The dimerization domain of the HIV-1 capsid protein binds a capsid protein-derived peptide: a biophysical characterization

The type 1 HIV presents a conical capsid formed by approximately 1500 units of the capsid protein, CA. Homodimerization of CA via its C-terminal domain, CA-C, constitutes a key step in virion assembly. CA-C dimerization is largely mediated by reciprocal interactions between residues of its second alpha-helix. Here, we show that an N-terminal-acetylated and C-terminal-amidated peptide, CAC1, comprising the sequence of the CA-C dimerization helix plus three flanking residues at each side, is able to form a complex with the entire CA-C domain. Thermal denaturation measurements followed by circular dichroism (CD), NMR, and size-exclusion chromatography provided evidence of the interaction between CAC1 and CA-C. The apparent dissociation constant of the heterocomplex formed by CA-C and CAC1 was determined by several biophysical techniques, namely, fluorescence (using an anthraniloyl-labeled peptide), affinity chromatography, and isothermal titration calorimetry. The three techniques yielded similar values for the apparent dissociation constant, in the order of 50 microM. This apparent dissociation constant was only five times higher than was the dissociation constant of both CA-C and the intact capsid protein homodimers (10 microM).     

Kinetochore Localization of Spindle Checkpoint Proteins: Who Controls Whom?

The spindle checkpoint prevents anaphase onset until all the chromosomes have successfully attached to the spindle microtubules. The mechanisms by which unattached kinetochores trigger and transmit a primary signal are poorly understood, although it seems to be dependent at least in part, on the kinetochore localization of the different checkpoint components. By using protein immunodepletion and mRNA translation in Xenopus egg extracts, we have studied the hierarchic sequence and the interdependent network that governs protein recruitment at the kinetochore in the spindle checkpoint pathway. Our results show that the first regulatory step of this cascade is defined by Aurora B/INCENP complex. Aurora B/INCENP controls the activation of a second regulatory level by inducing at the kinetochore the localization of Mps1, Bub1, Bub3, and CENP-E. This localization, in turn, promotes the recruitment to the kinetochore of Mad1/Mad2, Cdc20, and the anaphase promoting complex (APC). Unlike Aurora B/INCENP, Mps1, Bub1, and CENP-E, the downstream checkpoint protein Mad1 does not regulate the kinetochore localization of either Cdc20 or APC. Similarly, Cdc20 and APC do not require each other to be localized at these chromosome structures. Thus, at the last step of the spindle checkpoint cascade, Mad1/Mad2, Cdc20, and APC are recruited at the kinetochores independently from each other.     

Synthesis and antiprotozoal evaluation of benzothiazolopyrroloquinoxalinones,analogues of kuanoniamine A

Boc-aminoethylindoloquinone 8, a key intermediate for the building of pentacyclic quinoneimines, analogues of kuanoniamine A, was synthesized by alkylation of 4,7-dimethoxyindole 3 with 1,2-dibromoethane followed by transformation into azide, reduction of the latter with trimethylphosphine in the presence of 2-(tert-butoxycarbonyloximino)-2-phenylacetonitrile and oxydative demethylation of the Boc-amine 6 with silver(II) oxide. Quinone 8 was then treated in situ with the thiazole o-quinodimethane 10 to afford a regioisomeric mixture of the tetracyclic quinones 11. Treatment of the mixture with trifluoroacetic acid and molecular sieves 4-A provided the corresponding quinoneimines 12. Separation of the regioisomers was performed by preparative thin-layer chromatography on silica gel. The structural assignment was made by 2D 1H-13C HMBC correlations performed on the less polar regioisomer 12b. In vitro anti-leishmanial assays showed that the tested compounds possess a good potency towards two Leishmania sp. as well as against a virulent strain of Toxoplasma gondii and without any cytotoxicity against THP-1 cells.     

CD4+/CD25+ regulatory cells inhibit activation of tumor-primed CD4+ T cells with IFN-gamma-dependent antiangiogenic activity, as well as long-lasting tumor immunity elicited by peptide vaccination

CD25(+) regulatory T (T reg) cells suppress the activation/proliferation of other CD4(+) or CD8(+) T cells in vitro. Also, down-regulation of CD25(+) T reg cells enhance antitumor immune responses. In this study, we show that depletion of CD25(+) T reg cells allows the host to induce both CD4(+) and CD8(+) antitumoral responses following tumor challenge. Simultaneous depletion of CD25(+) and CD8(+) cells, as well as adoptive transfer experiments, revealed that tumor-specific CD4(+) T cells, which emerged in the absence of CD25(+) T reg cells, were able to reject CT26 colon cancer cells, a MHC class II-negative tumor. The antitumoral effect mediated by CD4(+) T cells was dependent on IFN-gamma production, which exerted a potent antiangiogenic activity. The capacity of the host to mount this antitumor response is lost once the number of CD25(+) T reg cells is restored over time. However, CD25(+) T reg cell depletion before immunization with AH1 (a cytotoxic T cell determinant from CT26 tumor cells) permits the induction of a long-lasting antitumoral immune response, not observed if immunization is conducted in the presence of regulatory cells. A study of the effect of different levels of depletion of CD25(+) T reg cells before immunization with the peptide AH1 alone, or in combination with a Th determinant, unraveled that Th cells play an important role in overcoming the suppressive effect of CD25(+) T reg on the induction of long-lasting cellular immune responses.     

