MIRC

Fragments of chromosomal DNA from Lactobacillus paracasei subsp. paracasei CG11 (formerly Lactobacillus casei CG11) capable of functioning as promoters were isolated using the broad host range, promoter-probe vector pGKV210. Five such fragments designated P61, P79, P80, P116, and P144 were completely sequenced and analyzed. Fragment P61 had the highest transcriptional efficiency in Escherichia coli and Lactobacillus reuteri whereas P80 was the most active in Lactococcus lactis. In general, the orders of the transcriptional strengths were almost identical in E. coli and Lactobacillus reuteri but different from that in Lactococcus lactis. Mapping of the 5′ end of cat mRNA showed that different regions of fragments P79 and P144 were used as promoters in Lactococcus lactis than in E. coli and Lactobacillus reuteri. Analysis of these DNA sequences revealed that the putative −35 and −10 hexanucleotides resembled those of E. coli, Bacillus subtilis, and lactococci. The spacing between thes...

PEA3, a member of the Ets family of transcription factors, is a nuclear phosphoprotein capable of activating transcription. Mouse PEA3 comprises 480 amino acids and bears an ϳ85-amino acid ETS domain near its carboxyl terminus. Whereas analyses of bacterially expressed PEA3 revealed that the ETS domain is required for sequence-specific DNA binding, little is known of the functional domains in the protein required for its activity in mammalian cells. To this end, we defined the location of the PEA3 functional domains in COS cells. PEA3 bears a strong activation domain near its amino terminus, which is flanked by two regions that independently negatively regulate its activity. PEA3 expressed in COS cells was incapable of binding to DNA in vitro. However, DNA binding activity could be unmasked by incubation with a PEA3-specific antibody. Analyses of the DNA binding activity of PEA3 deletion mutants revealed that two regions flanking the ETS domain independently inhibited DNA binding; deletion of both regions was required to detect DNA binding in the absence of a PEA3-specific antibody. Under these conditions, the ETS domain was sufficient for sequence-specific DNA binding. These findings suggest that the activity of PEA3 is exquisitely controlled at multiple functional levels.

The CEF-4/9E3 chemokine gene is expressed constitutively in chicken embryo ®broblasts (CEF) transformed by the Rous sarcoma virus (RSV). This aberrant induction is controlled at the transcriptional and posttranscriptional levels. Transcriptional activation depends on multiple elements of the CEF-4 promoter composing a Src-responsive-Unit or SRU. The SRU includes a TPA responsive element, a PRDII/kB domain and a CAAT box. In this report, we identify C/EBPb as a component of the transacting factor interacting with the CAAT box of the CEF-4 promoter. In addition, we show that C/ EBPb binds to a second element located in proximity of the TRE. A mutation of this distal CAAT box impaired the activation of the CEF-4 promoter by pp60 v-src indicating that this element is also part of the SRU. Using the RCASBP retroviral vector, we expressed a dominant negative mutant of C/EBPb (designated D184-C/EBPb) in RSV-transformed CEF. D184-C/EBPb decreased the accumulation of the CEF-4 mRNA and activation of the CEF-4 promoter by pp60 v-src. The induction of the Cox-2 gene (CEF-147) was also reduced by D184-C/EBPb. The eect of the dominant negative mutant was observed within 1 h of the activation of a thermolabile pp60 v-src suggesting that C/EBPb is an early target of v-src transformation. The dominant negative mutant did not inhibit the transformation of CEF by RSV and in fact accentuated the transformed cell phenotype. Therefore, the activation of C/EBPb is important for the expression of v-src regulated genes but is not required for the in vitro transformation of CEF by pp60 v-src .

Breast tumors comprise an infrequent tumor cell population, termed breast tumor initiating cells (BTIC), which sustain tumor growth, seed metastases and resist cytotoxic therapies. Hence therapies are needed to target BTIC to provide more durable breast cancer remissions than are currently achieved. We previously reported that serotonergic system antagonists abrogated the activity of mouse BTIC resident in the mammary tumors of a HER2-overexpressing model of breast cancer. Here we report that antagonists of serotonin (5-hydroxytryptamine; 5-HT) biosynthesis and activity, including US Federal Food and Drug Administration (FDA)-approved antidepressants, targeted BTIC resident in numerous breast tumor cell lines regardless of their clinical or molecular subtype. Notably, inhibitors of tryptophan hydroxylase 1 (TPH1), required for 5-HT biosynthesis in select non-neuronal cells, the serotonin reuptake transporter (SERT) and several 5-HT receptors compromised BTIC activity as assessed by ...

Engineering T cells with chimeric antigen receptors (CARs) is an effective method for directing T cells to attack tumors, but may cause adverse side effects such as the potentially lethal cytokine release syndrome. Here the authors show that the T cell antigen coupler (TAC), a chimeric receptor that co-opts the endogenous TCR, induces more efficient anti-tumor responses and reduced toxicity when compared with past-generation CARs. TAC-engineered T cells induce robust and antigen-specific cytokine production and cytotoxicity in vitro, and strong anti-tumor activity in a variety of xenograft models including solid and liquid tumors. In a solid tumor model, TAC-T cells outperform CD28-based CAR-T cells with increased anti-tumor efficacy, reduced toxicity, and faster tumor infiltration. Intratumoral TAC-T cells are enriched for Ki-67 CD8 T cells, demonstrating local expansion. These results indicate that TAC-T cells may have a superior therapeutic index relative to CAR-T cells.