A critique of current practice of transvaginal pudendal nerve blocks: a prospective audit of understanding and clinical practice.

Pudendal nerve blocks are a pre-requisite to forceps delivery without regional anaesthesia. Their efficacy is dependent on introducing local anaesthetic in close proximity to the pudendal nerve and allowing sufficient time for its onset of action. An audit of 57 obstetricians evaluated their clinical technique against standards using both a questionnaire and adapted model pelvis. The majority of participants were unable to describe correctly the point of infiltration and were unaware of the lag time required to effect adequate analgesia. We identify a deficiency in training and describe a method by which training can be facilitated and assessed.

The surgical transversus abdominis plane block--a novel approach for performing an established technique.

Although the transversus abdominis plane (TAP) block has an established role in providing postoperative analgesia following caesarean section, the technique is not widely used by obstetric anaesthetists. The conventional TAP block is associated with significant technical difficulties and risk of peritoneal, hollow viscus and organ perforation. We report a much simpler technique in which the obstetric surgeon, during open surgery, is able to introduce the TAP block via an intra-abdominal approach, which is technically easier and also obviates the risks associated with the conventional TAP procedure. We believe our technique may be easier, safer and equally effective.

Linking endocytic cargo to clathrin: structural and functional insights into coated vesicle formation.

Clathrin-mediated endocytosis is the major process by which transmembrane proteins are internalized from the cell's limiting membrane into the first compartment of the endosomal system, the early endosome. From here, these transmembrane cargo proteins, which are of widely varying type and function, are trafficked to their required destination. Endocytosis plays, therefore, an important role in cell signalling, nutrient uptake, cellular homoeostasis and the interaction of the cell with its external environment. The formation of clathrin-coated endocytic vesicles requires the complex interplay of many proteins with each other and with the membrane itself. Their formation has served as a paradigm for formation of all types of transport vesicle, which move cargo between the various membrane-bound compartments of the cell. Clathrin-coated vesicles (CCVs) possess three layers: the inner membrane layer, in which the transmembrane cargo is embedded, linked to the outer clathrin lattice by a layer of cargo-binding adaptors and proteins that aid and regulate vesicle formation. Protein X-ray crystallography in combination with biochemical, biophysical and cell biological assays has been used to investigate the structure and function of some of the proteins that make up the middle layer of CCVs. These proteins are diverse in their functions, but are all modular in nature, consisting of folded domains joined by long unstructured linkers. Within these linkers are short motifs that interact with the folded domains of other components of the CCV formation machinery. Many of these folded domains also bind directly to the membrane. These interactions, whose molecular basis we have studied, have affinities in the low micromolar range, making them readily reversible and easily regulated. The mechanism of CCV formation is discussed in the light of this structural and biochemical data.

Synthesis of functional Ras lipoproteins and fluorescent derivatives.

For the study of biological signal transduction, access to correctly lipidated proteins is of utmost importance. Furthermore, access to bioconjugates that embody the correct structure of the protein but that may additionally carry different lipid groups or labels (i.e., fluorescent tags) by which the protein can be traced in biological systems, could provide invaluable reagents. We report here of the development of techniques for the synthesis of a series of modified Ras proteins. These modified Ras proteins carry a number of different, natural and non-natural lipid residues, and the process was extended to also provide access to a number of fluorescently labeled derivatives. The maleimide group provided the key to link chemically synthesized lipopeptide molecules in a specific and efficient manner to a truncated form of the H-Ras protein. Furthermore, a preliminary study on the biological activity of the natural Ras protein derivative (containing the normal farnesyl and palmitoyl lipid residues) has shown full biological activity. This result highlights the usefulness of these compounds as invaluable tools for the study of Ras signal transduction processes and the plasma membrane localization of the Ras proteins.

A third specificity-determining site in mu 2 adaptin for sequences upstream of Yxx phi sorting motifs.

Internalization signals of the Yxx phi type (phi = bulky hydrophobic side chain) interact with the mu 2 chain of AP-2 adaptors. Internalization activity is intolerant of non-conservative substitution of either the tyrosine or the phi side chains, which bind to hydrophobic pockets in mu 2 adaptin in a conformation described as 'a two pinned plug into a socket'. P-selectin, a type I transmembrane protein, contains the Yxx phi-like sequence YGVF in its cytoplasmic domain, but substitution of either the tyrosine or phenylalanine with alanine in the full-length protein causes only small changes in the rate of endocytosis. It is shown here that the sequence YGVF contained within a peptide corresponding to the 17 COOH-terminal amino acids of P-selectin binds to mu 2 adaptin in the same fashion previously seen for other Yxx phi motifs. In addition, the P-selectin peptide binds to a third hydrophobic pocket in mu 2 adaptin through a leucine at position Y-3 in the peptide. This structure suggests that some sequences can function as a 'three pinned plug', in which internalization activity is not critically dependent on any one of the three interacting side chains.

Simultaneous binding of PtdIns(4,5)P2 and clathrin by AP180 in the nucleation of clathrin lattices on membranes.

