Possible Role In Maintenance Research Articles

Structural insights into the activity and regulation of human Josephin-2

The MJD family of human deubiquitinating enzymes contains four members: Ataxin-3, the ataxin-3-like protein (AT3L), Josephin-1, and Josephin-2. All share a conserved catalytic unit known as the Josephin domain. Ataxin-3 and AT3L also contain extensive regulatory regions that modulate their functions, whereas Josephins-1 and -2 are substantially smaller, containing only the Josephin domain. To gain insight into how these minimal Josephins differ from their larger relatives, we determined the 2.3 Å X-ray crystal structure of human Josephin-2 and probed the enzyme’s substrate specificity. Several large disordered loops are seen in the structure, suggesting a highly dynamic enzyme. Josephin-2 lacks several allosteric sites found in ataxin-3, but its structure suggests potential regulation via ubiquitination of a loop adjoining the active site. The enzyme preferentially recognizes substrates containing K11, K48, and K63 linkages, pointing toward a possible role in maintenance of protein quality control.

Belatacept (CTLA4-Ig) Shows Minimal Suppression of CMV-Specific Memory T Cell (CMV-T) and Antibody (CMV-Ab) in CMV Sero (+) Kidney Transplant Recipients (Tx Pts).

Background: Belatacept-treated (Rx) pts are at high risk for viral infections and their complications. We also observed an increased rate of CMV viremia in kidney Tx pts with Belatacept-Rx. However, the infection was mild in sero (+) pts. Clearance of CMV infection depends on CMV-T and CMV-Ab levels in CMV sero (+) pts. CD28 co-signaling is not required for memory T cell activation, but accelerates the activation. CD28 expression on plasma cells was recently shown, suggesting a possible role in maintenance of plasma cell function including viral-Ab production. Here we examine the effect of CD28 signaling blockade on CMV-T and CMV-Ab levels in vitro and in kidney Tx pts with Belatacept-Rx. Methods: CMV sero (+) blood from 18 normals and 20 pts treated with Belatacept + MMF + steroids for median 10M were submitted for CMV-T-cytokine flow cytometry (CFC) & anti-CMV-IgG ELISA. In some experiments, CMV-T-CFC was performed w/ or w/o anti-CD28 Ab co-stimulator, or w/ or w/o Belatacept. A peptide mixture from CMV pp65 & CMV lysate were used for CMV-CD8+ (CMV-Tc) & CD4+ T (CMV-Th) detection, respectively. The results were expressed as IFNγ+ cell% in each cell population and CMV-T >0.2% was considered+. Results: CMV-Tc levels w/ or w/o anti-CD28 Ab in the CFC were similar (2.2±2.4% vs. 2.9±3.1%), while CMV-Th was slightly lower w/o the Ab than w/ (0.9±0.8% vs. 1.0±1.0%, p=0.2). When Belatacept (0-1000mg/ml) was added to the CMV-T-CFC, no change was observed in CMV-Tc (4.2 vs. 4.2% at 0 vs. 1000mg/ml) and CMV-Th (1.2 vs. 1.2%). Positivity for CMV-Tc or CMV-Th was detected even after 10M Belatacept treatment in all 9 pts with available CMV-T results (Tc+/Th+: 7/9 [78%], Tc+/Th-: 1/9 11%], Tc-/Th+: 1/9 [11%]), which is a similar trend in 18 CMV sero (+) normals (Tc+/Th+: 15/18 [83%], Tc+/Th-: 1/18 [6%], Tc-/Th+: 2/18 [11%]). Anti-CMV-IgG levels in these pts did not change even after 10M Rx (Index: 3.8±1.1 vs. 3.5±1.1) Conclusions: The effect of CD28 blockade on CMV-T is minimal, and CMV-Tc, CMV-Th activity and anti-CMV-IgG levels are intact even after 10M Belatacept-Rx in CMV sero (+) pts. These results may explain the mild CMV infection in CMV sero (+) kidney Tx pts treated with Belatacept. DISCLOSURES:Jordan, S.: Grant/Research Support, Genentech Roche, CSL-Behring grant support.

