Structural requirements of rabbit IgG F(ab')2 fragment for activation of the complement system through the alternative pathway—II. Ionic and indole groups

The structural features of rabbit IgG F(ab')₂ fragments required for complement activation through the alternative pathway have been investigated by means of chemical modification of specific groups. These included ionic (amino and carboxyl) and indole groups.Potassium cyanate, glycine-ethyl ester in the presence of EDC,2 and NBB were used as modifying agents of the amino, carboxyl and indole groups respectively. Three different aspects of the modified protein were analysed: the conformational integrity of the protein (by CD and antigenic reactivity), the antibody activity (by the haemaglutination test and radiobinding to the erythrocyte surface) and the capacity to activate complement through-the alternative pathway (by direct haemolysis in conditions in which the classical pathway was blocked).Changes in protein conformation caused by the chemical treatment applied were not detected by the techniques used. An enhancement of the antibody activity of the F(ab')₂ fragment against the sheep erythrocyte membrane was observed when it was amidated by glycine-ethyl ester and a decrease when it was carbamylated or two tryptophanyl residues were altered per molecule. The complement-activating capability was preserved after tryptophan modification. On the contrary, modification of amino groups produced a partial decrease in the complement-activating ability and almost total loss of the ability when 34 carboxyl groups were amidated.     

Biomechanical properties of carbodiimide crosslinked collagen: influence of the formation of ester crosslinks

There is a growing interest in the use of collagen matrices for tissue engineering. To prevent rapid degradation and to improve their mechanical properties, collagen matrices have been modified using different crosslinking agents. Among the different agents used, water soluble carbodiimides (such as N'-(3-dimethylaminopropyl)-N-ethylcarbodiimide, EDC) in combination with N-hydroxysuccinimide (NHS) are attractive systems, because no additional chemical entities are incorporated in the matrix. EDC/NHS crosslinking leads to amide bond formation between activated carboxyl groups and amine groups. Recently, we proposed that in addition to amide bond formation, ester links are also formed between activated carboxyl groups and hydroxyl groups. This was based on observations we made after development of a new method to quantify concentrations of carboxyl groups of collagen materials before and after crosslinking. The current study is directed to the influence of ester bond crosslinks formed after crosslinking of collagen with EDC/NHS on its physical-chemical and biomechanical properties. Reconstituted dermal bovine collagen patches (RDBC) were used as model material and were crosslinked with EDC/NHS. In one RDBC group, collagen amine groups were blocked with propionaldehyde prior to crosslinking, while in the other group unprocessed RDBC was crosslinked without additional matrix modifications. It was shown that after activation of collagen carboxyl groups with EDC and NHS, amide crosslinks as well as ester crosslinks with collagen hydroxyl groups were formed. It was furthermore demonstrated that the ester crosslinks of EDC/NHS-crosslinked RDBC could be removed by mild hydrolysis affording collagen matrices with improved mechanical properties.     

Agonist and antimineralocorticoid activities of spirolactones.

To investigate the mechanism of action of antimineralocorticoids, a series of spirolactone analogues was evaluated for both mineralocorticoid antagonist and agonist activity. Antagonist activity was assessed by inhibition of aldosterone stimulated sodium transport employing toad bladder short-circuit current (SCC) measurements. Agonist activity was assessed in the same system by the direct effect of spirolactones on SCC. Opening of the gamma-lactone ring of a spirolactone dramatically decreased antagonist activity in the compound studied. Several C-7 chi-substitutions resulted in either enhanced or diminished activity, whereas deletion of the C-10 methyl group (i.e., 19-nor compound) had only minimal effects on antagonist potency. Agonist activity was demonstrable for three of the analogues studied: the 19-nor compound, and those containing a C-7 chi-substitution with a carboxyl isopropyl ester or a C-6-7 cyclopropyl linkage. The functional activity in toad bladder was compared to previous measurements of the relative binding affinity of the same spirolactones for mineralocorticoid receptors in rat kidney. Although there was some correlation between binding to rat kidney receptors and antagonist activity in the toad bladder, the results did not coincide in the case of the three spirolactones that possessed partial agonist activity. Some of the discrepancy may have resulted from differences between mammalian and amphibian receptors; however, intrinsic agonist activity limits antagonist potency and thus may cause a divergence between binding and functional studies limited to antagonist activity alone. Binding affinity, although indicative of total activity, fails to distinguish agonist from antagonist potency.     