Adaptor protein 180 (AP180) and its homolog, clathrin assembly lymphoid myeloid leukemia protein (CALM), are closely related proteins that play important roles in clathrin-mediated endocytosis. Here, we present the structure of the NH2-terminal domain of CALM bound to phosphatidylinositol-4,5- bisphosphate [PtdIns(4,5)P2] via a lysine-rich motif. This motif is found in other proteins predicted to have domains of similar structure (for example, Huntingtin interacting protein 1). The structure is in part similar to the epsin NH2-terminal (ENTH) domain, but epsin lacks the PtdIns(4,5)P2-binding site. Because AP180 could bind to PtdIns(4,5)P2 and clathrin simultaneously, it may serve to tether clathrin to the membrane. This was shown by using purified components and a budding assay on preformed lipid monolayers. In the presence of AP180, clathrin lattices formed on the monolayer. When AP2 was also present, coated pits were formed.

A one-pot synthesis of novel N,N-dialkyl-S-glycosylsulfenamides.

An efficient new entry into N,N-dialkyl-S-glycosylsulfenamides is reported. The reaction of bis-activated alkyl halides in the presence of a secondary amine base with glycosylic S-acetyl derivatives (1-S-acetyl-1-thioaldoses or 2-S-acetyl-2-thioketoses) results in the formation of novel carbohydrate sulfonamides. These new carbohydrate-based sulfonamides may provide useful derivatives with biological activity, as well as provide reactive carbohydrate sulfonylating agents.

The structural basis for the recognition of acetylated histone H4 by the bromodomain of histone acetyltransferase gcn5p.

The bromodomain is an approximately 110 amino acid module found in histone acetyltransferases and the ATPase component of certain nucleosome remodelling complexes. We report the crystal structure at 1.9 A resolution of the Saccharomyces cerevisiae Gcn5p bromodomain complexed with a peptide corresponding to residues 15-29 of histone H4 acetylated at the zeta-N of lysine 16. We show that this bromodomain preferentially binds to peptides containing an N:-acetyl lysine residue. Only residues 16-19 of the acetylated peptide interact with the bromodomain. The primary interaction is the N:-acetyl lysine binding in a cleft with the specificity provided by the interaction of the amide nitrogen of a conserved asparagine with the oxygen of the acetyl carbonyl group. A network of water-mediated H-bonds with protein main chain carbonyl groups at the base of the cleft contributes to the binding. Additional side chain binding occurs on a shallow depression that is hydrophobic at one end and can accommodate charge interactions at the other. These findings suggest that the Gcn5p bromodomain may discriminate between different acetylated lysine residues depending on the context in which they are displayed.

The structure and function of the beta 2-adaptin appendage domain.

The heterotetrameric AP2 adaptor (alpha, beta 2, mu 2 and sigma 2 subunits) plays a central role in clathrin-mediated endocytosis. We present the protein recruitment function and 1.7 A resolution structure of its beta 2-appendage domain to complement those previously determined for the mu 2 subunit and alpha appendage. Using structure-directed mutagenesis, we demonstrate the ability of the beta 2 appendage alone to bind directly to clathrin and the accessory proteins AP180, epsin and eps15 at the same site. Clathrin polymerization is promoted by binding of clathrin simultaneously to the beta 2-appendage site and to a second site on the adjacent beta 2 hinge. This results in the displacement of the other ligands from the beta 2 appendage. Thus clathrin binding to an AP2-accessory protein complex would cause the controlled release of accessory proteins at sites of vesicle formation.

Structural insights into clathrin-mediated endocytosis.

The process of clathrin-mediated endocytosis from the plasma membrane has been the subject of many biological and biochemical investigations. Recent atomic resolution structures determined by X-ray crystallography now enable the molecular basis for the interactions of some components of the endocytic machinery to be understood in detail.

Clathrin coat construction in endocytosis.

Electron cryomicroscopy of the clathrin coat and X-ray crystallography of parts of the clathrin heavy chain combine to give a detailed picture of the clathrin molecule, assembled as a cage. Recently determined domain structures of other components of the endocytic machinery, particularly the mu2 subunit and the alpha-appendage domain of the AP2 adaptor complex, provide important information on the sequence of recognition events involved in the dynamic process of clathrin coat assembly.

Bioorganic synthesis of lipid-modified proteins for the study of signal transduction.

Biological membranes define the boundaries of the cellular compartments in higher eukaryotes and are active in many processes such as signal transduction and vesicular transport. Although post-translational lipid modification of numerous proteins in signal transduction is crucial for biological function, analysis of protein-protein interactions has mainly focused on recombinant proteins in solution under defined in vitro conditions. Here we present a new strategy for the synthesis of such lipid-modified proteins. It involves the bacterial expression of a carboxy-terminally truncated non-lipidated protein, the chemical synthesis of differently lipidated peptides representing the C terminus of the proteins, and their covalent coupling. Our technique is demonstrated using Ras constructs, which exhibit properties very similar to fully processed Ras, but can be produced in high yields and are open for selective modifications. These constructs are operative in biophysical and cellular assay systems, showing specific recognition of effectors by Ras lipoproteins inserted into the membrane surface of biosensors and transforming activity of oncogenic variants after microinjection into cultured cells.

