High Molecular Weight Derivatives Research Articles

Discovery and overproduction of novel highly bioactive pamamycins through transcriptional engineering of the biosynthetic gene cluster

BackgroundPamamycins are a family of highly bioactive macrodiolide polyketides produced by Streptomyces alboniger as a complex mixture of derivatives with molecular weights ranging from 579 to 705 Daltons. The large derivatives are produced as a minor fraction, which has prevented their isolation and thus studies of chemical and biological properties.ResultsHerein, we describe the transcriptional engineering of the pamamycin biosynthetic gene cluster (pam BGC), which resulted in the shift in production profile toward high molecular weight derivatives. The pam BGC library was constructed by inserting randomized promoter sequences in front of key biosynthetic operons. The library was expressed in Streptomyces albus strain with improved resistance to pamamycins to overcome sensitivity-related host limitations. Clones with modified pamamycin profiles were selected and the properties of engineered pam BGC were studied in detail. The production level and composition of the mixture of pamamycins was found to depend on balance in expression of the corresponding biosynthetic genes. This approach enabled the isolation of known pamamycins and the discovery of three novel derivatives with molecular weights of 663 Da and higher. One of them, homopamamycin 677A, is the largest described representative of this family of natural products with an elucidated structure. The new pamamycin 663A shows extraordinary activity (IC50 2 nM) against hepatocyte cancer cells as well as strong activity (in the one-digit micromolar range) against a range of Gram-positive pathogenic bacteria.ConclusionBy employing transcriptional gene cluster refactoring, we not only enhanced the production of known pamamycins but also discovered novel derivatives exhibiting promising biological activities. This approach has the potential for broader application in various biosynthetic gene clusters, creating a sustainable supply and discovery platform for bioactive natural products.

Cyclic desorption based efficacy of polyvinyl alcohol-chitosan variant resins for multi heavy-metal removal

The simultaneous removal of Cu, Pb and Fe from water bodies has been targeted in this work with polyvinyl alcohol (PVA) and chitosan (low, medium, and high molecular weight) derivative and with cyclic desorption efficacy target. For a varied range of adsorbent loading (0.2–2 g L−1), initial concentration (187.7–563.1 mg L−1 for Cu, 5.2–15.6 mg L−1 for Pb, and 61.85–185.55 mg L−1 for Fe), and resin contact time (5 to 720 min), batch adsorption-desorption studies were conducted. After first adsorption-desorption cycle, the optimum absorption capacity was 6.85 mg g−1 for Pb, 243.90 mg g−1 for Cu, and 87.72 mg g−1 for Fe for the high molecular weight chitosan grafted polyvinyl alcohol resin (HCSPVA). The alternate kinetic and equilibrium models were analyzed along with the interaction mechanism between metal ions and functional groups. The cyclic desorption studies were carried out with simple eluent systems such as HCl, HNO3, H2SO4, KOH, and NaOH. The experiments revealed that the HCSPVA derivative has been an impressive, reusable, and effective sorbent for the mitigation of Pb, Fe, and Cu in complex wastewater systems. This is due to its easy synthesis, excellent adsorption capacity, quick sorption rate, and remarkable regeneration capabilities.

Synthesis of High Molecular Weight Stereo-Di-Block Copolymers Driven by a Co-Initiator Free Catalyst.

Stereo-diblock copolymers of high molecular weight polylactide (PLA) were synthetized by the one pot-sequential addition method assisted by a heteroscorpionate catalyst without the need of a co-initiator. The alkyl zinc organometallic heteroscorpionate derivative (Zn(Et)(κ3-bpzteH)] (bpzteH = 2,2-bis(3,5-dimethylpyrazol-1-yl)-1-para-tolylethoxide) proved to assist in the mechanism of reaction following a coordination-insertion process. Kinetic studies along with the linear correlation between monomer and number average molecular weight (Mn) conversion, and the narrow polydispersities supported the truly living polymerization character of the initiator, whereas matrix-assisted laser desorption/Ionization-time of flight (MALDI-TOF) studies showed a very low order of transesterification. The high stereo-control attained for the afforded high molecular weight derivatives was revealed by homonuclear decoupled 1H NMR spectra and polarimetry measurements. The nanostructure of the PLA derivatives was studied by both wide-angle X-ray scattering (WAXS) and differential scanning calorimetry (DSC) and the stereocomplex phase of the PLA stereo-diblock copolymers was successfully identified.