Immobilisation du complexe de nickel de l'histidine L(+) sur des supports macromoléculaires hydrophiles

N-(4-vinylbenzyl)-L -histidine ( 5 ) and the corresponding methyl ester were synthesized by reacting L -histidine or L -histidine methyl ester with 4-formylstyrene. N-(4-vinylbenzyl)-L -histidine methyl ester was radically copolymerized with various amounts of 2-hydroxyethyl methacrylate and ethylene dimethacrylate, leading to resins of different degrees of hydrophilicity and crosslinking. After saponification of the histidine ester groups, the resins were chelated with Ni²⁺ salt in order to be used as polymeric catalysts in enantioselective ester hydrolysis reactions.     

A New Versatile Radical Addition on α, ω‐Dimethacrylate Poly(ethylene oxide)

Summary: The synthesis of α,ω‐di(2‐methyl‐6‐pyrenyl‐2‐succinimidylhexanoic ester)poly(ethylene oxide) (Py(S)‐EO_n‐(S)Py) was obtained by a new radical reaction between 4‐pyrenylbutanoate N‐hydroxysuccinimidyl ester and α,ω‐dimethacrylate poly(ethylene oxide). The reason for the choice of such bulky groups is a possible application of this reaction to the synthesis of polyrotaxane from polypseudorotaxane. Two reaction media were examined, heterogeneous in water at room temperature using persulfate as initiator, or homogeneous in DMSO at 60 °C using AIBN as initiator. Structural characterization of the functionalization products was carried out by SEC, MALDI TOF, and NMR spectroscopies. It was shown that two types of α,ω‐dipyrenyl PEO could be obtained, one corresponding to the expected product (Py(S)‐EO_n‐(S)Py), the other corresponding to the same structure but without the succinimidyl substituent (Py(H)‐EO_n‐(H)Py). It was also shown that in the soluble system macrocycles could be formed and this last aspect has been assigned to intramolecular interactions existing between the pyrenyl groups. An interesting aspect of this synthesis is the possibility to find conditions giving high yields without crosslinking and fairly reduced amount of coupling.

     

Development of lipophilic anticancer agents for the treatment of brain tumors by the esterification of water-soluble chlorambucil

The lipophilic derivatives of the anticancer alkylating agent chlorambucil, chlorambucil-methyl, -isopropyl and -tertiary butyl esters, were synthesized and administered i.v. to anesthetized rats. Plasma and brain concentrations of these agents and of their active metabolites, chlorambucil and phenylacetic mustard, then were determined by high-performance liquid chromatography between 5 and 60 min. Whereas large amounts of chlorambucil-tertiary butyl ester entered and were maintained in brain, lower amounts of chlorambucil-isopropyl ester and no chlorambucil-methyl ester were found in brain. The comparative brain/plasma concentration-time integral ratios of the total active agents generated from chlorambucil-tertiary butyl, -isopropyl and -methyl esters were 0.85, 0.12 and 0.06, respectively, compared to a ratio of 0.02 for chlorambucil. In vitro alkylating activity of each ester was compared to that of equimolar chlorambucil, by reaction with 4-(p-nitrobenzyl)pyridine. Each ester possessed high intrinsic alkylating activity, equal to 38.4, 57.0 and 69.9% of chlorambucil activity, for the -tertiary butyl, -isopropyl and -methyl esters, respectively. Therefore each is an active antineoplastic agent irrespective of whether or not chlorambucil is regenerated. The rates of ester hydrolysis of these derivatives to chlorambucil were measured in fresh rat blood and in liver and brain homogenates at 37°C. Chlorambucil-methyl and -isopropyl esters were hydrolysed quickly within 30 s in blood and liver, whereas chlorambucil-tertiary butyl ester was more stable with half-lives of approximately 7 h and 2 h, respectively. All proved to be relatively stable in brain homogenate. Steric hindrance around the ester linkage of chlorambucil-tertiary butyl ester reduces its affinity to and rate of hydrolysis by plasma and liver esterases, and allows it to accumulate within the brain. Chlorambucil-tertiary butyl ester maintains high levels in brain despite rapidly declining plasma concentrations and, due to these favorable pharmacokinetics and to its intrinsic anticancer activity, it possesses promising characteristics for the treatment of malignant brain tumors.     

