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Bombesin has been synthesized by the continuous flow solid-phase procedure on the derivatized Kieselguhr-supported polydimethylacrylamide resin. Preformed Fmoc-amino acid symmetrical anhydrides (Met, Leu, and Arg) and Fmoc-amino acid active esters were used for amine acylation. The Mtr and the Pmc groups have been alternatively used for masking the side chain function of Arg-3. The progress of the synthesis was monitored by different analytical methods including quantitative solid-phase Edman degradation. Cleavage from the resin and simultaneous formation of the C-terminal amide function were achieved with a methanolic ammonia solution yielding indistinguishable crude peptides which have been purified by HPLC and fully characterized. Preliminary pharmacological experiments indicated that the activity of the synthetic peptides is similar to that previously measured for other synthetic bombesins. For comparison bombesin has also been prepared by solid-phase synthesis on 4-methyl benhydrylamine resin using the Boc chemistry. The results of the two strategies are discussed and compared.

The two dipeptides AE (Lalanine–Lglutamic acid) and AK (Lalanine–Llysine), that constitute the “building blocks” of the 16-unit self-complementary amphiphilic oligopeptide EAK16, have been investigated by XPS (X-ray photoelectron spectroscopy) and NEXAFS (near-edge X-ray absorption fine structure) spectroscopy. Thin films of both dipeptides on TiO2, a distinguished biocompatible surface, were prepared by incubation from aqueous solutions. Thick films of dipeptides on inert Au substrates were also studied for comparison. The chemical structure and composition were investigated by XPS spectroscopy; furthermore, molecular orientation of dipeptides on TiO2 was checked by angular dependent NEXAFS measurements at both C–K and N–K edges. In order to yield some insight on adsorption geometry and molecular orientation MD (molecular dynamic) simulations were also carried out. The performed molecular and electronic characterization of AE and AK provides an excellent model for the interpretation of more complex peptide spectra.

Infectious HIV-1 requires gp160 cleavage by furin at the REKR511 downward arrow motif (site1) into the gp120/gp41 complex, whereas the KAKR503 (site2) sequence remains uncleaved. We synthesized 41mer and 51mer peptides, comprising site1 and site2, to study their conformation and in vitro furin processing. We found that, while the previously reported 19mer and 13mer analogues represent excellent in vitro furin substrates, the present extended sequences require heparin for optimal processing. Our data support the hypothesis of a direct binding of heparin with site1 and site2, allowing selective exposure/accessibility of the REKR sequence, which is only then optimally cleaved by furin.

We present a spectroscopic study of the structure of two peptides deposited on Au and TiO2: – PeptA, (EAK16-RGD) bringing the adhesive RGD sequence linked to EAK16; – PeptB, having RGD linked to a “scrambled” sequence of the EAK16 peptide. Previously reported NEXAFS investigations on thin films of the self-assembling peptide EAK16 deposited on Au and TiO2, revealed molecular order and orientation for both substrates. IR spectra show a β-sheet conformation for PeptA and a random structure for PeptB. Angular-dependent NEXAFS measurements reveal an ordered structure with preferential molecular orientation only for PeptA. XPS analysis indicates that PeptA is adsorbed on TiO2 in a larger amount than PeptB.

The aim of this work was to evaluate the long-term immunological and clinical impact of idiotype (Id) vaccination in multiple myeloma (MM) patients in first remission after high-dose chemotherapy. A total of 15 patients received a series of subcutaneous (s.c.) injections of autologous Id, conjugated to keyhole limpet hemocyanin (KLH) and in association with low doses of GM-CSF. The median duration of follow-up was 110 months from diagnosis. The vaccine induced immune responses that lasted almost 2 years after the end of treatment. Antibody responses included anti-KLH IgM and IgG (90% of patients), anti-KLH IgE (30%), anti-GM-CSF IgG (20%), anti-Id IgG (20%), and anti-Id IgE (30%). Id-specific delayed type hypersensitivity skin tests were positive in 85% of tested patients. Following vaccination, a progressive recovery of T-cell receptor (TCR) diversity was observed and the loss of oligoclonality was significantly correlated with the remission duration. Although Id/KLH conjugates did not eliminate the residual tumor burden, the median progression-free survival, and overall survival were 40 and 82 months, respectively. A retrospective case-matched analysis showed similar results in patients treated with IFN-alpha alone or in association with steroids. This vaccine formulation can overcome Id-specific immune tolerance by inducing clinical responses that are worthy of further investigation.