Antibacterial, Antibiofilm, and Antiadhesive Properties of Different Quaternized Chitosan Derivatives.

In the era of antimicrobial resistance, the identification of new antimicrobials is a research priority at the global level. In this regard, the attention towards functional antimicrobial polymers, with biomedical/pharmaceutical grade, and exerting anti-infective properties has recently grown. The aim of this study was to evaluate the antibacterial, antibiofilm, and antiadhesive properties of a number of quaternized chitosan derivatives that have displayed significant muco-adhesive properties and wound healing promotion features in previous studies. Low (QAL) and high (QAH) molecular weight quaternized chitosan derivatives were synthetized and further modified with thiol moieties or pendant cyclodextrin, and their antibacterial activity evaluated as minimal inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC). The ability of the derivatives to prevent biofilm formation was assessed by crystal violet staining. Both QAL and QAH derivatives exerted a bactericidal and/or inhibitory activity on the growth of P. aeruginosa and S. epidermidis. The same compounds also showed marked dose-dependent anti-biofilm activity. Furthermore, the high molecular weight derivative (QAH) was used to functionalize titanium plates. The successful functionalization, demonstrated by electron microscopy, was able to partially inhibit the adhesion of S. epidermidis at 6 h of incubation. The shown ability of the chitosan derivatives tested to both inhibit bacterial growth and/or biofilm formation of clinically relevant bacterial species reveals their potential as multifunctional molecules against bacterial infections.

Glucuronoarabinoxylan extracted by treatment with endoxylanase from different zones of growing maize root

Glucuronoarabinoxylan is a key tethering glucan in the primary cell wall of cereals. Glucuronoarabinoxylan was extracted from different zones of maize (Zea mays L.) roots using endoxylanase that specifically cleaves β-(1,4)-glycoside bond between two consequent unsubstituted xylose residues. Changes in polysaccharide structure during elongation growth were characterized. Glucuronoarabinoxylan extractable after the endoxylanase treatment consisted of high molecular weight (30-400 kDa) and low molecular weight (<10 kDa) fractions. The presence of high molecular weight derivatives indicated that part of the natural glucuronoarabinoxylan is not digestible by the endoxylanase. This could be due to the revealed peculiar structural features, such as high level of substitution of xylose, absence of unsubstituted xylose residues existing in sequence, and significant degree of acetylation. In maize root meristem the indigestible fraction was 98% of the total extracted glucuronoarabinoxylan. This portion decreases to 47% during elongation. Also, the average molecular weight of indigestible glucuronoarabinoxylan reduced twofold. These changes in the ratio of glucuronoarabinoxylan fragments with different structure during root cell growth could reflect a transition of polysaccharide from its separating (highly substituted indigestible glucuronoarabinoxylan) form to that binding to cellulose microfibrils or other glucuronoarabinoxylan molecules and, hence, retarding growth.

Mitomycin C-dextran conjugate: a novel high molecular weight pro-drug of mitomycin C.

A high molecular weight derivative of mitomycin C (MMC), mitomycin C-dextran conjugate (MMC-D) has been synthesized and its biological and pharmacological properties investigated. MMC is released from MMC-D in vitro with a half-life of 24 h. After intraperitoneal injection of MMC-D, free MMC could be detected in plasma and urine of mouse for 5--8 h, while MMC administered as a free form was eliminated rapidly. After MMC-D, given to mice bearing Ehrlich ascites carcinoma or B16 melanoma there was a reduction in toxicitst that the high molecular weight MMC-dextran derivative is a kind of pro-drug which persists in the body giving a sustained release of free MMC thus significantly increasing the antitumour activity of the parent drug.

