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M Hamdan

Publications and source records attributed to M Hamdan.

At least 37 records · Page 2Linked to original sources
PubMed1999

A(1) adenosine receptors in human neutrophils: direct binding and electron microscope visualization.

By occupying specific surface receptors, adenosine and adenosine analogues modulate neutrophil functions; in particular, functional and biochemical studies have shown that A(1) adenosine receptors modulate chemotaxis in response to chemotactic peptides. Until now, the characteristics of the specific agonist binding and the visualization of A(1) receptors in human neutrophils have not been investigated. In the present study, we used the agonist [(3)H] CHA for radioligand binding studies and a CHA-biotin XX probe in order to visualize the A(1) binding sites in human neutrophils, ultrastructurally, by conjugation with colloidal gold-streptavidin. [(3)H] CHA bound A(1) adenosine receptors with selectivity and specificity, although with a low binding capacity. Scatchard analysis showed a Kd value of 1.4 +/- 0.08 nM and a maximum density of binding sites of 7.1 +/- 0.37 fmol/mg of proteins. The good affinity and selectivity of the CHA-biotin XX probe for A(1) adenosine receptors allowed us to visualize them, after conjugation with colloidal gold-streptavidin, as electron-dense gold particles on the neutrophil surface and inside the cell. The internalization of the ligand-receptor complex was followed in a controlled temperature system, and occurred through a receptor-mediated pathway. The kinetics of the intracellular trafficking was fast, taking less than 5 min. These data suggest that the CHA-biotin XX-streptavidin-gold complex is a useful marker for the specific labelling of A(1) binding sites and to follow the intracellular trafficking of these receptors.

Adenosine↗
PubMed1999

Separation of double-stranded DNA in conventional and isoelectric buffers: studies on stability and separation performance.

In the capillary electrophoresis of double-stranded DNA in isoelectric buffers, worsening of resolution was observed in electropherograms as a function of time passed from the preparation of the separation solution, which consisted of 0.7% hydroxypropylcellulose, HPC, Mr 10(6), diluted in 150 mM histidine buffer. The DNA standards used were: kilobase pair-ladder, Marker V and Marker VI. In order to understand what happens in the histidine-HPC solution with ageing, the absorbance spectrum (200-500 nm), the conductivity and the pH of the solutions as a function of time were monitored. Fresh His gave a distinct peak at 206 nm. For all the solutions a significant diminution in the maximum absorbance value at 206 nm was observed as a function of ageing, with the concomitant appearance of a peak at 278 nm as the solutions became older. Also the conductivity increases dramatically with the ageing of the solutions and seemed to reach a plateau after ca. 40 days. In concomitance with the conductivity increments with time, the pH of the His solution (isoelectric point, pI=7.6) grew slowly up to pH 7.9; these combined data indicated that a new species contributing to the conductivity and altering the pH was formed from the His molecule, suggesting that His degraded in time. When the dipeptide His-Gly was used instead, a similar ageing phenomenon was observed, but with much reduced kinetics. Mass spectrometry, coupled to RP-HPLC, detected, in aged His solutions, in addition to intact His, two main degradation products: a 110.1 u species and a 93.2 u compound. The mass of the former coincides with the protonated species derived from the formation of a Schiff base on the alpha-amino group of His and subsequent decarboxylation without transformation of the final Schiff base into a chetonic group (a histamine-like molecule terminating with an imino, rather than with an amino group).

Buffers↗
PubMed1999

General experimental aspects of the use of isoelectric buffers in capillary electrophoresis.

