The distributional clines in P susceptibility causing by the P family transposable element in Drosophila melanogaster population of China

BACKGROUND: Recombinant human growth hormone (rhGH) has shown beneficial effects on cardiac function after myocardial infarction (MI) in rats. High-dose angiotensin II (AT1) receptor blockade in normal rats inhibited the hypertrophic effect of growth hormone (GH), therefore we investigated whether GH effects after MI would be enhanced by giving it in sequence after remodeling had been inhibited by prior AT1 blockade (losartan, L). METHODS AND RESULTS: Rats given losartan for 10 weeks after MI followed by rhGH for 2 weeks (2 mg/kg twice a day, GH plus losartan) were compared with rats given losartan for 10 weeks followed by placebo for 2 weeks (placebo plus losartan group) and with untreated controls (n = 17-20/group). Average MI sizes and left ventricular (LV) end diastolic (ED) dimensions (echocardiography) did not differ between groups. In GH and losartan, body weight (BW) was increased but left ventricular weight (LVW)/BW was reduced, and the LV fractional shortening and LV dP/dtmax (catheter tip micromanometer) were increased compared with the control group (20.3 vs 15.4% and 5579 vs 4699 mmHg/s, respectively, P < .05). The cardiac index also was significantly increased. In the placebo plus losartan group, the LVW/BW was also reduced and the cardiac index increased versus controls. Stroke volume was increased in GH plus losartan group compared with both placebo plus losartan and controls, and the systemic vascular resistance was significantly decreased only in the GH plus losartan group. The ED posterior wall thickness (noninfarcted wall) was increased in GH plus losartan compared with both control and placebo plus losartan. Left ventricular end diastolic pressure reduction was not significant in GH plus losartan group versus controls but was reduced in placebo plus losartan group, whereas LV relaxation (tau) was improved in both groups versus control rats. Thus, persistent remodeling effects caused by prior AT1 blockade undoubtedly contributed to some responses, but short-term GH given in sequence after chronic AT1 blockade had favorable actions on the failing heart and peripheral circulation by increasing LV wall thickness with partial reversal of unfavorable remodeling, lowering of vascular resistance, improvement of LV contractility, and enhanced LV systolic function and cardiac index relatively late after experimental MI.     

Major stress protein Hsp70 interacts with NF-kB regulatory complex in human T-lymphoma cells

Utilization of psychiatric in-patient care among 537 new patients was studied in the Department of Psychiatry in Oulu, Finland, during a 3-year follow-up period. Hospitalization during the second and third years of the follow-up was predicted by hospitalization and number of emergency out-patient contacts during the first year of the study, diagnosis of functional psychosis or personality disorder, and previous in-patient care. In total, 5% of the cohort fulfilled our criteria for 'revolving-door' patients. The 'revolving-door' phenomenon was associated with in-patient care at the first contact with the psychiatric services and diagnosis of psychosis or personality disorder. In total, 2% of the cohort became long-stay hospital patients, and this was predicted by psychosis diagnosis. The clinical implications of these findings are that increased attention should be paid to the first assessment of new patients and to the interaction between psychiatric services and patients during the first year of care.     

DNA-dependent protein kinase interacts with antigen receptor response element binding proteins NF90 and NF45

The DNA-dependent protein kinase (DNA-PK) is composed of a large catalytic subunit of approximately 470 kDa (DNA-PKcs) and the DNA-binding protein, Ku. Absence of DNA-PK activity confers sensitivity to x-rays and defects in both DNA double-strand break repair and V(D)J recombination. However the precise function of DNA-PK in DNA double-strand break repair is not known. Here we show, using electrophoretic mobility shift assays, that polypeptides in a fraction purified from human cells interact with DNA-PK and stabilize the formation of a complex containing DNA-PKcs-Ku and DNA. Five polypeptides in this fraction have been identified by amino-terminal sequence analysis and/or immunoblotting. These proteins are NF90 and NF45, which are the 90- and 45-kDa subunits of a protein known to bind specifically to the antigen receptor response element of the interleukin 2 promoter, and the alpha, beta, and gamma subunits of eukaryotic translation initiation factor eIF-2. We also show that NF90, NF45, and eIF-2 beta are substrates for DNA-PK in vitro. In addition, recombinant NF90 promotes formation of a complex between DNA-PKcs, Ku, and DNA, and antibodies to recombinant NF90 or recombinant NF45 immunoprecipitate DNA-PKcs in vitro. Together, our data suggest that NF90, in complex with NF45, interacts with DNA-PKcs and Ku on DNA and that NF90 and NF45 may be important for the function of DNA-PK.     

