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The interaction between ubiquitinated proteins and intracellular proteins harboring ubiquitin binding domains (UBDs) is critical to a multitude of cellular processes. Here, we report that Rabex-5, a guanine nucleotide exchange factor for Rab5, binds to Ub through two independent UBDs. These UBDs determine a number of properties of Rabex-5, including its coupled monoubiquitination and interaction in vivo with ubiquitinated EGFRs. Structural and biochemical characterization of the UBDs of Rabex-5 revealed that one of them (MIU, motif interacting with ubiquitin) binds to Ub with modes superimposable to those of the UIM (ubiquitin-interacting motif):Ub interaction, although in the opposite orientation. The other UBD, RUZ (Rabex-5 ubiquitin binding zinc finger) binds to a surface of Ub centered on Asp58(Ub) and distinct from the "canonical" Ile44(Ub)-based surface. The two binding surfaces allow Ub to interact simultaneously with different UBDs, thus opening new perspectives in Ub-mediated signaling.

The spindle assembly checkpoint (SAC) monitors chromosome attachment to spindle microtubules. SAC proteins operate at kinetochores, scaffolds mediating chromosome-microtubule attachment. The ubiquitous SAC constituents Mad1 and Mad2 are recruited to kinetochores in prometaphase. Mad2 sequesters Cdc20 to prevent its ability to mediate anaphase onset. Its function is counteracted by p31comet (formerly CMT2). Upon binding Cdc20, Mad2 changes its conformation from O-Mad2 (Open) to C-Mad2 (Closed). A Mad1-bound C-Mad2 template, to which O-Mad2 binds prior to being converted into Cdc20-bound C-Mad2, assists this process. A molecular understanding of this prion-like property of Mad2 is missing. We characterized the molecular determinants of the O-Mad2:C-Mad2 conformational dimer and derived a rationalization of the binding interface in terms of symmetric and asymmetric components. Mutation of individual interface residues abrogates the SAC in Saccharomyces cerevisiae. NMR chemical shift perturbations indicate that O-Mad2 undergoes a major conformational rearrangement upon binding C-Mad2, suggesting that dimerization facilitates the structural conversion of O-Mad2 required to bind Cdc20. We also show that the negative effects of p31comet on the SAC are based on its competition with O-Mad2 for C-Mad2 binding.

Different plasma membrane receptors are internalized through saturable/noncompetitive pathways, suggesting cargo-specific regulation. Here, we report that TTP (SH3BP4), a SH3-containing protein, specifically regulates the internalization of the transferrin receptor (TfR). TTP interacts with endocytic proteins, including clathrin, dynamin, and the TfR, and localizes selectively to TfR-containing coated-pits (CCP) and -vesicles (CCV). Overexpression of TTP specifically inhibits TfR internalization, and causes the formation of morphologically aberrant CCP, which are probably fission impaired. This effect is mediated by the SH3 of TTP, which can bind to dynamin, and it is rescued by overexpression of dynamin. Functional ablation of TTP causes a reduction in TfR internalization, and reduced cargo loading and size of TfR-CCV. Tyrosine phosphorylation of either TTP or dynamin prevents their interaction, pointing to a possible mechanism of exclusion of TTP from some CCP. Thus, TTP might represent one of the long sought for molecules that allow cargo-specific control of clathrin endocytosis.