Four acidic, isoelectric buffers, for peptide and protein separations, have been recently described and adopted in capillary zone electrophoresis: cysteic acid [Cys-A, isoelectric point (pI) 1.85], iminodiacetic acid (IDA, pI 2.23), aspartic acid (Asp, pI 2.77) and glutamic acid (Glu, pI 3.22). These four buffers allow to explore an acidic portion of the titration curves of macroions, covering about 1.6 pH units (from pH 1.85 to ca. 3.45), thus permitting resolution of compounds having coincident titration curves at a given pH value. Given the rather acidic pI values of these buffers, their long-term stability has been investigated, by monitoring pH and conductivity changes upon increasing storage times. When dissolved in plain water, all four buffers appear to give constant pH and conductivity readings up to 15 days; after that, the conductivity keeps steadily increasing in a similar fashion. The same parameters, when the same buffers are dissolved in 6 M urea, appear to be stable for only one week, with the conductivity progressively augmenting after this period. A similar behaviour is exhibited by histidine (pI 7.70), a neutral, isoelectric buffer adopted for separation of DNA fragments. By mass spectrometry, Cys-A shows minute amounts (ca. 1%) of a degradation product after ageing for 3 weeks; in the same time period, Glu is extensively degraded (20%). No degradation species could be detected in IDA and Asp solutions. It is additionally shown that the acidic buffers are not quite stationary in the electric field, but can be transported at progressively higher rates (according to the pI value) from the cathodic to the anodic vessel. This is due to the fact that, at their respective pI values, a fraction of the amphotere has to be negatively charged in order to provide counterions to the excess of protons due to bulk water dissociation. Guidelines are given for the proper use and storage of such buffers.

Buffers↗
PubMed1999

Synthesis and structure-activity relationship studies of new endothelin pseudopeptide analogues containing alkyl spacers.

We replaced the Asp18-Ile19 dipeptide of the C-terminal ET analogue Ph-Ph-CH2-O-N=CH-CO-Phe-Asp-Ile-Ile-Trp-OH by alkyl spacers of various lengths to investigate the role of the aminoacidic central portion of the molecule and to define the N-terminal and C-terminal pharmacophoric regions of this analogue. The side-chains of the central dipeptide have been shown to be irrelevant for the binding of the molecule to the receptor, but the distance between the two postulated sites of interaction of the ligand with the ETB receptor appears to be fundamental.

Animals↗
PubMed1999

Investigation of some covalent and noncovalent complexes by matrix-assisted laser desorption/ionization time-of-flight and electrospray mass spectrometry.

Covalent and noncovalent complexes involving bovine superoxide dismutase, bovine alpha-lactalbumin and two variants, A and B, of bovine beta-lactoglobulin have been examined by delayed extraction matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and electrospray mass spectrometry. Covalent complexation with acrylamide was obtained through the interaction of these proteins with acrylamide monomers while treatment with peroxynitrite yielded complexes with NO2. Some of the complexation sites with acrylamide were reliably identified by performing tryptic digestion followed by MALDI-TOF measurements in the reflection mode. These data demonstrate that the presence of free cysteine in the investigated sequences is not a precondition for the observation of Cys-acrylamide complexes. Although the bulk of the present work is dedicated to the characterization of Cys-acrylamide adducts, the preliminary data on noncovalent complexes between two of those proteins and 8-anilinonaphthalene-1-sulfonic acid (ANS) were easily observed under electrospray conditions, while under MALDI-TOF conditions only the complex with beta-lactoglobulin B was clearly evident. The presented data demonstrate the advantage of the parallel use of both ionization techniques for this type of investigation.

Acrylamide↗
PubMed1999

Probing the reactivity of S-S bridges to acrylamide in some proteins under high pH conditions by matrix-assisted laser desorption/ ionisation.

There is compelling evidence to suggest that cysteine-acrylamide adduct formation is a modification experienced by proteins separated by two-dimensional (2-D) gel electrophoresis. Whether the -SH group involved in such complexation is offered by a free or initially disulphide-linked cysteine residue remains an open question. To address this question a number of proteins containing free and/or disulphide-linked cysteine (Cys) residues have been incubated with acrylamide monomer and examined by delayed extraction matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF). These data provide strong evidence to suggest that the presence of free Cys in the investigated proteins is not the most important requirement for the observation of Cys-acrylamide adducts. Unambiguous confirmation of this deduction was obtained by analysing the tryptic digests of the same proteins by reflectron MALDI-TOF. The assignment of the adduction sites was facilitated by the mass accuracy attained for the monitored tryptic fragments and their agreement with the corresponding predicted masses reported in the Swiss-Prot database. The same data suggest that at high pH the cysteine pairing is flexible enough to allow initially S-S linked residues to complex with acrylamide. It is also plausible that the -NH(2) terminal blockage so often encountered in proteins electroblotted from 2-D maps could originate from carbamylation, and might not have anything to do with alkylation by free, unreacted acrylamide in polyacrylamide gels.

