Human herpesvirus 8 glycoprotein B (gB), gH, and gL can mediate cell fusion

Herpesvirus entry into cells and herpesvirus-induced cell fusion are related processes in that virus penetration proceeds by fusion of the viral envelope and cell membrane. To characterize the human herpesvirus 8 (HHV-8) glycoproteins that can mediate cell fusion, a luciferase reporter gene activation assay was used. Chinese hamster ovary (CHO) cells expressing the HHV-8 glycoproteins of interest along with a luciferase reporter gene under the control of the T7 promoter were cocultivated with human cells transfected with T7 RNA polymerase. Because HHV-8 glycoprotein B (gB) expressed in CHO cells localizes to the perinuclear region, a truncated form of gB (designated gB(MUT)) that lacks putative endocytosis signals was constructed by deletion of the distal 58 amino acids of the cytoplasmic tail. HHV-8 gB(MUT) was expressed efficiently on the surface of CHO cells. HHV-8 gB, gH, and gL could mediate the fusion of CHO cells with two different human cell types, embryonic kidney cells and B lymphocytes. Substituting gB(MUT) for gB significantly enhanced the fusion of CHO cells with human embryonic kidney cells but not B lymphocytes. Thus, two human cell types known to be susceptible to HHV-8 entry were also suitable targets for cell fusion induced by HHV-8 gB, gH, and gL. For human embryonic kidney cells and B cells at least, optimal fusion was noted with the expression of all three HHV-8 glycoproteins.     

Matrix metalloproteinases-2,-3,-7,-9 and-10, but not MMP-11, are differentially expressed in normal, benign tumorigenic and malignant human keratinocyte cell lines

In order to investigate the correlations between constitutive proteinase expression and the degree of tumorigenicity of cancer cells we have studied a model system of three keratinocyte cell lines. RT-PCR studies showed that the cell lines express the genes of matrix metalloproteinase-2, -3, -7, -9, -10 and -11, indicating that they are able to synthesize the corresponding enzymes. Actual MMP synthesis was proven by zymography and Western blotting. In conditioned media gelatinolytic activities or immunoreactive forms of MMP-2, -3, -7, -9, -10 and -11 were detected. The signal intensities showed that MMP secretion increases in the order HaCaT < A5 < or = II-4RT, whereas only MMP-11 is secreted by all cell lines in equal amounts. Intracellularly, enhanced levels of one or both of the tumorigenic variants were only found for MMP-3, -9 and -10, suggesting special functions of these intracellular MMP pools for the tumorigenic cell lines. For MMP-11 exclusive expression in stromal fibroblasts of tumor tissues is widely accepted; however, our results and three other recent reports demonstrate that this concept is not generally valid. In conclusion, the three keratinocyte cell lines investigated here represent an excellent model for studying constitutive expression and secretion of MMPs in correlation to the degree of in vivo tumorigenicity.     

In vitro reaction of radioactive 7beta, 8alpha, --dihydroxy--9alpha, 10alpha-epoxy--7,8,9,10--tetrahydrobenzo (A) pyrene and 7beta,8alpha--dihydroxy--9beta, 10beta--epoxy--7,8,9,10--tetrahydrobenzo (A) pyrene with DNA.

The $\text{in vitro}$ reaction of bacteriophage T7-DNA with the radioactive diastereomeric benzo(a)pyrene-diol-epoxides, (±) [${}^3\text{H}{}_9$, ${}^3\text{H}{}_{10}$]-7β,8α-dihydroxy-9α,10β-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene, and (±) [${}^3\text{H}{}_9$, ${}^3\text{H}{}_{10}$]-7β,8α-dihydroxy-9β,19β-epoxy-7,8,9,10-tetrahydrobenzo(1)pyrene, was investigated. Chromatographic analysis of digests of the DNA allowed the distinction of characteristic deoxynucleoside adduct peaks for the two benzo(a)pyrene-diol-epoxides. Our results, together with data from the literature, allow the identification of these adducts as mostly N₂-(10-7β,8α,9α-trihydroxy-7,8,9,10-tetrahydrobenzo(a)pyreney1)deoxyguanosine and N₂-(10-7β,8α,9β-trihydroxy-7,8,9,10-tetrahydrobenzo(a)pyreney1)deoxyguanosine, respectively. DNA-benzo(a)pyrene adducts with the same chromatographic properties were formed in mouse embryo fibroblasts upon treatment with benzo(a)pyrene.     

