Interactions of MCP1 with components of the replication machinery in mammalian cells

Eukaryotic DNA replication starts with the assembly of a pre-replication complex (pre-RC) at replication origins. We have previously demonstrated that Metaphase Chromosome Protein 1 (MCP1) is involved in the early events of DNA replication. Here we show that MCP1 associates with proteins that are required for the establishment of the pre-replication complex. Reciprocal immunoprecipitation analysis showed that MCP1 interacted with Cdc6, ORC2, ORC4, MCM2, MCM3 and MCM7, with Cdc45 and PCNA. Immunofluorescence studies demonstrated the co-localization of MCP1 with some of those proteins. Moreover, biochemical studies utilizing chromatin-immunoprecipitation (ChIP) revealed that MCP1 preferentially binds replication initiation sites in human cells. Interestingly, although members of the pre-RC are known to interact with some hallmarks of heterochromatin, our co-immunoprecipitation and immunofluorescence analyses showed that MCP1 did not interact and did not co-localize with heterochromatic proteins including HP1β and MetH3K9. These observations suggest that MCP1 is associated with replication factors required for the initiation of DNA replication and binds to the initiation sites in loci that replicate early in S-phase. In addition, immunological assays revealed the association of MCP1 forms with histone H1 variants and mass spectrometry analysis confirmed that MCP1 peptides share common sequences with H1.2 and H1.5 subtypes.     

Valuation,Categories and Attributes

Existing research on categories has only examined indirectly the value associated with being a member of a category relative to the value of the set of attributes that determine membership in that category. This study uses survey data to analyze consumers'' preferences for the "organic” label versus for the attributes underlying that label. We found that consumers generally preferred products with the category label to those with the attributes required for the organic label but without the label. We also found that the value accorded to the organic label increased with the number of attributes that an individual associated with the category. Category membership nevertheless still had greater value than even that of the sum of the attributes associated with it.     

Lectin histochemistry of mouse vagina during the estrous cycle

Estrous cycle-related histochemical changes in the vaginal epithelium of sexually mature female mice were studied with 30 fluorescein isothiocyanate (FITC)-labeled lectins. On the basis of the staining pattern the lectins were divided into five groups: I, seventeen lectins that reacted with mucinous surface layer of proestrus. This group comprised two subgroups: Ia, seven lectins that reacted exclusively with the mucinous layer, and Ib, ten lectins that reacted with mucinous cells and the underlying squamous epithelium of proestrus; II, two lectins that reacted with squamous epithelium of proestrus only but were unreactive with mucinous cells; III, three lectins that reacted in a phase-specific manner with squamous epithelium; IV, six lectins that showed increased luminal surface reactivity in diestrus and/or metestrus; and V, eleven lectins that were unreactive with vaginal epithelium. These data indicate that the cyclic changes in the morphology of the vaginal epithelium are accompanied by distinct lectin reactivity patterns.     

SecDFyajC forms a heterotetrameric complex with YidC

The Escherichia coli preprotein translocase is composed of a "preprotein conducting channel" domain that consists of the peripherally bound translocation ATPase SecA and the heterotrimeric SecYEG membrane protein complex. SecD, SecF, and YajC form another heterotrimeric complex that can associate with the SecYEG complex. YidC is an essential membrane protein that plays a role in the integration of newly synthesized membrane proteins, and has been shown to co-purify with SecYEG when all translocase components are overproduced. Here, we demonstrate that under conditions that YidC co-purifies with overproduced SecDFyajC it does not co-purify with overproduced SecYEG. Moreover, this interaction of YidC with the SecDFyajC complex is also found at chromosomal protein levels of SecD, SecF and YajC. Closer examination of the SecDFyajC-YidC complex showed that YidC binds to SecD and SecF, whereas YajC interacts only with SecF. As SecF and YajC have previously been shown to interact with SecY, we propose that these two proteins link the heterotetrameric SecDFyajC-YidC complex to the SecYEG complex.     

Serological evidence of alpha herpesvirus infection in sooty mangabeys

Contact between sooty mangabeys (SMs) and a pigtailed macaque prompted the serological screening of SMs for evidence of infection with B virus. Serological tests detected SM antibodies that reacted with B virus polypeptides. Additional testing was performed with sera from SMs with no previous contact with macaques. Results from these tests indicated that 56% (33/59) of the SMs had antibodies that reacted with B virus and SA8. SM antibodies also reacted with herpesvirus papio 2 and to a lesser extent with human alpha herpesviruses (HSV-1 and HSV-2). There was an age-related increase in the presence of these antibodies in SMs that was consistent with the serological pattern of reactivity observed in other nonhuman primate species infected with alpha herpesviruses. These data suggest that SMs may be a host for a herpesvirus that is antigenically similar to those viruses present in other Old World nonhuman primates.     