Initiation at the human Fragile X (FMR1) origin of chromosomal replication

Eukaryotic chromosomal DNA replication begins at origins marked by pre‐replication complexes (pre‐RCs) containing ORC, MCM2‐7, and other proteins. However, understanding of mammalian origins and their possible role in maintenance of genomic stability remains incomplete. We have mapped a replication origin 5' of the FMR1 gene, a heritable fragile site on the human X chromosome. As expected, the FMR1 origin binds to pre‐RC proteins in vivo. FMR1 alleles with heritable CGG repeat expansions suffer chromosome breaks and gaps when cells experience folate deficiency, but how the expansion leads to chromosome fragility is not clear.Recent clues from the human DNA helicase HDHB may lead to one answer for this question. In late M/G1, HDHB assembles in pre‐RCs at the laminB2, MCM4, and FMR1 origins, but not in flanking sequences. Based on siRNA and dominant negative approaches, HDHB is important for initiation. At G1/S, HDHB is released from the laminB2 and MCM4 origins and much of it is exported from the nucleus. However, a low level of HDHB is retained on bulk chromatin during S phase and this fraction is increased in cells exposed to agents that stall fork progression. To ask whether HDHB may localize at stalled replication forks even in unperturbed cells, we used ChIP to study the FMR1 origin region and the common fragile site FRA16D. HDHB was strongly enriched in S phase at both sites and not in flanking regions. Taken together, the data suggest that HDHB functions in initiation of replication at origins and in preventing or repairing stalled replication forks that arise in specific regions even in unperturbed cells.

Excretion of polyamines in alfalfa and tobacco suspension-cultured cells and its possible role in maintenance of intracellular polyamine contents

Changes in polyamines (PAs) in cells and cultivation media of alfalfa (Medicago sativa L.) and tobacco bright yellow 2 (BY-2) (Nicotiana tabacum L.) cell suspension cultures were studied over their growth cycles. The total content of PAs (both free and conjugated forms) was nearly 10 times higher in alfalfa, with high level of free putrescine (Put) (in exponential growth phase it represented about 65-73% of the intracellular Put pool). In contrast, the high content of soluble Put conjugates was found in tobacco cells (in exponential phase about 70% of the intracellular Put). Marked differences occurred in the amount of PAs excreted into the cultivation medium: alfalfa cells excreted at the first day after inoculation 2117.0, 230.5, 29.0 and 88.0 nmol g(-1) of cell fresh weight (FW) of Put, spermidine (Spd), spermine (Spm) and cadaverine (Cad), respectively, while at the same time tobacco cells excreted only small amount of Put and Spd (12.7 and 2.4 nmol g(-1) FW, respectively). On day 1 the amounts of Put, Spd, Spm and Cad excreted by alfalfa cells represented 21, 38, 12 and 15% of the total pool (intra- plus extra-cellular contents) of Put, Spd, Spm and Cad, respectively. In the course of lag-phase and the beginning of exponential phase the relative contents of extracellular PAs continually decreased (with the exception of Cad). On day 10, the extracellular Put, Spd, Spm and Cad still represented 11.3, 10.9, 2.1 and 27% of their total pools. The extracellular PAs in tobacco cells represented from day 3 only 0.1% from their total pools. The possible role of PA excretion into the cultivation medium in maintenance of intracellular PA contents in the cells of the two cell culture systems, differing markedly in growth rate and PA metabolism is discussed.

Deficiency of Caenorhabditis elegans RecQ5 homologue reduces life span and increases sensitivity to ionizing radiation

Gene expression and RNA interference phenotypes were investigated for a Caenorhabditis elegans homologue (Ce-RCQ-5) of human RecQ5 protein. Expression of the mRNA was observed by in situ hybridization from earliest embryogenesis and gradually decreased during late embryogenesis. Ce-RCQ-5 was immuno-localized in the nuclei of embryos, germ cells, and oocytes and also in the nuclei of various somatic cells of larvae and adults. Despite ubiquitous expression in postembryonic cells, RCQ-5 protein expression was highest in intestinal cells, which was confirmed by tagging the gene expression with green fluorescence protein. When endogenous Ce- rcq-5 gene expression was inhibited by RNA interference, no clear phenotypes were observed during development. However, C. elegans life span was reduced by 37% due to RNA interference of rcq-5 gene, suggesting its possible role in maintenance of genomic stability, as has been ascribed to other RecQ family DNA helicases. In addition, C. elegans became significantly more sensitive to ionizing radiation after inhibition of rcq-5 gene expression, indicating an involvement of C. elegans RCQ-5 in a cellular response to DNA damage, possibly in DNA repair.