Human xeroderma pigmentosum group A protein interacts with human replication protein A and inhibits DNA replication

Human replication protein A (RPA; also known as human single-stranded DNA binding protein, or HSSB) is a multisubunit complex involved in both DNA replication and repair. While the role of RPA in replication has been well studied, its function in repair is less clear, although it is known to be involved in the early stages of the repair process. We found that RPA interacts with xeroderma pigmentosum group A complementing protein (XPAC), a protein that specifically recognizes UV-damaged DNA. We examined the effect of this XPAC-RPA interaction on in vitro simian virus 40 (SV40) DNA replication catalyzed by the monopolymerase system. XPAC inhibited SV40 DNA replication in vitro, and this inhibition was reversed by the addition of RPA but not by the addition of DNA polymerase alpha-primase complex, SV40 large tumor antigen, or topoisomerase I. This inhibition did not result from an interaction between XPAC and single-stranded DNA (ssDNA), or from competition between RPA and XPAC for DNA binding, because XPAC does not show any ssDNA binding activity and, in fact, stimulates RPA's ssDNA binding activity. Furthermore, XPAC inhibited DNA polymerase alpha activity in the presence of RPA but not in RPA's absence. These results suggest that the inhibitory effect of XPAC on DNA replication probably occurs through its interaction with RPA.     

A novel RING finger protein, Vps8p, functionally interacts with the small GTPase, Vps21p, to facilitate soluble vacuolar protein localization

Genetic analyses of vacuolar protein sorting in Saccharomyces cerevisiae have uncovered a large number of mutants (vps) that missort and secrete soluble vacuolar hydrolases. Here we report the characterization of the gene product affected in one of these mutants, Vps8p. Polyclonal antiserum raised against a trpE-Vps8 fusion protein specifically detects a 134-kDa protein in labeled yeast cell extracts. Subcellular fractionation studies demonstrate that Vps8p is distributed between a low speed membrane pellet fraction and a high speed membrane pellet fraction. The lack of a hydrophobic domain in Vps8p suggests that Vps8p peripherally associates with a membrane(s). This association was found to depend on the function of Vps21p, a member of the Rab/Ypt/Sec4 family of small GTPases. In vps21 null mutant cells, Vps8p is found in the cytosol. In addition, overexpression of Vps21p partially suppresses a vps8 null mutant, indicating that Vps8p and Vps21p functionally interact. Vps8p contains a C-terminal cysteine-rich region that conforms to the H2 variant of the RING finger Zn2+ binding motif. Truncation of this C-terminal region partially compromises Vps8p function. While vps8 null mutant strains missort and secrete soluble vacuolar hydrolases, the integral vacuolar membrane protein, alkaline phosphatase (ALP), is sorted to the vacuole and matured normally. In addition, when vps8 mutants are combined with endocytic or late secretory pathway mutants (end3 or sec1, respectively), ALP is still delivered to the vacuole. These observations indicate that ALP is sorted to the vacuole in a Vps8p-independent manner, possibly via an alternative vesicle carrier.     

A novel nucleic acid-binding protein that interacts with human rad51 recombinase

Using the yeast two-hybrid system, we isolated a cDNA encoding a novel human protein, named Pir51, that strongly interacts with human Rad51 recombinase. Analysis in vitro confirmed the interaction between Rad51 and Pir51. Pir51 mRNA is expressed in a number of human organs, most notably in testis, thymus, colon and small intestine. The Pir51 gene locus was mapped to chromosome 12p13.1-13. 2 by fluorescence in situ hybridization. The Pir51 protein was expressed in Escherichia coli and purified to near homogeneity. Biochemical analysis shows that the Pir51 protein binds both single- and double-stranded DNA, and is capable of aggregating DNA. The protein also binds RNA. The Pir51 protein may represent a new member of the multiprotein complexes postulated to carry out homologous recombination and DNA repair in mammalian cells.     

