Allelic MHC class I chain related B (MICB) molecules affect the binding to the human cytomegalovirus (HCMV) unique long 16 (UL16) protein: Implications for immune surveillance

Unique long 16 (UL16) is a viral glycoprotein produced in a host cell infected with human cytomegalovirus (HCMV). It down regulates surface expression of MICB, one of the NKG2D ligands, by forming stable intracellular complexes and retained in the endoplasmic reticulum. Down expression of MICB renders cells less susceptible to NK cell lysis via the NKG2D receptor. Diverse UL16 sequences were identified from different strains of HCMV. MICB is known to be polymorphic. It is not known whether these polymorphisms affect the interactions between these molecules leading to alteration of the immune surveillance of HCMV. The soluble Fc fusion variant UL16 proteins from four laboratory and clinical isolates (AD169, Toledo, PH, and TR) were produced. Four allelic MICB alleles (008, 003, 004, and 00502) were cloned and stable cell lines expressing these MICB alleles were produced. The binding activities of variant UL16 to allelic MICB proteins were determined by flow cytometry. The variants of UL16 proteins did not affect the binding activities to allelic MICB proteins. However, diverse MICB alleles differentially bound UL16. We found that MICB*008 which contains methionine and asparagine at the amino acid positions 98 and 113, respectively, in the alpha 2 domain showed decreased binding activities to UL16 when compared to MICB*003, 004, and MICB*00502 containing isoleucine and aspartic acid, respectively. This finding may imply that MICB*008 is a protective allele and involved in the immune surveillance of HCMV infected patients.     

Lysophosphatidic acid receptor-5 negatively regulates cell motile and invasive activities of human sarcoma cell lines

LPA signaling via LPA receptors [LPA receptor-1 (LPA₁)–LPA₆] mediates the several cellular responses in cancer cells, including cell motility and invasion. In the present study, to investigate a role of LPA₅ in the cell motile and invasive activities of sarcoma cells, LPAR5 knockdown (HOSL5 and HT1080L5) cells were generated from human osteosarcoma HOS and fibrosarcoma HT1080 cells, respectively. In cell motility assays with cell culture inserts, HOSL5 and HT1080L5 cells indicated the high cell motile activities, compared with control cells. The cell invasive activities of HOSL5 and HT1080L5 cells were significantly higher than those of control cells. Moreover, the activities of matrix metalloproteinase (MMP)-2 and MMP-9 were measured by gelatin zymography. MMP-2 was significantly activated in HOSL5 cells, but not MMP-9. The elevated activities of MMP-2 and MMP-9 were found in HT1080L5 cells, in comparison with control cells. These results suggest that LPA signaling via LPA₅ negatively regulates the cell motile and invasive activities of human sarcoma cells.     

Cracking the enigmatic linker histone code

Recently, the existence of a 'histone code' has been proposed to explain the link between the covalent chemical modification of histone proteins and the epigenetic regulation of gene activity. Although the role of the four 'core' histones has been extensively studied, little is known about the involvement of the linker histone, histone H1 and its variants, in this code. For many years, few sites of chemical modification had been mapped in linker histones, but this has changed recently with the use of functional proteomic techniques, principally mass spectrometry, to characterize these modifications. The functionality of many of these sites, however, remains to be determined.     

Changes in D1-protein turnover and recovery of photosystem II activity precede accumulation of chlorophyll in plants after release from mineral stress