Calling is an honest indicator of paternal genetic quality in poison frogs

Several competing hypotheses have been put forward to explain why females of many species mate preferentially with males possessing the most conspicuous signals (e.g., ornaments, displays, or songs). We performed a laboratory experiment using two species of poison frogs, Dendrobates leucomelas and Epipedobates tricolor, to test the hypothesis that male calling performance is an honest indicator of parental quality. Our analyses are based on data from behavioral observations of mating activities of captive-reared individuals (and their offspring) that were housed in terraria for four consecutive breeding seasons. Male mating success increased with male calling rate and chirp duration in both species, suggesting that females preferred males with more elaborate calls. Because calling performance improved with age in D. leucomelas, female poison frogs that prefer males with more elaborate calls in the wild may end up mating with older males that have already proven their ability to survive. Females that mated with good callers obtained higher quality offspring. Eggs fertilized by males with high calling rates and long chirp durations had higher hatching success and produced tadpoles that were more likely to metamorphose into surviving frogs. As a consequence, females that mated with males with high calling performance obtained more surviving offspring per egg, compared to females that mated with poor callers. Collectively, our findings comply with the notion that female poison frogs prefer to mate with good callers because calling performance is a reliable predictor of offspring quality. The possible influence of maternal allocation and reasons for the strong effect size compared to previous studies are discussed.     

Mask of wax: Secretions of wax conceal aphids from detection by spider's eyes

Abstract

We investigated how insects use wax as a defence against visual predators, using a New Zealand salticid species, Marpissa marina, as the predator and Eriosoma lanigerum, an aphid that covers itself with wax, as the prey. For live‐prey testing, the predator was presented with two aphids, one with its wax covering intact and one with its wax removed. The predator ate more of the waxless than wax‐covered aphids. The predators were presented with two lures at a time: (1) one that was fully covered with wax (hid the aphid's head) compared with one that was without wax (waxless) or (2) one that was fully covered with wax compared with one that was only partially covered with wax (the head of the prey exposed), or (3) one that was waxless compared with one that was partially covered with wax. The predators stalked waxless prey more often than they stalked prey that was fully or partially covered with wax. When wax only partially covered the prey (i.e., when the prey's head was left exposed), the predator more often stalked than when the insect was fully covered. These findings suggest that the aphid's wax covering functions in part to hide prey‐identification cues from vision‐guided predators.     

Halide peroxidase in tissues that interact with bacteria in the host squid Euprymna scolopes

An enzyme with similarities to myeloperoxidase, the antimicrobial halide peroxidase in mammalian neutrophils, occurs abundantly in the light organ tissue of Euprymna scolopes, a squid that maintains a beneficial association with the luminous bacterium Vibrio fischeri. Using three independent assays typically applied to the analysis of halide peroxidase enzymes, we directly compared the activity of the squid enzyme with that of human myeloperoxidase. One of these methods, the diethanolamine assay, confirmed that the squid peroxidase requires halide ions for its activity. The identification of a halide peroxidase in a cooperative bacterial association suggested that this type of enzyme can function not only to control pathogens, but also to modulate the interactions of host animals with their beneficial partners. To determine whether the squid peroxidase functions under both circumstances, we examined its distribution in a variety of host tissues, including those that typically interact with bacteria and those that do not. Tissues interacting with bacteria included those that have specific cooperative associations with bacteria (i.e., the light organ and accessory nidamental gland) and those that have transient nonspecific interactions with bacteria (i.e., the gills, which clear the cephalopod circulatory system of invading microorganisms). These bacteria-associated tissues were compared with the eye, digestive gland, white body, and ink-producing tissues, which do not typically interact directly with bacteria. Peroxidase enzyme assays, immunocytochemical localization, and DNA-RNA hybridizations showed that the halide-dependent peroxidase is consistently expressed in high concentration in tissues that interact bacteria. Elevated levels of the peroxidase were also found in the ink-producing tissues, which are known to have enzymatic pathways associated with antimicrobial activity. Taken together, these data suggest that the host uses a common biochemical response to the variety of types of associations that it forms with microorganisms.     

Disparate binding of chaperone proteins by HLA-A subtypes

We examined chaperone association with subtypes of HLA-A68 differing at positions 116 and/or 70, and analyzed the surface expression of each A68 subtype. Our findings with A68 indicate that certain subtypes have inefficient association with the assembly complex and correspondingly high surface expression, dependent on the character of position 116. Specifically, poor association of A68 subtypes with the transporter associated with antigen processing correlated with a comparatively high level of W6/32(+) forms at the cell surface. This observation suggests that intracellular retention is a dominant function of the assembly complex and that natural differences in assembly complex interaction may dictate the level of surface expression of MHC class I molecules. We also found that position 116 was crucial for HLA-A68 subtype association with the assembly complex. Our data contrast with results we obtained previously with HLA-B7 in that an aspartic acid at position 116 abrogated chaperone association for HLA-A68, whereas it increased association for HLA-B7. In total, HLA-A molecules exhibit natural allele-specific distinctions in chaperone association that correlate with differences in cell surface expression and with the identity of amino acid position 116.     

