Anticoagulant activity of dextran derivatives. Part I: Synthesis and characterization

Substituted dextrans bearing carboxymethyl and benzylsulphonate groups have been prepared. These materials exhibit an antithrombic activity correlated with the ratio of each substituent. The highest activity is obtained when the dextran derivative contains more than 50% of carboxylic acid groups and simultaneously about 15% of benzylsulphonate functions.     

Role of the ventromedial nucleus of the thalamus in motor behaviour—I. Effects of focal injections of drugs

An assortment of drugs was injected into one or both ventromedial nuclei of the thalamus, to see how these influenced stereotypy, locomotion and posture in spontaneously behaving and actively rotating rats. Unilateral intrathalamic muscimol promoted weak ipsiversive circling, while bilateral treatment gave catalepsy. Similar injections of 4-amino-hex-5-enoic acid, which inhibits γ-aminobutyrate metabolism, raised γ-aminobutyrate levels in the ventromedial nuclei more than three-fold yet had none of these behavioural effects. The indirectly acting γ-aminobutyrate agonists flurazepam and cis-1,3-aminocyclohexane car☐ylic acid had little effect on posture and locomotion and, like muscimol and 4-amino-hex-5-enoic acid, elicited only very weak stereotypies. Procaine behaved like the γ-aminobutyrate antagonist bicuculline, provoking vigorous locomotor hyperactivity and teeth chattering if given uni- or bilaterally. Pretreatment of one ventromedial nucleus with muscimol or 4-amino-hex-5-enoic acid, and to a lesser extent flurazepam or cis-1,3-aminocyclohexane car☐ylic acid, gave rise to pronounced ipsilateral asymmetries when combined with a large systemic dose of apomorphine. Contraversive rotations were initiated by unilateral stereotaxic injection of muscimol into the substantia nigra pars reticulata, or with apomorphine from the supersensitive striatum in unilaterally 6-hydroxydopamine lesioned rats. Drug treatments in the ipsilateral ventromedial nucleus showed a similar rank order of potency at inhibiting these circling behaviours, seemingly by reducing apomorphine-induced posture and muscimol-induced hypermotility. The suppression of circling by muscimol in these tests was highlighted by introducing the compound into the ventromedial nucleus at the height of circling activity. Both types of circling stimulus lost the capacity to increase locomotion, but still caused head turning and stereotypy in rats made cataleptic with bilateral ventromedial muscimol. Treating one ventromedial thalamus with muscimol greatly intensified any pre-existing posture directed towards that side, and vice versa.

These data suggest that the ventromedial nucleus is not involved with the expression of stereotyped behaviours, but can profoundly influence posture and locomotion, especially in the presence of some other motor stimulus. The recovery of circus movements in rats with impaired ventromedial nucleus function implies this nucleus is not essential for the execution of circling in these models.     

The enkephalins are amongst the peptides hydrolyzed by purified acetylcholinesterase

We have shown that purified acetylcholinesterase has the ability to hydrolyze a number of peptides including the physiologically occurring enkephalins. The enkephalins lost both the amino- and car☐yl-terminal amino acids, but several other peptides were not degraded. The enzyme was purified using an affinity Chromatographic matrix that recognised one component of the active centre that is specific to cholinesterases, the anionic-binding site. The acetylcholinesterase was extracted from four tissues of diverse origin to minimise the risk of co-purifying a peptidase. The enzyme was essentially homogeneous on polyacrylamide gels, and there was only one protein that bound diisopropylfluorophosphate in the samples. The peptidase activity was not affected by the aminopeptidase inhibitor puromycin, but it was inhibited by acetylcholine at concentrations that also reduced the esterase activity. It was concluded that acetylcholinesterase also has the capacity for a novel type of hydrolysis of peptide bonds.

The ability of acetylcholinesterase to hydrolyse naturally occurring compounds of different chemical nature, like esters and peptides, may help explain the long-standing puzzle of why the enzyme is more widely distributed than acetylcholine, once thought to be its sole natural substrate. The localization of the enzyme probably more accurately reflects the distribution of all its substrates, although their identity remains to be determined.     

