-5R alpha gene transcription, whereas enhanced accumulation of mRNAs for IL-3R alpha and the beta-chain results from reduced mRNA degradation. We suggest from these experiments that in human blood eosinophils, IL-5R alpha gene transcription and IL-5R alpha mRNA metabolism can be regulated by mechanisms that are distinct from those used for IL-3R alpha and GM-CSFR alpha.

Cells, Cultured↗

PubMed1998

Two receptor tyrosine phosphatases of the LAR family are expressed in the developing leech by specific central neurons as well as select peripheral neurons, muscles, and other cells.

Receptor protein tyrosine phosphatases (rPTPs) are thought to play a crucial role in neuronal development, particularly in pathfinding by growing processes. We have cloned and sequenced two Hirudo medicinalis rPTPs that are homologous to the Drosophila and vertebrate rPTPs of the Leukocyte common antigen-related (LAR) subfamily. These Hirudo rPTPs, HmLAR1 and HmLAR2, are products of different, homologous genes, both containing two tandem intracellular phosphatase domains and ectodomains with three tandem Ig domains and different numbers of tandem fibronectin type III (FIII) domains. They are expressed in distinct patterns during embryogenesis. HmLAR1 mRNA is expressed by a subset of central and peripheral neurons and by several peripheral muscular structures, whereas HmLAR2 mRNA is expressed by a different subset of central neurons and by the peripheral, neuron-like Comb cells. HmLAR1 and HmLAR2 proteins are located on the neurites of central neurons. In addition, HmLAR2 is expressed on the cell body, processes, and growth cones of the Comb cells. Because of their CAM-like ectodomains and homology to proteins known to be involved in pathfinding and because they are expressed by different subsets of neurons, we hypothesize that HmLAR1 and HmLAR2 participate in navigational decisions that distinguish the sets of neurons that express them. Furthermore, we hypothesize that HmLAR2 is also involved in setting up the highly regular array of parallel processes established by the Comb cells. Lastly, we propose that the HmLAR1 ectodomain on peripheral muscle cells plays a role in target recognition via interactions with neuronal receptors, which might include HmLAR1 or HmLAR2.

Amino Acid Sequence↗

PubMed1998

Evidence for existence of tissue-specific regulation of the mammalian pyruvate dehydrogenase complex.

Tissue distribution and kinetic parameters for the four isoenzymes of pyruvate dehydrogenase kinase (PDK1, PDK2, PDK3 and PDK4) identified thus far in mammals were analysed. It appeared that expression of these isoenzymes occurs in a tissue-specific manner. The mRNA for isoenzyme PDK1 was found almost exclusively in rat heart. The mRNA for PDK3 was most abundantly expressed in rat testis. The message for PDK2 was present in all tissues tested but the level was low in spleen and lung. The mRNA for PDK4 was predominantly expressed in skeletal muscle and heart. The specific activities of the isoenzymes varied 25-fold, from 50nmol/min per mg for PDK2 to 1250nmol/min per mg for PDK3. Apparent Ki values of the isoenzymes for the synthetic analogue of pyruvate, dichloroacetate, varied 40-fold, from 0.2 mM for PDK2 to 8 mM for PDK3. The isoenzymes were also different with respect to their ability to respond to NADH and NADH plus acetyl-CoA. NADH alone stimulated the activities of PDK1 and PDK2 by 20 and 30% respectively. NADH plus acetyl-CoA activated these isoenzymes nearly 200 and 300%. Under comparable conditions, isoenzyme PDK3 was almost completely unresponsive to NADH, and NADH plus acetyl-CoA caused inhibition rather than activation. Isoenzyme PDK4 was activated almost 2-fold by NADH, but NADH plus acetyl-CoA did not activate above the level seen with NADH alone. These results provide the first evidence that the unique tissue distribution and kinetic characteristics of the isoenzymes of PDK are among the major factors responsible for tissue-specific regulation of the pyruvate dehydrogenase complex activity.