Calcium binding sequences in calmyrin regulates interaction with presenilin-2

Calmyrin is a myristoylated calcium binding protein that contains four putative EF-hands. Calmyrin interacts with a number of proteins, including presenilin-2 (PS2). However, the biophysical properties of calmyrin, and the molecular mechanisms that regulate its binding to different partners, are not well understood. By site-directed mutagenesis and Ca2+ binding studies, we found that calmyrin binds two Ca2+ ions with a dissociation constant of approximately 53 microM, and that the two C-terminal EF-hands 3 and 4 bind calcium. Using ultraviolet spectroscopy, circular dichroism (CD), and NMR, we found that Ca(2+)-free and -bound calmyrin have substantially different protein conformations. By yeast two-hybrid assays, we found that both EF-hands 3 and 4 of calmyrin must be intact for calmyrin to interact with PS2-loop sequences. Pulse-chase studies of HeLa cells transfected with calmyrin expression constructs indicated that wild-type (Wt) calmyrin has a half-life of approximately 75 min, whereas a mutant defective in myristoylation turns over more rapidly (half-life of 35 min). By contrast, the half-lives of calmyrin mutants with a disrupted EF-hand 3 or EF-hand 4 were 52 and 170 min, respectively. Using immunofluorescence staining of HeLa cells transfected with Wt and mutant calmyrin cDNAs, we found that both calcium binding and myristoylation are important for dynamic intracellular targeting of calmyrin. Double immunofluorescence microscopy indicated that Wt and myristoylation-defective calmyrin proteins colocalize efficiently and to the same extent with PS2, whereas calmyrin mutants defective in calcium binding display less colocalization with PS2. Our results suggest that calmyrin functions as a calcium sensor and that calcium binding sequences in calmyrin are important for interaction with the PS2 loop.     

Mutations of Epstein-Barr virus gH that are differentially able to support fusion with B cells or epithelial cells

The core fusion machinery of all herpesviruses consists of three conserved glycoproteins, gB and gHgL, suggesting a common mechanism for virus cell fusion, but fusion of Epstein-Barr virus (EBV) with B cells and epithelial cells is initiated differently. Fusion with B cells requires a fourth protein, gp42, which complexes with gHgL and interacts with HLA class II, the B-cell coreceptor. Fusion with an epithelial cell does not require gp42 but requires interaction of gHgL with a novel epithelial cell coreceptor. Epithelial cell fusion can be inhibited by gp42 binding to gHgL and by antibodies to gH that fail to block B-cell fusion. This suggests that regions of gHgL initiating fusion with each cell are separable from each other and from regions involved in fusion itself. To address this possibility we mapped the region of gH recognized by a monoclonal antibody to gH that blocks EBV fusion with epithelial cells but not B cells by making a series of chimeras with the gH homolog of rhesus lymphocryptovirus. Proteins with mutations engineered within this region included those that preferentially mediate fusion with B cells, those that preferentially mediate fusion with epithelial cells, and those that mediate fusion with neither cell type. These results support the hypothesis that the core fusion function of gH is the same for B cells and epithelial cells and that it differs only in the way in which it is triggered into a functionally active state.     

侵染广西烟草的中国番茄黄化曲叶病毒及其伴随的卫星DNA分子的基因组特征

从中国广西靖西的烟草病株上分离到病毒分离物G102和G103,用双生病毒特异性引物均扩增出约500bp的片段,两者序列同源性达99%。对G102基因组DNA-A全序列测定表明,其全长为2728个核苷酸,与中国番茄黄化曲叶病毒(TYLCCNV)同源性最高,达96.5%。进一步研究发现,G102和G103都伴随有长为1342个核苷酸的卫星DNA分子(DNAβ),这两个DNAβ分子的全序列与TYLCCNV的DNAβ同源性最高,分别为92.9%和93.4%。这是首次明确广西分离的TYLCCNV也伴随有卫星分子。     

Bacteraemia in free-ranging Hawaiian green turtles Chelonia mydas with fibropapillomatosis

