Final Archaeological Investigations at the Maynard-Burgess House (18AP64): An 1850-1980 African-American Household in Annapolis, Maryland

The Maynard-Burgess House was excavated by Archaeology in Annapolis from Fall, 1990 to Summer, 1992. The still-standing house is located at 163 Duke of Gloucester Street in Annapolis' Historic District and is today being restored by Port of Annapolis, Incorporated. Archaeological testing and excavation of the site was developed alongside architectural analyses and archival research as the initial phase of the home's restoration. The Maynard-Burgess House was continuously occupied by two African-American families, the Maynards and the Burgesses, from the 1850s until the late 1980s. The main block of the house was built between 1850 and 1858 by the household of John T. Maynard, a free African American born in 1810,and his wife Maria Spencer Maynard. Maynard descendants lived in the home until it was foreclosed in 1908 and subsequently sold to the family of Willis and Mary Burgess in 1915. Willis had been a boarder in the home in 1880, and his sister Martha Ready had married John and Maria's son John Henry. Burgess descendants lived at the home until its sale in 1990.

Contracting Freedom: Governance and East Indian Indenture in the British Atlantic, 1838-1917

This is a dissertation about identity and governance, and how they are mutually constituted. Between 1838 and 1917, the British brought approximately half a million East Indian laborers to the Atlantic to work on sugar plantations. The dissertation argues that contrary to previous historiographical assumptions, indentured East Indians were an amorphous mass of people drawn from various regions of British India. They were brought together not by their innate "Indian-ness" upon their arrival in the Caribbean, but by the common experience of indenture recruitment, transportation and plantation life. Ideas of innate "Indian-ness" were products of an imperial discourse that emerged from and shaped official approaches to governing East Indians in the Atlantic. Government officials and planters promoted visions of East Indians as "primitive" subjects who engaged in child marriage and wife murder. Officials mobilized ideas about gender to sustain racialized stereotypes of East Indian subjects. East Indian women were thought to be promiscuous, and East Indian men were violent and depraved (especially in response to East Indian women's promiscuity). By pointing to these stereotypes about East Indians, government officials and planters could highlight the promise of indenture as a civilizing mechanism. This dissertation links the study of governance and subject formation to complicate ideas of colonial rule as static. It uncovers how colonial processes evolved to handle the challenges posed by migrant populations.

The primary architects of indenture, Caribbean governments, the British Colonial Office, and planters hoped that East Indian indentured laborers would form a stable and easily-governed labor force. They anticipated that the presence of these laborers would undermine the demands of Afro-Creole workers for higher wages and shorter working hours. Indenture, however, was controversial among British liberals who saw it as potentially hindering the creation of a free labor market, and abolitionists who also feared that indenture was a new form of slavery. Using court records, newspapers, legislative documents, bureaucratic correspondence, memoirs, novels, and travel accounts from archives and libraries in Britain, Guyana, and Trinidad and Tobago, this dissertation explores how indenture was envisioned and constantly re-envisioned in response to its critics. It chronicles how the struggles between the planter class and the colonial state for authority over indentured laborers affected the way that indenture functioned in the British Atlantic. In addition to focusing on indenture's official origins, this dissertation examines the actions of East Indian indentured subjects as they are recorded in the imperial archive to explore how these people experienced indenture.

Indenture contracts were central to the justification of indenture and to the creation of a pliable labor force in the Atlantic. According to English common law, only free parties could enter into contracts. Indenture contracts limited the period of indenture and affirmed that laborers would be remunerated for their labor. While the architects of indenture pointed to contracts as evidence that indenture was not slavery, contracts in reality prevented laborers from participating in the free labor market and kept the wages of indentured laborers low. Further, in late nineteenth-century Britain, contracts were civil matters. In the British Atlantic, indentured laborers who violated the terms of their contracts faced criminal trials and their associated punishments such as imprisonment and hard labor. Officials used indenture contracts to exploit the labor and limit the mobility of indentured laborers in a manner that was reminiscent of slavery but that instead established indentured laborers as subjects with limited rights. The dissertation chronicles how indenture contracts spawned a complex inter-imperial bureaucracy in British India, Britain, and the Caribbean that was responsible for the transportation and governance of East Indian indentured laborers overseas.

Antibody deficiency due to a missense mutation in CD19 demonstrates the importance of the conserved tryptophan 41 in immunoglobulin superfamily domain formation.

