Can we infer peptide recognition specificity mediated by SH3 domains?

Protein interaction domain families that modulate the formation of macromolecular complexes recognize specific sequence or structural motifs. For instance SH3 and WW domains bind to polyproline peptides while SH2 and FHA domains bind to peptides phosphorylated in Tyr and Thr respectively. Within each family, variations in the chemical characteristics of the domain binding pocket modulate a finer peptide recognition specificity and, as a consequence, determine the selection of functional protein partners in vivo. In the proteomic era there is the need for reliable inference methods to help restricting the sequence space of the putative targets to be confirmed experimentally by more laborious experimental approaches. Here we will review the published data about the peptide recognition specificity of the SH3 domain family and we will propose a classification of SH3 domains into eight classes. Finally, we will discuss whether the available information is sufficient to infer the recognition specificity of any uncharacterized SH3 domain.     

Presence and location of endothelin and endothelin-binding sites in human nasal mucosa under normal and metaplastic conditions

The presence and site of production of endothelin-1 (ET-1) was investigated in biopsies obtained from the nasal mucosa of 10 healthy human subjects and 10 patients affected by chronic rhinitis. The presence and localization of receptors for ET-1 was also investigated. Bioptic fragments were examined by scanning electron microscopy. ET-1 was present in the vessels and in the respiratory epithelium of normal subjects, whereas in patients affected by epithelial metaplasia induced by chronic rhinitis, it was absent in the metaplastic epithelium and present in the endothelium and vascular wall. Receptors for ET (A- and B-receptor subtypes) were localized in the vessels of the nasal mucosa, both in normal and in pathological subjects. In particular, A-receptors were identified in the vascular wall, whereas B-receptors were mainly distributed in the endothelium. We suggest that ET-1 is involved in the homeostasis of nasal blood flow (shunting the blood toward the deep cavernous plexus and inducing mucosal swelling) by an autocrine and/or paracrine mechanism. Normal epithelium seems to be important in this mechanism, since it is able to produce ET. However, when pathologic conditions induce squamous or cuboidal metaplasia, the epithelium is no longer able to play this role.     

Microsatellite-AFLP for genetic mapping of complex polyploids.

In spite of the economical relevance of polyploid crops, genetic mapping of these species has been relatively overlooked. This is because of intrinsic difficulties such as the uncertainty of the chromosome behavior at meiosis I and the need for very large segregating populations. An important, yet underestimated issue, in mapping polyploids is the choice of the molecular marker system. An ideal molecular marker system for polyploid mapping should maximize the percentage of single dose markers (SDMs) detected and the possibility of recognizing allelic markers. In the present work, the marker index for genetic mapping (MIgm) of M-AFLP is compared with that of AFLP and SAMPL. M-AFLPs have the highest MIgm values (22 vs. 18.5 of SAMPL and 9.83 of AFLP) mostly because of their high power to detect polymorphism. Owing to their prevalent codominant inheritance, it is proposed that M-AFLP can be used for the preliminary identification of hom(e)ologous groups.     

Immunophenotypic properties and estrogen dependency of budding cell structures in the developing mouse mammary gland

Abstract. The initial phase of growth of the parenchymal component of the mouse mammary gland is ductal clongation, which is mainly accomplished by proliferating cells in a specialized structure termed end bud. End buds are composed of multiple layers of epithelial cells (so called body cells) which are capped by a single layer of morphologically unique cells termed cap cells.
We sought to examine the interrelationship between cap cells and other epithelial cell subclasses using a variety of antibodies to different keratin proteins and also antibodies to vimentin, actin and collagen IV. An extensive immunohistochemical characterization of the epithelial components of the developing and differentiating mammary gland demonstrated that cap cells were devoid of any immunohistochemically - detectable keratins but were positive for collagen IV. In contrast, the majority of cells in the end bud along with the luminal epithelial and myoepithelial cells were keratin positive. The body cells of the end bud were the only cells which were positive for antibody to keratin 6, a keratin which previously has been reported to be expressed in proliferating mammary epithelial cells. In addition, estrogen receptor was localized only to epithelial cells of ducts, alvcoli and body cells of end buds, but not to cap cells or myoepithelial cells. We interpret these results to suggest that cap cells are not totpotent stem cells but rather cells specialized in paving the way for ductal elongation as well as serving as precursors to myoepithelial cells.     

beta-Like globin RNA sequences in hemoglobin Lepore disease.

A Southern Italian patient homozygous for hemoglobin Lepore disease synthesizes approximately 3% Lepore delta beta-globin chains (relative to alpha chains) in the reticulocytes. Measurement of beta-like RNA sequences by hybridization to complementary DNA specific for beta-globin demonstrates a low level (1--2% relative to alpha sequences) of these sequences in cytoplasmic RNA from reticulocytes or spleen cells, suggesting that the Lepore gene is expressed into mRNA at a lower extent than normal alpha or beta genes; the comparison with the level of beta-like sequences found in nuclear RNA (6--8%) further supports this conclusion and indicates, in addition, that Lepore RNA might be degraded at a faster rate than normal. 2--3% beta-like sequences are found in nuclear RNA in three cases of homozygous beta0-thalassemia, setting the highest possible estimate for the delta-RNA level; this figure suggests that the 'delta-promoter'-dependent Lepore delta beta gene is somehow more actively expressed than the delta gene.     

