Cellular and humoral immune reconstitution after autologous peripheral blood stem cell transplantation (PBSCT).

Immune reconstitution after autologous peripheral blood stem cell transplantation (PBSCT) is of particular interest because of its importance for clinical outcome. Despite prolonged immunosuppression, especially of CD4(+) cells, few infections after neutrophil recovery occur. Only reactivation of varicella zoster virus (VZV) is more frequent in the first year after transplantation. From August 1997 to May 2001, we prospectively evaluated 38 patients prior to conditioning and during follow-up of 12 months post-transplant for virus antibodies [measles, mumps, rubella, polio, herpes simplex, varicella zoster, mononucleosis, cytomegalovirus (CMV)] and lymphocyte subpopulations by flow cytometry. CD3(+) T lymphocytes, CD8(+) T cells, and B-lymphocyte reconstitution in our study confirms previous reports. Complete CD4(+) lymphocyte reconstitution was not achieved in the 12 months post-transplant leading to a suppressed CD4/CD8 ratio. IgG antibody titers against measles, mumps, rubella, and polio were present in almost all patients pretransplant and during 12 months post-transplant, indicating persistent humoral immunity. CD3(+) and CD8(+) counts of patients with clinical VZV reactivation ( n=5) post-transplant were significantly higher (median: 1201/microl and 938/microl, respectively) than in patients without VZV reactivation (median: 594/microl and 482/microl, respectively) 6-12 months post-transplant. Positive CMV titers pretransplant ( n=19) were also correlated with higher CD3(+) and CD8(+) counts 3-6 months post-transplant (median: 1050/microl and 1056/microl, respectively) compared to CMV-negative patients (738/microl and 584/microl, respectively), although none of the patients suffered from CMV disease. Therefore, we conclude that persistent viral infections can contribute to the CD8(+) T-cell reconstitution after PBSCT by oligoclonal expansion of antigen-specific memory CD8(+) T cells.

Gene structure and larval expression of cnox-2Am from the coral Acropora millepora.

We have cloned a Hox-like gene, cnox-2Am, from a staghorn coral, Acropora millepora, an anthozoan cnidarian, and characterised its embryonic and larval expression. cnox-2Am and its orthologs in other cnidarians and Trichoplax most closely resemble the Gsx and, to a lesser extent, Hox 3/4 proteins. Developmental northern blots and in situ hybridisation are consistent in showing that cnox-2Am message appears in the planula larva shortly after the oral/aboral axis is formed following gastrulation. Expression is localised in scattered ectodermal cells with a restricted distribution along the oral/aboral body axis. They are most abundant along the sides of the cylindrical larva, rare in the oral region and absent from the aboral region. These cells, which on morphological grounds we believe to be neurons, are of two types; one tri-or multipolar near the basement membrane and a second extending projections in both directions from a mid-ectodermal nucleus. Anti-RFamide staining reveals neurons with a similar morphology to the cnox-2Am-expressing cells. However, RFamide-expressing neurons are more abundant, especially at the aboral end of the planula, where there is no cnox-2Am expression. The pattern of expression of cnox-2Am resembles that of Gsx orthologs in Drosophila and vertebrates in being expressed in a spatially restricted portion of the nervous system.

pADPRT-2: a novel mammalian polymerizing(ADP-ribosyl)transferase gene related to truncated pADPRT homologues in plants and Caenorhabditis elegans.

Until recently, poly(ADP-ribosyl)ation was supposed to be confined only to polymerizing(ADP-ribosyl)transferase/(ADP-ribose)polymerase (E.C. 2.4.2.30). Here, we present novel polymerizing(ADP-ribosyl)transferase homologues from mouse and man that lack all of the N-terminal DNA binding and BRCA1 C-terminus domains and will be designated polymerizing(ADP-ribosyl)transferase-2 as distinguished from the classical polymerizing(ADP-ribosyl)transferase (polymerizing(ADP-ribosyl)transferase-1). The murine polymerizing(ADP-ribosyl)transferase-2 gene shares three identical intron positions with its Caenorhabditis elegans (EMBL nucleotide sequence database Z47075) and one with the Arabidopsis thaliana homologue ('APP', GenBank database AF069298). Expression of the murine polymerizing(ADP-ribosyl)transferase-2 gene was elevated in spleen, thymus and testis and the corresponding poly(ADP-ribosyl)ation activity might account for most of the residual poly(ADP-ribosyl)ation observed in polymerizing(ADP-ribosyl)transferase-1(-/-) mice.

Nucleotide sequence of ub52 from the cnidarian Acropora millepora reveals high evolutionary conservation.