Acetyl Coenzyme A↗

PubMed1998

Starvation and diabetes increase the amount of pyruvate dehydrogenase kinase isoenzyme 4 in rat heart.

This study investigated whether conditions known to alter the activity and phosphorylation state of the pyruvate dehydrogenase complex have specific effects on the levels of isoenzymes of pyruvate dehydrogenase kinase (PDK) in rat heart. Immunoblot analysis revealed a remarkable increase in the amount of PDK4 in the hearts of rats that had been starved or rendered diabetic with streptozotocin. Re-feeding of starved rats and insulin treatment of diabetic rats very effectively reversed the increase in PDK4 protein and restored PDK enzyme activity to levels of chow-fed control rats. Starvation and diabetes also markedly increased the abundance of PDK4 mRNA, and re-feeding and insulin treatment reduced levels of the message to that of controls. In contrast with the findings for PDK4, little or no changes in the amounts of PDK1 and PDK2 protein and the abundance of their messages occurred in response to starvation and diabetes. The observed shift in the relative abundance of PDK isoenzymes probably explains previous studies of the effects of starvation and diabetes on heart PDK activity. The results indicate that control of the amount of PDK4 is important in long-term regulation of the activity of the pyruvate dehydrogenase complex in rat heart.

Animals↗

PubMed1998

Characterization of the synovial T cell response to various recombinant Yersinia antigens in Yersinia enterocolitica-triggered reactive arthritis. Heat-shock protein 60 drives a major immune response.

OBJECTIVE: In Yersinia enterocolitica-triggered reactive arthritis (Yersinia ReA), the synovial T cell response is primarily directed against bacterial components, which are mostly unknown. This study was performed to investigate the synovial proliferative T cell response to a panel of recombinant Yersinia antigens in patients with Yersinia ReA and in controls. METHODS: Synovial fluid mononuclear cells (SFMC) were obtained from 4 patients with Yersinia ReA and from 14 patients with arthritides of different etiology. SFMC were stimulated with 5 recombinant Yersinia antigens (the 19-kd urease beta subunit, 13-kd ribosomal L23 protein, 32-kd ribosomal L2 protein, 18-kd outer membrane protein H, and Y. enterocolitica heat-shock protein 60 [hsp60]), and with human, Chlamydia trachomatis, and Borrelia burgdorferi hsp60. Three T cell clones specific for Y. enterocolitica hsp60 were generated from 1 patient with Yersinia ReA. Antigen-induced cytokine release was measured by enzyme-linked immunosorbent assay. RESULTS: SFMC from all 4 patients with Yersinia ReA responded to each of the Yersinia antigens except the 13-kd protein. These antigens were also recognized by SFMC from a subgroup of patients with undifferentiated arthritis (n = 4), but not by SFMC from other patients with arthritis of different etiology (n = 10). Y. enterocolitica hsp60 induced the strongest proliferative response in all cases. Two types of hsp60-reactive T cell clones could be obtained. One clone responded to all hsp60 variants, including the human variant, and showed a type 2 T helper (Th2)-like cytokine-secretion pattern. In contrast, another clone with specificity for the bacterial hsp60 proteins, but not the human equivalent, reacted with a more Th1-like pattern. CONCLUSION: In Y. enterocolitica-triggered ReA, at least 4 immunodominant T cell antigens exist, which might be used in lymphocyte proliferation assays to identify patients with Yersinia ReA. The hsp60 is a strong antigen, inducing both bacteria-specific and potentially autoreactive CD4+ T cells of both the Th1 and Th2 type.

Adolescent↗

PubMed1998

Effect of interferon therapy on hepatitis C virus RNA in whole blood, plasma, and peripheral blood mononuclear cells.