EAK16 is a 16 amino acid peptide consisting of an alternation of polar and non-polar pending groups and of positively and negatively charged residues, that makes this material capable of self-assembling, producing an extended ordered structure. Thin films of EAK16 were prepared on TiO2, and An surfaces and investigated by surface-sensitive techniques such as XPS (X-ray photoelectron spectroscopy), NEXAFS (near edge X-ray absorption fine structure) and IR spectroscopies. XPS analysis allowed to check the chemical structure of the samples and to determine the film thickness. IR experiments yielded evidence of the beta-sheet conformation of the peptide backbone. Polarization dependent NEXAFS measurements allowed estimating the angle between the axis of the peptide backbone and the sample surface. (c) 2005 Elsevier B.V. All rights reserved.

Proteolytic activation of the HIV-1 envelope glycoprotein gp160 is selectively performed by the proprotein convertase furin at the C terminus of the sequence R508-E-K-R511 (site 1), in spite of the presence of another consensus sequence, Lys500-Ala-Lys-Arg503 (site 2). On the basis of the solution structural analysis of the synthetic peptide p498, spanning the gp160 sequence Pro498-Gly516, we previously suggested a possible role of an N-terminal helix in regulating the exposure and accessibility of the gp160 physiological cleavage site, enclosed in a loop. Here we report on the activity and conformation of the 23 residue peptide h-REKR, designed to exhibit a large N terminal helix, followed by the gp160 native sequence, Arg508-Gly516, h-REKR is digested by furin with high efficiency comparable to the full native p498. Circular dichroism analyses, in mixtures from pure water to 98% trifluoroethanol, outline a significant content of helical structure in the peptide conformation. The molecular model obtained from NMR data collected in trifluoroethanol/water, by means of DYANA and AMBER simulations, indeed has helical structure on a large N-terminal segment. Such a long helix does not seem to affect the loop conformation of the C-terminal site 1-containing sequence, which exhibits the same proton chemical shifts already observed for the full native p498.

We previously demonstrated that a 23-mer peptide (DB3) derived from the V3 loop of the surface glycoprotein of HIV-1 MN strain was able to bind to soluble CD4 and enhance HIV-1 infection. The mechanism and structural features required for these biological activities were studied by using shortened DB3 derivatives and DB3 analogs carrying single amino acid substitutions. We found that peptides in which the aromatic amino acid in position 15 or 16 had been replaced by an uncharged hydrophobic residue (DB3-115 and DB3-116), analogs in which positively charged amino acids were replaced by corresponding D-enantiomers, and shortened DB3-derivatives lost both enhancing activity and ability to bind to soluble CD4. Other peptide variants in which a positively charged amino acid was replaced by asparagine at positions 3 (DB3-N3), 6 (DB3-N6), and 19 (DB3-N19), respectively, retained both enhancing and binding activities, although with different efficiencies. The CD4 binder peptides DB3 and DB3-N19, but none of the CD4 nonbinder peptides, enhanced CD4 expression on peptidetreated cells as well as gp120 binding to both CD4+ cells and soluble CD4. These findings strongly suggest that the peptide/CD4 interaction induced an increase in both CD4 expression and CD4/gp120 binding affinity, which in turn mediated the enhancement of viral infection. A model of the structural conformation of DB3 peptide required for its biological activities is discussed.

In previous studies we have demonstrated that synthetic peptides, corresponding to sequences in the (307-330) region of the gp120 principal neutralizing domain of different HIV-1 isolates are specifically recognized by a site distinct from the high affinity gp120-binding site of CD4. Interestingly, a peptide designed from the HIV-1 MN strain is able to enhance viral infection, while a HTLV-IIIB derived analogue is at least ten-fold less efficient and no effect is shown by other tested peptides. This enhancing effect occurs in the early step of infection and it is not strain restricted. A correlation between structure and biological functions evidenced by CD, FT-IR, and preliminary mono and bidimensional NMR is presented in this paper. The experimental data are compared to the predictions obtained by theoretical calculations.