We report the cDNA sequence encoding Ub52 from the cnidarian Acropora millepora. As in other eukaryotes, the N-terminal region of the deduced amino-acid sequence is a ubiquitin moiety and the C-terminal region CEP52, a protein component of the large ribosomal subunit. A millepora Ub52 is highly homologous (> 95% identical) with the corresponding Drosophila and vertebrate sequences, the cnidarian sequence having higher identity with these 'higher' metazoan homologs than does the corresponding Caenorhabditis elegans protein. However, the cnidarian sequence contains two unique substitutions in the ubiquitin moiety, Ser22 and Thr28. Northern analysis indicates that A. millepora Ub52 is encoded by a small (< 800 nt) mRNA present in eggs and is first expressed after the early gastrula stage in embryonic development.

Mice lacking ADPRT and poly(ADP-ribosyl)ation develop normally but are susceptible to skin disease.

Poly(ADP-ribosyl)ation is catalyzed by NAD+: protein(ADP-ribosyl) transferase (ADPRT), a chromatin-associated enzyme which, in the presence of DNA breaks, transfers ADP-ribose from NAD+ to nuclear proteins. This post-translational modification has been implicated in many fundamental processes, like DNA repair, chromatin stability, cell proliferation, and cell death. To elucidate the biological function of ADPRT and poly(ADP-ribosyl)ation in vivo the gene was inactivated in the mouse germ line. Mice homozygous for the ADPRT mutation are healthy and fertile. Analysis of mutant tissues and fibroblasts isolated from mutant fetuses revealed the absence of ADPRT enzymatic activity and poly(ADP-ribose), implying that no poly(ADP-ribosyl)ated proteins are present. Mutant embryonic fibroblasts were able to efficiently repair DNA damaged by UV and alkylating agents. However, proliferation of mutant primary fibroblasts as well as thymocytes following gamma-radiation in vivo was impaired. Moreover, mutant mice are susceptible to the spontaneous development of skin disease as approximately 30% of older mice develop epidermal hyperplasia. The generation of viable ADPRT-/-mice negates an essential role for this enzyme in normal chromatin function, but the impaired proliferation and the onset of skin lesions in older mice suggest a function for ADPRT in response to environmental stress.

On the biological role of the nuclear polymerizing NAD+: protein(ADP-ribosyl) transferase (ADPRT): ADPRT from Dictyostelium discoideum and inactivation of the ADPRT gene in the mouse.

Two approaches have been used to elucidate the role of the nuclear polymerizing NAD+:protein(ADP-ribosyl)-transferase (ADPRT): i) comparison of the primary structure of Dictyostelium discoideum ADPRT derived from a 2 kb, partial cDNA sequence with the mammalian, fish, amphibian and insect counterparts revealed an overall homology of 25%. Whereas the automodification domain was not conserved at all, the NAD+ binding domain (aa 859-908) showed more than 70% identical amino acids in all species. Together with the similar enzymatic properties of the ADPRTs the genetic conservation underlined the notion that ADPRT plays a major role in various cellular processes; and ii) inactivation of the ADPRT gene in murine embryonic stem cells by homologous recombination led to mouse strains with a complete lack of nuclear poly(ADP-ribosyl)ation. These ADPRT mutant mice were viable and fertile indicating that ADPRT is dispensable in mouse development. Moreover, repair of UV and MNNG induced DNA damage was not affected in ADPRT/3T3 like fibroblasts, as measured by reactivation of in vitro damaged reporter plasmids and unscheduled DNA synthesis. However, about 30% of the ADPRT mutant mice developed pathological skin aberrations on a mixed 129/Sv x C57B1/6 genetic background. These mice will be extremely useful to define the precise biological role of poly(ADP-ribosyl)ation.

Reversibility of oxygen switch-off effect on Bacillus polymyxa nitrogenase.

The objective of this study was to analyse in vivo the effect of oxygen on the nitrogenase of Bacillus polymyxa. The culture technique employed in this study prevented spore formation by B. polymyxa during the entire period of exposure to acetylene. Under these conditions the acetylene-reduction assay allowed quantification of nitrogenase activity over long incubation periods (44 h). Nitrogenase activity was highest in cells harvested in the late logarithmic phase. At PO2 of 0.19 and 0.37 kPa, acetylene reduction was inhibited by 80 and 100%, respectively. This switch-off effect could be reversed through oxygen exhaustion, either by flushing the culture with N2 or by cellular respiration, suggesting a respiratory protection mechanism for the nitrogenase complex in B. polymyxa. Oxygen consumption measured by a closed-chamber respirometer showed a linear increase up to a PO2 of 0.2 kPa. Above 0.3 kPa a saturation in oxygen consumption was observed. Exposure to high oxygen pressures resulted in an irreversible loss of nitrogenase activity. The oxygen inhibition pattern was shown to be similar to that in other microaerophilic and anaerobic nitrogen-fixing microorganisms.