Results of phalloplasty.

We present our experience with a method of phalloplasty which can be applied by reconstructive surgeons to cases of penile loss from diverse etiologies. The complications with this method have been frequent, but not life-threatening. Achievement of the stated objectives is obtained in a sufficiently high proportion of cases to make this a method whorthwhile to use until a clearly superior method can be demonstrated.     

Effect of polyelectrolyte structure on protein-polyelectrolyte coacervates: coacervates of bovine serum albumin with poly(diallyldimethylammonium chloride) versus chitosan

Electrostatic interactions between synthetic polyelectrolytes and proteins can lead to the formation of dense, macroion-rich liquid phases, with equilibrium microheterogeneities on length scales up to hundreds of nanometers. The effects of pH and ionic strength on the rheological and optical properties of these coacervates indicate microstructures sensitive to protein-polyelectrolyte interactions. We report here on the properties of coacervates obtained for bovine serum albumin (BSA) with the biopolyelectrolyte chitosan and find remarkable differences relative to coacervates obtained for BSA with poly(diallyldimethylammonium chloride) (PDADMAC). Coacervation with chitosan occurs more readily than with PDADMAC. Viscosities of coacervates obtained with chitosan are more than an order of magnitude larger and, unlike those with PDADMAC, show temperature and shear rate dependence. For the coacervates with chitosan, a fast relaxation time in dynamic light scattering, attributable to relatively unrestricted protein diffusion in both systems, is diminished in intensity by a factor of 3-4, and the consequent dominance by slow modes is accompanied by a more heterogeneous array of slow apparent diffusivities. In place of a small-angle neutron scattering Guinier region in the vicinity of 0.004 A-1, a 10-fold increase in scattering intensity is observed at lower q. Taken together, these results confirm the presence of dense domains on length scales of hundreds of nanometers to micrometers, which in coacervates prepared with chitosan are less solidlike, more interconnected, and occupy a larger volume fraction. The differences in properties are thus correlated with differences in mesophase structure.     

Structural Basis of GD2 Ganglioside and Mimetic Peptide Recognition by 14G2a Antibody