Expression of an antigen homologous to the human CO17-1A/GA733 colon cancer antigen in animal tissues

The CO17-1A/GA733 antigen is associated with human carcinomas and some normal epithelial tissues. This antigen has shown promise as a target in approaches to passive and active immunotherapy of colorectal cancer. The relevance of animal models for studies of immunotherapy targeting this antigen in patients is dependent on the expression of the antigen on normal animal tissues. Immunohistoperoxidase staining with polyclonal rabbit antibodies to the human antigen revealed the human homologue on normal small intestine, colon and liver of mice, rats and non-human primates, whereas mouse monoclonal antibodies to the CO17-1A or GA733 epitopes on the human antigen did not detect the antigen. Polyclonal rabbit antibodies, elicited by the murine antigen homologue derived from recombinant baculovirus-infected insect cells, immunoprecipitated the antigen from mouse small intestine, colon, stomach, kidney and lung. The isolated recombinant murine protein bound polyclonal, but not monoclonal, antibodies to the human CO17-1A/GA733 antigen, and recombinant human antigen bound polyclonal antibodies elicited by the murine antigen homologue. Thus, the antigen homologue expressed by animal tissues is similar, but not identical, to the human antigen. These results have important implications for experimental active and passive immunotherapy targeting the CO17-1A/GA733 antigen.     

Cloning and characterization of NE-dlg: a novel human homolog of the Drosophila discs large (dlg) tumor suppressor protein interacts with the APC protein

We have cloned a cDNA for a novel human homolog of the Drosophila discs large (dig) tumor suppressor protein, termed NE-dlg (neuronal and endocrine dig). Northern blot analysis revealed that the gene is highly expressed in neuronal and endocrine tissues. Fluorescence in situ hybridization (FISH) and radiation hybrid mapping studies localized the NE-dlg gene to chromosome Xq13. We also found that the NE-dlg gene encoded a 100 kDa protein. Immunolocalization studies using an NE-dlg antibody showed that the protein tended to be expressed in non-proliferating cells, such as neurons, cells in Langerhans islets of the pancreas, myocytes of the heart muscles, and the prickle and functional layer cells of the esophageal epithelium. Proliferative cells, including various cultured cancer cell lines and basal cells in the esophageal epithelium, showed little expression of the NE-dlg protein. In addition, yeast two-hybrid screening and in vitro binding assays revealed that the NE-dlg interacted with the carboxyl-terminal region of the APC tumor suppressor protein. These data suggest that NE-dlg negatively regulates cell proliferation through its interaction with the APC protein.     

Identification of human T cell epitopes in the Mycobacterium leprae heat shock protein 70-kD antigen

In a number of pathogens, heat shock proteins (hsp) stimulate humoral and cellular immune responses despite significant sequence identity with host hsp. The 70-kD hsp of Mycobacterium leprae, which shares 47% identity with human hsp70 at the protein level, elicited a T cell response in most Myco. bovis (bacille Calmette-Guérin (BCG)) vaccinees as well as leprosy and tuberculosis patients and their contacts. In order to locate T cell epitopes, DNA fragments encoding portions of the 70-kD hsp were expressed in the vector pGEX-2T and tested for T cell reactivity in an in vitro proliferative assay. Cultures of peripheral blood mononuclear cells (PBMC) from BCG vaccinees indicated that the C-terminal half of the molecule contained multiple T cell epitopes, as the T cells from a majority of Myco. leprae hsp70-reactive individuals responded to C-344. Lower proportions of patients with paucibacillary leprosy (36%) and tuberculosis patients (16%) responded to C-344. The smaller C-142 fragment which includes the terminal 70 residues unique to Myco. leprae and is the target for the human antibody response elicited a cellular response in few patients and no vaccinees. In order to map T cell epitopes, two series of synthetic peptides encompassing the region 278-502 were prepared. Using overlapping 12mer and 20mer peptides, this region of the molecule was found to contain several potential T cell epitopes. The longer peptides gave a clearer indication of reactive sequences including regions of the molecule which were not identified with the 12mer peptides. Fine mapping of reactive peptide pools using the 12mer peptides identified two T cell epitopes. Although both were located in regions of the molecule shared with Myco. tuberculosis, one appeared to be cross-reactive with the equivalent human sequence, and thus has the potential to initiate autoimmune responses.     