Microstructure of the sialic acid moiety of N-glycolylneuraminyllactosylceramide and the elucidation of Hanganutziu and Deicher (HD) antigenicity

The carboxyl (-COOH) group of heterophile Hanganutziu and Deicher (HD) antigen-active ganglioside (N-glycolylneuraminyllactosylceramide) was esterified (-CO2CH3) by methyl iodide and then reduced to a primary alcohol (-CH2OH) by sodium borohydride. The intact molecule (commonly known as HD3) as well as its two derivatives were tested for HD antigen potency using four human pathologic sera containing HD antibodies. The methyl ester derivative (1-methyl-HD3) gave the same inhibition potency as HD3, but the reduced HD3 gave poor inhibition (1/66) compared to the intact HD3. The results show that reduction of the carboxyl group diminishes the inhibitory potency of HD3. This suggests that although the N-glycolyl (-CH2OH) group of HD3 is the most important determinant for manifestation of HD antigenicity, it is likely that the antibody recognizes both the N-glycolyl and carboxyl groups together when they form a hydrogen bond (-CH2OH---OOC-), aided by their possible proximity, and that substitution of either group therefore reduces the reaction of HD3 with HD antibody dramatically.     

Preparation of cross-linked hyaluronic acid film using 2-chloro-1-methylpyridinium iodide or water-soluble 1-ethyl-(3,3-dimethylaminopropyl)carbodiimide

In order to obtain much slower biodegradable films, which are often required for biomedical applications, we have developed a series of studies on heterogeneous cross-linking of hyaluronic acid (HA) films by using 2-chloro-1-methylpyridinium iodide (CMPI) or 1-ethyl-(3,3- dimethylaminopropyl)carbodiimide (EDC) as cross-linking reagents. From the in vitro degradation rate, we found that EDC cross-linked HA films completely dissolved in PBS at 37 °C during the period of 4-6 days. However, CMPI cross-linked HA films showed only a low percentage of weight loss over 30 days. This phenomenon could be explained from the mechanism of reaction between carboxyl group of HA and EDC. The latter reacted with carboxyl group to form an unstable intermediate O-acylurea, which showed a relatively low reactivity and quickly rearranged to form a stable N-acylurea. Thus, most of the EDC-activated carboxyl groups in HA were chemically transferred into N-acylurea or left as unreactive O-acylurea, and only a few of cross-linking bonds were formed between HA. On the other hand, the intermediate obtained from the reaction between carboxyl group and CMPI showed a relatively high reactivity and reacted with the hydroxyl group of the same and/or different molecules of HA to form an inter- and intramolecular esterification. Apparently, CMPI cross-linked HA films have a much higher cross-linking density and constructed a more rigid three-dimensional network. Therefore, it produced HA films, which dramatically increased its enzymatic stability in aqueous solution of hyaluronidase. The obtained results from elemental analyses, FT-IR spectra and NMR spectra also indicate that acylurea groups were introduced into EDC-cross-linked HA films.     

Immobilization of aminoglycosidic aminocyclitols antibiotic onto soap-free poly(MMA-EA-AA) latex particles

Monodispersed soap-free poly(MMA-EA-AA) latex particles with surface carboxyl groups were synthesized by emulsion polymerization of methyl methacrylate (MMA), ethyl acrylate (EA) and acrylic acid (AA) in aqueous medium, and streptomycin sulfate (SMS) was immobilized onto these particles using three different methods. A model experiment was designed to test the feasibility of the reaction between the carboxyl groups of polymer and the amino groups of the medicine. The covalent coupling between the latex particles and the medicine was confirmed by XPS. Results showed that the medicine molecules were located on the particle surface after immobilization, and the coupling efficiency of SMS in pre-adsorption method was higher than that in direct method. The highest coupling efficiency of this medicine was achieved using the spacer-arm method. It was demonstrated that the immobilized medicine had similar antimicrobial activity as the free form using Escherichia coli as an evaluating organism.     