Immunoglobulin superfamily (IgSF) domains are conserved structures present in many proteins in eukaryotes and prokaryotes. These domains are well-capable of facilitating sequence variation, which is most clearly illustrated by the variable regions in immunoglobulins (Igs) and T cell receptors (TRs). We studied an antibody-deficient patient suffering from recurrent respiratory infections and with impaired antibody responses to vaccinations. Patient's B cells showed impaired Ca(2+) influx upon stimulation with anti-IgM and lacked detectable CD19 membrane expression. CD19 sequence analysis revealed a homozygous missense mutation resulting in a tryptophan to cystein (W52C) amino acid change. The affected tryptophan is CONSERVED-TRP 41 located on the C-strand of the first extracellular IgSF domain of CD19 and was found to be highly conserved, not only in mammalian CD19 proteins, but in nearly all characterized IgSF domains. Furthermore, the tryptophan is present in all variable domains in Ig and TR and was not mutated in 117 Ig class-switched transcripts of B cells from controls, despite an overall 10% amino acid change frequency. In vitro complementation studies and CD19 western blotting of patient's B cells demonstrated that the mutated protein remained immaturely glycosylated. This first missense mutation resulting in a CD19 deficiency demonstrates the crucial role of a highly conserved tryptophan in proper folding or stability of IgSF domains.

Insolvency: ordering a sale of the family home

Comments on the Chancery Division ruling in Nicholls v Lan on whether the interests of a bankrupt husband's creditors prevailed over those of the wife, despite her circumstances being exceptional within the meaning of the Insolvency Act 1986 s.335A on account of her suffering from chronic schizophrenia, where the wife was the joint owner of another property which could be realised to buy out the trustee in bankruptcy's half share in the equity of the matrimonial home.

Legal myth-making: Medea and the legal representation of the feminine ‘other'

The Law operates by, and through, the creation of ideal benchmarks of conduct that are deemed to be representative of the behavioural norm. It is in this sense that it could be contended that the Law utilises, and relies on, myths in the same way as do other disciplines, notably psycho-analysis. It is possible to go even further and argue that the use of a created narrative mythology is essential to the establishment of a defined legal benchmark of behaviour by which the female defendant is assessed, judged and punished. While mythology expresses and symbolizes cultural and political behaviour, it is the Law that embodies and prescribes punitive sanctions. This element represents a powerful literary strand in classical mythology. This may be seen, for instance, in Antigone’s appeal to the Law as justification for her conduct, as much as in Medea’s challenge to the Law though her desire for vengeance. Despite its image of neutral, objective rationality, the Law, in creating and sustaining the ideals of legally-sanctioned conduct, engages in the same literary processes of imagination, reason and emotion that are central to the creation and re-creation of myth. The (re-)presentation of the Medea myth in literature (especially in theatre) and in art, finds its echo in the theatre of the courtroom where wronged women who have refused to passively accept their place, have instead responded with violence. Consequently, the Medea myth, in its depiction of the (un)feminine, serves as a template for the Law’s judgment of ‘conventional’ feminine conduct in the roles of wife and mother. Medea is an image of deviant femininity, as is Lady Macbeth and the countless other un-feminine literary and mythological women who challenge the power of the dominant culture and its ally, the Law. These women stand opposed to the other dominant theme of both literature and Law: the conformist woman, the passive dupe, who are victims of male oppression – women such as Ariadne of Naxos and Tess of the D’Ubervilles – and who are subsequently consumed by the Law, much as Semele is consumed by the fire of Jupiter’s gaze upon her. All of these women, the former as well as the latter, have their real-life counterparts in the pages of the Law Reports. As Fox puts it, “these women have come to bear the weight of the cultural stereotypes and preconceptions about women who kill.”

The conserved Helix C region in the superfamily of interferon-a/interleukin-10-related cytokines corresponds to a high-affinity binding site for the HSP70 chaperone DnaK.