Past studies of free-ranging green turtles Chelonia mydas with fibropapillomatosis (FP) in Hawaii have shown that animals become immunosuppressed with increasing severity of this disease. Additionally, preliminary clinical examination of moribund turtles with FP revealed that some animals were also bacteraemic. We tested the hypothesis that bacteraemia in sea turtles is associated with the severity of FP. We captured free-ranging green turtles from areas in Hawaii where FP is absent, and areas where FP has been endemic since the late 1950s. Each turtle was given an FP severity score ranging from 0 (no tumours) to 3 (severely affected). A fifth category included turtles that were stranded ashore and moribund with FP. We found that the percentage of turtles with bacteraemia increased with the severity of FP, and that the majority of bacteria cultured were Vibrio spp. Turtles with severe FP were more susceptible to bactaeremia, probably in part due to immunosuppression. The pattern of bacteraemia in relation to severity of disease strengthens the hypothesis that immunosuppression is a sequel to FP.     

Telomere length of transferred lymphocytes correlates with in vivo persistence and tumor regression in melanoma patients receiving cell transfer therapy

Recent studies have indicated that adoptive immunotherapy with autologous antitumor tumor-infiltrating lymphocytes (TILs) following nonmyeloablative chemotherapy mediates tumor regression in approximately 50% of treated patients with metastatic melanoma, and that tumor regression is correlated with the degree of persistence of adoptively transferred T cells in peripheral blood. These findings, which suggested that the proliferative potential of transferred T cells may play a role in clinical responses, led to the current studies in which telomere length as well as phenotypic markers expressed on the administered TILs were examined. TILs that were associated with objective clinical responses following adoptive transfer possessed a mean telomere length of 6.3 kb, whereas TILs that were not associated with significant clinical responses were significantly shorter, averaging 4.9 kb (p < 0.01). Furthermore, individual TIL-derived T cell clonotypes that persisted in vivo following adoptive cell transfer possessed telomeres that were longer than telomeres of T cell clonotypes that failed to persist (6.2 vs 4.5 kb, respectively; p < 0.001). Expression of the costimulatory molecule CD28 also appeared to be associated with long telomeres and T cell persistence. These results, indicating that the telomere length of transferred lymphocytes correlated with in vivo T cell persistence following adoptive transfer, and coupled with the previous observation that T cell persistence was associated with clinical responses in this adoptive immunotherapy trial, suggest that telomere length and the proliferative potential of the transferred T cells may play a significant role in mediating response to adoptive immunotherapy.     

Detection and localization of a lectin on Actinomyces viscosus T14V by monoclonal antibodies

A cell-associated lectin activity that mediates lactose-inhibitable adherence of Actinomyces viscosus T14V has been localized to a specific population of fimbriae by the use of monoclonal antibodies. Nine monoclonal antibodies were produced that reacted with only 1 of 2 immunoelectrophoretically distinct fimbrial components on T14V. The fibrillar morphology of this component was revealed by the immunoelectronmicroscopic examination of bacteria incubated with the monoclonal antibodies. The lectin activity associated with these structures was detected when isolated fimbriae were cross-linked with monoclonal antibodies to form immune complexes with agglutination activity for neuraminidase-treated human erythrocytes, a reaction that was inhibited by lactose. Although the 9 monoclonal antibodies differed in their fine specificities, they reacted only with strains of A. viscosus and A. naeslundii that exhibited lactose-inhibitable adherence. These findings indicate that the lectin activity common to these bacteria resides on fimbriae that are antigenically related to those of T14V.     

Changes in DNase and DNA-polymerase activities in different tissues of chick with age.

The presence of acid and alkaline DNases in nuclei of chick brain cells has been demonstrated. The activities of these two DNases along with those of DNA-polymerases were assessed in chicken tissues with known varied cell proliferative capacities (eg., spleen, kidney and brain) at different ages. The results indicate that the acid and alkaline DNases are probably the 'house keeping' enzymes with a constitutive role in DNA repair process, the former with a DNA-repair process that is linked to cell proliferation (DNA synthesis) while the latter with the basal DNA-repair operations that must go on at all times without any regard to the cell division process. Chicken brain, unlike that of rat, possesses significant levels of aphidicolin sensitive DNA-polymerase(s) that are considered to be more replication oriented enzymes suggesting that the replication potential of adult and old avian brain cells may be different from that of a mammalian brain.     

Fold designability, distribution, and disease

Fold designability has been estimated by the number of families contained in that fold. Here, we show that among orthologous proteins, sequence divergence is higher for folds with greater numbers of families. Folds with greater numbers of families also tend to have families that appear more often in the proteome and greater promiscuity (the number of unique “partner” folds that the fold is found with within the same protein). We also find that many disease-related proteins have folds with relatively few families. In particular, a number of these proteins are associated with diseases occurring at high frequency. These results suggest that family counts reflect how certain structures are distributed in nature and is an important characteristic associated with many human diseases.