A Homozygous Nme7 Mutation Is Associated with Situs Inversus Totalis

We investigated the cause of situs inversus totalis (SIT) in two siblings from a consanguineous family. Genotyping and whole‐exome analysis revealed a homozygous change in NME7, resulting in deletion of an exon causing an in‐frame deletion of 34 amino acids located in the second NDK domain of the protein and segregated with the defective lateralization in the family. NME7 is an important developmental gene, and NME7 protein is a component of the γ‐tubulin ring complex. This mutation is predicted to affect the interaction of NME7 protein with this complex as it deletes the amino acids crucial for the binding. SIT associated with homozygous deletion in our patients is in line with Nme7^?/‐ mutant mice phenotypes consisting of congenital hydrocephalus and SIT, indicating a novel human laterality patterning role for NME7. Further cases are required to elaborate the full human phenotype associated with NME7 mutations.     

Telechelics 6. Oligostyrenes by radical polymerization

Styrene polymerized by AIBN as initiator results in telechelic α,ω-bis(1-cyano-1-methylethyl)oligostyrenes ( 1 ). The functionality, f_CN, was found to be 1,99 ± 0,05 in the molecular weight range up to 2000 and the M_w/M_n-values were found to be < 1,5 after complete consumption of the initiator. Using methyl 2,2′-azoisobutyrate as initiator, oligostyrenes with two ester endgroups were obtained. The conversion of styrene was limited by “dead end” conditions as described by Tobolsky. The efficiency was 0,9 under the preparative conditions used. The cyano endgroups were converted into amino endgroups by hydrogenation and into carboxyl endgroups by hydrolysis. Telechelic α,ω-bis(2-isocyanato-1,1-dimethylethyl)- and α,ω-bis(1-isocyanato-1-methylethyl)oligostyrenes 4 and 6 were obtained from the amino product 2 by the phosgene method and from the carboxyl product 3 by the Weinstock/Curtius reaction, respectively. Connecting these telechelic styrene blocks by a polyreaction results in polymers in the 20 000 – 50 000 molecular weight range.     

Protein carboxyl methylation and methyl ester turnover in density-fractionated human erythrocytes

A widely distributed methyltransferase modifies protein D-aspartyl and L-isoaspartyl residues which arise spontaneously as proteins age. Protein carboxyl methylation reactions were analyzed in human erythrocytes which had been separated on density gradients, a procedure which provides fractions enriched in older cells in the denser areas of the gradient. The total flux of methyl groups through the carboxyl methylation pathway was monitored by incubating cells from each fraction with L-[methyl-3H]methionine and measuring the formation of both protein [3H]methyl esters and [3H]methanol, derived from the hydrolysis of protein [3H]methyl esters in vivo. Cells isolated from denser areas of the gradient showed progressively higher rates of both protein carboxyl methylation and methanol production. In all cases, only 10-20% of the total methyl groups transferred were still present as intact protein [3H]methyl esters, consistent with the rapid hydrolysis of protein methyl esters in erythrocytes of all ages. The total flux of methyl groups through the carboxyl methylation pathway was approximately 3-fold higher in cells isolated from densest areas of the gradient compared to cells isolated from least dense areas of the gradient. Increases of a similar magnitude were observed in the numbers of both membrane protein carboxyl methyl esters and cytosolic protein carboxyl methyl esters. The only protein whose methylation was unchanged in denser cells was a 35,000 Da cytosolic protein. It has been proposed that protein carboxyl methyl esters are intermediates in either the repair or metabolism of structurally damaged proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Three-phase carboxylation reactions of poly(chloromethylstyrene) beads with sodium nitrite

