Oligomerization characteristics of cysteine string protein

CSP function is vital to synaptic transmission, however; the precise nature of its role remains controversial. Conflicting reports support either a role for CSP: (i) in exocytosis or (ii) in the regulation of transmembrane calcium fluxes. Here we have examined the self-association of CSP to form oligomers that are stable upon SDS-PAGE. To understand the structural requirements for CSP self-association a series of CSP deletion mutants were constructed, expressed, and purified. This analysis revealed an interesting pattern of oligomerization. Amino acids between 83 and 136 were observed to be important for self-association. The recombinant CSP oligomers as well as the CSP monomers directly associate with Ni(2+)-NTA agarose. Thus CSP-CSP interactions may be an important consideration for current working models of CSP chaperone activity at the synapse.     

Involvement of very short DNA tandem repeats and the influence of the RAD52 gene on the occurrence of deletions in Saccharomyces cerevisiae

Chromosomal rearrangements, such as deletions, duplications, or Ty transposition, are rare events. We devised a method to select for such events as Ura(+) revertants of a particular ura2 mutant. Among 133 Ura(+) revertants, 14 were identified as the result of a deletion in URA2. Of seven classes of deletions, six had very short regions of identity at their junctions (from 7 to 13 bp long). This strongly suggests a nonhomologous recombination mechanism for the formation of these deletions. The total Ura(+) reversion rate was increased 4.2-fold in a rad52Delta strain compared to the wild type, and the deletion rate was significantly increased. All the deletions selected in the rad52Delta context had microhomologies at their junctions. We propose two mechanisms to explain the occurrence of these deletions and discuss the role of microhomology stretches in the formation of fusion proteins.     

Genomic exploration of the hemiascomycetous yeasts: 8. Zygosaccharomyces rouxii

This paper reports the genomic analysis of strain CBS732 of Zygosaccharomyces rouxii, a homothallic diploid yeast. We explored the sequences of 4934 random sequencing tags of about 1 kb in size and compared them to the Saccharomyces cerevisiae gene products. Approximately 2250 nuclear genes, 57 tRNAs, the rDNA locus, the endogenous pSR1 plasmid and 15 mitochondrial genes were identified. According to 18S and 25S rRNA cladograms and to synteny analysis, Z. rouxii could be placed among the S. cerevisiae sensu lato yeasts.     

Condensation by DNA looping facilitates transfer of large DNA molecules into mammalian cells

Experimental studies of complete mammalian genes and other genetic domains are impeded by the difficulty of introducing large DNA molecules into cells in culture. Previously we have shown that GST–Z2, a protein that contains three zinc fingers and a proline-rich multimerization domain from the polydactyl zinc finger protein RIP60 fused to glutathione S-transferase (GST), mediates DNA binding and looping in vitro. Atomic force microscopy showed that GST–Z2 is able to condense 130–150 kb bacterial artificial chromosomes (BACs) into protein–DNA complexes containing multiple DNA loops. Condensation of the DNA loops onto the Z2 protein–BAC DNA core complexes with cationic lipid resulted in particles that were readily transferred into multiple cell types in culture. Transfer of total genomic linear DNA containing amplified DHFR genes into DHFR^– cells by GST–Z2 resulted in a 10-fold higher transformation rate than calcium phosphate co-precipitation. Chinese hamster ovarian cells transfected with a BAC containing the human TP53 gene locus expressed p53, showing native promoter elements are active after GST–Z2-mediated gene transfer. Because DNA condensation by GST–Z2 does not require the introduction of specific recognition sequences into the DNA substrate, condensation by the Z2 domain of RIP60 may be used in conjunction with a variety of other agents to provide a flexible and efficient non-viral platform for the delivery of large genes into mammalian cells.     