Nephroureterectomy with the excision of the ipsilateral ureteral orifice and bladder cuff has been considered the standard treatment of the urinary upper transitional cell carcinoma. With the advent of sophisticated techniques for the endo-urologic management of many benign urologic diseases of the upper tract, there has been growing enthusiasm for the application of these same techniques in the management of upper tract TCC, which is also supported by recent advances in the development of small calibre telescopes with improved optics and the development of small calibre adjunctive instruments and laser fibers. A large number of cases published in the literature has confirmed the safety and efficacy of percutaneous treatment in selected patients with upper tract TCC of low grade and stage. Between 1997 and 2005 we treated 62 pts (37 pelvic transitional cell carcinoma and 25 ureteral). 4 pts (5 renal units: 4 T1G2 and 1 TaG1) underwent percutaneous resection for a tumor in a solitary kidney (2 cases), one case for bilateral neoplasm, and in the other case the lesion was unilateral with chronic renal failure. After preoperative evaluation, (excretory urography, computerized tomography and ureteroscopy with biopsy to confirm the low stage and grade of the lesion) the tumor was resected using an Amplatz sheat of 26-30 Fr and a 24 Fr resectoscope to keep a low intra-caliceal pressure. The tumor base was biopsied and fulgurated After 48 h, contrastography to assure integrity of the urinary system was performed and Mitomycin C was infused over 24 h. Second-look nephroscopy with multiple biopsies was performed in all cases 7 days later and 8 Ch nephrostomy was placed. If the biopsies resulted negative the patient was submitted to 6 weekly endocavitary instillation of BCG through the nephrostomy tube. All pts at a mean follow up of 71 months were tumor free. One patient presented a bladder relapse after 83 months. No complication of percutaneous resection was observed. The endocavitary instillations were well tolerated. In our experience the percutaneous approach is safe and useful in neoplastic lesions of low grade and stage and should be considered as first line therapy in selected patients. Adjuvant topical therapy appears efficacious and some complications may be avoided by maintaining low intracavitary pressures during administration.

The molecular anatomy of cancer cells is being explored through unbiased approaches aimed at the identification of cancer-specific transcriptional signatures. An alternative biased approach is exploitation of molecular tools capable of inducing cellular transformation. Transcriptional signatures thus identified can be readily validated in real cancers and more easily reverse-engineered into signaling pathways, given preexisting molecular knowledge. We exploited the ability of the adenovirus early region 1 A protein (E1A) oncogene to force the reentry into the cell cycle of terminally differentiated cells in order to identify and characterize genes whose expression is upregulated in this process. A subset of these genes was activated through a retinoblastoma protein/E2 viral promoter required factor-independent (pRb/E2F-independent) mechanism and was overexpressed in a fraction of human cancers. Furthermore, this overexpression correlated with tumor progression in colon cancer, and 2 of these genes predicted unfavorable prognosis in breast cancer. A proof of principle biological validation was performed on one of the genes of the signature, skeletal muscle cell reentry-induced (SKIN) gene, a previously undescribed gene. SKIN was found overexpressed in some primary tumors and tumor cell lines and was amplified in a fraction of colon adenocarcinomas. Furthermore, knockdown of SKIN caused selective growth suppression in overexpressing tumor cell lines but not in tumor lines expressing physiological levels of the transcript. Thus, SKIN is a candidate oncogene in human cancer.

8p11 myeloproliferative syndrome (EMS) is a clinical-pathologic entity characterized by rearrangements involving the FGFR1 gene, which encodes a receptor tyrosine kinase. These rearrangements invariably lead to aberrant fusion proteins in which the kinase activity is constitutively turned on, with resulting oncogenic properties. In this article, we describe a new translocation in EMS, t(7;8)(q34;p11), in which the FGFR1 gene is fused to a previously unidentified partner, the TIF1 gene. We show that both the TIF1-FGFR1 and FGFR1-TIF1 fusion proteins have the potential to be translated as a result of the translocation. Thus, our data extend the involvement of FGFR1 in EMS and lend support to the concept that there is a precise correlation between genotype and phenotype in this disease.

The identification and study of evolutionarily conserved genomic sequences that surround disease-related genes is a valuable tool to gain insight into the functional role of these genes and to better elucidate the pathogenetic mechanisms of disease. We created the DG-CST (Disease Gene Conserved Sequence Tags) database for the identification and detailed annotation of human-mouse conserved genomic sequences that are localized within or in the vicinity of human disease-related genes. CSTs are defined as sequences that show at least 70% identity between human and mouse over a length of at least 100 bp. The database contains CST data relative to over 1088 genes responsible for monogenetic human genetic diseases or involved in the susceptibility to multifactorial/polygenic diseases. DG-CST is accessible via the internet at http://dgcst.ceinge.unina.it/ and may be searched using both simple and complex queries. A graphic browser allows direct visualization of the CSTs and related annotations within the context of the relative gene and its transcripts.

