Telomere length and telomerase activity in malignant lymphomas at diagnosis and relapse

Telomere length maintenance, in the vast majority of cases executed by telomerase, is a prerequisite for long-term proliferation. Most malignant tumours, including lymphomas, are telomerase-positive and this activity is a potential target for future therapeutic interventions since inhibition of telomerase has been shown to result in telomere shortening and cell death in vitro. One prerequisite for the suitability of anti-telomerase drugs in treating cancer is that tumours exhibit shortened telomeres compared to telomerase-positive stem cells. A scenario is envisioned where the tumour burden is reduced using conventional therapy whereafter remaining tumour cells are treated with telomerase inhibitors. In evaluating the realism of such an approach it is essential to know the effects on telomere status by traditional therapeutic regimens. We have studied the telomere lengths in 47 diagnostic lymphomas and a significant telomere shortening was observed compared to benign lymphoid tissues. In addition, telomere length and telomerase activity were studied in consecutive samples from patients with relapsing non-Hodgkin's lymphomas. Shortened, unchanged and elongated telomere lengths were observed in the relapse samples. The telomere length alterations found in the relapsing lymphomas appeared to be independent of telomerase and rather represented clonal selection random at the telomere length level. These data indicate that anti-telomerase therapy would be suitable in only a fraction of malignant lymphomas. © 2000 Cancer Research Campaig

Interactions in vivo between the Vif protein of HIV-1 and the precursor (Pr55GAG) of the virion nucleocapsid proteins

The abnormality of viral core structure seen in vif-defective HIV-1 grown in PBMCs has suggested a role for Vif in viral morphogenesis. Using an in vivo mammalian two-hybrid assay, the interaction between Vif and the precursor (Pr55GAG) of the virion nucleocapsid proteins has been analysed. This revealed the amino-terminal (aa 1–22) and central (aa 70–100) regions of Vif to be essential for its interaction with Pr55GAG, but deletion of the carboxy-terminal (aa 158–192) region of the protein had only a minor effect on its interaction. Initial deletion studies carried out on Pr55GAG showed that a 35-amino-acid region of the protein bridging the MA(p17)–CA(p24) junction was essential for its ability to interact with Vif. Site-directed mutagenesis of a conserved tryptophan (Trp21) near the amino terminus of Vif showed it to be important for the interaction with Pr55GAG. By contrast, mutagenesis of the highly conserved YLAL residues forming part of the BC-box motif, shown to be important in Vif promoting degradation of APOBEC3G/3F, had little or no effect on the Vif–Pr55GAG interaction

Identifying Determinants of Cullin Binding Specificity Among the Three Functionally Different Drosophila melanogaster Roc Proteins via Domain Swapping

BACKGROUND: Cullin-dependent E3 ubiquitin ligases (CDL) are key regulators of protein destruction that participate in a wide range of cell biological processes. The Roc subunit of CDL contains an evolutionarily conserved RING domain that binds ubiquitin charged E2 and is essential for ubiquitylation. Drosophila melanogaster contains three highly related Roc proteins: Roc1a and Roc2, which are conserved in vertebrates, and Roc1b, which is specific to Drosophila. Our previous genetic data analyzing Roc1a and Roc1b mutants suggested that Roc proteins are functionally distinct, but the molecular basis for this distinction is not known. METHODOLOGY/PRINCIPAL FINDINGS: Using co-immunoprecipitation studies we show that Drosophila Roc proteins bind specific Cullins: Roc1a binds Cul1-4, Roc1b binds Cul3, and Roc2 binds Cul5. Through domain swapping experiments, we demonstrate that Cullin binding specificity is strongly influenced by the Roc NH(2)-terminal domain, which forms an inter-molecular beta sheet with the Cullin. Substitution of the Roc1a RING domain with that of Roc1b results in a protein with similar Cullin binding properties to Roc1a that is active as an E3 ligase but cannot complement Roc1a mutant lethality, indicating that the identity of the RING domain can be an important determinant of CDL function. In contrast, the converse chimeric protein with a substitution of the Roc1b RING domain with that of Roc1a can rescue the male sterility of Roc1b mutants, but only when expressed from the endogenous Roc1b promoter. We also identified mutations of Roc2 and Cul5 and show that they cause no overt developmental phenotype, consistent with our finding that Roc2 and Cul5 proteins are exclusive binding partners, which others have observed in human cells as well. CONCLUSIONS: The Drosophila Roc proteins are highly similar, but have diverged during evolution to bind a distinct set of Cullins and to utilize RING domains that have overlapping, but not identical, function in vivo

The RNA Polymerase PB2 Subunit of Influenza A/HongKong/156/1997 (H5N1) Restrict the Replication of Reassortant Ribonucleoprotein Complexes