Human cytomegalovirus proteins pp65 and immediate early protein 1 are common targets for CD8+ T cell responses in children with congenital or postnatal human cytomegalovirus infection

Recombinant modified vaccinia Ankara- and peptide-based IFN-gamma ELISPOT assays were used to detect and measure human CMV (HCMV)-specific CD8(+) T cell responses to the pp65 (UL83) and immediate early protein 1 (IE1; UL123) gene products in 16 HCMV-infected infants and children. Age at study ranged from birth to 2 years. HCMV-specific CD8(+) T cells were detected in 14 (88%) of 16 children at frequencies ranging from 60 to >2000 spots/million PBMC. Responses were detected as early as 1 day of age in infants with documented congenital infection. Nine children responded to both pp65 and IE1, whereas responses to pp65 or IE1 alone were detected in three and two children, respectively. Regardless of the specificity of initial responses, IE1-specific responses predominated by 1 year of age. Changes in HCMV epitopes targeted by the CD8(+) T cell responses were observed over time; epitopes commonly recognized by HLA-A2(+) adults with latent HCMV infection did not fully account for responses detected in early childhood. Finally, the detection of HCMV-specific CD8(+) T cell responses was temporally associated with a decrease in peripheral blood HCMV load. Taken altogether, these data demonstrate that the fetus and young infant can generate virus-specific CD8(+) T cell responses. Changes observed in the protein and epitope-specificity of HCMV-specific CD8(+) T cells over time are consistent with those observed after other primary viral infections. The temporal association between the detection of HCMV-specific CD8(+) T cell responses and the reduction in blood HCMV load supports the importance of CD8(+) T cells in controlling primary HCMV viremia.     

Human dendritic cells activated via dectin-1 are efficient at priming Th17, cytotoxic CD8 T and B cell responses

Background

Dendritic cells capture antigens through PRRs and modulate adaptive immune responses. The type of adaptive immune T cell response generated is dependent upon the type of PRR activated by the microbes. Dectin-1 is a C-type lectin receptor present on dendritic cells.

Methodology/Principal Findings

Here we show that selective dectin-1 agonist Curdlan can activate human DCs and induce the secretion of large amounts of IL-23, IL-1β, IL-6 and low levels of IL-12p70 as determined by ELISA. The Curdlan-stimulated DCs are efficient at priming naïve CD4 cells to differentiate into Th17 and Th1 cells. Furthermore, these CD4 T cells induce differentiation of B cells to secrete IgG and IgA. In addition, Curdlan-stimulated DCs promote the expansion and differentiation of Granzyme and perforin expressing cytotoxic T lymphocyte that display high cytolytic activity against target tumor cells in vitro.

Conclusions/Significance

These data demonstrate that DCs stimulated through Dectin-1 can generate efficient Th, CTL and B cell responses and can therefore be used as effective mucosal and systemic adjuvants in humans.     

Structure-activity relationship of linear peptide Bu-His6-DPhe7-Arg8-Trp9-Gly10-NH2 at the human melanocortin-1 and -4 receptors: DPhe7 and Trp9 substitution

A series of pentapeptides, based on hMC4R pentapeptide agonist (Bu-His(6)-DPhe(7)-Arg(8)-Trp(9)-Gly(10)-NH(2)), was prepared in which either DPhe(7) or Trp(9) residue was systematically substituted. A number of interesting DPhe surrogates (D-Thi, D-3-CF(3)Phe, D-2-Nal and D-3,4-diClPhe) as well as Trp surrogates (2-Nal and Bta) were identified in this study.     

Structure activity studies of the melanocortin antagonist SHU9119 modified at the 6, 7, 8, and 9 positions

The melanocortin system is involved in the regulation of several diverse physiological pathways, including energy homeostasis. Several synthetic peptide analogs have been designed, synthesized, and pharmacologically characterized at the mouse melanocortin receptor subtypes MC1R, MC3R, MC4R, and MC5R. These peptides incorporate modifications of the melanocortin core amino acids His-Phe-Arg-Trp by using the cyclic lactam templates of the lead structures MTII and SHU9119. Analogs containing DNal(2') at position 7 resulted in partial agonist and antagonistic activities at the mMC3R while possessing full antagonistic activities at the mMC4R. Recently, the melanocortin-5 receptor (MC5R) has been demonstrated to have a role in the regulation of exocrine gland function. This study has characterized the following analogs of SHU9119 that possess antagonist activity at the MC5R: Ac-Nle-c[Asp-(1-Me)His(6)-DNal(2')(7)-Arg-Trp-Lys]-NH(2), pA(2) = 7. 1; Ac-Nle-c[Asp-(1-Me)His(6)-DNal(2')(7)-Arg-Nal(2')(9)-Lys]-NH(2), pA(2) = 7.2; and Ac-Nle-c[Asp-Trp(6)-DNal(2')(7)-Arg-Nal(2')(9)-Lys]-NH(2), pA(2) = 6. 6.     