New heparin-like insoluble materials: Part II

Heparin-like insoluble materials were prepared by reacting different α-amino acids with cross-linked chlorosulfonated polystyrene. The amount of thrombin inactivated by these resins, when suspended in plasma, is linearly dependent upon the polymer content for all the materials studied and upon the thrombin concentrations examined. The anticoagulant potency of each resin can be expressed as the activity of the sulfonate groups added to the activity of the α-amino acid sulfamide groups linked to polystyrene. The activity coefficients of substituting groups increase according to the following sequence: (1) −SO₂ But NH₂; (2) −SO ₃ ⁻ , −SO₂ Ala, −SO₂ Phe; (3) −SO₂ Met, −SO₂ OH Pro, −SO₂ Pro, −SO₂ Thr, −SO₂ Z Lys; (4) −SO₂ Glu.     

Anticoagulant activity of amino acid modified polystyrene resins: influence of the carboxylic acid function

In previous papers, we have described the preparation and heparin-like properties of insoluble modified polystyrene resins. We now report results obtained with amino acid sulphamide resins that are virtually devoid of sulphonate groups and with resins bearing both sulphonate groups as well as amino acid sulphamides with spacers of various lengths. The absence of sulphonate groups does not affect the anticoagulant activity of the former type of resin. The biologic activity of the latter type of resin is dependent upon the length of the spacer between the amino and carboxylic acid functions. Maximal anticoagulant potency is attained with a spacer consisting of three methylene groups. Biologic activity is reduced with spacers that are less than or greater than this critical size.     

Heparin-like activity of insoluble sulphonated polystyrene resins. Part I: Influence of the surface density, nature and binding of substituted anionic groups

It was previously demonstrated that copolystyrene (sulphonate-amino acid sulphamide) resins possessed an anticoagulant heparin-like activity in the presence of blood plasma. Taking into account the variable surfaces of swollen resins developed by these dry resins, it is now shown that the antithrombic activity of crosslinked sulphonated polystyrene is linearly dependent on the surface density of the sulphonate groups. This fact implies that the presence of such isolated groups is sufficient to obtain a catalytic site for increasing the rate of inactivation of thrombin by plasmatic proteins. It is also shown that replacing sulphonate groups either by directly backbone-bonded carboxylate groups or by methionine linked by amide bonds to polystyrene backbone is not sufficient to endow the resulting resins with a significant anticoagulant activity.     

Heparin-like activity of insoluble sulphonated polystyrene resins. Part III: Binding of dicarboxylic amino acids

It has been demonstrated previously that polystyrene sulphonate possesses anticoagulant properties and that the binding of some amino acids could enhance the heparin-like properties of such resins. These properties depend on the surface density of the active groups, the nature and binding of the group and on the net change borne by the polymer. In this paper, we describe the preparation of copolystyrene (sulphonate-dicarboxylic amino acid sulphamide) resins. By measuring their antithrombotic-surface-activity, we demonstrate that the activity developed by each carboxyl group is at least roughly the same as the activity of one sulphonate group, except in the case of aspartic acid sulphamide resin for which a cooperative effect is shown. The anticoagulant properties of resins bearing phosphonate or monocarboxylic amino acid sulphamides are also examined.     

血红素加氧酶在大肠杆菌中稳定表达的氨基酸范围的研究

目的　确定在原核细胞中呈可溶性稳定表达但不改变活性的血红素加氧酶氨基酸范围。方法　应用PCR技术构建了鼠血红素加氧酶 1(RHO 1)和人血红素加氧酶 2 (HHO 2 )的N端和C端部分氨基酸缺失的变异型RHO 1和HHO 2 ,将它们在大肠杆菌中表达并纯化后通过光谱扫描法测定变异型酶与野生型酶的催化活性 ,确定RHO 1和HHO 2中的N端和C端缺失不影响其可溶性表达及催化活性的氨基酸范围。结果　△N8RHO 1(N端缺失 8个氨基酸的RHO 1)和△C70RHO 1在大肠杆菌中表达为可溶性蛋白且催化活性与RHO 1野生型相同 ,△N2 0HHO 2和△C77HHO 2在大肠杆菌中也表达为可溶性蛋白且催化活性与HHO 2野生型相同 ,而△N9RHO 1和△C75RHO 1在大肠杆菌中表达为包涵体。结论　RHO 1和HHO 2的N端和C端部分氨基酸缺失的突变型酶对其催化功能没有影响 ,并可在大肠杆菌中稳定可溶性表达。     