The formation of carboxylic acid groups (carboxyl + ester groups) was found in a threephase reaction of crosslinked poly(chloromethylstyrene) beads with an excess amount of sodium nitrite in immiscible solvents of benzene and water with tetrabutylammonium bromide as a phase transfer catalyst (PTC), and the dependence of carboxylic acid yield on the experimental conditions was studied. The product contains hydroxyl groups and ester bonds besides carboxyl groups. It was tentatively considered that the carboxyl groups are produced by direct oxidation of chloromethylstyrene groups by nitrous anions. Nitrous anions do not only act as a nucleophile but also as an oxidative reagent in this case.     

Immobilization of aminoglycosidic aminocyclitols antibiotic onto soap-free poly(MMA-EA-AA) latex particles

Monodispersed soap-free poly(MMA-EA-AA) latex particles with surface carboxyl groups were synthesized by emulsion polymerization of methyl methacrylate (MMA), ethyl acrylate (EA) and acrylic acid (AA) in aqueous medium, and streptomycin sulfate (SMS) was immobilized onto these particles using three different methods. A model experiment was designed to test the feasibility of the reaction between the carboxyl groups of polymer and the amino groups of the medicine. The covalent coupling between the latex particles and the medicine was confirmed by XPS. Results showed that the medicine molecules were located on the particle surface after immobilization, and the coupling efficiency of SMS in pre-adsorption method was higher than that in direct method. The highest coupling efficiency of this medicine was achieved using the spacer-arm method. It was demonstrated that the immobilized medicine had similar antimicrobial activity as the free form using Escherichia coli as an evaluating organism.     

Preparation of cross-linked hyaluronic acid film using 2-chloro-1-methylpyridinium iodide or water-soluble 1-ethyl-(3,3-dimethylaminopropyl)carbodiimide

In order to obtain much slower biodegradable films, which are often required for biomedical applications, we have developed a series of studies on heterogeneous cross-linking of hyaluronic acid (HA) films by using 2-chloro-1-methylpyridinium iodide (CMPI) or 1-ethyl-(3,3-dimethylaminopropyl)carbodiimide (EDC) as cross-linking reagents. From the in vitro degradation rate, we found that EDC cross-linked HA films completely dissolved in PBS at 37 degrees C during the period of 4-6 days. However, CMPI cross-linked HA films showed only a low percentage of weight loss over 30 days. This phenomenon could be explained from the mechanism of reaction between carboxyl group of HA and EDC. The latter reacted with carboxyl group to form an unstable intermediate O-acylurea, which showed a relatively low reactivity and quickly rearranged to form a stable N-acylurea. Thus, most of the EDC-activated carboxyl groups in HA were chemically transferred into N-acylurea or left as unreactive O-acylurea, and only a few of cross-linking bonds were formed between HA. On the other hand, the intermediate obtained from the reaction between carboxyl group and CMPI showed a relatively high reactivity and reacted with the hydroxyl group of the same and/or different molecules of HA to form an inter- and intramolecular esterification. Apparently, CMPI cross-linked HA films have a much higher cross-linking density and constructed a more rigid three-dimensional network. Therefore, it produced HA films, which dramatically increased its enzymatic stability in aqueous solution of hyaluronidase. The obtained results from elemental analyses, FT-IR spectra and NMR spectra also indicate that acylurea groups were introduced into EDC-cross-linked HA films.     

miR-141-3p promotes proliferation and metastasis of nasopharyngeal carcinoma by targeting NME1