Interaction of nucleoplasmin with core histones

Nucleoplasmin is one of the most abundant proteins in Xenopus laevis oocytes, and it has been involved in the chromatin remodeling that takes place immediately after fertilization. This molecule has been shown to be responsible for the removal of the sperm-specific proteins and deposition of somatic histones onto the male pronuclear chromatin. To better understand the latter process, we have used sedimentation velocity, sedimentation equilibrium, and sucrose gradient fractionation analysis to show that the pentameric form of nucleoplasmin binds to a histone octamer equivalent consisting of equal amounts of the four core histones, H2A, H2B, H3, and H4, without any noticeable preference for any of these proteins. Removal of the histone N-terminal "tail" domains or the major C-terminal polyglutamic tracts of nucleoplasmin did not alter these binding properties. These results indicate that interactions other than those electrostatic in nature (likely hydrophobic) also play a critical role in the formation of the complex between the negatively charged nucleoplasmin and positively charged histones. Although the association of histones with nucleoplasmin may involve some ionic interactions, the interaction process is not electrostatically driven.     

Cooperative partition model of nystatin interaction with phospholipid vesicles

Nystatin is a membrane-active polyene antibiotic that is thought to kill fungal cells by forming ion-permeable channels. In this report we have investigated nystatin interaction with phosphatidylcholine liposomes of different sizes (large and small unilamellar vesicles) by time-resolved fluorescence measurements. Our data show that the fluorescence emission decay kinetics of the antibiotic interacting with gel-phase 1,2-dipalmitoyl-sn-glycero-3-phosphocholine vesicles is controlled by the mean number of membrane-bound antibiotic molecules per liposome, . The transition from a monomeric to an oligomeric state of the antibiotic, which is associated with a sharp increase in nystatin mean fluorescence lifetime from approximately 7-10 to 35 ns, begins to occur at a critical concentration of 10 nystatin molecules per lipid vesicle. To gain further information about the transverse location (degree of penetration) of the membrane-bound antibiotic molecules, the spin-labeled fatty acids (5- and 16-doxyl stearic acids) were used in depth-dependent fluorescence quenching experiments. The results obtained show that monomeric nystatin is anchored at the phospholipid/water interface and suggest that nystatin oligomerization is accompanied by its insertion into the membrane. Globally, the experimental data was quantitatively described by a cooperative partition model which assumes that monomeric nystatin molecules partition into the lipid bilayer surface and reversibly assemble into aggregates of 6 +/- 2 antibiotic molecules.     

A novel preclinical model of human malignant melanoma utilizing bioreactor rotating-wall vessels

Malignant melanoma poses a serious health risk which is becoming more crucial as the incidence of this disease steadily increases. The development of appropriate in vitro models that reflect the in vivo tumor environment is a key factor for the study of this malignancy. The local tumor microenvironment plays a critical role in the ability of tumor cells to proliferate and metastasize. While interactions among various cell types are known to be important for tumor growth, most in vitro models utilize only tumor cells, ignoring the importance of tumor-stroma interactions, as well as the contribution of immune cells, which may be important for potential therapies. In addition, the cellular architecture found in vivo, known to be involved in changes in gene expression, is not reflected in standard two-dimensional culture systems. In this study, we have utilized rotating-vessel bioreactors to culture minced human melanoma specimens, allowing the culture of three-dimensional structures which reflect the cellular architecture and heterogeneous composition of the tumor site in vivo. The viability of the pieces in culture can be maintained for 1-2 wk. Immunohistochemical analysis shows multiple cellular types similar to the in vivo situation. Therefore, this system provides a unique model of human melanoma that mimics the in vivo tumor environment much more closely than current culture methods. This novel system may be utilized to determine the mechanism of action of current therapy protocols, as well as to develop new treatment regimens.     

Successful immunotherapy with matrix metalloproteinase-derived peptides in adjuvant arthritis depends on the timing of peptide administration

We have recently found that matrix metalloproteinases (MMPs) are targets for T-cell and B-cell reactivity in experimental arthritis. In the present article, we investigate whether modulation of MMP-specific T-cell responses could influence the course of adjuvant arthritis (AA). Lewis rats were treated nasally with MMP peptides prior to or after AA induction. Administration of the MMP-10 or the MMP-16 peptide prior to AA induction reduced the arthritic symptoms. In contrast, administration of the MMP-10 peptide after AA induction aggravated the arthritic symptoms. The present study shows the possible usefulness of MMP peptides for immunotherapy. However, a clear understanding of proper timing of peptide administration is crucial for the development of such therapies.     

Proteomic characterization of the small subunit of Chlamydomonas reinhardtii chloroplast ribosome: identification of a novel S1 domain-containing protein and unusually large orthologs of bacterial S2, S3, and S5

To understand how chloroplast mRNAs are translated into functional proteins, a detailed understanding of all of the components of chloroplast translation is needed. To this end, we performed a proteomic analysis of the plastid ribosomal proteins in the small subunit of the chloroplast ribosome from the green alga Chlamydomonas reinhardtii. Twenty proteins were identified, including orthologs of Escherichia coli S1, S2, S3, S4, S5, S6, S7, S9, S10, S12, S13, S14, S15, S16, S17, S18, S19, S20, and S21 and a homolog of spinach plastid-specific ribosomal protein-3 (PSRP-3). In addition, a novel S1 domain-containing protein, PSRP-7, was identified. Among the identified proteins, S2 (57 kD), S3 (76 kD), and S5 (84 kD) are prominently larger than their E. coli or spinach counterparts, containing N-terminal extensions (S2 and S5) or insertion sequence (S3). Structural predictions based on the crystal structure of the bacterial 30S subunit suggest that the additional domains of S2, S3, and S5 are located adjacent to each other on the solvent side near the binding site of the S1 protein. These additional domains may interact with the S1 protein and PSRP-7 to function in aspects of mRNA recognition and translation initiation that are unique to the Chlamydomonas chloroplast.