Human globin chain separation by isoelectric focusing

Human globin chain separation, a key procedure in the study of hemoglobinopathies, is routinely performed by chromatography on carboxymethylcellulose. This method, though relatively easy and highly reliable, is expensive and time consuming. A new procedure, based on isoelectric focusing, is presented which allows the simultaneous separation of globin chains from multiple samples (at least 20 per gel slab). The method is rapid, inexpensive and can be easily carried out in clinical laboratories, and its high sensitivity allows the identification of radioactive bands even with minute amounts of labelled material. A new phenomenon, called the 'Nonidet P-40 effect', which greatly enhances the separation between gamma and beta chains by binding to these two chains and shifting their pI values in opposite directions, is described.     

Arguments against the prostatic origin of the R-3327 Dunning H tumor

The Dunning tumor, originally described as a carcinoma of the rat dorsal prostate, has for long been used as an experimental model of prostatic cancer. We have recently presented a number of morphological findings that are incompatible with the prostatic origin of the H-subline of the Dunning tumor. In this paper, biochemical and immunohistochemical markers of rat prostate and mammary gland are studied in the R-3327 Dunning H tumor. Pieces of the H tumor were inoculated in male or lactating female rats. The electrophoretic protein pattern of Dunning tumor extracts was more similar to that of the mammary gland than the dorsolateral prostate. Proteins selectively appearing after metabolic labeling in Dunning tumors grown in lactating rats corresponded to labeled proteins in mammary glands from the same animals. Secretory proteins typical of the lateral prostate (SVS II) and dorsal prostate (transglutaminase) could not be detected immunohistochemically in the Dunning tumor. Western blot studies of tumor extracts and slot blot analysis of RNA preparations from the tumor confirmed the absence of SVS II and prostate specific transglutaminase from the Dunning tumor. On the other hand, the presence of mammary gland proteins such as milk fat globule membrane proteins, lactoperoxidase and lactalbumin were detected in the Dunning tumor by immunohistochemistry and Western blotting, but were absent from the dorsolateral prostate. Transferrin-mRNA, expressed in the male urogenital tract and also in the liver and other tissues, was detected in the mammary gland and Dunning tumor, but not in the dorsolateral prostate. The absence of mammary gland secretory beta-casein in the Dunning tumor was related to the elevated Ha-ras oncogene expression in the tumor, previously reported to suppress casein expression. The findings clearly demonstrate that the prostate cannot be the origin of the Dunning tumor, presently being used in prostatic cancer research. The designation prostatic adenocarcinoma for this tumor is therefore invalid. Furthermore, the data support our view that mammary gland might be the origin of the Dunning tumor, although the derivation from the bulbourethral or the parotid glands cannot strictly be excluded.     

Characterization of the oriI and oriII Origins of Replication in Phage-Plasmid P4

In the Escherichia coli phage-plasmid P4, two partially overlapping replicons with bipartite ori sites coexist. The essential components of the oriI replicon are the alpha and cnr genes and the ori1 and crr sites; the oriII replicon is composed of the alpha gene, with the internal ori2 site, and the crr region. The P4 alpha protein has primase and helicase activities and specifically binds type I iterons, present in ori1 and crr. Using a complementation test for plasmid replication, we demonstrated that the two replicons depend on both the primase and helicase activities of the alpha protein. Moreover, neither replicon requires the host DnaA, DnaG, and Rep functions. The bipartite origins of the two replicons share the crr site and differ for ori1 and ori2, respectively. By deletion mapping, we defined the minimal ori1 and ori2 regions sufficient for replication. The ori1 site was limited to a 123-bp region, which contains six type I iterons spaced regularly close to the helical periodicity, and a 35-bp AT-rich region. Deletion of one or more type I iterons inactivated oriI. Moreover, insertion of 6 or 10 bp within the ori1 region also abolished replication ability, suggesting that the relative arrangement of the iterons is relevant. The ori2 site was limited to a 36-bp P4 region that does not contain type I iterons. In vitro, the alpha protein did not bind ori2. Thus, the alpha protein appears to act differently at the two origins of replication.     

Characterization of conjugative transposon Tn5251 of Streptococcus pneumoniae

Abstract Tn5251 belongs to the Tn916-Tn1545 family of conjugative transposons (CT) and was found integrated into CT Tn5252 , to form the composite element Tn5253 of Streptococcus pneumoniae . We show that Tn5251 is identical in structure and size to Tn916 . DNA sequence analysis of a 4,419-bp segment containing the tet(M) gene showed that only 73 nucleotides out of 4,419 were different in the the two CT. Essentially all differences (66 / 73) were clustered in a 688-bp segment of tet(M) , which was 90% identical to Tn916 and 100% identical to the tet(M) genes of Tn1545 from S. pneumoniae and pOZ101 from Neisseria gonorrhoeae . DNA sequence analysis of the Tn5251/Tn5252 junction fragments allowed us (i) to determine Tn5251 termini, (ii) to define the 6-bp coupling sequences flanking the CT, and (iii) to infer the structure of the integration site ( attB ) of Tn5251 into Tn5252 . Conjugal transfer of Tn5251 independent from Tn5253 could not be detected, even if we could show excision and formation of Tn5251 circular intermediates at a level of 5.4 copies per 10⁶ chromosomes.