The Plasmodium falciparum Ca(2+)-ATPase PfATP6: insensitive to artemisinin, but a potential drug target

The disease malaria, caused by the parasite Plasmodium falciparum, remains one of the most important causes of morbidity and mortality in sub-Saharan Africa. In the absence of an efficient vaccine, the medical treatment of malaria is dependent on the use of drugs. Since artemisinin is a powerful anti-malarial drug which has been proposed to target a particular Ca2+-ATPase (PfATP6) in the parasite, it has been important to characterize the molecular properties of this enzyme. PfATP6 is a 139?kDa protein composed of 1228 amino acids with a 39% overall identity with rabbit SERCA1a (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 1a). PfATP6 conserves all sequences and motifs that are important for the function and/or structure of a SERCA, such as two high-affinity Ca2+-binding sites, a nucleotide-binding site and a phosphorylation site. We have been successful in isolating PfATP6 after heterologous expression in yeast and affinity chromatography in a pure, active and stable detergent-solubilized form. With this preparation, we have characterized and compared with the eukaryotic SERCA1a isoform the substrate (Ca2+ and ATP) -dependency for PfATP6 activity as well as the specific inhibition/interaction of the protein with drugs. Our data fully confirm that PfATP6 is a SERCA, but with a distinct pharmacological profile: compared with SERCA1a, it has a lower affinity for thapsigargin and much higher affinity for cyclopiazonic acid. On the other hand, we were not able to demonstrate any inhibition by artemisinin and were also not able to monitor any binding of the drug to the isolated enzyme. Thus it is unlikely that PfATP6 plays an important role as a target for artemisinin in the parasite P. falciparum.     

Inhibition by 4-hydroxynonenal (HNE) of Ca2+ transport by SERCA1a: Low concentrations of HNE open protein-mediated leaks in the membrane

Exposure of sarcoplasmic reticulum membranes to 4-hydroxy-2-nonenal (HNE) resulted in inhibition of the maximal ATPase activity and Ca²⁺ transport ability of SERCA1a, the Ca²⁺ pump in these membranes. The concomitant presence of ATP significantly protected SERCA1a ATPase activity from inhibition. ATP binding and phosphoenzyme formation from ATP were reduced after treatment with HNE, whereas Ca²⁺ binding to the high affinity sites was altered to a lower extent. HNE reacted with SH groups, some of which were identified by MALDI-TOF mass spectrometry, and competition studies with FITC indicated that HNE also reacted with Lys⁵¹⁵ within the nucleotide binding pocket of SERCA1a. A remarkable fact was that both the steady-state ability of SR vesicles to sequester Ca²⁺ as well as the ATPase activity of SR membranes in the absence of added ionophore or detergent were sensitive to concentrations of HNE much smaller than those which affected the maximal ATPase activity of SERCA1a. This was due to increase in the passive permeability to Ca²⁺ of HNE-treated SR vesicles, an increase in permeability which did not arise from alteration of the lipid component of these vesicles. Judging from immunodetection with an anti-HNE antibody, this HNE-dependent increase in permeability probably arose from modification of proteins of about 150–170 kDa, present in very low abundance in longitudinal SR membranes (and in slightly larger abundance in SR terminal cisternae). HNE-induced promotion, via these proteins, of Ca²⁺ leakage pathways, might be involved in the general toxic effects of HNE.     

Detection of nascent and/or mature forms of oviductin in the female reproductive tract and post-ovulatory oocytes by use of a polyclonal antibody against recombinant hamster oviductin.

Oviductins belong to a family of glycoproteins that have been suggested to play several roles during the early processes of reproduction. Recently, a polyclonal antibody was raised against recombinant hamster oviductin (rhaOv(m)). Here the anti-rhaOv(m) antibody was used to investigate the sites of localization of oviductin in the female golden hamster. In the hamster oviduct, immunolabeling was restricted to the content of the Golgi saccules and secretory granules of the non-ciliated oviduct cells. After its release into the lumen, oviductin becomes associated with the zona pellucida of post-ovulatory oocytes. In unfertilized oocytes, oviductin was also detected in membrane invaginations along the oolemma and in some vesicles within the ooplasm. Furthermore, oviductin was detected over the microvilli and within multivesicular bodies of uterine epithelial cells. Western blotting analysis revealed the presence of oviductin in the hamster oviduct but not in the uterus or ovary. In the oviduct, the anti-rhaOv(m) antibody detected a polydispersed band corresponding to native oviductin (160-350 kD) and several lower molecular weight bands (<100 kD) corresponding to nascent and partially glycosylated forms of oviductin. The anti-rhaOv(m) antibody provides an additional tool for investigation into the cytochemical and biochemical properties of different forms of hamster oviductin in the female reproductive tract.     