Monoclonal antibodies targeting GD2 ganglioside (GD2) have recently been approved for the treatment of high risk neuroblastoma and are extensively evaluated in clinics in other indications. This study illustrates how a therapeutic antibody distinguishes between different types of gangliosides present on normal and cancer cells and informs how synthetic peptides can imitate ganglioside in its binding to the antibody. Using high resolution crystal structures we demonstrate that the ganglioside recognition by a model antibody (14G2a) is based primarily on an extended network of direct and water molecule mediated hydrogen bonds. Comparison of the GD2-Fab structure with that of a ligand free antibody reveals an induced fit mechanism of ligand binding. These conclusions are validated by directed mutagenesis and allowed structure guided generation of antibody variant with improved affinity toward GD2. Contrary to the carbohydrate, both evaluated mimetic peptides utilize a “key and lock” interaction mechanism complementing the surface of the antibody binding groove exactly as found in the empty structure. The interaction of both peptides with the Fab relies considerably on hydrophobic contacts however, the detailed connections differ significantly between the peptides. As such, the evaluated peptide carbohydrate mimicry is defined primarily in a functional and not in structural manner.Malignant transformation is universally accompanied by changes in cell surface glycosylation. A glycolipid, GD2 ganglioside (GD2)¹, is one of the most prominent tumor-associated antigens, ranking in the 12th position of the NCI prioritized list of cancer vaccine targets (1). GD2 is embedded in the outer plasma membrane with its ceramide tail (fatty acid coupled sphingosine). The sugar moiety is exposed to the extracellular milieu and is composed of glucose (Glc; linked to ceramide), galactose (Gal) and N-acetylgalactosamine (GalNAc). Two additional sialic acid residues (N-acetylneuraminic acid, NeuAc) branch form Gal and provide GD2 with a negative charge (Fig. 1). Overexpression of GD2 is well documented in neuroblastoma, melanoma, certain osteosarcomas, small cell lung cancers, and soft tissue sarcomas (2–4).Open in a separate windowFig. 1.Recognition of GD2 ganglioside by monoclonal antibody 14G2a at the cell surface. (top panel) Antigen combining region of 14G2a antibody recognizes the sugar moiety of GD2 ganglioside (yellow), which is exposed to the extracellular milieu. The lipid part of the ganglioside is buried inside the cell membrane. GD2 bound Fab structure determined in this study is shown in color. Fc fragment (PDB ID: 1igt) and membrane model derived from published data are shown in corresponding scale and colored gray. (bottom panel) Chemical structure of GD2 ganglioside and sugar ring nomenclature used throughout the study.The concept of therapeutic targeting of GD2 is currently most advanced in neuroblastoma, the most common extracranial tumor of childhood. Neuroblastoma is a heterogenous and complex disease. Spontaneous remissions are sometimes observed, but more than a half of the patients are diagnosed with a high-risk neuroblastoma of poor prognosis. This highlights the demand for treatment modalities that would offer major clinical benefits for this group of patients (5). High and stable presence of GD2 on cancer cells in neuroblastoma and limited expression on relevant normal tissues (i.e. neurons, peripheral nerve fibers and skin melanocytes) allows diagnosis, detection of metastases, treatment monitoring and, most importantly, targeting of the tumor itself.GD2-specific monoclonal antibodies have been extensively tested in clinics. This includes a mouse 14G2a antibody (IgG2a; derived from a mouse 14.18 antibody of IgG3 subclass), and improved modifications thereof including a chimeric antibody ch14.18, and recently a humanized antibody hu14.18K322A. Moreover, mouse 3F8 antibody (IgG3) and recently its humanized derivative hu3F8 were also evaluated. The antibodies were demonstrated to engage antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against neuroblastoma (5). Additionally, direct cytotoxic effects were observed in neuroblastoma models (6). The results of a randomized clinical trial published in 2010, evaluating ch14.18, interleukin-2 and granulocyte and macrophage-colony stimulating factor combined with a standard maintenance agent 13-cis retinoic acid demonstrated significant improvement of outcome in high-risk neuroblastoma patients (7). Based on these and further findings, the Food and Drug Administration (FDA) has just recently approved Unituxin (dinutuximab; ch14.18) combination therapy for high risk neuroblastoma (8). Therefore, the standard care treatment protocols may now be extended with monoclonal antibodies targeting GD2 for a better expected outcome.Antibodies against gangliosides other than GD2 are considered as potential therapeutic agents in different types of cancer. Ganglioside-specific antibodies are moreover involved in various types of autoimmune diseases (9). Nevertheless, the molecular mechanism of ganglioside recognition remains unknown because not a single crystal structure of antibody–ganglioside complex has been determined to date. In particular, it is not known how the specificity against GD2 is achieved in antibodies evaluated in clinics. Although crystal structures of empty ME36.1 antibody specific for GD2 and GD3 (10) and empty 3F8 antibody specific for GD2 (11) were determined, the conclusions concerning GD2 binding have to be treated with caution because of general limitations in reliable prediction of binding modes of complex, flexible ligands in dynamic pockets.The success of GD2-specific antibodies in treatment of neuroblastoma fuels investigation on active immunization strategies. To overcome poor antigenicity of GD2, glycolipid surrogates including peptide mimetics are being developed. The idea of a peptide vaccine eliciting anticarbohydrate response has been precedented in the case of Group B Streptococcus polysaccharide (12). Multiple peptides mimicking GD2 in its binding to specific antibodies were selected using phage display (13, 14) and some have been demonstrated to elicit protective, GD2 directed response in preclinical studies. However, the structural basis of peptide-ganglioside mimicry and its relation to the potential of particular peptides to induce GD2 directed immune response remain unknown.Here, we analyze the interactions guiding ganglioside recognition by an antibody and the structural basis of peptide-ganglioside mimicry. The crystal structure of Fab fragment of 14G2a antibody in a complex with the sugar moiety of GD2 ganglioside is provided and the binding mode is discussed in detail. Structure of an empty 14G2a antibody is reported for reference. The major conclusions are verified by directed mutagenesis and antibody variant with increased affinity toward GD2 is developed using structure guided approach. The binding modes of two largely divergent peptide mimics of GD2 (15) at the antigen-binding site of 14G2a antibody are reported and compared with that of the carbohydrate. Mouse 14G2a antibody was chosen for this study because it contains the same antigen binding region as the ch14.18 chimeric antibody recently approved by FDA (8).     

The development of first Staphylococcus aureus SplB protease inhibitors: Phosphonic analogues of glutamine

Produced by Staphylococcus aureus, SplB belongs to the chymotrypsin-like serine protease family. Since the biological role of SplB protease is unknown, the design and application of its specific inhibitors may help to reveal the function of this enzyme. Until now no SplB inhibitors have been reported. Herein, we present the design and synthesis of novel α-aminophosphonic analogues of glutamine, as well as their peptidyl derivatives. The inhibitory effects of these compounds towards the newly discovered SplB serine protease from S. aureus are characterized. We have also investigated the influence of aromatic ester substituents on inhibitory potency towards SplB. One of the compounds-Cbz-Glu-Leu-Gln(P)(OC(6)H(4)-4-O-CH(3))(2)-displayed an apparent second-order inhibition rate value of 1400M(-1)s(-1).     