HSP70 chaperones mediate protein folding by ATP-dependent interaction with short linear peptide segments that are exposed on unfolded proteins. The mode of action of the Escherichia coli homolog DnaK is representative of all HSP70 chaperones, including the endoplasmic reticulum variant BiP/GRP78. DnaK has been shown to be effective in assisting refolding of a wide variety of prokaryotic and eukaryotic proteins, including the -helical homodimeric secretory cytokine interferon- (IFN-). We screened solid-phase peptide libraries from human and mouse IFN- to identify DnaK-binding sites. Conserved DnaK-binding sites were identified in the N-terminal half of helix B and in the C-terminal half of helix C, both of which are located at the IFN- dimer interface. Soluble peptides derived from helices B and C bound DnaK with high affinity in competition assays. No DnaK-binding sites were found in the loops connecting the -helices. The helix C DnaK-binding site appears to be conserved in most members of the superfamily of interleukin (IL)-10-related cytokines that comprises, apart from IL-10 and IFN-, a series of recently discovered small secretory proteins, including IL-19, IL-20, IL-22/IL-TIF, IL-24/MDA-7 (melanoma differentiation-associated gene), IL-26/AK155, and a number of viral IL-10 homologs. These cytokines belong to a relatively small group of homodimeric proteins with highly interdigitated interfaces that exhibit the strongly hydrophobic character of the interior core of a single-chain folded domain. We propose that binding of DnaK to helix C in the superfamily of IL-10-related cytokines may constitute the hallmark of a novel conserved regulatory mechanism in which HSP70-like chaperones assist in the formation of a hydrophobic dimeric "folding" interface.

Sequence and Structure Based Protein Folding Studies With Implications

As the expression of the genetic blueprint, proteins are at the heart of all biological systems. The ever increasing set of available protein structures has taught us that diversity is the hallmark of their architecture, a fundamental characteristic that enables them to perform the vast array of functionality upon which all of life depends. This diversity, however, is central to one of the most challenging problems in molecular biology: how does a folding polypeptide chain navigate its way through all of the myriad of possible conformations to find its own particular biologically active form? With few overarching structural principles to draw upon that can be applied to all protein architecture, the search for a solution to the protein folding problem has yet to produce an algorithm that can explain and duplicate this fundamental biological process. In this thesis, we take a two-pronged approach for investigating the protein folding process. Our initial statistical studies of the distributions of hydrophobic and hydrophilic residues within α-helices and β-sheets suggest (i) that hydrophobicity plays a critical role in helix and sheet formation; and (ii) that the nucleation of these motifs may result in largely unidirectional growth. Most tellingly, from an examination of the amino acids found in the smallest β-sheets, we do not find any evidence of a β-nucleating code in the primary protein sequence. Complementing these statistical analyses, we have analyzed the structural environments of several ever-widening aspects of protein topology. Our examination of the gaps between strands in the smallest β-sheets reveals a common organizational principle underlying β-formation involving strands separated by large sequential gaps: with very few exceptions, these large gaps fold into single, compact structural modules, bringing the β-strands that are otherwise far apart in the sequence close together in space. We conclude, therefore, that β-nucleation in the smallest sheets results from the co-location of two strands that are either local in sequence, or local in space following prior folding events. A second study of larger β-sheets both corroborates and extends these findings: virtually all large sequential gaps between pairs of β-strands organize themselves into an hierarchical arrangement, creating a bread-crumb model of go-and-come-back structural organization that ultimately juxtaposes two strands of a parental β-structure that are far apart in the sequence in close spatial proximity. In a final study, we have formalized this go-and-come-back notion into the concept of anti-parallel double-strandedness (DS), and measure this property across protein architecture in general. With over 90% of all residues in a large, non-redundant set of protein structures classified as DS, we conclude that DS is a unifying structural principle that underpins all globular proteins. We postulate, moreover, that this one simple principle, anti-parallel double-strandedness, unites protein structure, protein folding and protein evolution.

Inhibition of Secretion of Interleukin (IL)-12/IL-23 Family Cytokines by 4-Trifluoromethyl-celecoxib Is Coupled to Degradation via the Endoplasmic Reticulum Stress Protein HERP

Interleukin-12 (IL-12), p80, and IL-23 are structurally related cytokines sharing a p40 subunit. We have recently demonstrated that celecoxib and its COX-2-independent analogue 4-trifluoromethyl-celecoxib (TFM-C) inhibit secretion but not transcription of IL-12 (p35/p40) and p80 (p40/p40). This is associated with a mechanism involving altered cytokine-chaperone interaction in the endoplasmic reticulum (ER). In the present study, we found that celecoxib and TFM-C also block secretion of IL-23 (p40/p19 heterodimers). Given the putative ER-centric mode of these compounds, we performed a comprehensive RTPCR analysis of 23 ER-resident chaperones/foldases and associated co-factors. This revealed that TFM-C induced 1.5-3-fold transcriptional up-regulation of calreticulin, GRP78, GRP94, GRP170, ERp72, ERp57, ERdj4, and ERp29. However, more significantly, a 7-fold up-regulation of homocysteine-inducible ER protein (HERP) was observed. HERP is part of a high molecular mass protein complex involved in ER-associated protein degradation (ERAD). Using co-immunoprecipitation assays, we show that TFM-C induces protein interaction of p80 and IL-23 with HERP. Both HERP siRNA knockdown and HERP overexpression coupled to cycloheximide chase assays revealed that HERP is necessary for degradation of intracellularly retained p80 by TFM-C. Thus, our data suggest that targeting cytokine folding in the ER by small molecule drugs could be therapeutically exploited to alleviate in appropriate inflammation in autoimmune conditions.