Perfluorooctane Sulfonate (PFOS) Precursors Can Be Metabolized Enantioselectively: Principle for a New PFOS Source Tracking Tool

Perfluorooctane sulfonate (PFOS) is the most prominent perfluoroalkyl substance found in the serum of humans and wildlife, yet the major routes by which exposure occurs are not clear. An important issue facing both the scientific and chemical regulatory communities is the extent to which PFOS concentrations in biota are attributable to direct exposure versus metabolism of PFOS-precursors (higher molecular weight derivatives that can be biotransformed to PFOS). Given that certain branched PFOS-precursors are chiral, we hypothesized that nonracemic proportions of PFOS isomers in biological samples could be used as a marker of significant exposure to PFOS-precursors. In this proof-of-principle study we examined the enantiomer-specific biotransformation of a high-purity model PFOS-precursor isomer: C(6)F(13)C*F(CF(3))SO(2)N(H)CH(2)(C(6)H(4))OCH(3) (named 1m-PreFOS hereafter, and whereby * indicates the chiral carbon center). A method for the enantiospecific separation of a compound with a long perfluoroalkyl chain and a chiral center was developed and applied to evaluate the enantioselectivity of 1m-PreFOS biotransformation in human liver microsomes. Gradient elution in reversed-phase mode on a Chiralpak IC column permitted the near-baseline separation of the two enantiomers (E1 and E2, nomenclature based on retention order) in 65 min. Microsome incubations demonstrated that E1 and E2 were metabolized at significantly different rates; k(E1) = 6.5(+/-0.3) x 10(-2) min(-1) (half-life = 10.6 min) and k(E2) = 5.2(+/-0.3) x 10(-2) min(-1) (half-life = 13.3 min), respectively. These results suggest that tracking of PFOS exposure sources by enantiomeric fractionation is feasible, and that new analytical methods for the enantioselective analysis of PFOS isomers in human and environmental samples should be developed.

A new source of Proanthocyanidins with antiarthritic activity: Purple marshlocks (Comarum palustre L.) rhizome and roots

Purple marshlocks ( Comarum palustre ) is a wellknown and promising medicinal plant that is widely used in traditional medicine in the form of tinctures, decoctions, and extracts of rhizomes. It is used as a wound-healing and analgetic remedy for treatment of gastralgias, bronchitis, tuberculosis, and especially often for treatment of various diseases of joints. The commercially available dietary supplement Sabel’nikEvalar, which is manufactured in the form of tincture, pills, and cream, is efficiently used in cases of increased load on the locomotor system as a general healthimproving supplement, in complex therapy of rheumatoid arthrites, metabolic dystrophic osteoarthroses, arthritis, spondyloarthrosis, and vertebral osteochondrosis [1]. The chemical composition of C. palustre is highly diverse and includes the polyphenol complex, essential oils, gums, organic and phenolcarboxylic acids and their derivatives, saponins, and tannins (primarily condensed); the latter are the dominant components and account for approximately 10% [2‐4]. Condensed tannins, as was shown for many plant species, are highmolecular-weight derivatives of proanthocyanidins [5]. It was established that proanthocyanidins possess a broad spectrum of pharmacological activity and exhibit antioxidative (they are the best of known antioxidants), gastro- and cardioprotective, antisclerotic, and antiinflammatory properties [6‐10]. Since searching for new sources of proanthocyanidins is a topical problem, the goal of this work was to study the content of these compounds in the polyphenol complex of C. palustre and thereby to assess the possibility of broadening the spectrum of application of this plant as a medicine. The study was performed with three series of C. palustre samples collected in natural sites. Tannins were quantitated by a modified Porter method [11], which is based on acid hydrolysis of oligomeric and polymeric proanthocyanidins to anthocyanidins in the presence of a catalyst ( Fe 3+ ions). Although this method is highly sensitive, changes in the ratio of reagents and temperature conditions significantly decrease the yield of the colored reaction product and the measurement sensitivity [11]. When selecting the optimal conditions of extraction of total proanthocyanidins from various organs of C. palustre , we studied the influence of several factors, including the extracting solvent type

