Biological variation responses in fMLP-OMe analogs, introducing bulky protecting groups on the side-chain of hydrophilic residues at position 2.

for-Met-Ser(Bzl)-Phe-OMe, for-Met-Cys(Bzl)-Phe-OMe, for-Met-Tyr(Bzl)-Phe-OMe and for-Met-Lys(Z)-Phe-OMe were synthesized to investigate the importance of a bulky protecting group on the side-chain of a hydrophilic residue at position 2 on the biological activities of human neutrophils. Our results indicate that these compounds do not trigger a good chemotactic response, which, in any case, is not improved with respect to that induced by the analogs with the unprotected residues. Instead, both superoxide anion production and, particularly, lysozyme release are more efficient.     

Synthesis,conformation and biological activity of centrally modified pseudopeptidic analogues of For-Met-Leu-Phe-OMe

Summary. For-Met-βAlaψ[CSNH]-Phe-OMe (3), For-Met-βAlaψ[CH₂NH]-Phe-OMe (5), For-Met-NH-pC₆H₄-SO₂-Phe-OMe (8a), For-Met-NH-mC₆H₄-SO₂-Phe-OMe (8b) and the corresponding N-Boc precursors (2, 4, 7a, b) have been synthesized and their activity towards human neutrophils has been evaluated in comparison with that shown by the reference tripeptide For-Met-Leu-Phe-OMe (fMLF-OMe). Chemotaxis, lysozyme release and superoxide anion production have been measured. ¹H NMR titration experiments and IR spectra have been discussed in order to ascertain the preferred solution conformation adopted by the tripeptide 3 with particular reference to the presence of a folded conformation centred at the centrally positioned thionated β-residue.     

Isopeptide bonds in chemotactic tripeptides. Synthesis and activity of lysine-containing fMLF analogs.

In order to explore the properties of chemotactic N-formylpeptides containing isopeptide bonds within their backbones, a group of lysine-containing analogs of the prototypical chemotactic tripeptide N-formylmethionyl-leucyl-phenylalanine (fMLF) was synthesized. The new analogs were designed by adding to the HCO-Met or Boc-Met residue a dipeptide fragment made up of Lys and Phe residues joined through Lys N alpha or N epsilon bonds, in all possible combinations. Thus, the following six pairs of tripeptides were synthesized and examined for their bioactivity: RCO-Met-Lys(Z)-Phe-OMe (2a, b), RCO-Met-Lys(Z-Phe)-OMe (3a, b), Z-Lys(RCO-Met)-Phe-OMe (4a, b), Z-Phe-Lys(RCO-Met)-OMe (5a, b), RCO-Met-Phe-Lys(Z)-OMe (6a, b) and Z-Lys(RCO-Met-Phe)-OMe (7a, b), with R=OC(CH3)(3 )and R=H for compounds a and b, respectively. All the new models were characterized fully and their activity (chemotaxis, superoxide anion production and lysozyme release) on human neutrophils determined as agonists (compounds b) and antagonists (compounds a). All N-formyl derivatives 2b-7b are less potent than fMLF-OMe as chemoattractants, but compound 7b exhibits selective activity as superoxide anion producer. Derivatives 2a-7a do not show antagonistic activity towards fMLF induced chemotaxis and O(2)(-) production, however, all these compounds except 4a antagonize lysozyme release by 60%.     

Synthesis by conventional methods of human growth hormone peptide fragments. I.

The synthesis of two protected peptides which correspond to positions 139-146 and 147-156 of the HGH primary structure is described. These peptides prepared by the stepwise procedure, are: Boc-Phe-Lys(Z)-Gln-Thr(Bzl)-Ser(Bzl)-Lys(Z)-Phe-OMe and Boc-Asp(OBzl)-Asn-Ser(Bzl)-His(Dnp)-Asn(OBzl)-Asp(OBzl)-Ala-Leu-OBzl. All protected intermediates were isolated and characterized for homogeneity.     