Human cytotoxic T cell clones directed at autologous virus-transformed targets: further evidence for linkage of genetic restriction to T4 and T8 surface glycoproteins

Human cytotoxic T cell clones were generated against autologous EBV-transformed B lymphocytes. Whereas the majority of the clones expressed the T8 surface glycoproteins and showed a specificity for class I MHC gene products on the target cell, a minority expressed the T4 surface glycoprotein and demonstrated a class II specificity. Monoclonal antibodies to T4 and T8 inhibited cytotoxic effector function of reactive clones in a fashion analogous to their effect on alloreactive CTL clones. Each autoreactive T cell clone was cytotoxic for EBV-transformed B lymphocytes but not pokeweed mitogen-activated or resting autologous lymphocytes, suggesting a dual specificity for an MHC gene product as well as an antigen induced and/or encoded by virus. Taken together, the present findings provide further support for the notion that T4 and T8 serve as associative recognition elements on T lymphocytes for MHC gene products.     

Dicoumarol-induced 9-gamma-carboxyglutamic acid prothrombin: isolation and comparison with the 6-, 7-, 8-, and 10-gamma-carboxyglutamic acid isomers.

The role of gamma-carboxyglutamic acid (Gla) in prothrombin function can be effectively evaluated by characterizing dicoumarol-induced, Gla-deficient prothrombin structural isomers. In addition to the isolation of 8-, 7-, 6-, 5-, 3-, 2-, 1-, and 0-Gla isomers, we have now purified a variant prothrombin containing 9(8.80) Gla residues by barium citrate adsorption, elution, and finally by DEAE-cellulose and immunoaffinity chromatographies. Agar gel electrophoretic mobilities of the 9-Gla isomer and its fragment 1 were slower than those of the respective 10-Gla (normal) prothrombin and fragment 1, both in the absence and presence of Ca(II). In the presence of Ca(II), both 9- and 10-Gla fragments 1 moved slower than 8- and 7-Gla fragments 1. However, in the absence of metal ions, 9- and 7-Gla fragments 1 migrated at the same rate, but slower than 10- and 8-Gla fragments. Similarly, the 9-Gla fragment 1 electrofocused cathodically to 10- and 8-Gla, but comparably with 7-Gla fragment 1. The 9-Gla fragment 1 exhibited a Ca(II)-induced 44% decrease in the intrinsic fluorescence, compared with a 40% decrease in that of 10-Gla; 8-Gla fragment 1 revealed only 23% quenching. Ca(II)-dependent anti-normal prothrombin antibodies are not specific for 10-Gla prothrombin, since only a twofold molar excess of the 9-Gla isomer was required to displace equal amounts of labeled normal prothrombin. The most critical Gla residue for influencing the functional, thrombin-generating properties of prothrombin appears to be the one present in the 9-Gla isomer but absent in the 8-Gla variant, since 9-Gla prothrombin possesses four times the normal coagulant activity (78 versus 20%) of the 8-Gla isomer.     

Effect of fibroblast growth factors 1, 2, 4, 5, 6, 7, 8, 9, and 10 on avian chondrocyte proliferation.

It has been demonstrated that fibroblast growth factor receptors are key regulators of endochondral bone growth. However, it has not been determined what fibroblast growth factor ligand(s) (FGFs) are important in this process. This study sought to determine whether FGFs 1, 2, 4, 5, 6, 7, 8, 9, and 10 were capable of stimulating avian chondrocyte proliferation in vitro. We have found that FGFs 2, 4, and 9 strongly stimulate avian chondrocyte proliferation while FGFs 6 and 8 stimulate proliferation to a lesser extent. RT-PCR indicates that FGF-2 and FGF-4 are expressed in the postnatal avian epiphyseal growth plate (EGP) while FGF-8 and FGF-9 are not. Thus, FGF-2 and FGF-4 stimulate chondrocyte proliferation and are both present in the EGP. This suggests that FGF-2 and FGF-4 may be important ligands, in vivo, for the regulation of endochondral bone growth. These observations coupled with our observation that multiple avian FGF receptors (Cek1, Cek2, Cek3, and FREK) are expressed in proliferative chondrocytes highlights the complexity of FGF signaling pathways in postnatal endochondral bone growth.     

