Induction of indoleamine 2,3-dioxygenase in primary human macrophages by HIV-1

Increased kynurenine pathway metabolism has been implicated in the aetiology of the AIDS dementia complex (ADC). The rate limiting enzyme for this pathway is indoleamine 2,3-dioxygenase (IDO). We tested the efficacy of different strains of HIV-1 (HIV1-BaL, HIV1-JRFL and HIV1-631) to induce IDO in cultured human monocyte-derived macrophages (MDM). A significant increase in both IDO protein and kynurenine synthesis was observed after 48 h in MDM infected with the brain derived HIV-1 isolates, laboratory adapted (LA) HIV1-JRFL, and primary isolate HIV1-631. In contrast, almost no kynurenine production or IDO protein was evident in MDM infected with the high replicating macrophage tropic LA strain, HIV1-BaL. The induction of IDO and kynurenine synthesis by HIV1-JRFL and HIV1-631 declined to baseline levels by day-8 post-infection. Together, these results indicate that only selected strains of HIV-1 are capable of inducing IDO synthesis and subsequent oxidative tryptophan catabolism in MDM.     

Characterization of the Saccharomyces cerevisiae cyclic nucleotide phosphodiesterase involved in the metabolism of ADP-ribose 1",2"-cyclic phosphate

ADP-ribose 1″,2″-cyclic phosphate (Appr>p) is produced in yeast and other eukaryotes as a consequence of tRNA splicing. This molecule is converted to ADP-ribose 1″-phosphate (Appr-1″p) by the action of the cyclic nucleotide phosphodiesterase (CPDase). Comparison of the previously cloned CPDase from Arabidopsis with proteins having related cyclic phosphodiesterase or RNA ligase activities revealed two histidine-containing tetrapeptides conserved in these enzyme families. Using the consensus phosphodiesterase signature, we have identified the yeast Saccharomyces cerevisiae open reading frame YGR247w as encoding CPDase. The bacterially expressed yeast protein, named Cpd1p, is able to hydrolyze Appr>p to Appr-1″p. Moreover, as with the previously characterized Arabidopsis and wheat CPDases, Cpd1p hydrolyzes nucleosides 2′,3′-cyclic phosphates (N>p) to nucleosides 2′-phosphates. Apparent K_m values for Appr>p, A>p, U>p, C>p and G>p are 0.37, 4.97, 8.91, 12.18 and 14.29 mM, respectively. Site-directed mutagenesis of individual amino acids within the two conserved tetrapeptides showed that H₄₀ and H₁₅₀ residues are essential for CPDase activity. Deletion analysis has indicated that the CPD1 gene is not important for cellular viability. Likewise, overexpression of Cpd1p had no effect on yeast growth. These results do not implicate an important role for Appr>p or Appr-1″p in yeast cells grown under standard laboratory conditions.     

Sperm status and DNA dose play key roles in sperm/ICSI‐mediated gene transfer in caprine

In relation to the growing recent interest in the establishment of sperm‐mediated gene transfer (SMGT) technology as a convenient and effective method for the simple production of transgenic animals, in this study the possibility of using SMGT to produce transgenic caprine embryos was investigated for the first time. Buck sperm were directly incubated with different concentrations (0–500 ng) of pcDNA/his/Lac‐Z plasmid and used for IVF or ICSI. Sperm used for ICSI were categorized into motile or live‐immotile group before being injected into oocytes. In a separate experiment, dead sperm prepared by repeated freezing/thawing were used for DNA‐incubation before ICSI. Sham injection was carried out by intracytoplasmic injection of approximately the same volume of media containing different doses of DNA using an ICSI needle. Transgene expression and transmission were detected by X‐Gal staining and PCR analysis of developed embryos, respectively. A reasonable blastocyst rate was observed in all the groups. Only embryos in the sham group were negative for transgene transmission. Transgene expression was completely dependent on the delivery technique and status of sperm, and was only observed in the live‐immotile and dead ICSI groups. The results of this study showed that the technique (IVF vs. ICSI vs. sham injection), sperm status (motile vs. live‐immotile vs. dead) and to some extent DNA concentration affect embryo development, transgene transmission and expression. Mol. Reprod. Dev. 77:868–875, 2010. © 2010 Wiley‐Liss, Inc.     