Acrylamide↗
PubMed1999

Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry for monitoring alkylation of beta-lactoglobulin B exposed to a series of N-substituted acrylamide monomers.

Delayed-extraction matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry, in both linear and reflectron modes, has been used to examine the alkylation of bovine beta-lactoglobulin-bound cysteines exposed to various molar concentrations (0.5-30 mM) of acrylamide and a number of its N-substituted monomers. These measurements were conducted at pH approximately 9.5, and showed that at 0.5 mM all monomers (except N-acryloylaminopropanol) resulted in a measurable alkylation of at least one cysteine out of five. At higher concentrations (15 mM) all investigated monomers resulted in substantial alkylation which, for some, involved all five cysteine residues. Reflectron MALDI-TOF measurements of a number of alkylated protein digests revealed that, at low molar ratios, the alkylation site is influenced by the identity of the monomer. For example acrylamide and N, N-dimethylacrylamide attacked Cys(160) as well as one of the three cysteines within the tryptic fragment [102-124], while other investigated monomers did not involve Cys(160). The implications of the present data for two-dimensional (2D) gel electrophoresis, and their eventual correlation to the toxicity of the investigated monomers, are discussed.

Acrylamides↗
PubMed1998

Fluorescent probes for adenosine receptors: synthesis and biology of N6-dansylaminoalkyl-substituted NECA derivatives.

New fluorescent ligands for adenosine receptors are described; these compounds were obtained by the insertion, in the N6 position of NECA (a potent adenosine agonist), of dansylaminoalkyl moieties with alkyl spacers of increasing carbon chain length (from 3 to 12). Among them, the compound with a C6 alkyl spacer proved to be the most interesting one, showing a marked selectivity for the A1 receptor subtype; furthermore, in fluorescence microscopy assays it proved to be able to visualize and localize this receptor subtype at the level of the molecular layer of the rate cerebellar cortex.

Adenosine-5'-(N-ethylcarboxamide)↗
PubMed1998

On-line liquid chromatography/electrospray tandem mass spectrometry to investigate acrylamide adducts with cysteine residues: implications for polyacrylamide gel electrophoresis separations of proteins.

Adduction between acrylamide and cysteine residues is a post-translational modification associated with proteins separated by gel electrophoresis. In the present article, three model peptides containing 2-4 cysteine residues were reduced with dithiothreitol, incubated with acrylamide monomers and examined by on-line liquid chromatography coupled to electrospray tandem mass spectrometry. Each of the solutions examined in this work revealed the presence of four distinct components: the free peptide, two different peptide-acrylamide 1:1 adducts involving two cysteine residues at different positions within the same sequence, and the peptide-acrylamide 1:2 adducts. The use of liquid chromatography allowed the separation of components which differed only by the site of complexation of acrylamide, while the application of tandem mass spectrometry furnished reliable sequencing information permitting the identification of most cysteine residues involved in such complexation.

Acrylamides↗
PubMed1998

Liquid chromatography/tandem mass spectrometry of synthesis products associated with the viral protein US11.

US11 is a small basic protein composed of 161 amino acid residues, and is among the most abundant viral proteins in cells infected with herpes simplex viruses HSV1 and HSV2. The amino acid sequence [91-121] is considered essential for the binding of this protein with RNA. Automated solid phase synthesis of this fragment resulted in a crude reaction mixture containing the desired sequence as well as a number of unknown side products. On-line liquid chromatography/electrospray mass spectrometry (LC/ES-MS) and LC/ES tandem mass spectrometry (MS/MS) allowed the identification of the separated components and furnished relevant sequencing information. The unusual sequences of the monitored components, which consist of a tandemly repeated three-amino-acid motif with the sequence Arg-X-Pro, where X is an acidic or uncharged polar amino acid residue, yielded product ion spectra lacking substantial sequence information and rich in fragment ions manifesting the neutral losses 17, 42 and 60 u.