Guard cell photosynthesis is critical for stomatal turgor production,yet does not directly mediate CO2‐ and ABA‐induced stomatal closing

Stomata mediate gas exchange between the inter‐cellular spaces of leaves and the atmosphere. CO₂ levels in leaves (Ci) are determined by respiration, photosynthesis, stomatal conductance and atmospheric [CO₂]. [CO₂] in leaves mediates stomatal movements. The role of guard cell photosynthesis in stomatal conductance responses is a matter of debate, and genetic approaches are needed. We have generated transgenic Arabidopsis plants that are chlorophyll‐deficient in guard cells only, expressing a constitutively active chlorophyllase in a guard cell specific enhancer trap line. Our data show that more than 90% of guard cells were chlorophyll‐deficient. Interestingly, approximately 45% of stomata had an unusual, previously not‐described, morphology of thin‐shaped chlorophyll‐less stomata. Nevertheless, stomatal size, stomatal index, plant morphology, and whole‐leaf photosynthetic parameters (PSII, qP, qN, F_V′/F_M′) were comparable with wild‐type plants. Time‐resolved intact leaf gas‐exchange analyses showed a reduction in stomatal conductance and CO₂‐assimilation rates of the transgenic plants. Normalization of CO₂ responses showed that stomata of transgenic plants respond to [CO₂] shifts. Detailed stomatal aperture measurements of normal kidney‐shaped stomata, which lack chlorophyll, showed stomatal closing responses to [CO₂] elevation and abscisic acid (ABA), while thin‐shaped stomata were continuously closed. Our present findings show that stomatal movement responses to [CO₂] and ABA are functional in guard cells that lack chlorophyll. These data suggest that guard cell CO₂ and ABA signal transduction are not directly modulated by guard cell photosynthesis/electron transport. Moreover, the finding that chlorophyll‐less stomata cause a ‘deflated’ thin‐shaped phenotype, suggests that photosynthesis in guard cells is critical for energization and guard cell turgor production.     

The 6S- and 6R-diastereomers of 5, 10-dideaza-5, 6, 7, 8-tetrahydrofolate are equiactive inhibitors of de novo purine synthesis

The diasteromers of 5,10-dideaza-5,6,7,8-tetrahydrofolate (DDATHF) differing in chirality about carbon 6 were resolved and studied as inhibitors of folate-dependent processes in mouse leukemia cells. Both diastereomers of DDATHF were found to be potent inhibitors of leukemia cell growth due to effects on de novo purine synthesis. Cell growth inhibition by these compounds was prevented by 5-formyltetrahydrofolate in a dose-dependent manner. This indicated that the effects of the DDATHF diastereomers were due to inhibition of folate-dependent processes. Metabolite reversal experiments indicated that 5'-phosphoribosylglycinamide formyltransferase was the major site of action of these compounds in mouse cells. Another site in de novo purine synthesis was affected at higher concentrations of diastereomer B in L1210 cells. Low concentrations of both diastereomers were found to inhibit pure L1210 5'-phosphoribosylglycinamide formyltransferase competitively with the folate substrate. The two diastereomers were also efficient substrates for mouse liver folylpolyglutamate synthetase. We conclude that the 6R- and 6S-diastereomers of DDATHF are remarkably similar and equiactive antimetabolites inhibitory to de novo purine synthesis and that the biochemical processes involved in their cytotoxicity display little stereochemical specificity.     

Characterization of the Neurospora crassa Cell Fusion Proteins,HAM-6, HAM-7, HAM-8, HAM-9, HAM-10, AMPH-1 and WHI-2

Intercellular communication of vegetative cells and their subsequent cell fusion is vital for different aspects of growth, fitness, and differentiation of filamentous fungi. Cell fusion between germinating spores is important for early colony establishment, while hyphal fusion in the mature colony facilitates the movement of resources and organelles throughout an established colony. Approximately 50 proteins have been shown to be important for somatic cell-cell communication and fusion in the model filamentous fungus Neurospora crassa. Genetic, biochemical, and microscopic techniques were used to characterize the functions of seven previously poorly characterized cell fusion proteins. HAM-6, HAM-7 and HAM-8 share functional characteristics and are proposed to function in the same signaling network. Our data suggest that these proteins may form a sensor complex at the cell wall/plasma membrane for the MAK-1 cell wall integrity mitogen-activated protein kinase (MAPK) pathway. We also demonstrate that HAM-9, HAM-10, AMPH-1 and WHI-2 have more general functions and are required for normal growth and development. The activation status of the MAK-1 and MAK-2 MAPK pathways are altered in mutants lacking these proteins. We propose that these proteins may function to coordinate the activities of the two MAPK modules with other signaling pathways during cell fusion.