We have constructed a series of plasmids, the pEMBL family, characterized by the presence of 1) the bla gene as selectable marker, 2) a short segment coding for the alpha-peptide of beta-galactosidase and containing a multiple cloning sites polylinker, 3) the intragenic region of phage F1. pEMBL plasmids have the property of being encapsidated as single stranded DNA, upon superinfection with phage F1. These vectors have been used successfully for DNA sequencing with the dideoxy-method, and can be used for any other purpose for which M13 derivatives are used. However, the pEMBL plasmids have the advantage of being smaller than M13 vectors, and the purification of the DNA is simpler. In addition, and most importantly, long inserts have a higher stability in pEMBL plasmids than M13 vectors.

alpha 1‐acid glycoprotein (alpha AGP) is a well‐characterized human plasma protein. Its structural properties have been studied for many years but little is known about its function. Amino acid sequence analysis of purified human alpha AGP from plasma pooled from several individuals showed considerable heterogeneity. We have cloned the genomic DNA segment encoding alpha AGP and we show that it contains three adjacent alpha AGP coding regions, AGP‐A, B and B‘, identical in exon–intron organization but with slightly different coding potential. These results account for the heterogeneity observed by protein sequencing. Southern blot analysis indicates that the cloned cluster contains all the alpha AGP coding sequences present in the human genome. The larger majority of alpha AGP mRNA in human liver is transcribed from AGP‐A, whose promoter and cap site have been determined while the level of AGP‐B and B’ mRNA in human liver is very low. Using Hep3B hepatoma cells as a model system for the in vitro study of the acute phase reaction, we show that only AGP‐A is strongly induced by treatment with culture medium of LPS stimulated monocytes.

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Stretch-growth has been defined as a process that extends axons via the application of mechanical forces. In the present article, we used a protocol based on magnetic nanoparticles (NPs) for labeling the entire axon tract of hippocampal neurons, and an external magnetic field gradient to generate a dragging force. We found that the application of forces below 10 pN induces growth at a rate of 0.66 6 0.02 mm h 21 pN 21 . Calcium imaging confirmed the strong increase in elongation rate, in comparison with the condition of tip-growth. Enhanced growth in stretched axons was also accompanied by endoplasmic reticulum (ER) accumulation and, accordingly, it was blocked by an inhibition of translation. Stretch-growth was also found to stimulate axonal branching, glutamatergic synaptic transmission, and neuronal excitability. Moreover, stretched axons showed increased microtubule (MT) density and MT assembly was key to sustaining stretch-growth, suggesting a possible role of tensile forces in MT translocation/assembly. Additionally, our data showed that stretched axons do not respond to BDNF signaling, suggesting interference between the two pathways. As these extremely low mechanical forces are physiologically relevant, stretch-growth could be an important endogenous mechanism of axon growth, with a potential for designing novel strategies for axonal regrowth.

The proline-rich domain of synaptojanin 1, a synaptic protein with phosphatidylinositol phosphatase activity, binds to amphiphysin and to a family of recently discovered proteins known as the SH3p4/8/13, the SH3-GL, or the endophilin family. These interactions are mediated by SH3 domains and are believed to play a regulatory role in synaptic vesicle recycling. We have precisely mapped the target peptides on human synaptojanin that are recognized by the SH3 domains of endophilins and amphiphysin and proven that they are distinct. By a combination of different approaches, selection of phage displayed peptide libraries, substitution analyses of peptides synthesized on cellulose membranes, and a peptide scan spanning a 252-residue long synaptojanin fragment, we have concluded that amphiphysin binds to two sites, PIRPSR and PTIPPR, whereas endophilin has a distinct preferred binding site, PKRPPPPR. The comparison of the results obtained by phage display and substitution analysis permitted the identification of proline and arginine at positions 4 and 6 in the PIRPSR and PTIPPR target sequence as the major determinants of the recognition specificity mediated by the SH3 domain of amphiphysin 1. More complex is the structural rationalization of the preferred endophilin ligands where SH3 binding cannot be easily interpreted in the framework of the "classical" type I or type II SH3 binding models. Our results suggest that the binding repertoire of SH3 domains may be more complex than originally predicted.SH3 domains bind to proline-rich peptides that fold into a polyproline type 2 helix. Many SH3-binding proteins contain relatively long proline-rich domains (PRD) 1 with multiple potential SH3 interaction sites (1-4). Given the relatively low specificity of peptide recognition mediated by SH3 domains, it is not clear whether all these interactions, which are identified in vitro, are of functional significance. A second question that arises is whether SH3 domains bind rather unspecifically to many sites along the PRD or rather form specific complexes by binding to unique and distinct sites.Dynamin, synaptojanin, and synapsin, three proteins that are concentrated in the pre-synaptic region of nerve terminals, bear proline-rich regions that bind to diverse SH3-containing proteins. Synapsin I is the main synaptic ligand of the SH3 domain of the adapter protein Grb2 in vitro (2). Recently it has been reported that the same proline-rich D region of synapsin I interacts with c-Src and stimulates its tyrosine kinase activity (5). The physiological significance of these interactions is not clear yet. In contrast, strong evidence supports the notion that disruption of the interaction between amphiphysin and the PRD of dynamin impairs synaptic vesicle endocytosis (6). Dynamin is a GTPase that forms a collar at the neck of forming endocytic vesicles and participates in the fission process that results in the formation of free vesicles (7). Several other SH3-containing proteins have been shown to bind to dynamin in vitro (1, 8 -10).Syna...

