Binding modes of Tec family kinase SH3 domains: structural characteristics with implications on enzymatic regulation

Src Homology 3 domains are found ubiquitously throughout the known proteome. Because they have no catalytic activity and are able to bind specific peptide sequences containing a PXXP motif, they are thought to be used generally as coupling domains. While the significance of some SH3 mediated protein interactions may be slight, others have a great deal of importance. Namely, SH3 domains are thought to be responsible for binding mechanisms involved in regulating activity in some enzymes. The Tec family of protein tyrosine kinases each contain a single SH3 domain. Additionally, four of the members, Tec, Btk, Itk and Rlk include at least one polyproline peptide sequence known to bind SH3 domains. We have explored some of the characteristics of the interactions between these SH3 domains and their respective proline ligands. Specifically, ligand affinity and ligand availability were considered as possible mediating factors in binding. Our data suggests that the affinity of a polyproline ligand for its SH3 domain is a major factor in determining whether the interaction is intra- or intermolecular. Findings presented here lay a foundation for future work in probing regulatory mechanisms by SH3 domains

Sensitive and selective amplification of methylated DNA sequences using helper-dependent chain reaction in combination with a methylation-dependent restriction enzyme

We have developed a novel technique for specific amplification of rare methylated DNA fragments in a high background of unmethylated sequences that avoids the need of bisulphite conversion. The methylation-dependent restriction enzyme GlaI is used to selectively cut methylated DNA. Then targeted fragments are tagged using specially designed ‘helper’ oligonucleotides that are also used to maintain selection in subsequent amplification cycles in a process called ‘helper-dependent chain reaction’. The process uses disabled primers called ‘drivers’ that can only prime on each cycle if the helpers recognize specific sequences within the target amplicon. In this way, selection for the sequence of interest is maintained throughout the amplification, preventing amplification of unwanted sequences. Here we show how the method can be applied to methylated Septin 9, a promising biomarker for early diagnosis of colorectal cancer. The GlaI digestion and subsequent amplification can all be done in a single tube. A detection sensitivity of 0.1% methylated DNA in a background of unmethylated DNA was achieved, which was similar to the well-established Heavy Methyl method that requires bisulphite-treated DNA.Funding for open access charge: Commonwealth Scientific and Industrial Research Organisation (CSIRO), Australia. National Health & Medical Research Counci

Transformations in Death: The Archaeology of Funerary Practices and Personhood in the Bronze Age Levant

Burials in the Middle and Late Bronze Age Levant (ca. 2000-1200 B.C.E.) exhibit a high degree of mortuary diversity and experimentation with body disposal methods in primary, secondary, and co-mingled inhumations. Previous research has explained the process of multiple-successive burial as a result of the functional need to make room inside of a re-used burial space such as a chamber tomb. This explanation misses the opportunity to investigate the social and ritual meaning of repeated, close contact with the corporeal remains of the deceased after death and burial. Indeed, written sources from the ancient Near East attest to the existence of posthumous persons such as ancestors, ghosts, and the deified dead. Therefore, this dissertation poses four research questions to investigate the roles of the dead body and person in funerary rituals of the second millennium B.C.E. Levant: (1) What is the status of the dead after burial? (2) What roles do the corpse play in the funerary sequence? (3) What do textual sources and mortuary practices reveal about relationships between the post-mortem body and person? (4) Under what circumstances does personhood continue or transform after death? The treatment of the dead body determines the posthumous social roles of the deceased after burial and explains the high degree of burial diversity of the Middle and Late Bronze Age Levant. This work combines theoretical frameworks of personhood and embodiment with methods derived from mortuary archaeology, specifically funerary taphonomy, biological profiles of age and sex, and distributions of burial type, architecture, context, and grave goods. Drawing from a broad mortuary dataset from across the Levant, including three mortuary case studies from Tel Megiddo (Israel), patterns of deposition and corpse modification in residential burials are identified. These results are compared with contemporaneous textual evidence drawn from a variety of sources across the Levant and Mesopotamia which address the dead body and person. This study presents a new model of the extended funerary sequence in residential burials of the second millennium B.C.E., asserting that the bodies of the dead were treated differently according to their membership within the household. It is also argued that repeated fragmentation and intermixing of skeletal remains in residential burials was the primary pathway to achieving ancestor status after death and burial. The results provide new insight into the society of the Bronze Age Levant, a context in which the household encompassed the living and the dead and served as the primary social unit

