Intact Chain Research Articles

Fluorescence lifetime properties of near-infrared cyanine dyes in relation to their structures

Structurally diverse near-infrared (NIR) absorbing polymethine dyes were prepared and their fluorescence lifetimes (FLT) were evaluated in relation to their structural features. Comparative FLT analysis based on the modification of methine chain length and heterocyclic system showed that indolium or benzo[ e]indolium heptamethine dyes exhibited longer FLT than the benzo[ c, d]indolium trimethine dye. Modification of heterocyclic system alone with an intact chain length showed that indolium-based heptamethine dyes showed approximately 30% longer FLT than the benzo[ e]indolium-based dyes. In general, the FLT of polymethine dyes increased from polar to non-polar solvents. In addition, correlation study between the theoretical and the experimental FLT for indocyanine green (ICG) suggests that the lack of structural rigidity for these cyanine dyes is primarily responsible for the loss of the excited state energy via non-radiative pathway.

Cloning and Characterization of the DNA Region Responsible for Megacin A-216 Production in Bacillus megaterium 216

Upon induction, Bacillus megaterium 216 produces the bacteriocin megacin A-216, which leads to lysis of the producer cell and kills B. megaterium and a few other bacterial species. The DNA region responsible for megacinogeny was cloned in B. megaterium. The nucleotide sequence of a 5,494-bp-long subfragment was determined, and the function of the genes on this fragment was studied by generating deletions and analyzing their effects on MegA phenotypes. An open reading frame (ORF) encoding a 293-amino-acid protein was identified as the gene (megA) coding for megacin A-216. BLAST searches detected sequence similarity between megacin A-216 and proteins with phospholipase A2 activity. Purified biologically active megacin A-216 preparations contained three proteins. Mass spectrometry analysis showed that the largest protein is the full-length translation product of the megA gene, whereas the two shorter proteins are fragments of the long protein created by cleavage between Gln-185 and Val-186. The molecular masses of the three polypeptides are 32,855, 21,018, and 11,855 Da, respectively. Comparison of different megacin preparations suggests that the intact chain as well as the two combined fragments can form biologically active megacin. An ORF located next to the megA gene and encoding a 91-amino-acid protein was shown to be responsible for the relative immunity displayed by the producer strain against megacin A-216. Besides the megA gene, at least two other genes, including a gene encoding a 188-amino-acid protein sharing high sequence similarity with RNA polymerase sigma factors, were shown to be required for induction of megacin A-216 expression.

Conformational Stabilities of the Structural Repeats of Erythroid Spectrin and Their Functional Implications

The two polypeptide chains of the erythroid spectrin heterodimer contain between them 36 structural repeating modules, which can function as independently folding units. We have expressed all 36 and determined their thermal stabilities. These vary widely, with unfolding transition mid-points (T(m)) ranging from 21 to 72 degrees C. Eight of the isolated repeats are largely unfolded at physiological temperature. Constructs comprising two or more adjacent repeats show inter-repeat coupling with coupling free energies of several kcal mol(-1). Constructs comprising five successive repeats from the beta-chain displayed cooperativity and strong temperature dependence in forced unfolding by atomic force microscopy. Analysis of aligned sequences and molecular modeling suggests that high stability is conferred by large hydrophobic side chains at position e of the heptad hydrophobic repeats in the first helix of the three-helix bundle that makes up each repeat. This inference was borne out by the properties of mutants in which the critical residues have been replaced. The marginal stability of the tertiary structure at several points in the spectrin chains is moderated by energetic coupling with adjoining structural elements but may be expected to permit adaptation of the membrane to the large distortions that the red cell experiences in the circulation.