Fifty-two patients with chronic hepatitis C virus (HCV) infection were treated with standard doses of interferon alfa-2b. During treatment, HCV RNA detection was studied in samples of whole blood (WB), plasma (Pl), and peripheral blood mononuclear cells (PBMCs). Individuals were classified as sustained responders (SRs), complete responders with relapse (CRs), partial responders (PRs), or nonresponders (NRs) according to normalization of serum alanine transaminase (ALT) during treatment and follow-up. Before treatment, 100% of WB samples and more than 95% of Pl and PBMC samples were positive for HCV RNA. During treatment, there was progressive clearance of HCV RNA from Pl and PBMCs in SRs and CRs, but CRs had significantly more positive WB samples during and following treatment (P <.0001). At 6 months, only 10% of CR patients were positive by Pl assay, but 50% were positive by WB assay (P <.01). In the PR group, all WB samples remained positive throughout treatment, although 25% to 40% of PBMC and Pl samples became negative for HCV RNA during the first 2 months of therapy (WB > Pl or PBMC; P < .001). However, at later times during treatment most Pl and PBMC samples in the PR group were positive. Samples from the NR group showed no clearance of HCV RNA from WB, Pl, or PBMC fractions. These data document the increased sensitivity of WB assays for detecting HCV RNA in the peripheral blood of patients during interferon therapy. Furthermore, our findings suggest that WB analysis of HCV RNA may be a useful parameter to monitor in determining the end point of interferon therapy.

Adult↗

PubMed1998

Human mesotheliomas contain the simian virus-40 regulatory region and large tumor antigen DNA sequences.

BACKGROUND: A cohort (20%) of patients with mesothelioma will not have an exposure to asbestos. Recently, a DNA tumor virus (simian virus 40) has been shown to cause hamster mesotheliomas; we previously described simian virus 40-like DNA amino terminus sequences in 29 of 48 mesotheliomas. We analyzed an additional 42 mesotheliomas to determine (1) whether our initial observations were durable and (2) the extent to which the simian virus 40 genome is present in mesotheliomas. METHODS: Genomic DNA was extracted from snap frozen mesothelioma tumor samples and from the simian virus 40-induced hamster mesothelioma tumor H9A. Polymerase chain reaction primers were used to amplify various simian virus 40 large T-antigen regions including a 105-base pair amino terminus fragment, a 281-base pair carboxyl terminus fragment, and a 310-base pair fragment of the enhancer promoter region. Endonuclease digestions and Southern blotting were used to verify the expected product. RESULTS: Thirty of the 42 (71%) samples amplified T-antigen amino sequences, and specificity was verified by Southern hybridization. Sixteen of 42 samples (38%) amplified the appropriate size fragment for the carboxyl terminus, and digestion with BsaB1 matched that of H9A. Twenty-two of 42 samples (52%) amplified simian virus 40 regulatory sequences and Fok1 digestion matched that of the hamster control tumor. Sequence analysis (4 patients) revealed 100% homology with the regulatory region of simian virus 40 strain 776. CONCLUSIONS: These data suggest an association between the simian virus 40 virus and human mesothelioma that could be exploited for diagnostic/therapeutic options including early detection and potential vaccination strategies.

Animals↗

PubMed1998

General practitioners' attitudes to psychiatric and medical illness.

BACKGROUND: General practitioners are increasingly involved in the care of patients with long-term psychiatric disorders. We have previously reported that general practitioners are less willing to treat patients with schizophrenia than those without such a diagnosis, but this may have been attributable to a reluctance to treat patients with any psychiatric or chronic illness. We, therefore, examined general practitioners' attitudes to patients with chronic psychiatric or medical illnesses. METHODS: A random sample of 260 local general practitioners were each sent one of our case vignettes which were identical apart from mention of a previous diagnosis of schizophrenia, depression, diabetes or no illness. The general practitioners were asked to indicate their level of agreement with 13 attitudinal statements based on the vignette. RESULTS: One hundred and sixty-six (66%) of the general practitioners responded to the case vignettes. Those responding to the vignette about the patient with schizophrenia were less happy to have that patient on their practice list and were more concerned about the risk of violence and the child's welfare. Those responding to the depression vignette were more likely to offer the patient antidepressants or counselling; and those who replied to the diabetes case were most likely to refer the patient to a hospital specialist. These differences were not attributable to the personal or practice characteristics of the general practitioners. CONCLUSIONS: Patients with schizophrenia arouse concerns in general practitioners that are not simply due to those patients suffering from a psychiatric or chronic illness. Our results suggest that some patients with schizophrenia may find it difficult to register with a general practitioner and receive the integrated community-based health care service they require. Psychiatrists should provide education and support to general practitioners who look after patients with schizophrenia.