Hydroxyl-modified epoxy resins: Some technical and analytical aspects

The modification of epoxy resins by reactions involving their hydroxyl groups is described. For example, reactions with enol ethers, acrylonitrile, ethyl acetoacetate and certain other carboxylic esters, or triethyl orthoformate, gave products which had reduced reactivities towards various hardeners, and which therefore gave lower peak temperatures on cure and/or longer usable lives. Suitable modification of epoxy resins with various other reagents(certain acid anhydrides, epichlorohydrin, or N-hydroxymethylacrylamide) introduces additional useful functional groups. The uses of a diisocyanate and of α-naphthyl isocyanate to modify epoxy resins or their mixtures prior to GPC analysis are also mentioned.     

Cellular interactions and degradation of aliphatic poly(ester amide)s derived from glycine and/or 4-amino butyric acid

A series of poly(ester amide)s derived from amino acid (glycine or 4-amino butyric acid), diol (1,6-hexanediol or 1,4-butanediol) and sebacoyl chloride were prepared by interfacial polymerization. FT-IR analysis indicated that for poly(ester amide)s derived from glycine, only amide-amide hydrogen bonds and hydrogen-bonded C=O ester groups were established, whereas the poly(ester amide)s derived from 4-amino butyric acid contained amide-amide hydrogen bonds and amide-ester hydrogen bonds, including NH groups and C=O ester groups in free state. The biodegradability was estimated by weight residue of poly(ester amide) films in pH 6 buffer solution with papain at 37 degrees C. The poly(ester amide) films derived from glycine demonstrated significantly improved degradability compared to the poly(ester amide) films derived from 4-amino butyric acid. This difference of degradation rate could be explained by the bonding state in C=O ester groups. The cellular interaction of the poly(ester amide)s was studied by measuring the proliferation of human dermal fibroblasts on the polymer films. The cells proliferated significantly faster on poly(ester amide) films derived from 4-amino butyric acid than on poly(ester amide) films derived from glycine. These results suggest that the poly(ester amide) prepared in this study may serve as a potential cell-compatible biomedical material.     

Reusable heterogeneous catalysts of α-amino acid racemization, 1. Immobilization of 4-hydroxy-3-formylbenzenesulfonic acid (5-sulfosalicylaldehyde) on strongly basic anion exchangers

Strongly basic anion-exchange resins in OH^? form, partially neutralized with 4-hydroxy-3-formylbenzenesulfonic acid (5-sulfosalicylaldehyde) (SSA) could be shown to catalyse the racemization of neutral and basic optically active α-amino acids in the presence of copper (II) ions at moderate temperatures (25-45 °C). Under these conditions (S)-asparagine is racemized without hydrolysis of its amide groups. The heterogeneous racemization of (S)-alanine is accelerated by a factor of 880 as compared to a homogeneous reaction. The efficiency of heterogeneous catalysis depends on (1) the extent of neutralization of anion exchanger with SSA, (2) the type of the resin used and (3) the quantity of Cu(II) ions in the reaction mixture. The experimental data are in accordance with the fact that heterogeneous racemization of amino acids proceeds via the formation of a reactive copper(II)-Schiff's base complex. The heterogeneous catalysts can be recycled. The anion exchangers can be regenerated and, if necessary, re-used.     

cis-Dichlorodiammineplatinum alters GABAergic structures in the immature rat cerebellum

It has been reported that injection of the antitumoral drug cis-dichlorodiammineplatinum at 10 days of life affects cerebellar development in rats. After a single dose of 5 μg/g of body weight, the formation of granule cells is decreased and the maturation of postmitotic neurons is slowed down. A substantial time after treatment, reduced cell packing density of the internal granule layer and atrophy of the molecular layer can be observed. In addition, there is degeneration of some Purkinje cells and Golgi neurons. In spite of all these alterations, the regular architecture of the cerebellar folia is retained in many places. In the present study, we used immunocytochemistry with an immune serum raised against glutamic acid decar☐ylase to further characterize the cis-dichlorodiammineplatinum-induced alterations of GABAergic neurons. The aim was to examine cerebellar development and to test for factors controlling the settling of GABAergic circuits. At all post-treatment intervals, most of the Purkinje and Golgi neurons and molecular layer interneurons showed stronger anti-glutamic acid decar☐ylase immunoreactivity than in controls; this may have been due to altered fixation because of cis-dichlorodiammineplatinum-induced damages to the blood vessels; but could also reflect cellular retention of the enzyme, maybe due to cis-dichlorodiammineplatinum-induced damage of the microtubular apparatus.

