New method for determining cystine in leukocytes and fibroblasts.
(49/1728)
BACKGROUND: Cystinosis is a rare inborn error of cystine transport, leading to accumulation of cystine in the lysosomes. To diagnose cystinosis and monitor treatment with cysteamine, adequate measurements of cystine concentrations in leukocytes and cultured fibroblasts are required. METHODS: Cells were sonicated in the presence of excess N-ethylmaleimide to prevent oxidation of cysteine to cystine and disulfide exchange reactions of cystine with available sulfhydryl moieties. Cystine was measured as cysteine after reduction with sodium borohydride and derivatization with monobromobimane, followed by separation with automated HPLC and fluorescence detection. RESULTS: The assay was linear to 200 micromol/L cysteine. Within-run and day-to-day (total) imprecision (CV) was <5%, and the detection limit was 0.3 micromol/L. Added cysteine, up to 200 micromol/L, was completely removed, and recovery of added cystine was 69-86%. Cystine was stable for at least 2 months in leukocytes frozen in liquid nitrogen and stored at -80 degrees C CONCLUSIONS: Oxidation of cysteine to cystine and disulfide exchange reactions of cystine with sulfhydryl moieties are prevented by N-ethylmaleimide. The detection limit for the determination of cystine is adequate to measure cystine in leukocytes and cultured fibroblasts for diagnosis of cystinosis and monitoring treatment with cysteamine. (+info)
DNA replication in quiescent cell nuclei: regulation by the nuclear envelope and chromatin structure.
(50/1728)
Quiescent nuclei from differentiated somatic cells can reacquire pluripotence, the capacity to replicate, and reinitiate a program of differentiation after transplantation into amphibian eggs. The replication of quiescent nuclei is recapitulated in extracts derived from activated Xenopus eggs; therefore, we have exploited this cell-free system to explore the mechanisms that regulate initiation of replication in nuclei from terminally differentiated Xenopus erythrocytes. We find that these nuclei lack many, if not all, pre-replication complex (pre-RC) proteins. Pre-RC proteins from the extract form a stable association with the chromatin of permeable nuclei, which replicate in this system, but not with the chromatin of intact nuclei, which do not replicate, even though these proteins cross an intact nuclear envelope. During extract incubation, the linker histones H1 and H1(0) are removed from erythrocyte chromatin by nucleoplasmin. We show that H1 removal facilitates the replication of permeable nuclei by increasing the frequency of initiation most likely by promoting the assembly of pre-RCs on chromatin. These data indicate that initiation in erythrocyte nuclei requires the acquisition of pre-RC proteins from egg extract and that pre-RC assembly requires the loss of nuclear envelope integrity and is facilitated by the removal of linker histone H1 from chromatin. (+info)
Alteration of cGMP metabolism during chondrogenic differentiation of chondroprogenitor-like EC cells, ATDC5.
(51/1728)
Guanosine 3',5'-cyclic monophosphate (cGMP) has been recently reported to be involved in bone formation. ATDC5 cells were used to investigate cGMP metabolism during chondrogenic differentiation. Natriuretic peptide receptor (NPR)-A and NPR-B coupled with guanylate cyclase (GC) mediate biological functions of NPs, whereas NPR-C uncoupled with GC is thought to be the clearance receptor for NPs. The amounts of NPR-A, NPR-B, and CNP transcripts were increased but the amount of NPR-C transcripts was decreased in association with the chondrogenic differentiation of ATDC5 cells. CNP, a specific ligand for NPR-B lets ATDC5 cells accumulate great amounts of cGMP, revealing NPR-B as a dominant biological receptor through differentiation. cGMP hydrolytic activities of PDE1 and PDE5 existed in ATDC5 cells, and the activity of PDE1, which is stimulated by Ca(2+) and calmodulin (CaM) was major of them. Total cGMP hydrolytic activities as well as the amounts of PDE1 and PDE5 transcripts were enhanced during chondrogenic differentiation. Therefore, cGMP production and hydrolysis, cGMP metabolism was considered to be activated in association with chondrogenic differentiation of ATDC5 cells. These observations may lead to a better understanding of cGMP in the chondrocytes where bone formation occurs. (+info)
Source, catabolism and role of the tetrapeptide N-acetyl-ser-asp-lys-Pro within the testis.
(52/1728)
The tetrapeptide N-Acetyl-Seryl-Aspartyl-Lysyl-Proline (AcSDKP) is a natural regulator of hematopoietic stem cell proliferation. The present study was aimed at investigating the presence and the role of AcSDKP in rat testis. Specific immunoreactivity was always observed in the interstitial tissue at all stages of testicular development and in elongated spermatids at 45 days of age and in adults. In accordance with the interstitial labeling, high AcSDKP levels were detected in Leydig cell and testicular macrophage culture media and cell extracts, as well as in the testicular interstitial fluid (TIF). Much lower concentrations were found in peritubular cells and Sertoli cells cultures, whereas very low concentrations were present in cultured spermatocytes and spermatids. In contrast to the slight degradation rate of AcSDKP observed in the spermatocyte and spermatid culture media, no catabolism of the peptide was seen in testicular somatic cell culture medium. Furthermore, the degradation rate of AcSDKP was much lower in TIF than in peripheral blood plasma. Despite the very strong evidence indicating that Leydig cells and testicular macrophages produce AcSDKP, the selective destruction of these cells did not result in any change in AcSDKP levels in TIF or in plasma. This suggests a compensatory mechanism ensuring constant levels of the peptide in TIF when interstitial cells are absent. Finally, in vitro, in the presence of AcSDKP, significantly more [(3)H]thymidine incorporation was found in A spermatogonia. In conclusion, this study establishes the presence of very high concentrations of AcSDKP in rat testis and demonstrates its Leydig cell and testicular macrophage origin. The presence of AcSDKP in the TIF and its stimulatory effect on thymidine incorporation in spermatogonia very strongly suggest its implication in the paracrine control of spermatogenesis. (+info)
The A-kinase-anchoring protein AKAP95 is a multivalent protein with a key role in chromatin condensation at mitosis.