The effect of crosslinking by bis(3,5-dibromosalicyl) fumarate on the autoxidation of hemoglobin

Bis(3,5-dibromosalicyl) fumarate was used to crosslink hemoglobin both in the oxy and deoxy states. This double headed diaspirin was known to crosslink oxy Hb A selectively between Lys 82 beta 1 and Lys 82 beta 2 (Walder, J. A., et al. (1979) Biochemistry 18, 4265) and deoxy Hb A between Lys 99 alpha 1 and Lys 99 alpha 2 (Chatterjee R. Y., et al. (1986) J. Biol. Chem. 261, 9929). The autoxidation at 37 degrees C of oxy alpha 99 crosslinked hemoglobin was found to be 1.8 times as fast as that of Hb A while that of the oxy beta 82 crosslinked hemoglobin was only 1.2 times as fast. After 5 hours the formation of methemoglobin in the alpha crosslinked Hb A is 21.3% compared to 10.8% in beta crosslinked Hb A and 6.4% in Hb A. These results may effect the proposed use of alpha 99 crosslinked hemoglobin as a blood substitute by demonstrating the need for protection from autoxidation during storage.     

Cooperative ligand binding of crosslinked hemoglobins at very high temperatures

Human hemoglobin was reacted with the bifunctional reagent bis(3,5-dibromosalicyl) fumarate to yield a derivative (Hb alpha alpha) crosslinked between the two alpha-chains; when the reaction was carried out with HbA already crosslinked between the two beta-chains by 2-nor-2-formylpyridoxal 5'-phosphate, a doubly crosslinked derivative (Hb alpha alpha beta beta) was obtained. We have observed that both modified hemoglobins are extremely stable up to temperatures of at least 85 degrees C. The carbon monoxide binding kinetics of both crosslinked hemoglobins, studied at temperatures between 15 and 85 degrees C, by means of stopped flow and flash photolysis techniques, show that the ligand-linked allosteric transition is maintained even at the highest temperatures. These results are also relevant to the mechanism of thermal unfolding of human hemoglobin, since they show that dissociation into alpha beta dimers (and exposure of the relatively hydrophobic dimer-dimer interfaces) is an obligatory step in the irreversible denaturation of deoxy and carbon monoxy hemoglobin.     

A comparison of human mesenchymal stem cell osteogenesis in poly(ethylene glycol) hydrogels as a function of MMP-sensitive crosslinker and crosslink density in chemically defined medium

This study investigated osteogenesis of human mesenchymal stem cells encapsulated in matrix-metalloproteinase (MMP)-sensitive poly(ethylene glycol) (PEG) hydrogels in chemically defined medium (10 ng/ml bone morphogenic factor-2). Thiol-norbornene photoclick hydrogels were formed with CRGDS and crosslinkers of PEG dithiol (nondegradable), CVPLS-LYSGC (P1) or CRGRIGF-LRTDC (P2; dash indicates cleavage site) at two crosslink densities. Exogenous MMP-2 degraded P1 and P2 hydrogels similarly. MMP-14 degraded P1 hydrogels more rapidly than P2 hydrogels. Cell spreading was greatest in P1 low crosslinked hydrogels and to a lesser degree in P2 low crosslinked hydrogels, but not evident in nondegradable and high crosslinked MMP-sensitive hydrogels. Early osteogenesis (Alkaline phosphatase [ALP] activity) was accelerated in hydrogels that facilitated cell spreading. Contrarily, late osteogenesis (mineralization) was independent of cell spreading. Mineralized matrix was present in P1 hydrogels, but only present in P2 high crosslinked hydrogels and not yet present in nondegradable hydrogels. Overall, the low crosslinked P1 hydrogels exhibited an accelerated early and late osteogenesis with the highest ALP activity (Day 7), greatest calcium content (Day 14), and greatest collagen content (Day 28), concomitant with increased compressive modulus over time. Collectively, this study demonstrates that in chemically defined medium, hydrogel degradability is critical to accelerating early osteogenesis, but other factors are important in late osteogenesis.     