High-throughput MS-based protein phenotyping: Application to haptoglobin

A high-throughput affinity capture and reduction approach was developed for phenotype and post-translational modification analysis of a complexed globular protein, haptoglobin (Hp), directly from human plasma. Hp was selectively retrieved utilizing anti-Hp antibodies immobilized onto affinity pipette tips, eluted onto a formatted mass spectrometer target for reduction of Hp alpha-chains (Hpalpha1 and Hpalpha2) and subjected to subsequent MALDI-MS analysis. The affinity capture and reduction approach was originally developed from a pre-extraction reduction methodology that was optimized to an affinity capture post-reduction technique for intact Hp alpha-chain variant analysis, phenotype classification and ensuing post-translational variant detection. Three common Hp phenotypes (1-1, 2-1 and 2-2) were assigned according to detection of Hpalpha1 and/or Hpalpha2 reduced intact chain(s) average mass(es). The affinity capture post-reduction approach was scaled for high-throughput Hp alpha-chain phenotype analysis from a normal plasma cohort. The entire sample cohort was successfully analyzed and phenotyped using the developed approach. Additionally, Hp post-translational variants were detected and assigned via accurate MS analyses. The results of this study suggest use of the methodology in future analyses of other similarly complexed proteins and in normal versus disease cohort population proteomics studies.

“CRAB” Findings in a Patient With Fatigue and a History of Poor Appetite

demonstrated the presence of aberrant plasma cells constituting 64% of all cells observed in the bone marrow. Imaging studies of her axial skeleton revealed lytic lesions in both humeri and femurs, and in the skull, a compression fracture of the T12 vertebra, and a plasmacytoma in her left-side sixth rib. Laboratory testing revealed normal serum electrolytes, calcium, and creatinine levels. The patient started a prolonged chemotherapeutic regimen that included 3 cycles of vincristine, adriamycin, and dexamethasone (VAD), combination therapy with Thalidomide and Decadron, and Decadron maintenance therapy. Each regimen provided only a short-term reduction in the size of her serum Mspike and in her urine free light chain excretion rate. As a prelude to an autologous stem cell transplant, the patient underwent a repeat bone marrow biopsy, SPE, and UPE. The bone marrow biopsy demonstrated 27% aberrant plasma cells, while the SPE again showed an M-spike consistent with IgG kappa (0.38 g/dL); however, the UPE of a sample from a 24-hour urine collection was negative for any intact or free light chain immunoglobulins (data not shown). The patient underwent stem cell mobilization followed by bone marrow myeloablation using high-dose melphalan therapy and stem cell transfusion. She tolerated the procedure well, requiring only administration of packed red blood cells and platelets to maintain an adequate cell count of these formed elements while engraftment was occurring.

Insect cell conditioned medium contains an endoglycosidase able to liberate chondroitin sulfate chains from aggrecan

Culture medium obtained from baculovirus-infected High Five insect cells contains an endoglycosidase activity capable of releasing chondroitin sulfate chains from aggrecan, decorin and biglycan. Release appears to occur by cleavage within the linkage region of the chondroitin sulfate chain, but not all chains are amenable to release. Culture medium from Sf9 insect cells does not contain this activity. The endoglycosidase may become a useful reagent for biochemical research for releasing intact chondroitin sulfate chains from proteoglycans. It may also represent an impediment to such research when baculovirus systems are used to generate recombinant proteoglycans.

Use of the excluded protecting group (EPG) method for peptide synthesis

The excluded protecting group (EPG) method has been used for the solution synthesis of several peptides including Merrifield's Model Tetrapeptide, linear antamanide and an analogue of magainin-1, [Ala(19), Asn(22)]magainin-1. In the approach reported, the C-terminal amino acid is esterified to the 2-position of cholestane as the [2s,3s]iodohydrin ester and the penultimate amino acid added to the aminoacyl-steroid as the Fmoc-pentafluorophenyl-ester. The Fmoc group is removed with Et(2)NH/DMF ( approximately 15% v/v) and, after evaporation to approximately 10 mL, the solution chromatographed on Sephadex LH-20 in DMF. The dipeptidyl-steroid elutes as the free amine well separated from other reaction mixture components. Fractions containing the dipeptide, as determined by counting and TLC, are pooled and reacted with the next Fmoc-amino acid-pentafluorophenyl ester in the sequence. Repetition of the deprotection/purification/reaction cycle yields the fully protected peptide. On completion of the synthesis, the cholestane iodohydrin ester is selectively removed by treatment with Zn degrees /AcOH to yield the peptide with intact alpha-amino and side chain protecting groups. Global deprotection is achieved with HF. All intermediates from the syntheses reported were characterized. The magainin analogue was shown to have full biologic activity. The Fmoc iodohydrin esters of 16 of the 20 proteogenic amino acids have been prepared and characterized for use as the C-terminal amino acids in other EPG syntheses.