Tumor necrosis factor alpha stimulates cathepsin K and V activity via juxtacrine monocyte–endothelial cell signaling and JNK activation

Inflammation and damage promote monocyte adhesion to endothelium and cardiovascular disease (CVD). Elevated inflammation and increased monocyte-endothelial cell interactions represent the initial stages of vascular remodeling associated with a multitude of CVDs. Cathepsins are proteases produced by both cell types that degrade elastin and collagen in arterial walls, and are upregulated in CVD. We hypothesized that the inflammatory cytokine tumor necrosis factor alpha (TNFα) and monocyte binding would stimulate cathepsins K and V expression and activity in endothelial cells that may be responsible for initiating local proteolysis during CVD. Confluent human aortic endothelial cells were stimulated with TNFα or THP-1 monocyte co-cultures, and multiplex cathepsin zymography was used to detect changes in levels of active cathepsins K, L, S, and V. Direct monocyte-endothelial cell co-cultures stimulated with TNFα generated maximally observed cathepsin K and V activities compared to either cell type alone (n = 3, p < 0.05) by a c-Jun N-terminal kinase (JNK)-dependent manner. Inhibition of JNK with SP6000125 blocked upregulation of cathepsin K activity by 49 % and cathepsin V by 81 % in endothelial cells. Together, these data show that inflammatory cues and monocyte-endothelial cell interactions upregulate cathepsin activity via JNK signaling axis and identify a new mechanism to target toward slowing the earliest stages of tissue remodeling in CVD.     

Substrate specificity of Staphylococcus aureus cysteine proteases--Staphopains A, B and C

Human strains of Staphylococcus aureus secrete two papain-like proteases, staphopain A and B. Avian strains produce another homologous enzyme, staphopain C. Animal studies suggest that staphopains B and C contribute to bacterial virulence, in contrast to staphopain A, which seems to have a virulence unrelated function. Here we present a detailed study of substrate preferences of all three proteases. The specificity of staphopain A, B and C substrate-binding subsites was mapped using different synthetic substrate libraries, inhibitor libraries and a protein substrate combinatorial library. The analysis demonstrated that the most efficiently hydrolyzed sites, using Schechter and Berger nomenclature, comprise a P2–Gly↓Ala(Ser) sequence motif, where P2 distinguishes the specificity of staphopain A (Leu) from that of both staphopains B and C (Phe/Tyr). However, we show that at the same time the overall specificity of staphopains is relaxed, insofar as multiple substrates that diverge from the sequences described above are also efficiently hydrolyzed.     

Experiences and perceptions of oral health and oral health care among a sample of older New Zealanders

doi: 10.1111/j.1741‐2358.2010.00402.x Experiences and perceptions of oral health and oral health care among a sample of older New Zealanders Background: Most research on older people’s oral health has been quantitative. A need for more in‐depth understanding of the oral health of that age group has pointed to a need for more qualitative investigations. Objective: To explore experiences and perceptions of oral health and oral health care among an ethnically‐mixed sample of older New Zealanders. Methods: In‐depth interviews were conducted with 24 older people in two communities in New Zealand’s South Island. Thematic analysis of transcribed data was undertaken. Results: Three main themes that emerged were: (1) the processes of negotiating a tension between cost and convenience of access; (2) the experiential constraining of oral health maintenance; and (3) trusting in dental professionals. These serve to organise processes such as normalising, justifying and social comparisons that create an equilibrium or tolerance and acceptance of what might otherwise be considered to be relatively poor oral health. Conclusions: We identified a number of shared experiences which affect older people’s ability to maintain their oral health in the face of material and social barriers to oral health care. Because expectations were generally lower, there was greater concordance between experience and expectation, and people tended to be fairly satisfied with their oral health and the care they had received.     

Dynamics of a novel centromeric histone variant CenH3 reveals the evolutionary ancestral timing of centromere biogenesis

The centromeric histone H3 variant (CenH3) serves to target the kinetochore to the centromeres and thus ensures correct chromosome segregation during mitosis and meiosis. The Dictyostelium H3-like variant H3v1 was identified as the CenH3 ortholog. Dictyostelium CenH3 has an extended N-terminal domain with no similarity to any other known proteins and a histone fold domain at its C-terminus. Within the histone fold, α-helix 2 (α2) and an extended loop 1 (L1) have been shown to be required for targeting CenH3 to centromeres. Compared to other known and putative CenH3 histones, Dictyostelium CenH3 has a shorter L1, suggesting that the extension is not an obligatory feature. Through ChIP analysis and fluorescence microscopy of live and fixed cells, we provide here the first survey of centromere structure in amoebozoa. The six telocentric centromeres were found to mostly consist of all the DIRS-1 elements and to associate with H3K9me3. During interphase, the centromeres remain attached to the centrosome forming a single CenH3-containing cluster. Loading of Dictyostelium CenH3 onto centromeres occurs at the G2/prophase transition, in contrast to the anaphase/telophase loading of CenH3 observed in metazoans. This suggests that loading during G2/prophase is the ancestral eukaryotic mechanism and that anaphase/telophase loading of CenH3 has evolved more recently after the amoebozoa diverged from the animal linage.