Actin filament barbed-end capping proteins are essential for cell motility, as they regulate the growth of actin filaments to generate propulsive force. One family of capping proteins, whose prototype is gelsolin, shares modular architecture, mechanism of action, and regulation through signalling-dependent mechanisms, such as Ca(2+) or phosphatidylinositol-4,5-phosphate binding. Here we show that proteins of another family, the Eps8 family, also show barbed-end capping activity, which resides in their conserved carboxy-terminal effector domain. The isolated effector domain of Eps8 caps barbed ends with an affinity in the nanomolar range. Conversely, full-length Eps8 is auto-inhibited in vitro, and interaction with the Abi1 protein relieves this inhibition. In vivo, Eps8 is recruited to actin dynamic sites, and its removal impairs actin-based propulsion. Eps8-family proteins do not show any similarity to gelsolin-like proteins. Thus, our results identify a new family of actin cappers, and unveil novel modalities of regulation of capping through protein-protein interactions. One established function of the Eps8-Abi1 complex is to participate in the activation of the small GTPase Rac, suggesting a multifaceted role for this complex in actin dynamics, possibly through the participation in alternative larger complexes.

A new complex rearrangement involving chromosome bands 5q13, 12p13, 22q11, and 3q12 was identified and characterized in a patient with acute myeloid leukemia. Fluorescence in situ hybridization showed the involvement of the ETV6 gene in 12p13. ETV6 primers were specifically designed for 3'- and 5'-RACE-PCR experiments, which led to the identification of the other two rearranged genes. The derivative chromosome 5 harbored a fusion of the ETV6 sequence with that of the LOC115548 gene. The two genes were placed in opposite orientation and did not encode a fusion protein. On the derivative chromosome 12, ETV6 was fused to the MN1 gene on chromosome 22. Also in this case, the insertion, within the MN1 sequence, of a portion of chromosome 3 prevented the formation of a fusion protein. Finally, the derivative chromosome 22 contained the 3' portions of both LOC115548 and MN1, and no fusion transcript with coding potential could be predicted. In conclusion, all chromosome breakpoints led to the truncation of the three involved genes in the absence of predicted fusion proteins. This study lends further support to the hypothesis that gene disruption resulting in either loss of function or haploinsufficiency may be relevant in acute myeloid leukemia pathogenesis., (Copyright 2004 Wiley-Liss, Inc.)

Metanephric adenomas are benign tumors frequently found post-mortem (from 7% to 22% of autopsies) which originate from distal tubules; they are generally small in dimensions (smaller than 1 cm) and located in the renal cortex. Etiology is unknown, even though they could be associated with smoke, tubular nephrosclerosis, dialysis. An endocrinal relationship was proposed, because of its more frequent incidence in female (2:1). Metanephric adenoma is an uncommon kind of renal adenoma with no malignant potentiality: from the clinical and diagnostic viewpoint its own greater importance depends on the probability of diagnostic misunderstanding with Wilms' tumor; furthermore its echographic, tomographic and arteriographic characteristics are often similar to small renal adenocarcinoma ones (100). Polycytemia is frequently associated to metanephric adenoma as paraneoplastic syndrome and, more rarely, abdominal mass, abdominal pain, hematuria and hypertension (140). The most important study on metanephric adenoma is the one realized by E.K. Mostofi, including 50 cases from the Department of Genitourinary Pathology, Armed Forces Institute of Pathology, Washington, D.C., published in 1995; in this study half of findings was incidental; mean dimension of tumor about 5,5 cm and in 50% it could be seen a distinct capsule sourrounding the tumor (in the remaining cases the capsule was discontinuos or absent) (210). An important radiological characteristic is that metanephric adenomas are more frequent calcificated than other histotypes and, from the pathological viewpoint, the most important differential element seems to be the smaller dimensions of its cells with hyperchromatic nuclei in the absence or lack in mitotic activity and in the absence of chromosome aberrations. In the case of difficult histological diagnosis, cytogenetic and immunohistochemical analysis can be useful. In conclusion, because it is impossible to distinguish in the preoperatory between the metaneprhic adenomas and the other tumoral types on the bases of the symptoms, dimensions or radiographic appearance, it is mandatory to treat it as malignant eteroformations in a therapeutical strategy, when it is possible.