Functional analysis of the C-terminal domain of the WbaP protein that mediates initiation of O antigen synthesis in Salmonella enterica

WbaP catalyzes the transfer of galactose-1-phosphate onto undecaprenyl phosphate (Und-P). The enzyme belongs to a large family of bacterial membrane proteins required for initiation of the synthesis of O antigen lipopolysaccharide and polysaccharide capsules. Previous work in our laboratory demonstrated that the last transmembrane helix and C-terminal tail region of WbaP (WbaP(CT)) are sufficient for enzymatic activity. Here, we demonstrate the cytoplasmic location of the WbaP C-terminal tail and show that WbaPCT domain N-terminally fused to thioredoxin (TrxA-WbaP(CT)) exhibits improved protein folding and enhanced transferase activity. Alanine replacement of highly conserved charged or polar amino acids identified seven critical residues for enzyme activity in vivo and in vitro. Four of these residues are located in regions predicted to be a-helical. These regions and their secondary structure predictions are conserved in distinct WbaP family members, suggesting they may contribute to form a conserved catalytic center.

First report of the use of a saxitoxin-protein conjugate to develop a DNA aptamer to a small molecule toxin

Saxitoxin (STX) is a low molecular weight neurotoxin mainly produced by certain marine dinoflagellates that, along with its family of similarly related paralytic shellfish toxins, may cause the potentially fatal intoxication known as paralytic shellfish poisoning. Illness and fatality rates are low due to the effective monitoring programs that determine when toxins exceed the established regulatory action level and effectuate shellfish harvesting closures accordingly. Such monitoring programs rely on the ability to rapidly screen large volumes of samples. Many of the screening assays currently available employ antibodies or live animals. This research focused on developing an analytical recognition element that would eliminate the challenges associated with the limited availability of antibodies and the use of animals. Here we report the discovery of a DNA aptamer that targets STX. Concentration-dependent and selective binding of the aptamer to STX was determined using a surface plasmon resonance sensor. Not only does this work represent the first reported aptamer to STX, but also the first aptamer to any marine biotoxin. A novel strategy of using a toxin-protein conjugate for DNA aptamer selection was successfully implemented to overcome the challenges associated with aptamer selection to small molecules. Taking advantage of such an approach could lead to increased diversity and accessibility of aptamers to low molecular weight toxins, which could then be incorporated as analytical recognition elements in diagnostic assays for foodborne toxin detection. The selected STX aptamer sequence is provided here, making it available to any investigator for use in assay development for the detection of STX.

Re-Envisioning the Artist Hero Through Two Cole Porter Biopics

Songwriter Cole Porter is unusual in having had two biopics based on his life: Night and Day (1946) starring Cary Grant, and De-Lovely (2004), starring Kevin Kline. The differences in the treatment of the character of Cole Porter between the films are striking, and indicate a change in the way that society envisions its artists, and the very act of creativity. Night and Day was conceived partly as a showcase of Porter's songs, but also as a means of providing inspiration to soldiers returning wounded from World War II, based on Porter's recovery from a traumatic riding accident. It depicts Porter as an everyman following a trajectory of achievement, from having little to great success, which was positioned as easy to emulate. De-Lovely, on the other hand, is about the relationship between Porter and his wife Linda, and the way that his creativity was influenced by his changing relationships with various people. Drawing on the work on biopics of scholars such as G.F.Custen, together with research into the shifting ideas of how creativity operates and is popularly understood, this article uses these biopics as case studies to examine the representation of changing concepts of the artist and the act of creativity through Hollywood film. It also considers how these changing conceptions and representations connect to shifts in American society.