Properties of Antigenomic HDV Ribozyme Consisting of Two RNA Chains

B : LS ribozyme, a trans-variant of naturally occurring HDV ribozyme, has been constructed. The ribozyme consists of a substrate-containing LS chain and an enzyme B chain and differs from previously constructed trans-ribozymes in the length and nucleotide sequence of its oligonucleotide chains (34 and 33 bp, respectively). The chains readily associate with each other at a room temperature while the LS cleavage reaction at this temperature is negligible slow, which allowed us to investigate the association of the intact chains. At the same time the self-cleavage rate constant for the trans-ribozyme B : LS at 50 degrees C is close to those for the previously studied permuted cis-ribozymes, especially LSB variant. In addition, the dependence on the reaction conditions (Mg2+ concentration, pH, temperature) of the trans-ribozyme was similar to that of cis-ribozyme. Similar to other trans-ribozymes, B : LS ribozyme demonstrates the ability for multiple use of the enzyme B-chain with an excess of the substrate LS chain. The kinetics model of self-cleavage reaction for B : LS is presented in http://www.cardio.ru/labgen/RZ_r.html. Taken together, our results show that the original trans-variant of HDV ribozyme can be used as a model for the investigation of self-cleavage process of HDV ribozymes.

New insights into the alternating sequences of heparan sulfate

Introduction The sulfated domains (S‐domains) within HS are thought to be the primary binding site for protein ligands (Gallagher and Lyon, 2000). Studies on the binding of multimeric chemokines to HS have suggested that N‐acetylated sequences (NAc‐domains) act to space the S‐domains correctly so binding can occur at more than one HS binding site within a protein (Lortat‐Jacob et al. 1995; Stringer et al. 2002). So far, little functional significance has been ascribed to either alternating N‐sulfate/NAc or lone N‐sulfate types of sequence, although alternating sequences are thought to occur at the borders between S‐domains and NAc‐domains, forming transition zones between the two. Existing scission techniques used to excise oligosacchrides from the intact chain for structural and functional analysis disrupt the transition zones (heparinase III), or the S‐domains (heparinase I) or both (low pH nitrous acid).Materials and methods Radiolabelled HS was isolated from cultured 3T3 fibroblasts HS. Its patterns of degradation using bacteriophage K5 lyase as well as conventional heparinases I/III and nitrous acid were compared. K5 lyase‐resistant fragments were characterized by size exclusion chromatography and strong‐anion exchange HPLC.Results The work presented demonstrates how the enzyme K5‐lyase specifically cleaves only within NAc‐domains requiring sequences of at least five consecutive N‐acetylated disaccharides within the HS chain. The enzyme leaves intact S‐domains together with their flanking transition zones in situ.Discussion The characterization of oligosaccharides generated by K5 lyase cleavage of the intact HS chain provides new insights into the domain structure of HS.

Etiopathogenetic aspects of recurrent cholesteatoma development

The causes of cholesteatoma development have been interpreted in different ways. The object of our study was to present the most frequent causes of recurring cholesteatomas on the basis of our investigations. The prospective study carried out at the Institute for Otorhinolaryngology and Maxillofacial Surgery included 120 patients treated surgically for cholesteatomas. Seventy-five patients were cured after the first operation, while 45 were reoperated after recurrent cholesteatoma.The modified student t test was used to establish the difference between statistically significant groups (small independent sample proportion). The cholesteatoma was often localized in attic (25.3%) and was spreading into antrum (28%). Another group of cholesteatoma was more frequently present in mesotympanum. The small marginal perforations in back quadrants were noted more frequently in the second group (26.7%). Likewise, 40% of the patients from this group had intact chain of auditory ossicles. Destruction of all three auditory ossicles was more often in the first group. Polypoid mucosa was less detected in the first group (18.7%) in relation to the second group (46.7%). Canal wall-up tympanoplasty was used more commonly in the second group of patients.In patients treated surgically for cholesteatoma, the recurrence of the condition may be caused by different factors such as development of cholesteatoma in posterior parts of cavum, polypoid mucosa, localization of cholesteatoma in meso- and retrotympanum and obscured cavity recesses, intact chain of auditory ossicles and the application of close tympanoplasty.