Exogenously and endogenously originated signals are propagated within the cell by functional and physical networks of proteins, leading to numerous biological outcomes. Many protein-protein interactions take place between binding domains and short peptide motifs. Frequently, these interactions are inducible by upstream signaling events, in which case one of the two binding surfaces may be created by a posttranslational modification. Here, we discuss two protein networks. One, the EH-network, is based on the Eps15 homology (EH) domain, which binds to peptides containing the sequence Asp-Pro-Phe (NPF). The other, which we define as the monoubiquitin (mUb) network, relies on monoubiquitination, which is emerging as an important posttranslational modification that regulates protein function. Both networks were initially implicated in the control of plasma membrane receptor endocytosis and in the regulation of intracellular trafficking routes. The ramifications of these two networks, however, appear to extend into many other aspects of cell physiology as well, such as transcriptional regulation, actin cytoskeleton remodeling, and DNA repair. The focus of this review is to integrate available knowledge of the EH- and mUb networks with predictions of genetic and physical interactions stemming from functional genomics approaches.

EPS8 codes for a protein essential in Ras to Rac signaling leading to actin remodeling. Three genes highly homologous to EPS8 were discovered, thereby defining a novel gene family. Here, we report the genomic structure of EPS8 and the EPS8-related genes in human and mouse. We performed BLASTN searches against the Celera Human Genome and Mouse Fragments Database. The mouse fragments were manually assembled, and the organization of both human and mouse genes was reconstructed. The gene structures in Celera annotations of the human and mouse genomes were compared to outline correspondences and divergences. We also compared the EPS8 family gene structures predicted by Celera with those predicted by NCBI. Moreover, we performed a virtual analysis of the expression of the EPS8 gene family members by using the SAGEmap Database in NCBI. Finally, we analyzed the domain organization of the gene products and their evolutionary conservation to define novel putative domains, thereby helping to predict novel modality of action for the members of this gene family. The data obtained will be instrumental in directing further experimental functional characterization of these genes.

The increasing popularity of DNA chip technology for the study of gene expression is producing, for each experiment, a sizable quantity of numerical data to analyse and an accompanying large number of gene identifiers that should be associated with the relevant biological annotation. We describe here a website at IFOM (FIRC Institute of Molecular Oncology) where we release regularly updated annotation tables for the most used Affymetrix oligonucleotide DNA chips and for the whole Research Genetics 46K clone collection for cDNA arrays. These tables are synchronised with every new release of the mouse and human UniGene databases (NCBI; National Center for Biotechnology Information), allowing fast and easy preliminary annotation of DNA array experiments. We also report some comparative evidence about the importance of biological database synchronisation and cross-references in the process of generating annotation tables for DNA chips.

Objective: The use of "porcine acellular matrix", obtained from small intestine submucosa, could simplify the repair of long urethral strictures, whereas single stage techniques can be carried out only by means of grafts, as buccal mucosa; or flaps, as prepucial skin. To our knowledge we report the first use of porcine intestine submucosa in urethroplastic surgery., Materials and Methods: From May 2001 to December 2001, five urethral reconstructions were completed using "porcine acellular matrix". Four male patients had urethral strictures longer than 10 cm. After circumcision and penile degloving, we extended the surgical approach to the perineum-scrotal region by a midline longitudinal incision. The urethra was exposed, dissected from corpora cavernosa, then rotated of 180 degrees and on this side longitudinally incised throughout all the stenotic length. Urethroplasty was accomplished with matrix tissue modelled according to the length of the stenosis and grafted by a 5-0 polyglycolic acid running suture. The enlarged urethra was then derotated, laying the graft dorsally, closed to corpora cavernosa, to prevent pouching. A further graft was accomplished in a female patient with a 3 cm long urethral stricture. All urethroplasties were stented for 14 days. No complication developed., Results: After 1 month urethral patency was satisfactory compared with preoperatory images and the urinary flow was normal. After 2 months the urethra was endoscopically verified: it was easy to appreciate the homogeneous transformation of the graft into the native tissue. At 6-month follow-up radiological and urodynamic outcome is still satisfactory in all patients., Conclusions: According to our preliminary experience "porcine acellular matrix" is a promising approach for the repair of long urethral strictures. Its safety and effectiveness encourage us to treat more cases in male and female patients.