A protein required for nuclear-protein import, Mog1p, directly interacts with GTP-Gsp1p, the Saccharomyces cerevisiae ran homologue

We previously isolated 25 temperature-sensitive gsp1 alleles of Saccharomyces cerevisiae Ran homologue, each of which possesses amino acid changes that differ from each other. We report here isolation of three multicopy suppressors-PDE2, NTF2, and a gene designated MOG1-all of which rescued a growth defect of these gsp1 strains. The gsp1 suppression occurred even in the absence of GSP2, another S. cerevisiae GSP1-like gene. Previously, NTF2 was reported to suppress gsp1 but not PDE2. Mog1p, with a calculated molecular mass of 24 kDa, was found to be encoded by the yeast ORF YJR074W. Both MOG1 and NTF2 suppressed a series of gsp1 alleles with similar efficiency, and both suppressed gsp1 even with a single gene dose. Consistent with the high efficiency of gsp1 suppression, Mog1p directly bound to GTP, but not to GDP-Gsp1p. The disruption of MOG1 made yeast temperature-sensitive for growth. Deltamog1, which was suppressed by overexpression of NTF2, was found to have a defect in both classic and nonclassic nuclear localization signal-dependent nuclear-protein imports, but not in mRNA export. Thus, Mog1p, which was localized in the nucleus, is a Gsp1p-binding protein involved in nuclear-protein import and that functionally interacts with Ntf2p. Furthermore, the finding that PDE2 suppressed both gsp1 and rna1-1 indicates that the Ran GTPase cycle is regulated by the Ras-cAMP pathway.     

Huntingtin-associated protein 1 (HAP1) interacts with the p150Glued subunit of dynactin

Huntington's disease (HD) is an inherited neurodegenerative disease caused by expansion of a polyglutamine repeat in the HD protein huntingtin. Huntingtin's localization within the cell includes an association with cytoskeletal elements and vesicles. We previously identified a protein (HAP1) which binds to huntingtin in a glutamine repeat length-dependent manner. We now report that HAP1 interacts with cytoskeletal proteins, namely the p150 Glued subunit of dynactin and the pericentriolar protein PCM-1. Structural predictions indicate that both HAP1 and the interacting proteins have a high probability of forming coiled coils. We examined the interaction of HAP1 with p150 Glued . Binding of HAP1 to p150 Glued (amino acids 879-1150) was confirmed in vitro by binding of p150 Glued to a HAP1-GST fusion protein immobilized on glutathione-Sepharose beads. Also, HAP1 co-immunoprecipitated with p150 Glued from brain extracts, indicating that the interaction occurs in vivo . Like HAP1, p150 Glued is highly expressed in neurons in brain and both proteins are enriched in a nerve terminal vesicle-rich fraction. Double label immunofluorescence experiments in NGF-treated PC12 cells using confocal microscopy revealed that HAP1 and p150 Glued partially co-localize. These results suggest that HAP1 might function as an adaptor protein using coiled coils to mediate interactions among cytoskeletal, vesicular and motor proteins. Thus, HAP1 and huntingtin may play a role in vesicle trafficking within the cell and disruption of this function could contribute to the neuronal dysfunction and death seen in HD.     