After seven weeks of a combined magnesium and sulphur deficiency, spinach (Spinacea oleracea L.) plants showed a substantial accumulation of inactivated photosystem II (PSII) centres as indicated by a 40% decrease of the chlorophyll (Chl) fluorescence parameter F_v/F_m (F_v being the yield of variable fluorescence and F_m the yield of maximal fluorescence when all reaction centres are closed) together with a severe loss of leaf Chl content of 75%. The responses of the photosynthetic apparatus were examined when the deficient plants were transferred back to a rich nutrient medium. During the first 24 h of the recovery phase, thylakoid protein synthesis measured as incorporation of [¹⁴C]leucine per unit of Chl increased substantially. The synthesis rate of the D1 reaction-centre polypeptide of PSII, which in the deficient plants was reduced to 50% of the non-deficient control, was stimulated eight- to ninefold. D1-protein content, which in the deficient plants was reduced to 40% of the non-deficient control, started to increase 2 d later. Thus, D1-protein degradation was also enhanced. The increased D1-protein turnover led to a rapid repair of the existing PSII centres as indicated by the rise of F_v/F_m. It was completed at day 7 of the recovery phase. At day 2 of the recovery phase, the synthesis of other thylakoid proteins such as the D2 protein, cytochrome b ₅₅₉, CP 47 and the 33-kDa polypeptide of the water-splitting system, became stimulated. This process resulted in an accumulation of new PSII centres. During the first week, formation of new PSII centres was not associated with an increase in leaf Chl content. The Chl content of the recovering leaves only started to increase when the ratio of PSII polypeptides versus LHCII (light-harvesting complex of PSII), which was substantially diminished in the deficient plants, became comparable to that of the control. The recovery process was accompanied by substantial changes in thylakoid protein phosphorylation. Their relevance to thylakoid protein turnover and stability is discussed.Abbreviations Chl chlorophyll - cyt cytochrome - F_o yield of intrinsic fluorescence when all PSII centres are open in the dark - F_m yield of maximal fluorescence when all reaction centres are closed - F_m fluorescence yield when all reaction centres are closed (after a saturating flash) under steady-state conditions - F_v yield of variable fluorescence, (difference between F_oand F_m) - F yield of variable fluorescence under steady state conditions - LHC light-harvesting complex - PQ plastoquinone - Q_A primary quinone acceptor of PSII - Q_B secondary quinone acceptor of PSII - q_P photochemical quenching - q_n non-photochemical quenching The authors like to thank Dipl. Biol. Britta Untereiser for determining the chlorophyll fluorescence quenching factors. This work was supported by grants from the Bundesminister für Forschung und Technologie, the Project Europäisches Forschungszentrum and the German Israeli Foundation in cooperation with Prof. I. Ohad, Hebrew University, Jerusalem, Israel.     

Cloning of the hamster p16 gene 5' upstream region and its aberrant methylation patterns in pancreatic cancer

The hamster model of pancreatic carcinogenesis is useful for understanding the development of human pancreatic cancer. However, there is only a small amount of hamster genetic information available for analyzing the gene alterations in hamster pancreatic cancers. Here, we determined the nucleotide sequence of the 5' upstream region of the hamster p16 gene using a suppression polymerase chain reaction method combined with gene-specific primers. Based on this sequence, we analyzed the methylation status of the 5' region by bisulfite sequencing in three normal pancreatic tissues and five pancreatic duct adenocarcinomas (PDAs). All five PDAs were highly methylated in the 5' upstream region and showed reduced expressions of the p16 gene, while the three normal samples were demethylated. The method described in this study is a highly effective and rapid technique for determining the 5' upstream region, and is applicable to epigenetic studies of the methylation status of this region.     

Phosphorylation of PS II polypeptides inhibits D1 protein-degradation and increases PS II stability

To study the significance of Photosystem (PS) II phosphorylation for the turnover of the D1 protein, phosphorylation was compared with the synthesis and content of the D1 protein in intact chloroplasts. As shown by radioactive labelling with [³²P_i] phosphorylation of PS II polypeptides was saturated at light intensities of 125 mol m^-2 s^-1. Under steady state conditions, in intact chloroplasts D1 protein, once it was phosphorylated, was neither dephosphorylated nor degraded in the light. D1 protein-synthesis was measured as incorporation of [¹⁴C] leucine. As shown by non-denaturing gel-electrophoresis followed by SDS-PAGE newly synthesised D1 protein was assembled to intact PS II-centres and no free D1 protein could be detected. D1 protein-synthesis was saturated at light intensities of 500 mol m^-2 s^-1. The content of D1 protein stayed stable even after illumination with 5000 mol m^-2 s^-1 showing that D1 protein-degradation was saturated at the same light intensities. The difference in the light saturation points of phosphorylation and of D1 protein-turnover indicates a complex regulation of D1 protein-turnover by phosphorylation. Separation of the phosphorylated and dephosphorylated D1 protein by LiDS-gelelectrophoresis combined with radioactive pulse-labelling with [¹⁴C] leucine and [³²P_i] revealed that D1 protein, synthesised under steady state conditions in the light, did not become phosphorylated but instead was rapidly degraded whereas the phosphorylated form of the D1 protein was not a good substrate for degradation. According to these observations phosphorylation of the D1 protein creates a pool of PS II centres which is not involved in D1 to these observations phosphorylation of the D1 protein creates a pool of PS II centres which is not involved in D1 protein-turnover. Fractionation of thylakoid membranes confirms that the phosphorylated, non-turning over pool of PS II-centres was located in the central regions of the grana, whereas PS II-centres involved in D1 protein-turnover were found exclusively in the stroma-lamellae and in the grana-margins.Abbreviations chl chlorophyll - F_v yield of variable fluorescence, difference between F_m, the maximal fluorescence yield at saturating light, when all reaction-centres are closed, and F_o, the fluorescence yield in the dark, when all reaction-centres are open - LHC light harvesting complex - PFD photon flux density - PS photosystem     