The Eps15 homology (EH) domain was originally identified as a motif present in three copies at the NH2-termini of Eps15 and of the related molecule Eps15R. Both of these molecules are substrates for the tyrosine kinase activity of the epidermal growth factor receptor and hence the name 'Eps15 homology' or EH domain [Wong et al. (1994) Oncogene 9, 1591-1597; Wong et al. (1995) Proc. Natl. Acad. Sci. USA 92, 9530-9534; Fazioli et al. (1993) Mol. Cell. Biol. 13, 5814-5828] was derived. The motif was subsequently found in several proteins from yeast to nematode, thus establishing its evolutionary conservation. Initial studies with filter-binding assays and phage-displayed libraries demonstrated its protein:protein interaction abilities and identified specific ligands. Subsequently, structural analyses established the molecular bases of recognition between EH domains and cognate peptides. To date, several EH-containing and EH-binding proteins have been identified, which establish in the cell a network of protein:protein interactions, defined as the EH network. This network coordinates cellular functions connected with endocytosis, actin remodeling and intracellular transduction of signals.

Background: The patients who undergo radical pelvic surgery often found that sexual function is impaired. In this research hypothesis, we evaluated the efficacy of alternative therapy to conventional PGE 1 injections, such as the association of Sildenafil and L-Arginine. This association in based on the principle that L-Arginine, the precursor of nitric oxide, improves the effect of Sildenafil, which is effective in the presence of nitric oxide., Methods: The experimental plan was to make a comparative study of 2 random groups of patients selected from those undergoing radical cystectomies and prostatectomies over the past three years. 116 patients were illegible (64 prostatectomies and 52 cystectomies). The fìrst random group was treated with Sildenafil alone and the second with Sildenafil and L-Arginine. The efficacy of treatment was evaluated by the Buckling test (pressure threshold of cavernous flexation at penile axial rigidity) once-after ambulatorial administration and then by telephonic interview (subjective evaluation) after home administration., Results: The starter dose was 50 mg and was inefficient in both groups (Buckling test between 0 and 250). 100-mg doses gave significant results (Buckling test > 500) in both groups, especially the second. Cardiopathic patients, diabetics and patients with retinal disorders were excluded from the study. The mean age of patients was 65 years., Conclusions: The resumption of relatively satisfactory sexual activity was demonstrated using non-invasive pharmacological treatment.

Background: Patients undergoing radical pelvic floor surgery are often find that sexual function is impaired. In this research hypothesis, we evaluated the efficacy of alternative therapy to conventional PGE 1 injections, such as the association of Sildenafil and L-Arginine. This association is based on the principle that L-Arginine, the precursor of nitric oxide, improves the effect of Sildenafil, which is effective in the presence of nitric oxide., Methods: The experimental plan was to make a comparative study between 2 random groups of patients selected from those undergoing radical cystectomies and prostatectomies over the past three years. 116 patients were eligible (64 prostatectomies and 52 cystectomies). The first random group was treated with Sildenafil alone and the second with Sildenafil and L-Arginine. The efficacy of treatment was evaluated using the Buckling test (pressure threshold of cavernous flexation at penile axial rigidity) once after ambulatorial administration and then by telephone interview (subjective evaluation) after administration at domicile., Results: The starter dose was 50 mg and was inefficient in both groups (Buckling test between 0 and 250). 100-mg doses gave significant results (Buckling test >500) in both groups, especially the second. Cardiopathic patients, diabetics and patients with retinal disorders or who were unmotivated were excluded from the study. The mean age of patients was 65., Conclusions: The resumption of relatively satisfactory sexual activity was demonstrated using non-invasive pharmacological treatment.