Three-dimensional in vitro follicle growth: overview of culture models,biomaterials, design parameters and future directions

In vitro ovarian follicle culture is a new frontier in assisted reproductive technology with tremendous potential, especially for fertility preservation. Folliculogenesis within the ovary is a complex process requiring interaction between somatic cell components and the oocyte. Conventional two-dimensional culture on tissue culture substrata impedes spherical growth and preservation of the spatial arrangements between oocyte and surrounding granulosa cells. Granulosa cell attachment and migration can leave the oocyte naked and unable to complete the maturation process. Recognition of the importance of spatial arrangements between cells has spurred research in to three-dimensional culture system. Such systems may be vital when dealing with human primordial follicles that may require as long as three months in culture. In the present work we review pertinent aspects of in vitro follicle maturation, with an emphasis on tissue-engineering solutions for maintaining the follicular unit during the culture interval. We focus primarily on presenting the various 3-dimensional culture systems that have been applied for in vitro maturation of follicle:oocyte complexes. We also try to present an overview of outcomes with various biomaterials and animal models and also the limitations of the existing systems.     

Human CD5 protects circulating tumor antigen-specific CTL from tumor-mediated activation-induced cell death

We previously characterized several tumor-specific T cell clones from PBL and tumor-infiltrating lymphocytes of a lung cancer patient with identical TCR rearrangements and similar lytic potential, but with different antitumor response. A role of the TCR inhibitory molecule CD5 to impair reactivity of peripheral T cells against the tumor was found to be involved in this process. In this report, we demonstrate that CD5 also controls the susceptibility of specific T cells to activation-induced cell death (AICD) triggered by the tumor. Using a panel of tumor-infiltrating lymphocytes and PBL-derived clones expressing different levels of CD5, our results indicate that T lymphocyte AICD in response to the cognate tumor is inversely proportional to the surface expression level of CD5. They also suggest a direct involvement of CD5 in this process, as revealed by an increase in tumor-mediated T lymphocyte AICD following neutralization of the molecule with specific mAb. Mechanistically, our data indicate that down-regulation of FasL expression and subsequent inhibition of caspase-8 activation are involved in CD5-induced T cell survival. These results provide evidence for a role of CD5 in the fate of peripheral tumor-specific T cells and further suggest its contribution to regulate the extension of CTL response against tumor.     

Acute promyelocytic leukemia: new issues on pathogenesis and treatment response

Pathogenesis of acute promyelocytic leukemia appears to be one of the best understood among human malignancies. The ability of retinoic acid (RA) and arsenic trioxide to directly target the oncogenic promyelocytic leukemia-retinoic receptor A (PML-RARA) fusion protein also made this disease the first model for oncogene-targeted therapies. A set of recent data has significantly increased the complexity of our view of acute promyelocytic leukemia pathogenesis, as well as of therapeutic response. This review summarizes and discusses these findings, which yield novels questions and models.     

Mutations in Either TUBB or MAPRE2 Cause Circumferential Skin Creases Kunze Type

Circumferential skin creases Kunze type (CSC-KT) is a specific congenital entity with an unknown genetic cause. The disease phenotype comprises characteristic circumferential skin creases accompanied by intellectual disability, a cleft palate, short stature, and dysmorphic features. Here, we report that mutations in either MAPRE2 or TUBB underlie the genetic origin of this syndrome. MAPRE2 encodes a member of the microtubule end-binding family of proteins that bind to the guanosine triphosphate cap at growing microtubule plus ends, and TUBB encodes a β-tubulin isotype that is expressed abundantly in the developing brain. Functional analyses of the TUBB mutants show multiple defects in the chaperone-dependent tubulin heterodimer folding and assembly pathway that leads to a compromised yield of native heterodimers. The TUBB mutations also have an impact on microtubule dynamics. For MAPRE2, we show that the mutations result in enhanced MAPRE2 binding to microtubules, implying an increased dwell time at microtubule plus ends. Further, in vivo analysis of MAPRE2 mutations in a zebrafish model of craniofacial development shows that the variants most likely perturb the patterning of branchial arches, either through excessive activity (under a recessive paradigm) or through haploinsufficiency (dominant de novo paradigm). Taken together, our data add CSC-KT to the growing list of tubulinopathies and highlight how multiple inheritance paradigms can affect dosage-sensitive biological systems so as to result in the same clinical defect.