Telomere associations in interphase nuclei: possible role in maintenance of interphase chromosome topology.

The relative sizes of individual telomeres in cultured human cells under conditions of cell cycling, replicative quiescence, cell transformation and immortalization were determined using quantitative fluorescence in situ hybridization (Q-FISH) with a telomere-specific peptide nucleic acid (PNA) probe. Results obtained from analysis of telomere length profiles (TLPs), which display the distribution of relative telomere lengths for individual cells, confirmed telomere length heterogeneity at the single cell level and proportional shortening of telomere length during replicative aging of virus-transformed cells. TLPs also revealed that some telomeric ends of chromosomes are so closely juxtaposed within interphase nuclei that their fluorescent signals appear as a single spot. These telomeric associations (TAs) were far more prevalent in interphase nuclei of noncycling normal and virus-transformed cells than in their cycling counterparts. The number of interphase TAs per nucleus observed in late-passage E6/E7-transformed cells did not increase during progression to crisis, suggesting that telomere shortening does not increase the frequency of interphase TAs. Furthermore, interphase TAs were rarely observed in rapidly cycling, telomerase-positive, immortalized cells that exhibit somewhat shortened, but stabilized, telomere length through the activity of telomerase. Our overall results suggest that the number of interphase TAs is dependent more on whether or not cells are cycling than on telomere length, with TAs being most prominent in the nuclei of replicatively quiescent cells in which nonrandom (even preferred) chromosome spatial arrangements have been observed. We propose that interphase TAs may play a role in the generation and/or maintenance of nuclear architecture and chromosome positional stability in interphase nuclei, especially in cells with a prolonged G(1)/G(0) phase and possibly in terminally differentiated cells.

CD5 Is A Potential Selecting Ligand for B-Cell Surface Immunoglobulin: A Possible Role in Maintenance and Selective Expansion of Normal and Malignant B Cells

Although the function of CD5 on B cells is unknown, previous studies suggested that CD5 interaction with VH framework regions of surface immunoglobulins (Igs) may contribute to survival and expansion of B cells. Here we used B-chronic lymphocytic leukemia (B-CLL) cells and transformed B-cell lines from normal and B-CLL patients to study CD5-Ig interactions. Immobilized Ig binds and permits isolation of CD5 from lysates of CD5-expressing cell lines. Immunoglobulins or Fab fragments of different VH families varied in their effectiveness as inhibitors of anti-CD5 staining of CLL cells, appendix and tonsil tissue sections. Human Ig also binds to purified recombinant CD5. We show here for the first time that the unconventional Ig-CD5 interaction maps to the extracellular CD5-D2 domain whereas conventional epitopes recognized by anti-CD5 antibodies are localized in the Dl domain of CD5. We propose that interactions of VH framework regions with CD5 as a ligand may maintain, select or expand normal, autoimmune or transformed B cells and also contribute to skewing of the normal VH repertoire.

Sequence of interleukin‐2 isolated from human placental poly A RNA: Possible role in maintenance of fetal allograft

There are several cell types within the placenta that produce cytokines which can contribute to the regulatory mechanisms that ensure normal pregnancy. The immunological milieu at the maternofetal interface is considered to be crucial for survival of the fetus. Interleukin-2 (IL-2) is expressed by the syncytiotrophoblast, the cell layer between the mother and the fetus. IL-2 appears to be a key factor in maintenance of pregnancy. Therefore, it was important to determine the sequence of human placental interleukin-2. Direct sequencing of human placental IL-2 cDNA was determined for the coding region. Subclone sequencing was carried out for the 5′- and 3′-untranslated regions (5′-UTR and 3′-UTR). The 5′-UTR for human placental IL-2 cDNA is 294 bp, which is 247 nucleotides longer than that reported for cDNA IL-2 derived from T cells. The sequence of the coding region is identical to that reported for T cell IL-2, while sequence analysis of the polymerase chain reaction (PCR) product showed that the cDNA from the 3′ end was the same as that reported for cDNA from T cells. Human placental IL-2 cDNA is 1,028 base pairs (excluding the poly A tail), which is 247 bp longer at the 5′ end than that reported for IL-2 T cell cDNA. Therefore, the extended 5′-UTR of the placental IL-2 cDNA may be a consequence of alternative promoter utilization in the placenta. © 1996 Wiley-Liss, Inc.