PurposeThis study aimed to investigate the expression and biological function of miR-141-3p in nasopharyngeal carcinoma (NPC) via targeting neoplasm metastasis 1 (NME1).Materials and methodsThe expression of miR-141-3p and NME1 in 5-8F, C666-1, CNE-1, CNE-2, 6-10B and NP69 nasopharyngeal epithelial cells were detected using real-time Polymerase Chain Reaction (real-time PCR) and western blot, respectively. Cell proliferation was detected using Cell Counting Kit-8 (CCK-8), and the metastasis was detected using Transwell. The binding of miR-141-3p to NME1 was detected by dual luciferase reporter gene detection system. The effects of miR-141-3p on tumor growth were also determined in vivo.ResultsThe results showed that the expression of miR-141-3p significantly increased in various tumor cell lines and the expression of NME1 was higher in NP69 cells and lower in 5-8F cells, which had significant negative correlation. Furthermore, the expression of NME1 was significantly reduced after transfection of miR-141-3p and miR-141-3p promoted cell proliferation and metastasis. The double luciferase reporter gene detection system confirmed that NME1 was the target gene of miR-141-3p. Knockout of NME1 promoted the proliferation and metastasis of NP69 or 6-10B cells and the activation of p-Akt, which were abrogated by miR-141-3p. In vivo, the tumor volumes and weights in the miR-141-3p group significantly increased followed by down-regulation of NME1 and activation of p-Akt.ConclusionsWe confirmed that miR-141-3p promotes the proliferation and metastasis of NPC by targeting NME1.     

Development of technology for linking photosensitizers to a model monoclonal antibody

A procedure is described whereby the photosensitizer, benzoporphyrin derivative monoacid ring A (BPD-MA) was covalently linked to a model monoclonal antibody in a manner which is reproducible, quantifiable, and retains both the biological activity of the antibody and the cytotoxicity of the photosensitizer. Preliminary steps involved the linkage of BPD-MA to a modified polyvinyl alcohol (PVA) backbone, followed by conjugation to the antibody using heterobifunctional linking technology. Briefly, polyvinyl alcohol (MW ca. 10,000) was modified with 2-fluoro-1-methyl pyridinium toluene-4-sulfonate and 1,6-hexanediamine to produce side chains containing free amino groups. The free carboxyl group of BPD-MA was utilized to conjugate photosensitizer molecules to modified PVA using a standard carbodiimide reaction. Final linkage of the PVA-BPD to a model monoclonal antibody involved further substitution of the carrier with 3-mercaptopropionic acid and carbodiimide to introduce 3-4 sulfhydryl residues per carrier molecule, and introduction of sulfo-m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester residues to the monoclonal (3-4 residues/molecule). Conjugation was effected by reaction of the two species at pH 5.5 for 18 h. Detailed methodology and tests for efficacy of the procedure are provided.     

Carboxyl-terminated poly(vinyl alcohol). Chain transfer of methyl propionate to vinyl acetate

Poly(vinyl acetate) was prepared at 60°C with benzoyl peroxide by using methyl[carbonyl-¹⁴C] propionate as chain transfer agent and solvent. The resulting polymer was hydrolysed to poly(vinyl alcohol) (PVAL) having carboxyl end groups and the average number of carboxyl groups per chain was unambiguously determined by comparing the activity of the polymer to that of the methyl propionate. An average density of 1,5 carboxyl groups per chain was found for poly(vinyl alcohol) prepared from poly(vinyl acetate) of DP 80 – 90. Other methods of end group analysis for carboxyl groups in PVAL are discussed.     

Die vielfältigen additionsprodukte von phenylen-, naphthylen-, biphenylylen- und methylendiphenylendimethacrylaten mit radikalen aus 2,2'-azoisobutyronitril

Reactions of phenylene, naphthylene, biphenylylene, and methylenediphenylene dimethacrylate ( 1–4 ) with 2,2'-azoisobutyronitrile (AIBN) were carried out without solvent, with butyllithium or sodium dihydronaphthylide in solution, and with AIBN in a great excess of refluxing benzene. Besides insoluble (crosslinked) polymers soluble polymers were obtained, among them cyclopolymers of type 9 are present as it was shown by hydrolysis, and subsequent reaction with diazomethane yielding poly(methyl methacrylate). Reactions in high dilution gave products 5 of suppressed polymerisation, of intramolecular head to tail and tail to tail addition of methacrylic acid ester groups of type 6 or 7 , respectively, cyclodimers 8 , and polymers 10 . It was shown that the yield of the different products is influenced by the concentration of the ester, by the mole ratio of ester and AIBN, and by the distance of the phenolic oxygens in the molecule.