Ultraviolet light-induced crosslinking of mRNA to proteins.

Irradiation of intact or EDTA-dissociated L-cell polyribosomes with 254 nm UV light at doses of 1-2 x 10(5) ergs/mm2 extensively crosslinks mRNA to proteins. The crosslinked mRNA-protein complexes can be isolated on the basis of buoyant density in urea-containing CS2SO4 gradients that dissociate non-covalent complexes. Crosslinking of mRNA can also be assayed by phenolchloroform extraction. mRNA recovered from the crosslinked complexes by digestion with proteinase K has the same electrophoretic mobility in polyacrylamide gels as unirradiated mRNA. Therefore, irradiation does not either crosslink RNA molecules to RNA molecules or break phosphodiester bonds. With these methods it has been found that more than 70% of high molecular weight polydisperse mRNA, but only 25-40% of histone mRNA, can be crosslinked to protein. On the basis of buoyant density the histone mRNA-protein complex had a protein content of 26%, whereas the mean protein content of most non-histone mRNA-protein complexes was 65%. It is concluded that most mRNA in polyribosomes is in close contact with proteins, and that histone mRNA can be crosslinked to many fewer proteins that most other mRNAs.     

Thermal stability of hemoglobin crosslinked in the T-state by bis(3,5-dibromosalicyl) fumarate

Bis(3,5-dibromosalicyl) fumarate was used to crosslink oxyhemoglobin between Lys 82 beta 1 and Lys 82 beta 2 (Walder, J. A., et al. (1979) Biochemistry 18, 4265) and deoxyhemoglobin between Lys 99 alpha 1 and Lys 99 alpha 2 (Chatterjee R.Y., et al. (1986) J. Biol. Chem. 261, 9929). Thermal denaturations demonstrated that alpha crosslinked hemoglobin (alpha 99XLHb A) has the same stability as the beta crosslinked one (beta 82XLHb A). Both alpha and beta crosslinked methemoglobins have a denaturation temperature in 0.9 M guanidine of 57 degrees C compared to 41 degrees C of Hb A. The second product from the T-state crosslinking reaction was found to be crosslinked between the beta chains by chain separation and amino acid analysis. The possible positions for this crosslink are limited to the bisphosphoglycerate binding site in the three-dimensional structure. Its stability is comparable to that of the alpha 99XLHb A or beta 82XLHb A. These modified hemoglobins are potential blood substitutes.     

Physicochemical properties of alkaline serine proteases in alcohol

The alkaline proteases subtilisin Carlsberg and alcalase possess substantial enzymatic activity even when dissolved in ethanol. The crude enzymes were purified by gel filtration and the main fractions suspended in ethanol to give a translucent suspension. Both the supernatant and the resuspended precipitate after high-speed centrifugation were found to have enzymatic activities. The solubility of subtilisin Carlsberg in anhydrous ethanol was found to be 45.1g/ml and that of alcalase was 48.1g/ml by Coomassie blue dye-binding method using bovine serum albumin as a standard. In the presence of water, the solubility of both enzymes increased with water content. The stability of enzymes incubated in ethanol was assayed by their amidase and transesterase activities using Ala-Ala-Pro-Phe-pNA as substrate in phosphate buffer (pH8.2) and Moz-Leu-OBzl as substrate in anhydrous ethanol, respectively. The soluble enzymes have a half-life of about 36 hr and that of suspended enzymes about 50 hr in the amidase activity assay, whereas the same soluble enzymes have a half-life of about several hours and that of suspended enzymes 1 h by the transesterase activity assay. The stability of both enzymes decreased as water concentration increased. The diastereoselectivity of the enzyme-catalyzed hydrolysis of diastereo pairs of tetrapeptide esters,l-Ala-l-Ala-(d-orl-)Pro-l-Phe-OMe andl-Ala-l-Ala-(d-orl-)Ala-l-Phe-OMe, in phosphate is as high as that of the transesterification of these substrates in ethanol. It is concluded that active sites and selectivity of alkaline serine proteases in anhydrous alcohol are probably very similar to those in aqueous solution in spite of the fact that a lower reactivity is usually associated with the enzymes in nonaqueous solvents.     

