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Ongoing clinical trials for treatment of beta-globinopathies by gene therapy involve the transfer of the beta-globin gene, which requires integration of three to four copies per genome in most target cells. This high proviral load may increase genome toxicity, potentially limiting the safety of this therapy and relegating its use to total body myeloablation. We hypothesized that introducing an additional hypersensitive site from the locus control region, the complete sequence of the second intron of the beta-globin gene, and the ankyrin insulator may enhance beta-globin expression. We identified a construct, ALS20, that synthesized significantly higher adult hemoglobin levels than those of other constructs currently used in clinical trials. These findings were confirmed in erythroblastic cell lines and in primary cells isolated from sickle cell disease patients. Bone marrow transplantation studies in beta-thalassemia mice revealed that ALS20 was curative at less than one copy per genome. Injection of human CD34+cells transduced with ALS20 led to safe, long-term, and high polyclonal engraftment in xenograft experiments. Successful treatment of beta-globinopathies with ALS20 could potentially be achieved at less than two copies per genome, minimizing the risk of cytotoxic events and lowering the intensity of myeloablation.

Innate lymphoid cells (ILCs) play important functions in immunity and tissue homeostasis, but their development is poorly understood. Through the use of single-cell approaches, we examined the transcriptional and functional heterogeneity of ILC progenitors, and studied the precursor–product relationships that link the subsets identified. This analysis identified two successive stages of ILC development within T cell factor 1-positive (TCF-1+) early innate lymphoid progenitors (EILPs), which we named ‘specified EILPs’ and ‘committed EILPs’. Specified EILPs generated dendritic cells, whereas this potential was greatly decreased in committed EILPs. TCF-1 was dispensable for the generation of specified EILPs, but required for the generation of committed EILPs. TCF-1 used a pre-existing regulatory landscape established in upstream lymphoid precursors to bind chromatin in EILPs. Our results provide insight into the mechanisms by which TCF-1 promotes developmental progression of ILC precursors, while constraining their dendritic cell lineage potential and enforcing commitment to ILC fate. Bhandoola and colleagues show that the transcription factor TCF-1 restrains the dendritic cell lineage potential in innate lymphoid cell progenitors.

The phospholipase A2(PLA2) activity of peroxiredoxin (Prdx)6 has important physiological roles in the synthesis of lung surfactant and in the repair of peroxidized cell membranes. These functions require the activity of a lysophospholipid acyl transferase as a critical component of the phospholipid remodeling pathway. We now describe a lysophosphatidylcholine acyl transferase (LPCAT) activity for Prdx6 that showed a strong preference for lysophosphatidylcholine (LPC) as the head group and for palmitoyl CoA in the acylation reaction. The calculated kinetic constants for acylation were Km18 μM and Vmax30 nmol/min/mg protein; the Vmaxwas increased 25-fold by phosphorylation of the protein while Kmwas unchanged. Study of recombinant protein in vitro and in mouse pulmonary microvascular endothelial cells infected with a lentiviral vector construct indicated that amino acid D31 is crucial for LPCAT activity. A linear incorporation of labeled fatty acyl CoA into dipalmitoyl phosphatidylcholine (PC) indicated that LPC generated by Prdx6 PLA2activity remained bound to the enzyme for the reacylation reaction. Prdx6 is the first LPCAT enzyme with demonstrated cytoplasmic localization. Thus, Prdx6 is a complete enzyme comprising both PLA2and LPCAT activities for the remodeling pathway of PC synthesis or for repair of membrane lipid peroxidation.

Caldesmon (CaD), a component of smooth muscle thin filaments, binds actin, tropomyosin, calmodulin, and myosin and inhibits actin-activated ATP hydrolysis by smooth muscle myosin. Internal deletions of the chicken CaD functional domain that spans from amino acids (aa) 718 to 731, which corresponds to aa 512–530 including the adjacent aa sequence in mouse CaD, lead to diminished CaD-induced inhibition of actin-activated ATP hydrolysis by myosin. Transgenic mice with mutations of five aa residues (Lys523to Gln, Val524to Leu, Ser526to Thr, Pro527to Cys, and Lys529to Ser), which encompass the ATPase inhibitory determinants located in exon 12, were generated by homologous recombination. Homozygous (−/−) animals did not develop, but heterozygous (+/−) mice carrying the expected mutations in the CaD ATPase inhibitory domain (CaD mutant) matured and reproduced normally. The peak force produced in response to KCl and electrical field stimulation by the detrusor smooth muscle from the CaD mutant was high compared with that of the wild type. CaD mutant mice revealed nonvoiding contractions during bladder filling on awake cystometry, suggesting that the CaD ATPase inhibitory domain suppresses force generation during the filling phase and this suppression is partially released by mutations in 50% of CaD in heterozygous. Our data show for the first time a functional phenotype, at the intact smooth muscle tissue and in vivo organ levels, following mutation of a functional domain at the COOH-terminal region of CaD.

MicroRNAs play important roles in regulating development at both transcriptional and posttranscriptional levels. Here, we report 29 microRNAs from mouse testis that are differentially expressed as the prepubertal testis differentiates to the adult testis. Using computational analyses to identify potential microRNA target mRNAs, we identify several possible male germ cell target mRNAs. One highly conserved sequence in the 3′-untranslated region (UTR) of transition protein 2 (Tnp2) mRNA, a testis-specific and posttranscriptionally regulated mRNA in postmeiotic germ cells, is complementary to Mirn122a. Mirn122ais enriched in late-stage male germ cells and is predominantly on polysomes. Mirn122a,but not another noncomplementary microRNA, inhibits the activity of a luciferase reporter construct containing the 3′-UTR of Tnp2.Site-directed mutations of Mirn122aindicate that base pairing of the 5′-region of Mirn122ato its complementary site in the 3′-UTR of Tnp2mRNA is essential for the downregulation of luciferase activity. Real-time reverse transcription-polymerase chain reaction and ribonuclease protection assays reveal that the Mirn122a-directed decrease of the Tnp2reporter gene activity results from mRNA cleavage. We propose that specific microRNAs, such as Mirn122a,could be involved in the posttranscriptional regulation of mRNAs such as Tnp2in the mammalian testis.