Identification of endogenous gibberellins in strawberry, including the novel gibberellins GA123, GA124 and GA125.

Extracts of carboxylic acids from immature fruits of strawberry (Fragaria x ananassa Duch. cv. Elsanta) were analysed for gibberellins by combined gas chromatography-mass spectrometry. The following previously characterised gibberellins were identified by comparison of their mass spectra and Kovats retention indices (KRIs) with those of standards or published data: GA1, GA3, GA5, GA8, GA12, GA17, GA19, GA20, GA29, GA44, GA48, GA49, GA53, GA77, GA97, GA111 and GA112. Evidence for endogenous 1-epi GA61 (GA119) and 11alpha-OH-GA12 was also obtained. In addition, a number of putative GAs were detected. Of these, three were shown to be 12alpha-hydroxy-GA53, 12alpha-hydroxy-GA44, and 12alpha-hydroxy-GA19 by comparison with authentic compounds prepared by rational synthesis, and have been allocated the descriptors GA123, GA124 and GA125, respectively.

Catalytic mechanism of phosphorylase kinase probed by mutational studies.

The contributions to catalysis of the conserved catalytic aspartate (Asp149) in the phosphorylase kinase catalytic subunit (PhK; residues 1-298) have been studied by kinetic and crystallographic methods. Kinetic studies in solvents of different viscosity show that PhK, like cyclic AMP dependent protein kinase, exhibits a mechanism in which the chemical step of phosphoryl transfer is fast and the rate-limiting step is release of the products, ADP and phosphoprotein, and possibly viscosity-dependent conformational changes. Site-directed mutagenesis of Asp149 to Ala and Asn resulted in enzymes with a small increase in K(m) for glycogen phosphorylase b (GPb) and ATP substrates and dramatic decreases in k(cat) (1.3 x 10(4) for Asp149Ala and 4.7 x 10(3) for Asp149Asn mutants, respectively). Viscosometric kinetic measurements with the Asp149Asn mutant showed a reduction in the rate-limiting step for release of products by 4.5 x 10(3) and a significant decrease (possibly as great as 2.2 x 10(3)) in the rate constant characterizing the chemical step. The date combined with the crystallographic evidence for the ternary PhK-AMPPNP-peptide complex [Lowe et al. (1997) EMBO J. 6, 6646-6658] provide powerful support for the role of the carboxyl of Asp149 in binding and orientation of the substrate and in catalysis of phosphoryl transfer. The constitutively active subunit PhK has a glutamate (Glu182) residue in the activation segment, in place of a phosphorylatable serine, threonine, or tyrosine residue in other protein kinases that are activated by phosphorylation. Site-directed mutagenesis of Glu182 and other residues involved in a hydrogen bond network resulted in mutant proteins (Glu182Ser, Arg148Ala, and Tyr206Phe) with decreased catalytic efficiency (approximate average decrease in k(cat)/K(m) by 20-fold). The crystal structure of the mutant Glu182Ser at 2.6 A resolution showed a phosphate dianion about 2.6 A from the position previously occupied by the carboxylate of Glu182. There was no change in tertiary structure from the native protein, but the activation segment in the region C-terminal to residue 182 showed increased disorder, indicating that correct localization of the activation segment is necessary in order to recognize and present the protein substrate for catalysis.

A structural explanation for the binding of multiple ligands by the alpha-adaptin appendage domain.

The alpha subunit of the endocytotic AP2 adaptor complex contains a 30 kDa "appendage" domain, which is joined to the rest of the protein via a flexible linker. The 1.9 A resolution crystal structure of this domain reveals a single binding site for its ligands, which include amphiphysin, Eps15, and epsin. This domain when overexpressed in COS7 fibroblasts is shown to inhibit transferrin uptake, whereas mutants in which interactions with its binding partners are abolished do not. DPF/W motifs present in appendage domain-binding partners are shown to play a crucial role in their interactions with the domain. A single site for binding multiple ligands would allow for temporal and spatial regulation in the recruitment of components of the endocytic machinery.

Peptide conjugates as tools for the study of biological signal transduction.

Today, many biological phenomena are being investigated and understood in molecular detail, and organic chemistry is increasingly being directed towards biological phenomena. This review is intended to highlight this interplay of organic chemistry and biology, using biological signal transduction as an example. Lipo-, glyco-, phospho- and nucleoproteins play key roles in the processes whereby chemical signals are passed across cell membranes and further to the cell nucleus. For the study of the biological phenomena associated with these protein conjugates, structurally well-defined peptides containing the characteristic linkage region of the peptide backbone with the lipid, the carbohydrate or the phosphoric acid ester can provide valuable tools. The multi-functionality and pronounced acid- and base-lability of such compounds renders their synthesis a formidable challenge to conventional organic synthesis. However, the recent development of enzymatic protecting groups, provides one of the central techniques which, when coupled with classic chemical synthesis, can provide access to these complex and sensitive biologically relevant peptide conjugates under particularly mild conditions and with high selectivity.