Interchain and intrachain crosslinking of actin thiols by a bifunctional thiol reagent.

From 1,9-nonylenedithiol and Ellman's Reagent the bifunctional asymmetric disulfide n-nonylene-1,9-bis-[5-dithio-(2-nitrobenzoic acid)] (NBDN) was prepared. By monovalent reaction with cysteine-374 the crosslinker could be introduced into monomeric actin, with release of one equivalent of yellow 2-nitro-5-thiobenzoate (NTB). From the monovalent actin derivative we prepared a crosslinked actin dimer (Cys-374-Cys-374') as well as a monomer with a crosslink between Cys-374 and Cys-10. Neither crosslinked actin species was able to polymerize the crosslinked monomer even in the presence of phalloidin. The crosslinked monomer polymerized on the addition of dithiothreitol, thus providing the first unpolymerizable actin species whose polymerizability can be restored under mild conditions. We suggest the use of NBDN as a thiol-specific crosslinker that reacts under spectrophotometric control and can be removed by the addition of thiols.     

Dichotomous effect of monocyte Fc receptor interaction on anti-CD3-induced immunoglobulin synthesis

Monoclonal antibodies against the TCR/CD3 complex are capable of activating T cells which in turn may induce immunoglobulin synthesis in B cells under appropriate conditions. Here we present evidence that distinct immune responses, induced by four commonly used TCR/CD3 mAb (Leu4, OKT3, BMA030, BMA031) were related to the mAb interaction with monocyte Fc receptors for IgG. Depending on their isotype and on the technique by which they were crosslinked, TCR/CD3 mAb induced variable IgM and IgG synthesis in PBMC: If the mAb were crosslinked by monocyte IgG-Fc receptors they induced a high Ig production, while crosslinking the same mAb by plastic-bound goat anti-mouse antibodies (panning) failed to do so. Nevertheless, both crosslinking techniques triggered a strong proliferation and IL-2, IL-4, and IFN gamma lymphokine gene expression. The lack of Ig production under panning conditions was due to an additional IgG-Fc receptor interaction with monocytes: (a) If namely mAb F(ab')2 fragments, or mAb isotypes unable to bind to monocyte Fc receptors (IgG2b, IgG1 in nonresponders) were crosslinked by panning, both a good proliferation as well as Ig production ensued; (b) if TCR/CD3 mAb isotypes which could additionally bind to monocyte Fc receptor (IgG2a) were crosslinked, no Ig production occurred; (c) if mAb F(ab')2 fragments were crosslinked with a second anti-T cell antibody of IgG2a isotype, which could bind to monocyte Fc receptors, Ig synthesis was reduced. Interestingly enough, this diminishing effect, due to monocyte Fc receptor interaction, was only observed if CD4-positive cells were proliferating, but not if CD8-positive cells were activated.     

Glutaraldehyde crosslinking of red blood cells by using a hemodialyzer

Summary Human red blood cells (RBC) were crosslinked with glutaraldehyde (GA) by using a hemodialyzer which is used as an artificial kidney. Human RBC, which was in a flow of 2 ml/min, was extensively crosslinked with 50 mM GA solution of 10 ml/min flow rate. The crosslinked RBC showed high stability against osmotic pressure. The oxygen transport activity of the crosslinked RBC was similar to unmodified RBC. This crosslinking method could be used for the development of an efficient reactor which produces a stable and active RBC.     