The Shc protein family is characterized by the (CH2)-PTB-CH1-SH2 modularity. Its complexity increased during evolution from one locus in Drosophila (dShc), to at least three loci in mammals (shc, rai and sli). The three mammalian loci encode, because of alternative initiation codon usage and splicing pattern, at least six Shc-like proteins. Genetic and biological evidence indicates that the mammalian Shc isoforms regulate functions as diverse as growth (p52/p46Shc), apoptosis (p66Shc) and life-span (p66Shc). Available structure-function data and analysis of sequence similarities of Shc-like genes and proteins suggest complex diversification of Shc functions during evolution. Notably, Ras activation, the best-characterized Shc activity, appears to be a recent evolutionary acquisition.

Membrane receptors are internalized either constitutively or upon ligand engagement. Whereas there is evidence for differential regulation of the two processes, little is known about the molecular machinery involved. Previous studies have shown that an unidentified kinase substrate is required for endocytosis of the epidermal growth factor receptor (EGFR), the prototypical ligand-inducible receptor, but not of the transferrin receptor (TfR), the prototypical constitutively internalized receptor. Eps15, an endocytic protein that is tyrosine phosphorylated by EGFR, is a candidate for such a function. Here, we show that tyrosine phosphorylation of Eps15 is necessary for internalization of the EGFR, but not of the TfR. We mapped Tyr 850 as the major in vivo tyrosine phosphorylation site of Eps15. A phosphorylation-negative mutant of Eps15 acted as a dominant negative on the internalization of the EGFR, but not of the TfR. A phosphopeptide, corresponding to the phosphorylated sequence of Eps15, inhibited EGFR endocytosis, suggesting that phosphotyrosine in Eps15 serves as a docking site for a phosphotyrosine binding protein. Thus, tyrosine phosphorylation of Eps15 represents the first molecular determinant, other than those contained in the receptors themselves, which is involved in the differential regulation of constitutive vs. regulated endocytosis.

Eps15 is a substrate for the tyrosine kinase of the epidermal growth factor receptor (EGFR) and is characterized by the presence of a novel protein:protein interaction domain, the EH domain. Eps15 also stably binds the clathrin adaptor protein complex AP-2. Previous work demonstrated an essential role for eps15 in receptor-mediated endocytosis. In this study we show that, upon activation of the EGFR kinase, eps15 undergoes dramatic relocalization consisting of 1) initial relocalization to the plasma membrane and 2) subsequent colocalization with the EGFR in various intracellular compartments of the endocytic pathway, with the notable exclusion of coated vesicles. Relocalization of eps15 is independent of its binding to the EGFR or of binding of the receptor to AP-2. Furthermore, eps15 appears to undergo tyrosine phosphorylation both at the plasma membrane and in a nocodazole-sensitive compartment, suggesting sustained phosphorylation in endocytic compartments. Our results are consistent with a model in which eps15 undergoes cycles of association:dissociation with membranes and suggest multiple roles for this protein in the endocytic pathway.

The Eps homology (EH) domain is a recently described protein binding module that is found, in multiple or single copies, in several proteins in species as diverse as human and yeast. In this work, we have investigated the molecular details of recognition specificity mediated by this domain family by characterizing the peptide-binding preference of 11 different EH domains from mammal and yeast proteins. Ten of the eleven EH domains could bind at least some peptides containing an Asn-Pro-Phe (NPF) motif. By contrast, the first EH domain of End3p preferentially binds peptides containing an His-Thr/Ser-Phe (HT/SF) motif. Domains that have a low affinity for the majority of NPF peptides reveal some affinity for a third class of peptides that contains two consecutive amino acids with aromatic side chains (FW or WW). This is the case for the third EH domain of Eps15 and for the two N-terminal domains of YBL47c. The consensus sequences derived from the peptides selected from phage-displayed peptide libraries allows for grouping of EH domains into families that are characterized by different NPF-context preference. Finally, comparison of the primary sequence of EH domains with similar or divergent specificity identifies a residue at position +3 following a conserved tryptophan, whose chemical characteristics modulate binding preference.