S100A2 is a BRCA1/p63 coregulated tumour suppressor gene with roles in the regulation of mutant p53 stability

Here, we show for the first time that the familial breast/ovarian cancer susceptibility gene, BRCA1, along with interacting ΔNp63 proteins, transcriptionally upregulate the putative tumour suppressor protein, S100A2. Both BRCA1 and ΔNp63 proteins are required for S100A2 expression. BRCA1 requires ΔNp63 proteins for recruitment to the S100A2 proximal promoter region, while exogenous expression of individual ΔNp63 proteins cannot activate S100A2 transcription in the absence of a functional BRCA1. Consequently, mutation of the ΔNp63/p53 response element within the S100A2 promoter completely abrogates the ability of BRCA1 to upregulate S100A2. S100A2 shows growth control features in a range of cell models. Transient or stable exogenous S100A2 expression inhibits the growth of BRCA1 mutant and basal-like breast cancer cell lines, while short interfering RNA (siRNA) knockdown of S100A2 in non-tumorigenic cells results in enhanced proliferation. S100A2 modulates binding of mutant p53 to HSP90, which is required for efficient folding of mutant p53 proteins, by competing for binding to HSP70/HSP90 organising protein (HOP). HOP is a cochaperone that is required for the efficient transfer of proteins from HSP70 to HSP90. Loss of S100A2 leads to an HSP90-dependent stabilisation of mutant p53 with a concomitant loss of p63. Accordingly, S100A2-deficient cells are more sensitive to the HSP-90 inhibitor, 17-N-allylamino-17-demethoxygeldanamycin, potentially representing a novel therapeutic strategy for S100A2- and BRCA1-deficient cancers. Taken together, these data demonstrate the importance of S100A2 downstream of the BRCA1/ΔNp63 signalling axis in modulating transcriptional responses and enforcing growth control mechanisms through destabilisation of mutant p53.

Probing the Unfolded Configurations of a β-Hairpin Using Sketch-Map

This work examines the conformational ensemble involved in β-hairpin folding by means of advanced molecular dynamics simulations and dimensionality reduction. A fully atomistic description of the protein and the surrounding solvent molecules is used, and this complex energy landscape is sampled by means of parallel tempering metadynamics simulations. The ensemble of configurations explored is analyzed using the recently proposed sketch-map algorithm. Further simulations allow us to probe how mutations affect the structures adopted by this protein. We find that many of the configurations adopted by a mutant are the same as those adopted by the wild-type protein. Furthermore, certain mutations destabilize secondary-structure-containing configurations by preventing the formation of hydrogen bonds or by promoting the formation of new intramolecular contacts. Our analysis demonstrates that machine-learning techniques can be used to study the energy landscapes of complex molecules and that the visualizations that are generated in this way provide a natural basis for examining how the stabilities of particular configurations of the molecule are affected by factors such as temperature or structural mutations.

Role of the AP2 beta-appendage hub in recruiting partners for clathrin-coated vesicle assembly

Adaptor protein complex 2 alpha and beta-appendage domains act as hubs for the assembly of accessory protein networks involved in clathrin-coated vesicle formation. We identify a large repertoire of beta-appendage interactors by mass spectrometry. These interact with two distinct ligand interaction sites on the beta-appendage (the "top" and "side" sites) that bind motifs distinct from those previously identified on the alpha-appendage. We solved the structure of the beta-appendage with a peptide from the accessory protein Eps15 bound to the side site and with a peptide from the accessory cargo adaptor beta-arrestin bound to the top site. We show that accessory proteins can bind simultaneously to multiple appendages, allowing these to cooperate in enhancing ligand avidities that appear to be irreversible in vitro. We now propose that clathrin, which interacts with the beta-appendage, achieves ligand displacement in vivo by self-polymerisation as the coated pit matures. This changes the interaction environment from liquid-phase, affinity-driven interactions, to interactions driven by solid-phase stability ("matricity"). Accessory proteins that interact solely with the appendages are thereby displaced to areas of the coated pit where clathrin has not yet polymerised. However, proteins such as beta-arrestin (non-visual arrestin) and autosomal recessive hypercholesterolemia protein, which have direct clathrin interactions, will remain in the coated pits with their interacting receptors.

General, PDB-based collective variables for protein folding

New, automated forms of data-analysis are required in order to understand the high-dimensional trajectories that are obtained from molecular dynamics simulations on proteins. Dimensionality reduction algorithms are particularly appealing in this regard as they allow one to construct unbiased, low-dimensional representations of the trajectory using only the information encoded in the trajectory. The downside of this approach is that different sets of coordinates are required for each different chemical systems under study precisely because the coordinates are constructed using information from the trajectory. In this paper we show how one can resolve this problem by using the sketch-map algorithm that we recently proposed to construct a low-dimensional representation of the structures contained in the protein data bank (PDB). We show that the resulting coordinates are as useful for analysing trajectory data as coordinates constructed using landmark configurations taken from the trajectory and that these coordinates can thus be used for understanding protein folding across a range of systems.