Calcium-induced localization of calcium-activated neutral proteinase on plasma membranes

The location of calcium-activated neutral proteinase (CANP) was determined in human erythrocytes by crosslinking CANP to co-localizing proteins using a photolabeling bifunctional reagent, 4,4'-dithiobisphenylazide (DTBPA). The crosslinked products were selectively isolated by immunoprecipitation with a polyclonal anti-CANP antibody and analyzed by SDS-polyacrylamide gel electrophoresis after cleavage of the crosslinkage. In the calcium-free incubation medium the main proteins crosslinked with CANP were cytosolic proteins such as hemoglobin. In the presence of calcium ions, on the other hand, membrane skeletal proteins such as spectrin, band 4.1, 4.2 and 6 proteins as well as band 3 were crosslinked with CANP. Addition of calcium ionophore further increased the amount of crosslinked membrane proteins. These results suggest that in the absence of calcium ions CANP exists diffusely in the cytoplasm and is crosslinked with cytoplasmic hemoglobin nonspecifically while in the presence of calcium ions CANP associated with membrane where it is crosslinked specifically with the lining proteins. Thus it is demonstrated biochemically that the localization of CANP is dynamic depending on the presence of calcium ions.     

Ca2+-induced structural change in the Ca2+Mg2+ domain of troponin C detected by crosslinking

Rabbit muscle troponin C was selectively modified at Cys-98 by 1,3-difluoro-4,6-dinitrobenzene. The second function of the bifunctional reagent was triggered at alkaline pH in the presence and absence of Ca²⁺. The crosslinked troponin C was hydrolyzed by trypsin and the peptides containing a dinitrobenzene moiety were isolated. When troponin C was crosslinked in the presence of Ca²⁺, the single dinitrobenzene-containing peptide was Gly-89-Arg-100, in which Cys-98 was crosslinked with Lys-90. When crosslinking was performed in the absence of Ca²⁺, beside the above peptide two additional peptides containing dinitrobenzene were found. One of these peptides is made up of two fragments, Ser-91-Arg-100 and Asn-105-Arg-120, crosslinked between Cys-98 and Tyr-109. The second peptide, Ala-121-Lys-140, contains modified Lys-136, presumably crosslinked with His-135. The data indicate that the distances between the α-carbon of Cys-98 and those of Lys-90, Tyr-109, Lys-136 and probably the α-carbon distance His-125-Lys-136, do not exceed 14 Å. Comparison with the X-ray structure of troponin C (Herzberg, O, and James, M.N.G. (1985) Nature 313, 653–659) indicates that some of the above distances increase on Ca²⁺-binding.     

Preparation of superabsorbent cellulosic hydrogels

In this work, milled softwood (SW) bleached kraft fibers were crosslinked by esterification with poly(vinyl methyl ether-co-maleic acid) (PVMEMA) and polyethylene glycol (PEG). The effects of fiber length, crosslinking reaction time, and dosage of PVMEMA on water absorption and retention value (WAARV) for the crosslinked fibers were determined. The results show that as the softwood fiber length is mechanically decreased from 2.41 to 0.50 mm and employing a weight ratio of fiber to polymers equivalent to 1.00:1.28 the WAARV increased from 86.50 to 189.20 g/g. Analysis of the crosslinked fibers by SEM and light microscope indicated that the polymers and fibers form a crosslinked fibrous matrix. FT-IR spectroscopy was employed to detect the ester linkage between PVMEMA and PEG/SW kraft pulp fibers. The results suggested that the ester crosslinked pulps exhibit excellent water absorbent properties and have the potential of utilizing milled bleached SW kraft fibers, such as refiner dust or pulp fines, for novel water absorbent applications.     