eps15R was identified because of its relatedness to eps15, a gene encoding a tyrosine kinase substrate bearing a novel protein-protein interaction domain, called EH. In this paper, we report a biochemical characterization of the eps15R gene product(s). In NIH-3T3 cells, three proteins of 125, 108, and 76 kDa were specifically recognized by anti-eps15R sera. The 125-kDa species is a bona fide product of the eps15R gene, whereas p108 and p76 are most likely products of alternative splicing events. Eps15R protein(s) are tyrosine-phosphorylated following epidermal growth factor receptor activation in NIH-3T3 cells overexpressing the receptor, even at low levels of receptor occupancy, thus behaving as physiological substrates. A role for eps15R in clathrin-mediated endocytosis is suggested by its localization in plasma membrane-coated pits and in vivo association to the coated pits' adapter protein AP-2. Finally, we demonstrate that a sizable fraction of eps15R exists in the cell as a complex with eps15 and that its EH domains exhibit binding specificities that are partially distinct from those of eps15. We propose that eps15 and eps15R are multifunctional binding proteins that serve pleiotropic functions within the cell.

EH is a recently identified protein-protein interaction domain found in the signal transducers Eps15 and Eps15R and several other proteins of yeast nematode. We show that EH domains from Eps15 and Eps15R bind in vitro to peptides containing an asparagine-proline-phenylalanine (NPF) motif. Direct screening of expression libraries with EH domains yielded a number of putative EH interactors, all of which possessed NPF motifs that were shown to be responsible for the interaction. Among these interactors were the human homolog of NUMB, a developmentally reguated gene of Drosophila, and RAB, the cellular cofactor of the HIV REV protein. We demonstrated coimmunoprecipitation of Eps15 with NUMB and RAB. Finally, in vitro binding of NPF-containing peptides to cellular proteins and EST database screening established the existence of a family of EH-containing proteins in mammals. Based on the characteristics of EH-containing and EH-binding proteins, we propose that EH domains are involved in processes connected with the transport and sorting of molecules within the cell.

The multiple-dose pharmacokinetics and safety of a new potential antidementia compound, CL 275,838, were examined in two randomized, double-blind, parallel-group, placebo-controlled studies. The Alzheimer Disease Assessment Scale (ADAS) was employed to preliminarily assess the patients' cognitive and the behavioral profiles. In the first study, nine patients with Alzheimer type or vascular dementia were treated for 2 weeks with daily doses of 50 mg. In the second study, nine other patients, selected with the same inclusion/exclusion criteria and treated following the same experimental design, were given 100 mg day(minus sign1) CL 275,838. At the lower dose, no side effects were detected; in the 100-mg day(minus sign1) study, mild drowsiness (one patient) and moderate agitation (two patients) were observed. Laboratory tests showed no changes in either study, apart from a slight, transient increase in serum bilirubin in one patient given 100 mg. At the dose of 50 mg, no patients showed any modification on the ADAS, whereas three patients given 100 mg showed some improvement. During the 2 weeks of oral dosing, predose plasma concentrations of the parent compounds and its metabolites II and IV increased but not in proportion to the dose, although at both doses, accumulation was essentially complete within 10 days. At 50 mg, the mean steady-state C(max) and C(ss) of the parent compound were similar to those reached in young males after a comparable regimen. Mean steady-state metabolite-to-parent drug ratios were higher in patients than in healthy individuals, although there was wide variation in our patient group. Mean washout t(1/2) of the parent compound (34 h) and its metabolites II (37 h) and IV (41 h) were longer than in young men and were similar in all cases to those observed after single doses in healthy elderly subjects. After 100 mg, mean C(max) and C(ss) of the unchanged compound rose more than proportionally and the apparent t(1/2) tended to rise, compared with the lower dose. Mean steady-state oral clearance decreased, changing the metabolite-to-parent drug ratios, suggesting nonlinear kinetics after several relatively high oral doses. This might explain why the higher dose was less well tolerated.