Preparation of 1,3,5-thiadiazine-2-thione derivatives of chitosan and their potential antioxidant activity in vitro

Three new kinds of 1,3,5-thiadiazine-2-thione derivatives of chitosan with two different molecular weight (SATTCS1, SATTCS2, TITTCS1, TITTCS2, CITTCS1 and CITTCS2) have been prepared. Their structures were characterized by IR spectroscopy. The substitution degree of derivatives calculated by elemental analyses was 0.47, 0.42, 0.41, 0.38, 0.41 and 0.36, respectively. The result shows that substitution degree of derivatives was higher with lower molecular weight. The antioxidant activity was studied using an established system, such as hydroxyl radical scavenging, superoxide radical scavenging and reducing power. Antioxidant activity of the 1,3,5-thiadiazine-2-thione derivatives of chitosan were stronger than that of chitosans and antioxidant activity of low molecular weight derivatives were stronger than that of high molecular weight derivatives. It is a potential antioxidant in vitro.

Molecular mechanism of tamoxifen‐induced calcium influx in human platelets

Tamoxifen, a non steroidal estrogen antagonist, is the most widely used drug for treatment and prevention of breast cancer. However, tamoxifen can cause thrombosis by an unknown mechanism. Using fura-2 to measure intracellular calcium in freshly isolated human platelets, we found that tamoxifen and its major metabolite 4-hydroxy tamoxifen at 5–10 uM promote calcium influx into platelets. Tamoxifen also potentiates action of platelet agonists (thrombin, vasopressin, ADP) to raise calcium. Tamoxifen effect was not attenuated by estrogen receptor antagonist ICI 182780 or COX inhibitor aspirin, but was blocked by the PLC inhibitor U-73122. Tamoxifen-induced calcium elevation was blocked by the lipid-soluble antioxidant vitamin E, but was unaffected by water-soluble vitamin C and quercetin. Tamoxifen effect was also blocked by the NADPH inhibitor apocynin and by wortmannin, an inhibitor of PI3 kinase, an upstream effector of NADPH oxidase. High molecular weight derivatives of 4-hydroxy tamoxifen conjugated to poly-methacrylic acid also promoted calcium elevation in platelets. An anti-estrogenic drug raloxifene, which is structurally related to tamoxifen, does not affect platelet calcium entry. Therefore tamoxifen may affect membrane-bound PLC, NADPH oxidase and PI3 kinase, leading to the activation of store-operated calcium channels, calcium entry into platelets, platelet activation and aggregation, which may be an underlying mechanism of tamoxifen-induced thrombosis. (Supported by grants from CHRB of Virginia and Cancer Research and Prevention Foundation to YD and Army Breast Cancer Research Program to RW).

Formation of high-molecular-weight protein adducts by methyl docosahexaenoate peroxidation products

In the present study, the formation of modified proteins by methyl docosahexaenoate (DHA) peroxidation products in the presence of a metal-catalyzed oxidation system was investigated. Metal-catalyzed oxidation of mixtures containing bovine serum albumin (BSA) and DHA led to formation of two high molecular weight derivatives of BSA. One had a mass of 71.5 kDa as determined by two-dimensional electrophoresis, matrix assisted laser desorption and ionization mass spectrometer (MALDI MS) analysis. The other was estimated to be 93 kDa by SDS-PAGE electrophoresis. The exposure of BSA to DHA also led to the generation of carbonyl groups. Oxygen radical scavengers could inhibit these modifications induced by DHA peroxidation. Furthermore, there was little difference of the peptides mass fingerprinting between the two kinds of modified high-molecular-weight proteins. These results suggest that oxygen radicals formed during lipid peroxidation are involved in the formation of protein derivatives. Our study may be important in the understanding the specific role of docosahexaenoic acid in the formation of modified proteins during aging and its related diseases.