The proteotytic systems of lactic acid bacteria

Proteolysis in dairy lactic acid bacteria has been studied in great detail by genetic, biochemical and ultrastructural methods. From these studies the picture emerges that the proteolytic systems of lactococci and lactobacilli are remarkably similar in their components and mode of action. The proteolytic system consists of an extracellularly located serine-proteinase, transport systems specific for di-tripeptides and oligopeptides (> 3 residues), and a multitude of intracellular peptidases. This review describes the properties and regulation of individual components as well as studies that have led to identification of their cellular localization. Targeted mutational techniques developed in recent years have made it possible to investigate the role of individual and combinations of enzymes in vivo. Based on these results as well as in vitro studies of the enzymes and transporters, a model for the proteolytic pathway is proposed. The main features are: (i) proteinases have a broad specificity and are capable of releasing a large number of different oligopeptides, of which a large fraction falls in the range of 4 to 8 amino acid residues; (ii) oligopeptide transport is the main route for nitrogen entry into the cell; (iii) all peptidases are located intracellularly and concerted action of peptidases is required for complete degradation of accumulated peptides.     

Proteins crosslinked to messenger RNA by irradiating polyribosomes with ultraviolet light.

A set of proteins crosslinked to L-cell mRNA by irradiating polyribosomes with 254 nm ultraviolet light has been identified. 35S-methionine-labeled crosslinked mRNA-protein complexes were isolated by chromatography on oligo(dT)-cellulose under conditions that prevented non-covalent binding of proteins to RNA and to the column. After enzymatic removal of the RNA the proteins were analyzed in sodium dodecylsulfate/polyacrylamide gels. Six proteins having molecular weights of 98,000, 78,000, 75,000, 68,000, 62,000, and 52,000 were crosslinked to mRNA whether intact polyribosomes or EDTA- released mRNA-protein complexes were irradiated. Digestions with specific RNAases and chromatography on oligo(dT)-cellulose were used to show that a protein of 78,000 daltons was the only one crosslinked to poly(A), and the other proteins were crosslinked to sequences other than poly(A). However, a 78,000 dalton protein was also crosslinked to a sequence other than poly(A).     

NADPH-cytochrome P-450 reductase-cytochrome b5 interactions: crosslinking of the phospholipid vesicle-associated proteins by a water-soluble carbodiimide

Detergent-solubilized and purified rabbit liver microsomal NADPH-cytochrome P-450 reductase and cytochrome b5 were coreconstituted into phospholipid vesicles. When the proteoliposomes were incubated with a water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), a new higher-molecular-weight band was seen by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The band was purified by chromatography on DEAE-Sepharose CL-6B, 2'5'-ADP-Sepharose 4B, and Sephadex G-100. The heme absorption spectrum and fluorophotometric assay of flavin of the purified material demonstrate that this product is a 1:1 crosslinked complex containing one molecule each of the flavoprotein and cytochrome. Proteolysis of the crosslinked form indicates that the hydrophilic catalytic domains participate in the covalent attachment, and that the hydrophobic membrane-attachment peptide is necessary for the protein interaction. The purified crosslinked derivative showed no activities for reduction of either cytochrome c or ferricyanide. About half of the enzyme-associated flavin was reduced rapidly by NADPH, as was 20-30% of the crosslinked cytochrome, indicating that, in at least some of the complexes, the flavin-mediated pathway for reduction of cytochrome by pyridine nucleotide was intact. These data suggest that the output- rather than the input-electron transfer site(s) in the flavoprotein was (were) blocked by the covalently attached cytochrome.     

Crosslinking of alginic acid/chitosan matrices using polycarboxylic acids and their utilization for sodium diclofenac release

Three different ratios of alginic acid/chitosan matrices of ratios 3/1, 1/1 and 1/2, respectively, were crosslinked in their dry state using citric acid (CA)/sodium hypophosphite (SHP) at different conditions controlling the crosslinking process such as citric acid concentration, citric acid/sodium hypophosphite molar ratio as well as time and temperature of reaction. Results indicate that such matrices were crosslinked efficiently on curing at 180 °C for 9 min in presence of CA/SHP ratio 1 and the citric acid concentration of 0.6 based on the weight of any matrices. The crosslinked matrices were characterized by investigating their swelling properties, FT-IR and thermalgravimetric analysis. Furthermore, such crosslinked matrices were tested as drug release for sodium diclofenac.