Adult↗

PubMed1998

Provision of positive and negative selections in retroviral vectors containing the cytosine deaminase gene.

The E. coli cytosine deaminase (CD) provides a negative selection system for suicide gene therapy as CD transfectants are eliminated following 5-fluorocytosine (5FC) treatment. Here we report a positive selection system for the CD gene using 5-fluorouracil (5FU) and cytosine in selection medium to screen for CD-positive transfectants. It is based on the relief of 5FU toxicity by uracil which is converted from cytosine via CD catalysis, as uracil competes with the toxic 5FU in subsequent pyrimidine metabolism. Hence, a retroviral vector containing the CD gene may provide both positive and negative selections after gene transfer. The CD transfectants selected with the positive selection system showed susceptibility to 5FC in subsequent negative selection in vitro and in vivo. Therefore, this dual selection system is useful not only for combination therapy with transgene and CD gene, but can also act to eliminate selectively transduced cells after the transgene has furnished its effects or upon undesired conditions if 5FC is applied for negative selection in vivo.

Animals↗

PubMed1998

Molecular dissection of developmental behavior of plant height in rice (Oryza sativa L.).

A doubled haploid population of 123 lines from IR64/Azucena was used to dissect the developmental behavior and genotype by environment interaction for plant height by conditional and unconditional quantitative trait loci (QTL) mapping methods in rice. It was shown that the number of QTL detected was different at various measuring stages. Some QTL could be detected at all stages and some only at one or several stages. More QTL could be found on the basis of time-dependent measures of different stages. By conditional QTL mapping of time-dependent measures, it is possible to reveal dynamic gene expression for quantitative traits. Mapping QTL for genetic main effects and GE interaction effects could help us in understanding the nature of QTL x environment interaction for the development of quantitative traits.

Adaptation, Physiological↗

PubMed1998

Germline transcription and recombination of a murine VDJmudeltagamma1 transgene.

To investigate the regulation of Ig switch recombination, we have constructed mice with a 56 kb VDJmudeltagamma1 transgene. This transgene included an anti-nitrophenyl VDJ segment, Smu, Cmu, Cdelta, Igamma1, Sgamma1, Cgamma1 and the Cgamma1 membrane exons from the murine Igh(a) haplotype. Two founder lines were produced, with very similar characteristics. Transgenic B cells expressed normal amounts of Cmu (which is >95% transgenic), Cdelta and other cell surface markers, and normal amounts of VDJ and Cmu RNA. Gamma1 germline transcription of the transgenes is properly regulated since stable transcripts were not expressed in B cells treated with lipopolysaccharide (LPS) alone, nor in thymus or non-lymphoid tissues, but were expressed after treatment of B cells with LPS + IL-4 or CD40L + IL-4. B cells from both lines of transgenic mice expressed transgenic gamma1a after in vitro culture with CD40L + IL-4, but not after culture with CD40L alone. However, the CD40L + IL-4 induced IgG1 precursor frequency is much lower for VDJmudeltagamma1 transgenic B cells (1:240-760) than for non-transgenic B cells (1:9). Analysis of DNA from transgenic hybridomas indicated that switch recombination can take place in switch (S) regions, but can also take place outside S regions. These results indicate that targeting of switch recombinase to S regions must include regulation beyond the S regions themselves and correct germline transcription. This additional regulation might include cis-acting elements or appropriate spacing or arrangement of the recombining elements.

Animals↗