High-Throughput Analysis of Hemoglobin from Neonates Using Matrix-assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

The risks associated with sickle cell and other untreated hemoglobinopathies have led to mandatory neonatal screenings in 41 of the 50 states in the US (1). Preliminary screenings are often performed by electrophoresis at alkaline pH on cellulose acetate, with follow-up analysis of abnormal samples by acid electrophoresis on citrate agar (2). Alternatively, isoelectric focusing (IEF) or HPLC can be used in the screenings (3), but other techniques offer advantages. In the past decade, new mass spectrometry (MS) approaches, including matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS, have been applied to the study of proteins (4). MALDI-TOF MS offers a potent means of analyzing constituent chains of hemoglobin A (HbA) directly from samples as small as a single red blood cell (5). Mass accuracy and resolution are sufficient to determine the presence of point mutations within chains differing in mass by as little as ∼20 Da. This ability allows detection of the β6Glu→Val variant (∼30 Da lower in molecular mass) during direct analysis of the intact chains. Moreover, the site of mutation can be determined by combining MALDI-TOF MS with specific enzymatic digestion, i.e., “mass mapping”. Thus, MALDI-TOF MS has the potential to be used in place of conventional approaches (6) for HbAS/HbS screening in the general population. To date, however, the mass spectrometric approaches have not been taken to the high-throughput mode necessary for efficient population screening, nor have they been applied to neonatal blood samples, which contain roughly equal portions of HbA and HbF. We report here progress in the development of a high-throughput mass spectrometric screening approach capable of screening the hemoglobin of neonates at rates of ∼100 samples/h. In a blind analysis, 96 neonate blood samples were prepared for both intact protein molecular mass determination and mass mapping using parallel robotics (96-well format) and analyzed …

Electrospray tandem mass spectrometry of intact beta-chain hemoglobin variants.

In this report, we present data to illustrate how human hemoglobin (Hb) variants can be identified by electrospray tandem mass spectrometry (MS/MS) of the intact Hb chains following the one-step dilution of whole blood. MS/MS spectra were recorded on a series of intact beta-chain human Hb variants. The resultant spectra were interpreted, and using the information gleaned from the fragmentation patterns of known variants, two unknown beta-chain variants were characterized solely by this mass spectrometric method. Fragment ions that serve to identify beta-chain variants were identified. The fragmentation patterns of the intact beta-chain [M + 18H]18+ ions showed classical facile cleavages adjacent to acidic residues and N-terminal to proline residues, with Thr50-Pro51 being the most prominent cleavage site. Abundant product ions were formed by peptide bond cleavage in the regions close to the termini of the beta chain, the central region being less well-represented in the MS/MS spectra. Nearly 50% of the beta-chain primary structure could be determined by MS/MS of the intact chain. However, analysis of the Hb variants where mutations have occurred in the inner region (residues 58-111) of the beta globin proved to be difficult and required mass spectrometric analysis of their tryptic peptides for a complete identification.

Genotyping single nucleotide polymorphisms using intact polymerase chain reaction products by electrospray quadrupole mass spectrometry.

Both single nucleotide polymorphisms (SNPs) and mutations are commonly observed in the gene encoding the tumor suppressor protein, p53. SNPs occur at specific locations within genes whereas mutations may be distributed across large regions of genes. When determining nucleotide differences, mass spectrometry is the only method other than Sanger sequencing which offers direct structural information. Electrospray ionization (ESI) quadrupole mass spectrometry (MS) analysis of intact polymerase chain reaction (PCR) products was performed following a simple purification and on-line heating to limit ion adduction. The PCR products were amplified directly from genomic DNA rather than plasmids, as in our previous work. Two known polymorphisms of the p53 gene were genotyped. A cytosine (C) or guanine (G) transversion, designated C <--> G (G <--> C on the opposite strand), were each detected by a 40.0 Da change upon ESI quadrupole MS analysis. Using known PCR products as standards, the genotypes determined for 10 human samples corresponded with restriction fragment length polymorphism (RFLP) analysis. Cytosine/thymine (T) transitions, designated C <--> T (G <--> A on the opposite strand), were also genotyped by ESI-MS. This SNP is discriminated by a 15.0 Da change on one strand (C <--> T) and a 16.0 Da change on the other (G <--> A). Appropriate sample preparation and instrumental configuration (including heated sample inlet syringe and MS source), to limit adducts, are both vital for successful ESI quadrupole MS analysis of intact PCR products.