A sequence in the N-terminal region of human uracil-DNA glycosylase with homology to XPA interacts with the C-terminal part of the 34-kDa subunit of replication protein A

Uracil-DNA glycosylase releases free uracil from DNA and initiates base excision repair for removal of this potentially mutagenic DNA lesion. Using the yeast two-hybrid system, human uracil-DNA glycosylase encoded by the UNG gene (UNG) was found to interact with the C-terminal part of the 34-kDa subunit of replication protein A (RPA2). No interaction with RPA4 (a homolog of RPA2), RPA1, or RPA3 was observed. A sandwich enzyme-linked immunosorbent assay with trimeric RPA and the two-hybrid system both demonstrated that the interaction depends on a region in UNG localized between amino acids 28 and 79 in the open reading frame. In this part of UNG a 23-amino acid sequence has a significant homology to the RPA2-binding region of XPA, a protein involved in damage recognition in nucleotide excision repair. Trimeric RPA did not enhance the activity of UNG in vitro on single- or double-stranded DNA. A part of the N-terminal region of UNG corresponding in size to the complete presequence was efficiently removed by proteinase K, leaving the proteinase K-resistant compact catalytic domain intact and fully active. These results indicate that the N-terminal part constitutes a separate structural domain required for RPA binding and suggest a possible function for RPA in base excision repair.     

Induction of homologous immune response to Rickettsia tsutsugamushi Boryong with a partial 56-kilodalton recombinant antigen fused with the maltose-binding protein MBP-Bor56

Although the 56-kDa protein of Rickettsia tsutsugamushi has been presumed to play important roles in generating protective immunity against scrub typhus, studies of this protein have been impeded. We used the recombinant 56-kDa protein of R. tsutsugamushi Boryong fused with the maltose-binding protein of Escherichia coli (MBP-Bor56) to analyze its ability to induce protective immunity in a C3H/HeDub murine model. Intraperitoneal immunization of mice with MBP-Bor56 resulted in an increase in the 50% minimal lethal dose of more than 160 times compared with that for the control mice. Splenic mononuclear cells from the mice immunized with MBP-Bor56 showed a dose-dependent pattern of lymphocyte proliferation response and secreted gamma interferon and interleukin-2 when stimulated with irradiated R. tsutsugamushi Boryong, which is a cytokine profile of Th1 cells. High titers of antibody to R. tsutsugamushi were also demonstrated by indirect immunofluorescent-antibody testing. These findings suggest that the 56-kDa protein of R. tsutsugamushi is one of the candidates for a vaccine against scrub typhus.     

Mas6p can be cross-linked to an arrested precursor and interacts with other proteins during mitochondrial protein import

Mas6p is an integral membrane protein of the yeast mitochondrial inner membrane, which is essential for mitochondrial protein import (1). To determine whether Mas6p is directly involved in recognizing precursors or translocating them across the inner membrane, we asked if Mas6p was in close proximity to precursor proteins being imported into mitochondria. We report here that Mas6p can be chemically cross-linked to an imported protein arrested in transit through the mitochondrial inner membrane. Antiserum to Mas6p specifically immunoprecipitates one of several different mitochondrial proteins that are cross-linked to blocked precursors. Our results strongly suggest that Mas6p physically interacts with precursors during their translocation into the matrix. In addition, at least two other mitochondrial proteins that are each cross-linked to arrested precursors can be coimmunoprecipitated along with Mas6p under non-denaturing conditions. These observations provide evidence for a complex of proteins including Mas6p, each of which interacts with mitochondrial precursors during import.     

Interaction of the p53-regulated protein Gadd45 with proliferating cell nuclear antigen

GADD45 is a ubiquitously expressed mammalian gene that is induced by DNA damage and certain other stresses. Like another p53-regulated gene, p21WAF1/CIP1, whose product binds to cyclin-dependent kinases (Cdk's) and proliferating cell nuclear antigen (PCNA), GADD45 has been associated with growth suppression. Gadd45 was found to bind to PCNA, a normal component of Cdk complexes and a protein involved in DNA replication and repair. Gadd45 stimulated DNA excision repair in vitro and inhibited entry of cells into S phase. These results establish GADD45 as a link between the p53-dependent cell cycle checkpoint and DNA repair.