After seven days, large roundish immunoreactive varicosities were present in the molecular layer adjacent to the Purkinje cell dendritic poles. These varicosities, which were not observed in control animals, may be terminals of Purkinje cell axon recurrent collaterals contributing to the supraganglionic plexus, whose abnormal development would compensate for the reduced inhibitory inputs from inhibitory interneurons and/or Purkinje cells, which degenerated at early post-treatment intervals. At later post-treatment intervals (15 and 21 days), there were also alterations in the pericellular basket at the Purkinje cell axon hillock, which was poorly developed in or absent from the majority of cells. The finding was confirmed by morphological observation of basket cells in Golgi-Cox preparation and immunocytochemistry with an antibody raised against 200,000 mol. wt phosphorylated neurofilaments.

It is concluded that early changes in anti-glutamic acid decar☐ylase immunoreactivity of neurons may be due to a direct interference of the drug with the cellular metabolic pathways. The late anomalies in the anti-glutamic acid decar☐ylase immunoreactivity appear to be secondary to changes in the tissue cytoarchitecture rather than being primary cis-dichlorodiammineplatinum-induced lesions of the cells.     

Vinyl polymerization by metal complexes. VI. On the initiation mechanism of vinyl polymerization by the α-amino acid ester/copper(II) system

Polymerization of acrylonitrile and methyl methacrylate was studied in the presence of an α-amino acid ester and copper(II) ion in dimethyl sulfoxide solution. The initiation mechanism of the polymerization by the α-amino acid ester/copper(II) system was refered to the complex forming properties of the system, confirmed by spectroscopic measurements. The polymerization of both monomers was influenced by the kind of the α-alkyl group of the α-amino acid esters, but it was not affected by the kind of the ester groups. The carboxylic group of the esters was found to affect only the polymerization of methyl methacrylate. Based on the results, the character of the free radicals was discussed.     

Anticoagulant hydrogels derived from crosslinked dextran. Part II: Mechanism of thrombin inactivation

Sephadex derivatives bearing carboxymethyl, sulphonated benzylamine and amino acid groups exhibit heparin-like behaviour as demonstrated by the kinetic study of the thrombin inactivation in the presence of antithrombin III. Furthermore, whatever the chemical composition of these hydrogels, the diffusion coefficient of thrombin remained approximately constant and could not be connected with the variation of the antithrombin activity of the resins. Hence, in the heparin-like mechanism, the diffusion rate of thrombin inside the beads of hydrogels was not the limiting step. In fact, the swelling ratio (varying according to the chemical composition of these biomaterials) was involved in the anticoagulant properties of the resins.     

Synthesis and Characteristics of Biodegradable Epoxy–Polyester Resins Cured with Glutaric Anhydride

Biodegradable epoxy–polyester resins were synthesized in two steps: (1) the synthesis of the polyesters with allyl pendant groups, and (2) the epoxidation of the allyl groups in the polyesters. Polyesters with allyl pendant groups were synthesized by the melt copolymerization of succinic anhydride (SA) and allyl glycidyl ether (AGE) in the presence of benzyltrimethylammonium chloride (BTMAC) as the catalyst. The functionality of some polyesters was reduced by replacing a part of AGE with butyl glycidyl ether (BGE). The epoxidation of the polyesters obtained was carried out using m‐chloroperbenzoic acid (MCPBA). The multifunctional epoxy–polyester resins were cured with different amounts of glutaric anhydride (GA). The course of the curing process was monitored using DSC method. The following parameters of the process were determined: heat of curing, onset, maximal and end temperatures. Glass transition temperatures (T_g s) of the cured resins were determined by dynamic mechanical thermal analysis (DMTA). The influence of the composition of the initial resins and amount of GA on the properties of the cured products, such as density, hardness, T_g values, swelling and water sorption, were examined. Epoxy–polyester resins were subjected to the test of accelerated hydrolytic degradation in an aqueous phosphate buffer of pH = 7.4 at 70°C.     