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Protein kinase A (PKA) and the nuclear A-kinase-anchoring protein AKAP95 have previously been shown to localize in separate compartments in interphase but associate at mitosis. We demonstrate here a role for the mitotic AKAP95-PKA complex. In HeLa cells, AKAP95 is associated with the nuclear matrix in interphase and redistributes mostly into a chromatin fraction at mitosis. In a cytosolic extract derived from mitotic cells, AKAP95 recruits the RIIalpha regulatory subunit of PKA onto chromatin. Intranuclear immunoblocking of AKAP95 inhibits chromosome condensation at mitosis and in mitotic extract in a PKA-independent manner. Immunodepletion of AKAP95 from the extract or immunoblocking of AKAP95 at metaphase induces premature chromatin decondensation. Condensation is restored in vitro by a recombinant AKAP95 fragment comprising the 306-carboxy-terminal amino acids of the protein. Maintenance of condensed chromatin requires PKA binding to chromatin-associated AKAP95 and cAMP signaling through PKA. Chromatin-associated AKAP95 interacts with Eg7, the human homologue of Xenopus pEg7, a component of the 13S condensin complex. Moreover, immunoblocking nuclear AKAP95 inhibits the recruitment of Eg7 to chromatin in vitro. We propose that AKAP95 is a multivalent molecule that in addition to anchoring a cAMP/PKA-signaling complex onto chromosomes, plays a role in regulating chromosome structure at mitosis. (+info)
Cell cycle-regulated transcription by the human immunodeficiency virus type 1 Tat transactivator.
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Cyclin-dependent kinases are required for the Tat-dependent transition from abortive to productive elongation. Further, the human immunodeficiency virus type 1 (HIV-1) Vpr protein prevents proliferation of infected cells by arresting them in the G(2) phase of the cell cycle. These findings suggest that the life cycle of the virus may be integrally related to the cell cycle. We now demonstrate by in vitro transcription analysis that Tat-dependent transcription takes place in a cell cycle-dependent manner. Remarkably, Tat activates gene expression in two distinct stages of the cell cycle. Tat-dependent long terminal repeat activation is observed in G(1). This activation is TAR dependent and requires a functional Sp1 binding site. A second phase of transactivation by Tat is observed in G(2) and is TAR independent. This later phase of transcription is enhanced by a natural cell cycle blocker of HIV-1, vpr, which arrests infected cells at the G(2)/M boundary. These studies link the HIV-1 Tat protein to cell cycle-specific biological functions. (+info)
A role for single-stranded templates in cell-free adeno-associated virus DNA replication.
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Assays have been described in which duplex adeno-associated virus (AAV) DNA can be replicated in HeLa cell extracts with exogenous AAV Rep protein. These assays appear to mimic the AAV DNA replication that occurs in the cell, including the ability of extracts from adenovirus (Ad)-infected cells to replicate duplex AAV DNA templates more efficiently than extracts from uninfected cells can. We showed previously that the Ad-infected extract was able to support a more processive replication than the uninfected extract. When the Ad single-stranded DNA binding protein (Ad-DBP) was added to an uninfected extract, DNA replication became processive. Based on a strand displacement replication model, we hypothesized that the Ad-DBP was stabilizing the displaced single-stranded DNA during strand displacement replication. In this report, we show that in Ad-infected extracts most of the newly replicated duplex DNA is converted into a single-stranded form shortly after synthesis. Using the results of assays for the replication of single-stranded AAV DNA, we show that these single-stranded molecules serve as templates for additional replication. In addition, we identify a class of molecules which are likely to be intermediates of replication on single-stranded templates. We discuss a possible role for replication of single-stranded molecules in the infected cell. (+info)
Protection against methylation-induced cytotoxicity by DNA polymerase beta-dependent long patch base excision repair.
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Using a plasmid-based uracil-containing DNA substrate, we found that the long patch base excision repair (BER) activity of a wild-type mouse fibroblast extract was partially inhibited by an antibody to DNA polymerase beta (beta-pol). This suggests that beta-pol participates in long patch BER, in addition to single-nucleotide BER. In single-nucleotide BER, the deoxyribose phosphate (dRP) in the abasic site is removed by the lyase activity of beta-pol. Methoxyamine (MX) can react with the aldehyde of an abasic site, making it refractory to the beta-elimination step of the dRP lyase mechanism, thus blocking single-nucleotide BER. MX exposure sensitizes wild-type, but not beta-pol null mouse embryonic fibroblasts, to the cytotoxic effects of methyl methanesulfonate (MMS) and methylnitrosourea. Expression of beta-pol in the null cells restores the ability of MX to modulate sensitivity to MMS. The beta-pol null cells are known to be hypersensitive to MMS and methylnitrosourea, and in the presence of MX (i.e. under conditions where single-nucleotide BER is blocked) the null cells are still considerably more sensitive than wild-type. The data are consistent with a role of beta-pol in long patch BER, which helps protect cells against methylation damage-induced cytotoxicity. (+info)