Binding modes of Tec family kinase SH3 domains: structural characteristics with implications on enzymatic regulation

Src Homology 3 domains are found ubiquitously throughout the known proteome. Because they have no catalytic activity and are able to bind specific peptide sequences containing a PXXP motif, they are thought to be used generally as coupling domains. While the significance of some SH3 mediated protein interactions may be slight, others have a great deal of importance. Namely, SH3 domains are thought to be responsible for binding mechanisms involved in regulating activity in some enzymes. The Tec family of protein tyrosine kinases each contain a single SH3 domain. Additionally, four of the members, Tec, Btk, Itk and Rlk include at least one polyproline peptide sequence known to bind SH3 domains. We have explored some of the characteristics of the interactions between these SH3 domains and their respective proline ligands. Specifically, ligand affinity and ligand availability were considered as possible mediating factors in binding. Our data suggests that the affinity of a polyproline ligand for its SH3 domain is a major factor in determining whether the interaction is intra- or intermolecular. Findings presented here lay a foundation for future work in probing regulatory mechanisms by SH3 domains.</p

Funerary Practices and Personhood: Bronze Age Burial Assemblages from Tel Megiddo, Israel

Men's Basketball statistics; game bookHead Coach Rick MajerusMen's BasketballUtah 70 Wisc.-Milwaukee 5

Transformations in Death: The Archaeology of Funerary Practices and Personhood in the Bronze Age Levant

Burials in the Middle and Late Bronze Age Levant (ca. 2000-1200 B.C.E.) exhibit a high degree of mortuary diversity and experimentation with body disposal methods in primary, secondary, and co-mingled inhumations. Previous research has explained the process of multiple-successive burial as a result of the functional need to make room inside of a re-used burial space such as a chamber tomb. This explanation misses the opportunity to investigate the social and ritual meaning of repeated, close contact with the corporeal remains of the deceased after death and burial. Indeed, written sources from the ancient Near East attest to the existence of posthumous persons such as ancestors, ghosts, and the deified dead. Therefore, this dissertation poses four research questions to investigate the roles of the dead body and person in funerary rituals of the second millennium B.C.E. Levant: (1) What is the status of the dead after burial? (2) What roles do the corpse play in the funerary sequence? (3) What do textual sources and mortuary practices reveal about relationships between the post-mortem body and person? (4) Under what circumstances does personhood continue or transform after death? The treatment of the dead body determines the posthumous social roles of the deceased after burial and explains the high degree of burial diversity of the Middle and Late Bronze Age Levant. This work combines theoretical frameworks of personhood and embodiment with methods derived from mortuary archaeology, specifically funerary taphonomy, biological profiles of age and sex, and distributions of burial type, architecture, context, and grave goods. Drawing from a broad mortuary dataset from across the Levant, including three mortuary case studies from Tel Megiddo (Israel), patterns of deposition and corpse modification in residential burials are identified. These results are compared with contemporaneous textual evidence drawn from a variety of sources across the Levant and Mesopotamia which address the dead body and person. This study presents a new model of the extended funerary sequence in residential burials of the second millennium B.C.E., asserting that the bodies of the dead were treated differently according to their membership within the household. It is also argued that repeated fragmentation and intermixing of skeletal remains in residential burials was the primary pathway to achieving ancestor status after death and burial. The results provide new insight into the society of the Bronze Age Levant, a context in which the household encompassed the living and the dead and served as the primary social unit