The covalent attachment of an adhesive peptide, reproducing the 351–359 sequence of human vitronectin, to oxidized titanium surfaces was investigated by XPS spectroscopy. The peptide enhances osteoblast adhesion to titanium, the most used biomaterial for implants and prostheses. Core level spectra of the TiO2 surface and of the biomimetic surface were investigated. Novel selective covalent immobilization of (351–359) HVP was carried out by treatment of the TiO2 surface with (3-aminopropyl) triethoxysilane, glutaric anhydride and a side chain protected peptide sequence presenting only a free terminal amino group, followed by side chain deprotection. An alternative strategy for covalent attachment consists in photoactivation of physisorbed (351–359) HVP directly on the TiO2 surface; samples were incubated with HVP solution and subsequently irradiated with UV light. A comparison with the results previously obtained for non-selective HVP immobilization will be discussed.

The HIV-1 envelope glycoprotein gp120 interacts consecutively with CD4 and CCR5 to mediate the entry of R5-HIV-1 strains into target cells. The N-terminus of CCR5, which contains several sulfated tyrosines, plays a critical role in gp120-CCR5 binding and, consequently, in viral entry. Here, we demonstrate that a tyrosine sulfated peptide, reproducing the entire N-terminal extracellular region of CCR5, its unsulfated analogue, and a point-mutated peptide are unable to inhibit R5-HIV-1 mediated infection, competing with the entire CCR5 in the formation of gp120-CD4-CCR5 complex. Surprisingly, these peptides show the capability of enhancing HIV-1 infection caused by X4 strains through the up-regulation of both CD4 and CXCR4 receptors.

Motivation: Microarray expression profiling appears particularly promising for a deeper understanding of cancer biology and to identify molecular signatures supporting the histological classification schemes of neoplastic specimens. However, molecular diagnostics based on microarray data presents major challenges due to the overwhelming number of variables and the complex, multiclass nature of tumor samples. Thus, the development of marker selection methods, that allow the identification of those genes that are most likely to confer high classification accuracy of multiple tumor types, and of multiclass classification schemes is of paramount importance. Results: A computational procedure for marker identification and for classification of multiclass gene expression data through the application of disjoint principal component models is described. The identified features represent a rational and dimensionally reduced base for understanding the basic biology of diseases, defining targets for therapeutic intervention, and developing diagnostic tools for the identification and classification of multiple pathological states. The method has been tested on different microarray data sets obtained from various human tumor samples. The results demonstrate that this procedure allows the identification of specific phenotype markers and can classify previously unseen instances in the presence of multiple classes. Availability: Matlab source codes are available from the authors. Contact: silvio.bicciato@unipd.it Supplementary information: http://www.dpci.unipd.it/PersPages/SBicciato/SIMCArray.html * To whom correspondence should be addressed.

A key step in the infection of T cells by HIV-1 is the interaction between the cellular receptor CD4 and the viral envelope protein gp120. Various compound of potential pharmacological value have been tested for their ability to interfere with this interaction with aim of identifying drugs that reduce virus infectivity. In particular, monoclonal antibodies directed agianst the gp120-binding domain of CD4 and gp120 derived synthetic peptides which bind to CD4 can be used to modulate the immunoresponse and block HIV-1 infectivity. We report an affinity chromatography assay, which, in combination with epitope-specific anti-CD4 mAbs, may allow the rapid identification of ligands recognized by CD4 and their cognate binding sites. This method should facilitate the design of analogues exhibiting a higher affinity or selectivity for the protein thus restricting the number of potentially useful compounds to be tested in more sophisticated assays.

We show that the nonlinear analysis of nearly sinusoidal oscillators can be performed by a very simple symbolic program, whereby the periodic oscillation as well as its frequency is determined in symbolic form in terms of both linear and nonlinear circuit parameters. The program is an useful design tool, as it enables the harmonic content and the frequency sensitivity to circuit parameters to be obtained in analytic form.