Amino Acid Sequence↗
PubMed1998

Liquid chromatography/tandem mass spectrometry to monitor acrylamide adducts with bovine beta-lactoglobulin B.

Complexation of acrylamide with bovine beta-lactoglobulin B and some of its tryptic fragments have been examined by liquid chromatography coupled to tandem mass spectrometry. Such complexation was investigated both in the presence and in the absence of dithiothreitol as a reducing agent. Under the latter conditions, the intact protein exhibited a single cysteine-acrylamide complex which both the present work and previous studies attribute to Cys160. The involvement of this particular residue is tentatively attributed to an intramolecular disulphide exchange which results in its disengagement from the S-S bridge to offer a free SH group for reaction with the acrylamide monomer. In the absence of dithiothreitol, both free and complexed cysteine-containing tryptic fragments were present, while in its presence, one of the tryptic fragments, which contains three cysteine residues was fully absent, instead a part of this fragment containing two cysteines complexed with two acrylamide monomers was observed. The absence of any analytical information in the literature regarding the latter complexes underlines the potential of liquid chromatography coupled to mass spectrometry in the characterization of this commonly occurring modification.

Acrylamides↗
PubMed1998

"Cristal tachycardias": origin of right atrial tachycardias from the crista terminalis identified by intracardiac echocardiography.

OBJECTIVES: We sought to use intracardiac echocardiography (ICE) to identify the anatomic origin of focal right atrial tachycardias and to define their relation with the crista terminalis (CT). BACKGROUND: Previous studies using ICE during mapping of atrial flutter and inappropriate sinus tachycardia have demonstrated an important relation between endocardial anatomy and electrophysiologic events. Recent studies have suggested that right atrial tachycardias may also have a characteristic anatomic distribution. METHODS: Twenty-three consecutive patients with 27 right atrial tachycardias were included in the study. ICE was used to facilitate activation mapping in relation to endocardial structures. A 20-pole catheter was positioned along the CT under ICE guidance. ICE was also used to assist in guiding detailed mapping with the ablation catheter in the right atrium. RESULTS: Of 27 focal right atrial tachycardias, 18 (67%, 95% confidence interval [CI] 46% to 83%) were on the CT (2 high medial, 8 high lateral, 6 mid and 2 low). ICE identified the location of the tip of the ablation catheter in immediate relation to the CT in all 18 cases. The 20-pole mapping catheter together with echocardiographic visualization of the CT provided a guide to the site of tachycardia origin along this structure. Radiofrequency ablation was successful in 26 (96%) of 27 (95% CI 81% to 100%) right atrial tachycardias. CONCLUSIONS: This study demonstrates that approximately two thirds of focal right atrial tachycardias occurring in the absence of structural heart disease will arise along the CT. Recognition of this common distribution may potentially facilitate mapping and ablation of these tachycardias.

Adolescent↗
PubMed1998

A structure-activity study of a C-terminal endothelin analogue.

We report a structure-activity study of an endothelin (ET) analogue, obtained by introduction of a non-aminoacidic portion on the C-terminal ET pentapeptide. The peptidic moiety was modified with systematic replacement of each residue by alanine (Ala scan); further modifications were performed at the C-terminus. The biological activity was analyzed at both ET(A) and ET(B) receptor subtypes, showing that the two C-terminal residues (Ile-Trp) are very important for the activity. On the contrary, the aminoacidic central portion of the molecule appears to be much more tolerant toward modifications.

Amino Acid Sequence↗
PubMed1998

Bryostatin 1 down-regulates mdr1 and potentiates vincristine cytotoxicity in diffuse large cell lymphoma xenografts.