Comparability of multiple data types from the bering strait region: Cranial and dental metrics and nonmetrics,mtDNA, and Y‐chromosome DNA

Different data types have previously been shown to have the same microevolutionary patterns in worldwide data sets. However, peopling of the New World studies have shown a difference in migration paths and timings using multiple types of data, spurring research to understand why this is the case. This study was designed to test the degree of similarity in evolutionary patterns by using cranial and dental metric and nonmetric data, along with Y‐chromosome DNA and mtDNA. The populations used included Inuits from Alaska, Canada, Siberia, Greenland, and the Aleutian Islands. For comparability, the populations used for the cranial and molecular data were from similar geographic regions or had a shared population history. Distance, R and kinship matrices were generated for use in running Mantel tests, PROTEST analyses, and Procrustes analyses. A clear patterning was seen, with the craniometric data being most highly correlated to the mtDNA data and the cranial nonmetric data being most highly correlated with the Y‐chromosome data, while the phenotypic data were also linked. This patterning is suggestive of a possible male or female inheritance, or the correlated data types are affected by the same or similar evolutionary forces. The results of this study indicate cranial traits have some degree of heritability. Moreover, combining data types leads to a richer knowledge of biological affinity. This understanding is important for bioarchaeological contexts, in particular, peopling of the New World studies where focusing on reconciling the results from comparing multiple data types is necessary to move forward. Am J Phys Anthropol 154:334–348, 2014. © 2014 Wiley Periodicals, Inc.     

Scribble Modulates the MAPK/Fra1 Pathway to Disrupt Luminal and Ductal Integrity and Suppress Tumour Formation in the Mammary Gland

Polarity coordinates cell movement, differentiation, proliferation and apoptosis to build and maintain complex epithelial tissues such as the mammary gland. Loss of polarity and the deregulation of these processes are critical events in malignant progression but precisely how and at which stage polarity loss impacts on mammary development and tumourigenesis is unclear. Scrib is a core polarity regulator and tumour suppressor gene however to date our understanding of Scrib function in the mammary gland has been limited to cell culture and transplantation studies of cell lines. Utilizing a conditional mouse model of Scrib loss we report for the first time that Scrib is essential for mammary duct morphogenesis, mammary progenitor cell fate and maintenance, and we demonstrate a critical and specific role for Scribble in the control of the early steps of breast cancer progression. In particular, Scrib-deficiency significantly induced Fra1 expression and basal progenitor clonogenicity, which resulted in fully penetrant ductal hyperplasia characterized by high cell turnover, MAPK hyperactivity, frank polarity loss with mixing of apical and basolateral membrane constituents and expansion of atypical luminal cells. We also show for the first time a role for Scribble in mammalian spindle orientation with the onset of mammary hyperplasia being associated with aberrant luminal cell spindle orientation and a failure to apoptose during the final stage of duct tubulogenesis. Restoring MAPK/Fra1 to baseline levels prevented Scrib-hyperplasia, whereas persistent Scrib deficiency induced alveolar hyperplasia and increased the incidence, onset and grade of mammary tumours. These findings, based on a definitive genetic mouse model provide fundamental insights into mammary duct maturation and homeostasis and reveal that Scrib loss activates a MAPK/Fra1 pathway that alters mammary progenitor activity to drive premalignancy and accelerate tumour progression.