The parallel monitoring of the expression profiles of thousands of genes seems particularly promising for a deeper understanding of cancer biology and to identify molecular signatures supporting the histological classification schemes of neoplastic specimens. A computational procedure for feature extraction and classification of gene expression data through the application of principal component analysis and of the soft independent modeling of class analogy approach (SIMCA) is described. The identified features contain critical information about gene-phenotype relationships observed during changes in cell physiology. They represent a rational and dimensionally reduced base for understanding the basic biology of the onset of diseases, defining targets of therapeutic intervention, and developing diagnostic tools for the identification and classification of pathological states. The proposed method has been tested on the childhood round blue cell tumors study presented by Khan et al. [2001]. The analysis of the SIMCA model allows the identification of specific phenotype markers and provides the assignment to multiple classes for previously unseen instances.

Remyelination is an extremely efficient process in the adult rodent central nervous system yet the source of new oligodendroglia that appear following primary demyelination is still subject to much debate. Using a reliable marker for oligodendroglial progenitor cells in vivo, the NG2 chondroitin sulphate proteoglycan, we have evaluated the response of endogenous NG2(+) cells in the adult rat brain stem and cerebellum to inflammatory demyelinating lesions in an experimentally induced animal model of multiple sclerosis (MS), antibody augmented experimental allergic encephalomyelitis (ADEAE). We have manipulated T-cell mediated EAE in Lewis rats by injecting in addition, either anti-myelin/oligodendroglial glycoprotein (MOG) antibodies to induce inflammatory demyelination, or non-specific mouse immunoglobulins to induce an inflammatory response without demyelination. We have examined the relationship of NG2(+) progenitor cells to microglia (OX-42(+)), astrocytes (GFAP(+)) and mature oligodendroglia (CNP(+)), in the normal and demyelinated CNS. In the normal CNS NG2-expressing cells are closely intermingled with other glia but represent a distinct cell population. A prominent inflammatory response, identified by the presence of large perivascular and periventricular accumulations of reactive OX42(+) macrophages/microglia, occurred in animals with ADEAE at 7-9 days post injection (DPI), coinciding with severe clinical symptoms. In animals injected with anti-MOG antibodies inflammation was followed by the appearance of large areas of demyelination at 11-14 DPI, at which point the animals had recovered clinically. The response of NG2(+) cells was different depending on whether the inflammation was accompanied by demyelination. In the presence of inflammation, NG2(+) cells responded by an increase in immunoreactivity and an alteration in their morphology, exhibiting enlarged cell bodies and an increased number of intensely stained processes. In areas of demyelination NG2(+) cells had fewer intensely stained processes reminiscent of progenitor cells seen during development. Quantitative analysis revealed a 3-fold increase in the number of NG2(+) cells in demyelinated lesions at 11 DPI, whereas no change was observed in areas of inflammation in the absence of demyelination. Mitotic figures were only seen in NG2(+) cells in areas of demyelination. NG2(+) cell numbers appeared to return to control levels following remyelination. These results suggest that endogenous oligodendroglial progenitors divide and/or migrate, in response to signals triggered by demyelinating rather than inflammatory events, to generate a large progenitor population sufficient to promote the rapid and successful remyelination observed in this model.

We previously demonstrated that a 23-amino-acid peptide derived from the V3 loop of the surface glycoprotein of human immunodeficiency virus (HIV-1) strain MN was able to bind soluble CD4 and to enhance HIV-1 infection. Further studies suggested that the peptide/CD4 interaction induces an increase in both CD4 expression and CD4/gp120 binding affinity. To facilitate identification of the complementary binding site for the peptide on cellular CD4, we designed an analogue carrying a single fluorescein moiety. The synthesis of this modified analogue presented several problems because of the presence of several amino acids in the sequence carrying potentially reactive groups in their side-chains, and the necessity of introducing only one marker per molecule in a position that would not affect biological activity. The side-chain of Lys19 was selected because separate studies demonstrated that its substitution with an uncharged amino acid does not reduce the peptide's biological activity. We compared the merits of various synthetic protocols used to condense the fluorescent marker with the peptide. Biological assays indicated that the presence of the fluorescein moiety did not compromise peptide binding to CD4; furthermore, binding of the labeled analogue was not abolished by trypsin treatment, suggesting that the peptide may interact with both CD4 and additional trypsin-resistant binding sites on the cell surface. Finally, we verified the preservation of HIV infection enhancing ability in the labeled peptide.