Unmasking a hyaluronan-binding site of the BX(7)B type in the H3 heavy chain of the inter-alpha-inhibitor family.

The inter-alpha-inhibitor (I alpha I) family gathers together several plasma protease inhibitors such as I alpha I and pre-alpha-inhibitor (P alpha I) that are variously assembled from a set of polypeptide chain precursors designated H1P to H3P. In addition to their protease inhibitory activity, a major physiological function of I alpha I family members is hyaluronan (HA) binding and HA-dependent stabilization of the extracellular matrix surrounding various cell types. Also, binding of HA to these molecules has been shown to be an important event in tumor cell proliferation and rheumatoid arthritis. However, how HA and I alpha I family members first recognize each other has so far remained elusive. The so-called BX7B domain found in some HA-binding proteins is an HA-binding site in which B represents a basic amino-acid residue and X represents any nonacidic residue. This domain has now been identified in the N-terminal end of H3P that is a precursor of P alpha I. A series of wild-type or mutant recombinant H3P chains produced with a mouse cDNA expressed in Escherichia coli allowed us to demonstrate that this domain binds HA in a noncovalent fashion. Furthermore, unmasking this HA-binding activity required most of H3P to be trimmed off at its C-terminal end. The latter observation was confirmed with a natural, mature H3 chain purified from human plasma. Indeed, a thermolysin-generated, N-terminal fragment of this H3 chain strongly bound HA whereas the intact H3 chain did not. Therefore, in vivo, the HA-binding activity of the mature H3 chain within P alpha I may vary with the folding and/or fragmentation of this protein.

Abnormal immunoglobulin synthesis in monoclonal immunoglobulin light chain and light and heavy chain deposition disease

The Congo red-binding fibrils of AL amyloidosis are the most common form of monoclonal immunoglobulin tissue deposition (MIDD). Nonetheless, the less structured deposits found in light chain deposition disease (LCDD) and the similar, but distinct, deposits of light and heavy chain deposition disease (LHCDD) and heavy chain deposition disease (HCDD) can produce significant clinical pathology. Analyses of immunoglobulin synthesis by bone marrow cells obtained from 7 patients with LCDD and LHCDD demonstrated the production of excess light chains in all and the presence of incomplete light chains or heavy chain fragments in 5, regardless of the presence of an intact monoclonal protein or related subunit in the serum or urine.Our data indicate that, as is the case with the fibrillar deposits of AL amyloid, the non-fibrillar forms of monoclonal Ig deposition (LCDD and LHCDD) can be associated with the presence of immunoglobulin fragments in bone marrow cells. In some instances these appeared to be synthetic in origin, although rapid intracellular proteolysis or a combination of both could not be excluded. In either case the fragments may be more susceptible to tissue deposition, with subsequent organ compromise, than intact Ig chains.

Keratan sulfates from bovine tracheal cartilage

Intact keratan sulfate chains derived from bovine tracheal cartilage have been examined using both one-dimensional methods and the two-dimensional experiments COSY-45 and TOCSY for homonuclear shift correlations and a modified COLOC (correlated spectroscopy for long-range couplings) approach for 13C-1H shift correlations. Partial 1H and 13C NMR signal assignments for residues within the intact polymer chain are reported; data derived from the repeat region signals and from chain cap residues are assigned by comparison with published data derived from oligosaccharides obtained through cleavage of keratan sulfate polymer chains using keratanase and keratanase II and are discussed in detail. The one-dimensional spectra for both 1H and 13C nuclei contain highly crowded signal clusters for which data analysis is not directly possible. COSY-45 analysis allow the correlation and assignment of many proton resonances located within the 3.4-4.8 p.p.m. chemical shift region while from the C/H correlation spectrum data are assignable for some signals within the complex set of carbon resonances which fall in the region between 68 and 86 p.p.m., This work using material from tracheal cartilage has permitted the first detailed combined 1H and 13C NMR examination of the primary keratan sulfate polymer structure; this sequence forms the basis for the more complex members of the keratan sulfate family present in other tissues such as articular cartilage and cornea where further residues such as (alpha1-3)-linked fucose and (alpha2-6)-linked N-acetylneuraminic acid are also present. This nondestructive method of analysis complements the currently available degradative methods for structure determination which may then subsequently be utilized.