Effect of Carbamylation of Lysine ε-Amino Groups on the Activity of Blood Group Specific Glycoproteins

Carbamylation of ε-amino groups of lysine of human blood group MM glycoprotein, some of its precursors and the blood group A B antigens gave products with 41% - 91%. ε-amino group substitution. Even the most extensive carbamylation led to only marginal changes in the circular dichroic (CD) spectra of these substances and none in sedimentation coefficients studied. Nevertheless, carbamylation resulted in either increased or unchanged or decreased inhibitory activity of all blood group antigens tested depending solely on the source of the hemagglutinin used. Carbamylation of ε-amino groups of these blood group glycoproteins therefore leads to minor conformational changes, not involved with the primary blood group specificity, which is recognized by a large proportion but not by all corresponding antibodies and lectins.     

Synthesis and aqueous solution behavior of water‐soluble polyurethane (IPDI‐PPG‐DMPA) resin

Two types of polyurethane (PU) resins were synthesized by polyaddition of isophorone diisocyanate (IPDI), poly (propylene glycols: PPG‐750 and PPG‐2000), and 2,2‐bis(hydroxymethyl)propionic acid (DMPA). Chemical structure of PU was confirmed by ¹H and ¹³C NMR analysis. The PU resins were found to be water‐dispersible or soluble by neutralization of carboxylic acid groups in DMPA. Aggregate formation behavior of the PU and solubilizing ability for hydrophobic probe (pyrene) were investigated at 100% neutralization. Average aggregate sizes of the PU resins measured by photon correlation spectroscopy showed large values (ca. 25–120 nm). The aggregate sizes were strongly affected by the concentration of the resin and total ionic strength in the aqueous phase. The PU resins showed superior solubilizing ability for pyrene in comparison with that of conventional short chain surfactant. Additionally, critical micelle concentration (CMC) of the PU resins obtained from pyrene fluorescence method showed extremely low values (< 10^–3 mol/dm³ water) and their aggregate numbers were 7.2 and 6.6 for PUR‐750 and PUR‐2000, respectively.     

Modifizierte ionenaustauscherharze — synthese und eigenschaften, 1. Sulfomethylierte styrol-divinylbenzol-harze

Sulfomethylated resins are prepared by polymer analogous reactions, starting from macroporous poly(styrene-co-divinylbenzene) matrices. Different reaction paths are discussed and used in the synthesis. Sulfomethylation can be achieved by reaction of a chloromethylated resin with dimethyl sulfide and sodium sulfonate or alternatively by oxidation of polymer-bound thiol groups. Both methods give high conversions as shown by IR spectra and titration of the sulfonic acid groups. Poly[1-(4-hydroxysulfomethylphenyl)ethylene] (3) is obtained by reaction of poly[1-(4-hydroxyphenyl)ethylene] ( 2 ) resin with formaldehyde/sodium sulfonate. The thermal stability, catalytic activity, and ion exchange equilibria of the sulfomethylated resin are investigated.     

Aminolysis of α-hydroxy acid esters with α-amino acid salts; first step in the synthesis of optically active 2,5-morpholinediones

A process for preparing optically active 2,5-morpholinediones 1 is described starting from optically active α-amino acids and optically active α-hydroxy esters. The process involves three steps: In the first step the sodium salt of an α-amino acid is condensed with an α-hydroxy ester to yield an optically active N-(α-hydroxy acyl)-α-amino acid 4 . In the second step the hydroxy acid obtained is subjected to esterification with ethanol. In the third step the N-(α-hydroxy acyl)-α-amino acid ethyl ester 5 is subjected to cyclization with an acidic catalyst. For some examples the cyclization was also performed directly from the hydroxy acid 4 . Morpholinediones 1 are useful as precursors for the preparation of poly(depsipeptides) 2 and copolymers containing depsipeptide repeating units.