Background: Chemokines and chemokine receptors have been shown to play a critical role in HIV infection. Chemokine receptors have been identified as coreceptors for viral entry into susceptible target cells, and several members of the βchemokine subfamily of cytokines, MIP-1α, MIP-1β, and RANTES, have been identified as the major human immunodeficiency virus (HIV)-suppressive factors produced by activated CD8+ T lymphocytes. In macrophages, HIV-1 infection itself was shown to upregulate the production of MIP-1αand MIP-1β. In the present study, we address the mechanisms by which HIV-1 infection regulates βchemokine responses in macrophages and lymphocytes. Material and Methods: To address whether nitric oxide (NO), generated as a consequence of HIV-1 infection, regulates βchemokine responses in monocyte/macrophages and/or macrophage-depleted peripheral blood mononuclear cells (PBMCs) these two cell populations were isolated from HIV seronegative donors, placed in culture, and infected with HIV-1 in either the presence or absence of exogenous activators (e.g. lipopolysaccharide, phytohemagglutinin), inhibitors of nitric oxide synthase (NOS), or chemical donors of NO. Cultures were analyzed for βchemokine responses by ELISA and RNase protection. Results: LPS-induced MIP-1αrelease is enhanced in HIV-1-infected, as compared to uninfected, monocyte/macrophage cultures, and this enhancing effect is partially blocked by the addition of inhibitors of NOS, and can be reproduced by chemical generators of NO even in the absence of HIV-1 infection. A similar strategy was used to demonstrate a role for NO in HIV-1-mediated induction of MIP-1αin unstimulated macrophage cultures. NOS inhibitors also decreased MIP-1αand MIP-1βproduction by phytohemagglutinin-stimulated monocyte-depleted PBMC cultures. Conclusions: These results indicate that NO amplifies MIP-1αresponses in activated macrophages and lymphocytes, and suggests that this pleiotropic molecule might function as an enhancing signal that regulates secretion of βchemokines during HIV-1 infection. These findings reveal a novel mechanism by which NO might regulate the anti-HIV activity of immune cells.

The synthesis of tumor necrosis factor-α has been suggested to be regulated at both the transcriptional and translational levels in response to stimulation by bacterial lipopolysaccharide, although the relative contribution of these two mechanisms has not been quantitatively evaluated. Here, using the murine monocytic cell line RAW 264.7 as a model system, we show that steady-state TNF-α mRNA levels increase ∼77-fold following treatment with lipopolysaccharide for 2 h and to a maximum of 164-fold after 8 h as measured by an RNase protection assay. The TNF-α gene transcription rate increases ∼5-fold following exposure to lipopolysaccharide for 2 h as measured by a nuclear run-on assay. TNF-α mRNA stability did not change in the presence of lipopolysaccharide. A ribosomal sedimentation assay and an RNA transfection assay revealed that the translation rate of endogenous as well as transiently transfected TNF-α mRNAs increases only ∼2–3-fold after stimulation with lipopolysaccharide for 2 h. Taken together, these results suggest that the large increase in the level of secreted TNF-α protein in RAW 264.7 cells is due primarily to activation of TNF-α gene transcription.

We present a method that allows positive selection and rapid analysis of mutations in Enterobacteriaceae. Mutations are detected in a 2630 bp selection cartridge inserted in two different bacterial mutlicopy plasmid vectors. Spontaneous mutations in Escherichia coli, Enterobacter cloacae and Citrobacter freundii include insertions, deletions and point mutations. The small size of the target sequence facilitates rapid analysis of DNA rearrangements by cleavage with restriction enzymes and of any type of mutation by DNA sequence analysis. While in E. coli insertions of the mobile elements IS1, IS2 and IS5 were readily found, insertions of putative new transposable elements were detected in Enterobacter cloacae. The selection cartridge can thus serve as a tool for studying the spectrum of insertion mutations in Enterobacteriaceae and probably other Gramnegative bacteria, and the dependency of this spectrum on physiological and environmental factors and the host's genetic background can be investigated.

Gene addition of a functional copy of β-globin and reactivation of fetal hemoglobin (HbF) are promising therapeutical approaches for β-globinopathies such as Sickle Cell Disease (SCD) and β-thalassemia (β-thal).

A Gram-positive bacterium with the ability to utilize o-toluidine as sole source of carbon and nitrogen was isolated from soil. The organism was identified as Rhodococcus rhodochrous Sb 4. 3-Methylcatechol and the meta-fission product of 3-methylcatechol were identified as metabolites. A pathway for the degradation of o-toluidine is proposed.

A bacterium, which utilizes p-toluidine as sole source of carbon and energy was isolated from soil. The bacterium was identified as Pseudomonas testosteroni. From enzymatic studies we propose the following pathway for the degradation of p-toluidine:p-toluidine is oxidatively converted to 4-methyl-catechol, which is then cleaved by a meta-pyrocatechase to 2-hydroxy-5-methyl-cis-cis-muconate semialdehyde.