Formation of the metabolic intermediate 2,4,4-trimethyl-2-pentanol during incubation of a Sphingomonas sp. strain with the xeno-estrogenic octylphenol.

Degradation of branched octylphenol was studied in a bacterial culture of a Sphingomonas sp. strain. Octylphenol is considered to be the most stable degradation intermediate formed from the corresponding nonionic octylphenol polyethoxylates surfactants during biological wastewater treatment. Since octylphenol can exert estrogenic effects in wildlife, a detailed study of its biodegradation is warranted. The aerobic microbiological transformation of octylphenol was examined with and without the addition of the easily assimilable sodium acetate. In both cases the formation of the metabolite 2,4,4-trimethyl-2-pentanol, representing the intact alkyl chain as a tertiary alcohol, was observed. Since the octylphenol degradation rate was not affected by the presence of acetate, this strain did not show any diauxic metabolic behaviour when incubated with octylphenol and sodium acetate as the sources of carbon and energy. As a result of the biotransformation of octylphenol, its estrogenic potency was removed because it is the phenolic moiety that interacts with the estrogen receptors. This feature opens perspectives for the use of this strain in the framework of an adequate treatment of wastewater with high levels of alkylphenol polyethoxylates.

Strategies for analysis and structure characterization of glycans/proteoglycans by capillary electrophoresis. Their diagnostic and biopharmaceutical importance.

Proteoglycans are key biological macromolecules that, via their glycan constituents, participate and regulate several cellular events and physiopathological processes. Refined structures of their highly anionic glycan chains, involving sulphation pattern and uronic acid distribution through the polymeric chain, determine the interactions of proteoglycans with matrix effector molecules and are responsible for numerous effects. Analysis and structural characterization of glycans are, therefore, essential in understanding the biological functions of proteoglycans. Capillary electrophoresis with its high resolving power and sensitivity may successfully be used for the fine chemical characterization of components present in low amounts and to overcome limitations due to low amount/volume of biologic samples available. This technique is also friendly to the user and to the environment, since a very small amount of solvents (a few microL) is required. In this review the strategies used to analyse and characterize the structure of glycan chains of proteoglycans are summarized. They involve capillary electrophoretic analysis of depolymerized acidic glycan chains using specific enzymes and analysis of intact chains. The importance of this type of analysis in biologic samples and tissues and its possible diagnostic and biopharmaceutical use are also discussed.

Orthotopic cartilage transplantation. Morphologic, angiography and histochemical studies of the vitality of free septum transplants

Orthotopic cartilage transplantation is a technique frequently used in modern septal surgery. The prerequisite for a stable long-term result is viability of the transplanted cartilage. Therefore, we studied the healing process histologically, angiographically, and histochemically. We found a characteristic picture. Due to chondronal structure of the cartilage, the healing process varied in time and location. Reintegration took place by chondroneogenesis, commencing at the inner perichondrium. Reintegration depended directly on the distance of the cartilage cells to the surrounding vessels. Histochemically, we found an intact respiratory chain in the mitochondria and thus, we were able to demonstrate the preservation of viability in orthotopic transplanted cartilage.

Sialylation of the Sialic Acid Binding Lectin Sialoadhesin Regulates Its Ability to Mediate Cell Adhesion

The macrophage-specific cell surface receptor sialoadhesin, which is a member of the newly recognized family of sialic acid binding lectins called siglecs, binds glycoprotein and glycolipid ligands containing a2-3–linked sialic acid on the surface of several leukocyte subsets. Recently, the sialic acid binding activity of the siglec CD22 has been demonstrated to be regulated by sialylation of the CD22 receptor molecule. In the present work, we show that desialylation of in vivo macrophage sialylconjugates enhances sialoadhesin-mediated lectin activity. Herein, we show that receptor sialylation of soluble sialoadhesin inhibits its binding to Jurkat cell ligands, and that charge-dependent repulsion alone cannot explain this inhibition. Furthermore, we show that the inhibitory effect of sialic acid is partially dependent on the presence of an intact exocyclic side chain. These results, in conjunction with previous findings, suggest that sialylation of siglecs by specific glycosyltransferases may be a common mechanism by which siglec-mediated adhesion is regulated.