Sequence of interleukin-2 isolated from human placental poly A+ RNA: possible role in maintenance of fetal allograft.

There are several cell types within the placenta that produce cytokines which can contribute to the regulatory mechanisms that ensure normal pregnancy. The immunological milieu at the maternofetal interface is considered to be crucial for survival of the fetus. Interleukin-2 (IL-2) is expressed by the syncytiotrophoblast, the cell layer between the mother and the fetus. IL-2 appears to be a key factor in maintenance of pregnancy. Therefore, it was important to determine the sequence of human placental interleukin-2. Direct sequencing of human placental IL-2 cDNA was determined for the coding region. Subclone sequencing was carried out for the 5'- and 3'-untranslated regions (5'-UTR and 3'-UTR). The 5'-UTR for human placental IL-2 cDNA is 294 bp, which is 247 nucleotides longer than that reported for cDNA IL-2 derived from T cells. The sequence of the coding region is identical to that reported for T cell IL-2, while sequence analysis of the polymerase chain reaction (PCR) product showed that the cDNA from the 3' end was the same as that reported for cDNA from T cells. Human placental IL-2 cDNA is 1,028 base pairs (excluding the poly A tail), which is 247 bp longer at the 5' end than that reported for IL-2 T cell cDNA. Therefore, the extended 5'-UTR of the placental IL-2 cDNA may be a consequence of alternative promoter utilization in the placenta.

Complexes of soluble HLA antigens and anti-HLA autoantibodies in human sera: possible role in maintenance of self-tolerance.

The MHC plays an essential role in regulating the process of self-nonself discrimination. T cells recognize nonself antigens only in the context of self-MHC gene products. It is not clear, however, whether B cells are also endowed with immune receptors for self-MHC antigens. We have explored the existence of antibodies against self-MHC antigens (HLA) in the human by analyzing the specificity of anti-HLA antibodies developed by a population of 727 dialysis patients who had been monitored monthly over a period of 3-78 months. Anti-HLA autoantibodies were identified by serum screening in 119 patients. Twenty-five of these 119 patients had not been exposed to alloantigens before, indicating that the production of anti-HLA autoantibodies is not necessarily stimulated by allogeneic HLA antigens. Cross-matching of sera with autologous lymphocytes, confirmed the autoreactive nature of these anti-HLA antibodies which were of IgM or of IgG isotype in approximately equal numbers of patients. The fine specificities of anti-HLA autoantibodies was ascertained in studies which showed that antibodies can be adsorbed, and the eluted, from the membranes of target cells carrying then relevant HLA antigen. Since the antibodies characterized in our studies may be functionally active in vivo we examined the possibility that their level is controlled by anti-idiotypic antibodies or by soluble HLA antigens. We found that the titer of anti-HLA autoantibodies increased significantly if soluble HLA antigens were depleted from the serum. Our data suggest that circulating HLA antigens form immune complexes with anti-HLA autoantibodies and contribute to the maintenance of self-tolerance by inhibiting antibody binding to membrane HLA antigens. The finding that the immune repertoire includes B cells with receptors for self-MHC opens new perspectives for the study of network perturbations in autoimmune diseases.

Isolation of Poly α-L-Glutamic Acid from the Oviduct of the Domestic Fowl and a Possible Role in Maintenance of Fertility

IT has been known for a considerable time1 that avian sperm, while in the oviduct, is capable of survival and of fertilizing ova for periods of up to 3 weeks in the domestic fowl and for longer periods in the turkey. In vitro, its fertilizing capacity is lost within a few hours. Examination of the oviduct of the domestic fowl after insemination has shown that sperm are congregated at two points, the infundibulum and the utero-vaginal junction2,3, the sperm being found in invaginations known as “crypts” or “spermnests”4, where they are apparently also protected from the action of spermicidal agents5.