The H-Ala-Arg-(Ala)6-Lys-OH sequence is a biologically interesting ‘difficult sequence’ presenting Nα-Fmoc deprotection and coupling problems. Different chemical conditions and synthetic strategies have been tested in order to overcome the problems due to sequence-dependent interactions. In particular, it was confirmed that different solvents in the deprotection step did not provide any significant improvement, but the use of a more efficient base in the deprotection mixture avoided insufficient unblocking of Nα-protecting group; problems due to partial coupling in the last steps of the synthesis were solved by double coupling techniques. Moreover, the synthesis of the model peptide was carried out using both ‘continuous flow’ and ‘batch’ techniques. The present results demontrate that on-line monitoring of the deprotection step by absorbance measurements represents a very effective tool to detect the onset of internal aggregations during the synthesis. © Munksgaard 1997.

We give an iterative procedure for calculating the steady-state output waveform of nearly sinusoidal nonlinear feedback-oscillators whose order as well as the shape and the degree of the nonlinearity is assumed to be of the greatest generality. The procedure inherits its foundation from the classical perturbation theory and makes it possible to determine both the frequency and the complex amplitude of the harmonics of the oscillation by very simple recurrence formulas, by which a complete analytical representation of the nonlinear oscillation is obtained. These asymptotic formulas are valuable for both the analysis and synthesis purposes. The application of the above formulas to some typical oscillator circuits is given to show their usefulness in practical problems.

Doping profiles in bipolar devices are numerically calculated taking into account the actual space charge produced during the diffusion process, which is described by standard models based upon effective diffusivities. The influence of this charge on the diffusion of dopants was examined for arsenic/boron double diffusion in order to evaluate the errors on profiles obtained using the well-known approximation of space-charge neutrality. Numerical results show that this approximation satisfactorily predicts practical doping profiles except for short-time low-temperature diffusion. >

A peptide corresponding to the native (1-66) sequence of horse heart cytochrome c has been synthesized by stepwise automated solid-phase methods on PAM resin. The course of the synthesis has been monitored by several analytical methods including quantitative ninhydrin and Edman degradation. After HF cleavage, the peptide has been purified by a combination of semipreparative ion-exchange and RP-HPLC. The homogeneity of the purified synthetic peptide has been determined by different criteria including HPLC, amino-acid composition, electrophoresis, antibody binding, tryptic and chymotryptic peptide mapping. After deprotection of the Acm-Cys residues and CNBr cleavage of the Met65-Glu66 peptide bond with simultaneous transformation of the Met65 residue into the activated C-terminal [Hse65]lactone, this purified synthetic peptide has been utilized for conformation-assisted joining experiments in combination with synthetic (66-104) to produce fully synthetic [Hse65]apocytochrome c. This latter, after mitochondria-mediated stereospecific heme insertion, has given a functional molecule corresponding to native horse heart holocytochrome c.

An approximation method for calculating the periodic oscillations of nearly sinusoidal oscillators is presented which is based upon the classical pertubation method. Both the frequency and the amplitude of the harmonics of the oscillation are calculated to a first approximation by very simple relations which are useful for deriving a design criterion. To demonstrate the possibilities of the proposed method, some illustrative examples are provided.

Bioactivation of pro-proteins by limited proteolysis is a general mechanism in the biosynthesis of hormones, receptors and viral protein precursors. This proceeds by cleavage of peptide bonds at the level of single or pairs of basic residues in the proforms. Examination of a number of cleavage loci in various precursors failed to reveal any consensus primary sequence around the dibasic cleavage sites. Thus it has been proposed, on the basis of secondary structure predictions [Rholam, M., Nicolas, P. and Cohen, P. (1986) FEBS Lett., 207, 1-6], that those basic residues which operate as signal loci for the proteolytic enzyme machinery are situated in, or next to, privileged precursor regions most often constituted by flexible and exposed motifs, e.g. beta-turns and/or loops. Peptides reproducing the N-terminal processing domain of the hormone precursor, pro-ocytocin-neurophysin, were examined by a combination of spectroscopical techniques including circular dichroism, infrared Fourier transform and one- and two-dimensional proton NMR. The results indicate that: (i) the region situated on the N terminus of the Lys-Arg doublet is organized as a beta-turn in solution; (ii) the sequential organization of the residues participating in the beta-turn determines the privileged relative orientation of the basic amino acid side chains and the subtype of turn; (iii) the peptide segment situated on the C-terminal side of the dibasic, corresponding to the N-terminal octapeptide of neurophysin, is organized as an alpha-helix.(ABSTRACT TRUNCATED AT 250 WORDS)