PDZ domains are protein-protein interaction modules that typically bind to short peptide sequences at the carboxyl terminus of target proteins. Proteins containing multiple PDZ domains often bind to different transmembrane and intracellular proteins, playing a central role as organizers of multimeric complexes. To characterize the rules underlying the binding specificity of different PDZ domains, we have assembled a novel repertoire of random peptides that are displayed at high density at the carboxyl terminus of the capsid D protein of bacteriophage . We have exploited this combinatorial library to determine the peptide binding preference of the seven PDZ domains of human INADL, a multi-PDZ protein that is homologous to the INAD protein of Drosophila melanogaster. This approach has permitted the determination of the consensus ligand for each PDZ domain and the assignment to class I, class II, and to a new specificity class, class IV, characterized by the presence of an acidic residue at the carboxyl-terminal position. Homology modeling and site-directed mutagenesis experiments confirmed the involvement of specific residues at contact positions in determining the domain binding preference. However, these experiments failed to reveal simple rules that would permit the association of the chemical characteristics of any given residue in the peptide binding pocket to the preference for specific amino acid sequences in the ligand peptide. Rather, they suggested that to infer the binding preference of any PDZ domain, it is necessary to simultaneously take into account all contact positions by using computational procedures. For this purpose we extended the SPOT algorithm, originally developed for SH3 domains, to evaluate the probability that any peptide would bind to any given PDZ domain.A large number of interactions in the cell are mediated by families of protein binding modules that are found repeatedly and in different combinations in several proteins. Typically these modules mediate protein-protein interactions through recognition of short peptides in the target protein (1). Several approaches, based upon the screening of repertoires of combinatorial peptides, have been developed to investigate the recognition specificity of these domain families. Phage display of small peptides of random sequence has been successfully used for the characterization of binding domains such as SH2, SH3, WW, EH, etc. (reviewed in Ref. 2). PDZ domains (identified as conserved elements in postsynaptic density protein PSD-95, Disc-large tumor suppressor Dlg, Zonula occludens protein ZO-1) differ from the remaining domains since they bind to specific carboxyl-terminal sequences of target proteins and/or dimerize with other PDZ domains (reviewed in Ref. 3). This peculiarity has limited the possibility of using "classical" peptide repertoires displayed by fusion to M13 coat proteins, since these display systems present random peptides by fusing them to the amino terminus of pIII or pVIII coat proteins. As a consequence, PDZ specificity ha...

We have determined the sequence coding for human alpha 1-acid glycoprotein from two independently isolated cDNA clones and a genomic clone. The aminoacid sequences deduced from the three clones, deriving from three different individuals, are identical. Southern blot analysis on human DNA indicates that there are at least two genes coding for alpha 1-AGP. We propose that alpha 1-AGP found in plasma is a mixture of the products of these two different genes. This is the simpler explanation for the heterogeneity in the aminoacid composition in purified alpha 1-AGP observed by Schmid et al. (1). DNA sequence comparison with cDNA clones coding for human alpha 1-antitrypsin and haptoglobin shows a conserved sequence within the 5' untranslated region which may play a role in the acute phase response.