CD4+CD25+ T regulatory cells dominate multiple immune evasion mechanisms in early but not late phases of tumor development in a B cell lymphoma model

Tumors use a complex set of direct and indirect mechanisms to evade the immune system. Naturally arising CD4(+)CD25(+)FoxP3(+) T regulatory (Treg) cells have been implicated recently in tumor immune escape mechanism, but the relative contribution of these cells to overall tumor progression compared with other immune evasion mechanisms remains to be elucidated. Using the A20 B cell lymphoma as a transplantable tumor model, we demonstrate that this tumor employs multiple direct (expression of immunoinhibitory molecule PD-L1, IDO, and IL-10, and lack of expression of CD80 costimulatory molecule) and indirect (down-regulation of APC function and induction of Treg cells) immune evasion mechanisms. Importantly, Treg cells served as the dominant immune escape mechanism early in tumor progression because the physical elimination of these cells before tumor challenge resulted in tumor-free survival in 70% of mice, whereas their depletion in animals with established tumors had no therapeutic effect. Therefore, our data suggest that Treg cells may serve as an important therapeutic target for patients with early stages of cancer and that more vigorous combinatorial approaches simultaneously targeting multiple immune evasion as well as immunosurveillance mechanisms for the generation of a productive immune response against tumor may be required for effective immunotherapy in patients with advanced disease.     

Comparative analysis of mutants in the mycothiol biosynthesis pathway in Mycobacterium smegmatis

The role of mycothiol in mycobacteria was examined by comparative analysis of mutants disrupted in the four known genes encoding the protein machinery needed for mycothiol biosynthesis. These mutants were sensitive to acid stress, antibiotic stress, alkylating stress, and oxidative stress indicating that mycothiol and mycothiol-dependent enzymes protect the mycobacterial cell against attack from various different types of stresses and toxic agents.     

Use of glycerol-containing media to study the intrinsic fluorescence properties of detergent-solubilized native or expressed SERCA1a

Rapid irreversible inactivation of Ca (2+)-free states of detergent-solubilized SERCA1a (sarco-endoplasmic reticulum calcium ATPase 1a) has so far prevented the use of Trp fluorescence for functional characterization of this ATPase after its solubilization in various detergents. Here we show that using 20-40% glycerol for protection makes this fluorescence characterization possible. Most of the ligand-induced Trp fluorescence changes previously demonstrated to occur for SERCA1a embedded in native sarcoplasmic reticulum membranes were observed in the combined presence of glycerol and detergent, although the results greatly depended on the detergent used, namely, octaethylene glycol mono- n-dodecyl ether (C 12E 8) or dodecyl maltoside (DDM). In particular, at pH 6, we found a C 12E 8-dependent unexpectedly huge reduction in SERCA1a affinity for Ca (2+). We suggest that a major reason for the different effects of the two detergents is that high concentrations of C 12E 8, but not of DDM, slow down the E2 to E1 transition in solubilized and delipidated SERCA1a. Independently of the characterization of the specific effects of various detergents on SR vesicles, our results open the way to functional characterization by Trp fluorescence of heterologously expressed and purified mutants of SERCA1a in the presence of detergent, without their preliminary reconstitution into liposomes. As an example, we used the E309Q mutant to demonstrate our previous suspicion that Ca (2+) binding to Site I of SERCA1a in fact slightly reduces Trp fluorescence, and consequently that the rise in this fluorescence generally observed when two Ca (2+) ions bind to WT SERCA1a mainly reflects Ca (2+) binding at Site II of SERCA1a.