The down-regulation of multidrug resistance (mdr1) gene expression as detected by competitive reverse transcription-PCR and the antitumor activity of bryostatin 1 (Bryo1) are investigated in a newly established cell line from a patient with relapsed diffuse large cell lymphoma (DLCL). The cell line (WSU-DLCL2) grows in liquid culture and forms s.c. tumors in mice with severe combined immune deficiency. WSU-DLCL2 is a mature B-cell line (IgG lambda) that is negative for EBV nuclear antigen, expresses the multidrug resistance phenotype, and has t(14;18)(q32;q21) plus other chromosomal aberrations. Exposure of the WSU-DLCL2 cells to Bryo1 in culture reversed the multidrug resistance phenotype within 24 h. A functional assay revealed a 4-fold increase in [3H]vincristine accumulation in Bryo1-treated cells compared with control. Vincristine (VCR), doxorubicin, Bryo1, and 1-beta-D-arabinofuranosylcytosine showed no clinically significant activity when given alone to WSU-DLCL2-bearing severe combined immune deficiency mice. However, when given 24 h before each cytotoxic agent, Bryo1 substantially increased the antitumor activity of VCR but not 1-beta-D-arabinofuranosylcytosine. There was a statistically significant (P < 0.001) decrease in the expression of P-glycoprotein in WSU-DLCL2 tumors taken from Bryo1-treated animals compared with untreated controls. In vivo, a competitive reverse transcription-PCR assay revealed decreased mdr1 RNA expression 24 h after Bryo1 treatment. These findings taken together indicate that Bryo1-induced down-regulation of mdr1 might be one mechanism by which Bryo1 potentiates VCR activity. The sequential use of both agents resulted in clinically significant antitumor activity in this lymphoma model.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Crudes of synthesis of neuropeptide Y and beta-amyloid peptide examined by liquid chromatography/electrospray tandem mass spectrometry.

The two peptides, neuropeptide Y (M(r) = 4254) and beta (1-39) amyloid (M(r) = 4230) are attracting pharmaceutical and biomedical interest for different yet equally important reasons. The first is recognized for its various physiological functions in the peripheral and central nervous system, while the second has been identified as one of the components of the cerebral amyloid deposits characteristic of Alzheimer's disease. High level purity of both peptides is considered of prime importance for correct interpretation of data obtained by biological and pharmaceutical assays and binding tests. Solid-phase synthesis of both peptides resulted in crudes of reaction containing both the target peptides as well as a number of undesired side products. The use of liquid chromatography/electrospray-tandem mass spectrometry (LC/ESI-MS/MS) furnished reliable information on the target peptides and provided sequencing information on a number of side products. Furthermore, LC/MS/MS data of doubly and triply protonated sequences yielded a number of C-terminal fragment ions exhibiting the loss of NH3 considered to be an important process for the understanding of fragmentation mechanism(s) of multiply protonated peptides.

Amino Acid Sequence↗
PubMed1996

Investigation of newly synthesized peptides by capillary zone electrophoresis/electrospray mass spectrometry.

A series of newly synthesized peptides (M(r) = 1600-2250 Da), corresponding to portions of the extracellular domain of human granulocyte-macrophage colony stimulating factor receptor alpha subunit have been examined by capillary zone electrophoresis/electrospray-mass spectrometry (CZE/ES-MS). The separation efficiency of CZE combined with the specificity of mass spectrometry allowed rapid and reliable identification of the target peptides and a number of associated side products. In solid-phase synthesis of peptides and small proteins such side products are inevitable, therefore the use of CZE and/or high-performance liquid chromatography combined with mass spectrometry is gaining a central role in such synthesis procedures.

Amino Acid Sequence↗
PubMed1996

Investigation of beta-amyloid peptide by on-line high-performance liquid chromatography coupled to electrospray ionization mass spectrometry.

beta(1-39) amyloid peptide is one of the components of the cerebral amyloid deposits that are characteristic of Alzheimer's disease. Solid-phase synthesis of this peptide resulted in a fairly complex crude product containing both the target peptide and a number of side products. High-performance liquid chromatography coupled to electrospray ionization mass spectrometry allowed rapid and reliable identification of both the desired peptide and most of the side products which were found to have relative molecular masses above and below that of the target peptide.

Amino Acid Sequence↗