The pharmacokinetics and safety of CL 275,838, a new potential memory-enhancing compound, were examined after 14 daily doses (50 and 100 mg) in 16 healthy male volunteers, age 20 to 59 years, in a randomized, double-blind, placebo-controlled, parallel group study. Trough blood samples (predose) were collected on days 2, 4, 7, 10, and 14, and further samples were drawn after the final dose (day 14) to define the multiple-dose kinetics of the parent compound and its metabolites II and IV. Intercurrent clinical events, vital functions, EEG, ECG, and cognitive tests (attention, verbal memory, and spatial memory) were considered as outcome measures of safety. Performance in cognitive tests was also studied to collect preliminary information on possible therapeutic action. Predose plasma concentrations of the parent compound and its two metabolites increased approximately in proportion to the dose, and accumulation was complete within 7 days, regardless of the dose. At steady state, mean Cmax and AUC of the parent compound and its two metabolites were dose related. Mean wash-out t1/2 was 18 to 20 hours for the parent compound, 22-23 hours for metabolite II, and 28-33 hours for metabolite IV; these elimination t1/2 are comparable for the two doses, and are similar to those observed in single-dose studies. For the 50-mg-dose group, predicted and observed average plasma concentrations (Css) of CL 275,838 and its two metabolites did not differ significantly.(ABSTRACT TRUNCATED AT 250 WORDS)

In the Department of Obstetric and Gynecology, Faculty of Medicine-Varese, between March 1991 and June 1992, 74 consecutive patients undergoing elective oncologic surgery were evaluated in order to rationalize the use of antibiotics to decrease the costs of infectious complications. We divided the patients into two groups: a high infection risk group (in which every patient was submitted to antibiotic prophylaxis) and a low infection risk group (in which we didn't use any antibiotic prophylaxis). Our findings indicate that selection criteria for HIR patients are probably correct and in this group AP is necessary. In the LIR group, 45.5% of patients was not submitted to any antibiotic therapy. It's necessary to test the real efficacy of an AP in LIR patients in whom we had not a important incidence of infectious complications. In the LIR group AP should not exceed Lit. 23,251 per patient to be cost-effective.

The pharmacokinetics and safety of CL 275,838, a potential cognition-enhancing compound, were studied after single escalating oral doses first in young healthy male volunteers and then in old (60-74 years) and very old (over 75 years) volunteers of both sexes. In all age groups absorption of CL 275,838 was rapid as assessed by the mean time to reach maximum plasma concentrations (Cmax) which averaged 1-2 hr, regardless of the dose administered. In young male volunteers both Cmax and area under the curve (AUC) increased proportionally with dose from 10 to 100 mg. Mean elimination half-lives (t1/2) of the parent compound (18-21 hr) and of its circulating metabolites II (20-22 hr) and IV (27-30 hr) were well comparable for the doses tested (50 and 100 mg). Age did not appreciably affect plasma Cmax of CL 275,838 or its two metabolites. Mean AUC and elimination half-life did not appreciably differ between old and very old subjects given 50 mg CL 275,838, with the limitations dictated by the small number of elderly subjects examined. Compared with younger volunteers receiving comparable doses, however, the elderly had higher mean plasma AUC of the unchanged compound and its two metabolites, although the parameter varied widely between subjects. The mean elimination t1/2 (+/- SD) was longer in the elderly (38.8 +/- 19.6, 50.5 +/- 24.5 and 41.7 +/- 12.1 hr, respectively, for the parent compound and its metabolites II and IV) than in the young subjects. The cause(s) of these variations and the possible clinical implications remain to be established.(ABSTRACT TRUNCATED AT 250 WORDS)

A chromatographic method for quantifying the potential memory-enhancing agent CL 275,838 (I) in human plasma with a limit of detection of 1.25 ng/ml is described. The procedure relies on isolation of the compounds from plasma constituents using the Sep-pack C18 cartridge, resolution by reverse-phase high pressure liquid chromatography (HPLC) and post-column oxidation of the eluate peak to from a derivative which can be measured by fluorescence detection. Peak height and compound I concentration were linearly related from 1.25 to 25 ng/ml. Intra- and inter-day validation studies indicated an acceptable precision and reproducibility of the method within the concentration range investigated, the overall coefficient of variation being less than 15%.