BACKGROUND: Genetic reassortment plays a critical role in the generation of pandemic strains of influenza virus. The influenza virus RNA polymerase, composed of PB1, PB2 and PA subunits, has been suggested to influence the efficiency of genetic reassortment. However, the role of the RNA polymerase in the genetic reassortment is not well understood. METHODOLOGY/PRINCIPAL FINDINGS: Here, we reconstituted reassortant ribonucleoprotein (RNP) complexes, and demonstrated that the PB2 subunit of A/HongKong/156/1997 (H5N1) [HK PB2] dramatically reduced the synthesis of mRNA, cRNA and vRNA when introduced into the polymerase of other influenza strains of H1N1 or H3N2. The HK PB2 had no significant effect on the assembly of the polymerase trimeric complex, or on promoter binding activity or replication initiation activity in vitro. However, the HK PB2 was found to remarkably impair the accumulation of RNP. This impaired accumulation and activity of RNP was fully restored when four amino acids at position 108, 508, 524 and 627 of the HK PB2 were mutated. CONCLUSIONS/SIGNIFICANCE: Overall, we suggest that the PB2 subunit of influenza polymerase might play an important role for the replication of reassortant ribonucleoprotein complexes

Primary parotid gland lymphoma: a case report

<p>Abstract</p> <p>Introduction</p> <p>Mucosa associated lymphoid tissue lymphomas are the most common lymphomas of the salivary glands. The benign lymphoepithelial lesion is also a lymphoproliferative disease that develops in the parotid gland. In the present case report, we describe one case of benign lymphoepithelial lesion with a subsequent low transformation to grade mucosa associated lymphoid tissue lymphoma appearing as a cystic mass in the parotid gland.</p> <p>Case presentation</p> <p>A 78-year-old Caucasian female smoker was referred to our clinic with a non-tender left facial swelling that had been present for approximately three years. The patient underwent resection of the left parotid gland with preservation of the left facial nerve through a preauricular incision. The pathology report was consistent with a low-grade marginal-zone B-cell non-Hodgkin lymphoma (mucosa associated lymphoid tissue lymphoma) following benign lymphoepithelial lesion of the gland.</p> <p>Conclusions</p> <p>Salivary gland mucosa associated lymphoid tissue lymphoma should be considered in the differential diagnosis of cystic or bilateral salivary gland lesions. Parotidectomy is recommended in order to treat the tumor and to ensure histological diagnosis for further follow-up planning. Radiotherapy and chemotherapy should be considered in association with surgery in disseminated forms or after removal.</p

The SOCS-Box of HIV-1 Vif Interacts with ElonginBC by Induced-Folding to Recruit Its Cul5-Containing Ubiquitin Ligase Complex

The HIV-1 viral infectivity factor (Vif) protein recruits an E3 ubiquitin ligase complex, comprising the cellular proteins elongin B and C (EloBC), cullin 5 (Cul5) and RING-box 2 (Rbx2), to the anti-viral proteins APOBEC3G (A3G) and APOBEC3F (A3F) and induces their polyubiquitination and proteasomal degradation. In this study, we used purified proteins and direct in vitro binding assays, isothermal titration calorimetry and NMR spectroscopy to describe the molecular mechanism for assembly of the Vif-EloBC ternary complex. We demonstrate that Vif binds to EloBC in two locations, and that both interactions induce structural changes in the SOCS box of Vif as well as EloBC. In particular, in addition to the previously established binding of Vif's BC box to EloC, we report a novel interaction between the conserved Pro-Pro-Leu-Pro motif of Vif and the C-terminal domain of EloB. Using cell-based assays, we further show that this interaction is necessary for the formation of a functional ligase complex, thus establishing a role of this motif. We conclude that HIV-1 Vif engages EloBC via an induced-folding mechanism that does not require additional co-factors, and speculate that these features distinguish Vif from other EloBC specificity factors such as cellular SOCS proteins, and may enhance the prospects of obtaining therapeutic inhibitors of Vif function

Characterization of the Interaction of Full-Length HIV-1 Vif Protein with its Key Regulator CBFβ and CRL5 E3 Ubiquitin Ligase Components

Human immunodeficiency virus-1 (HIV-1) viral infectivity factor (Vif) is essential for viral replication because of its ability to eliminate the host's antiviral response to HIV-1 that is mediated by the APOBEC3 family of cellular cytidine deaminases. Vif targets these proteins, including APOBEC3G, for polyubiquitination and subsequent proteasome-mediated degradation via the formation of a Cullin5-ElonginB/C-based E3 ubiquitin ligase. Determining how the cellular components of this E3 ligase complex interact with Vif is critical to the intelligent design of new antiviral drugs. However, structural studies of Vif, both alone and in complex with cellular partners, have been hampered by an inability to express soluble full-length Vif protein. Here we demonstrate that a newly identified host regulator of Vif, core-binding factor-beta (CBFβ), interacts directly with Vif, including various isoforms and a truncated form of this regulator. In addition, carboxyl-terminal truncations of Vif lacking the BC-box and cullin box motifs were sufficient for CBFβ interaction. Furthermore, association of Vif with CBFβ, alone or in combination with Elongin B/C (EloB/C), greatly increased the solubility of full-length Vif. Finally, a stable complex containing Vif-CBFβ-EloB/C was purified in large quantity and shown to bind purified Cullin5 (Cul5). This efficient strategy for purifying Vif-Cul5-CBFβ-EloB/C complexes will facilitate future structural and biochemical studies of Vif function and may provide the basis for useful screening approaches for identifying novel anti-HIV drug candidates