Introduction and Aims: New agents have been introduced as induction prior to autologous stem cell transplantation (ASCT) and as consolidation/maintenance thereafter to improve complete response (CR) rates. We evaluate bortezomib plus pegylatedlyposomal-doxorubicin and dexamethasone (PAD) as induction prior to reduced intensity ASCT, followed by consolidation with lenalidomide and prednisone (LP) and maintenance with lenalidomide alone (L). Materials and Methods: Newly diagnosed multiple myeloma patients aged 65-75 years were eligible. Induction consisted of four 21-day PAD cycles (bortezomib 1.3 mg/m2 days 1, 4, 8, 11; pegylated-lyposomal-doxorubicin 30 mg/m2 day 4; dexamethasone 40 mg days 1-4, 8-11, and 15-18). Patients were conditioned with tandem melphalan 100 mg/m2 (MEL100) followed by stem cell support. After ASCT patients received consolidation with four 28-day LP cycles (lenalidomide 25 mg days 1-21, prednisone 50 mg every other day) followed by lenalidomide maintenance (10 mg days 1-21). Primary objectives were safety (grade 3 nonhematologic toxicity 35%). Results: One-hundred and two patients entered the study. After PAD, 94% of patients achieved at least partial response (PR) and 59% at least very good partial response (VGPR) including 13% CR. After tandem MEL100, 88% of patients obtained at least VGPR and 41% CR. After LP all patients achieved PR, 88% at least VGPR including 53% CR. After a median follow-up of 14 months, 1-year progression-free survival (PFS) was 92%, and 1-year overall survival was 92%. PFS was not significantly affected by b2-microglobulin levels (P = .10), presence of chromosome 13 deletion (P = .5) or t(4;14)(P = .61). During PAD, grade 3/4 adverse events included thrombocytopenia (13%), neutropenia (11%), infections (18%), gastrointestinal toxicities (12%), peripheral neuropathy (11%) and deep vein thrombosis (DVT) (6%). During LP, grade 3/4 toxicities included neutropenia (18%), thrombocytopenia (6%), infections (6%) and DVT (6%). The other grade 3/4 toxicities occurred in less than 5% of patients. Conclusion: Bortezomib as induction, followed by lenalidomide as consolidation/maintenance is a highly effective regimen in elderly patients. Updated results will be presented at the meeting.

Two CD4 oligopeptides, corresponding to residues (37–53) and (37–55) of the V1 domain of CD4, which recent structural studies propose as the most likely binding site of HIV-1 gp120, have been chemically synthesized by solid-phase techniques, modified by the addition of two side-chain protected cysteines at both termini and purified by HPLC. Their ability to inhibit the infectivity of human immunodeficiency virus type 1 (HIV-1) (HTLV-IIIB, RF and GB8 strains) in different cell lines was monitored by the production of progeny virus, p24 and reverse transcriptase activity in the culture supernatants and by electron microscopy. The results indicated that the peptides inhibited HIV-1 infectivity in a dose-dependent fashion without any detectable cytotoxicity.

1. 1. The aminoacidic composition of the globin of the genus Rana has been analyzed. 2. 2. The resulting data and those from five other species of Ranidae , from one species of Pipidae and one of Bufonidae having been compared in pairs according to the formula of Harris and Teller gave origin to a “dissimilarity matrix”. 3. 3. In a “Phylo” computer program, consistent with Moore's model, the data of the input matrix have been used to find the mutual taxonomic relations of the 11 anuran species examined.