α-Synuclein and Parkin Contribute to the Assembly of Ubiquitin Lysine 63-linked Multiubiquitin Chains

Mutations in alpha-synuclein, Parkin, and UCH-L1 cause heritable forms of Parkinson disease. Unlike alpha-synuclein, for which no precise biochemical function has been elucidated, Parkin functions as a ubiquitin E3 ligase, and UCH-L1 is a deubiquitinating enzyme. The E3 ligase activity of Parkin in Parkinson disease is poorly understood and is further obscured by the fact that multiubiquitin chains can be formed through distinct types of linkages that regulate diverse cellular processes. For instance, ubiquitin lysine 48-linked multiubiquitin chains target substrates to the proteasome, whereas ubiquitin lysine 63-linked chains control ribosome function, protein sorting and trafficking, and endocytosis of membrane proteins. It is notable in this regard that ubiquitin lysine 63-linked chains promote the degradation of membrane proteins by the lysosome. Because both Parkin and alpha-synuclein can regulate the activity of the dopamine transporter, we investigated whether they influenced ubiquitin lysine 63-linked chain assembly. These studies revealed novel biochemical activities for both Parkin and alpha-synuclein. We determined that Parkin functions with UbcH13/Uev1a, a dimeric ubiquitin-conjugating enzyme, to assemble ubiquitin lysine 63-linked chains. Our results and the results of others indicate that Parkin can promote both lysine 48- and lysine 63-linked ubiquitin chains. alpha-Synuclein also stimulated the assembly of lysine 63-linked ubiquitin chains. Because UCH-L1, a ubiquitin hydrolase, was recently reported to form lysine 63-linked conjugates, it is evident that three proteins that are genetically linked to Parkinson disease can contribute to lysine 63 multiubiquitin chain formation.

In vitro Relaxometric and Luminescence Characterization of P792 (Gadomelitol, Vistarem®), an Efficient and Rapid Clearance Blood Pool MRI Contrast Agent

AbstractP792 (Gadomelitol, Vistarem®), a hydrophilic high molecular weight (MW = 6473 g/mol) derivative of DOTA‐Gd is a rapid clearance blood pool agent (RCBPA) characterized by high proton relaxivities r1 and r2 at 310 K in the current range of clinical imaging magnetic fields. Luminescence data of the EuIII complex, as expected for this type of DOTA derivative, agree with the presence of one water molecule in the first coordination sphere of the lanthanide ion. The stability of the water proton longitudinal relaxation rate in the presence of zinc(II) and phosphate ions showed that the transmetallation by ZnII ions is negligible. The temperature dependence of the water oxygen‐17 transverse relaxation rate gave a τM smaller than 100 ns at 310 K. No significant increase of longitudinal or transverse relaxation rates in serum or HSA solution was noticed, precluding association with high molecular weight blood components. The proton Nuclear Magnetic Relaxation Dispersion (NMRD) profile of the water solution showed a maximum of longitudinal relaxivity between 20 and 40 MHz (r1 ≈ 40 s–1 mM–1 at 310 K). Fitting of the proton NMRD curve by the classical outersphere and innersphere models, including or not additional second sphere water molecules, gave a rotational correlation time of approximately 2 to 3 ns and a large value of the electronic relaxation time at low field (τSO ≈ 500 ps). (© Wiley‐VCH Verlag GmbH &amp; Co. KGaA, 69451 Weinheim, Germany, 2005)

A monoclonal antibody specific to the granulocyte-derived elastase-fragment D species of human fibrinogen and fibrin: its application to the measurement of granulocyte-derived elastase digests in plasma

When granulocytes are stimulated under certain clinical conditions, elastase is released therefrom and digests fibrin(ogen) independently of the plasmin system, which may also be mobilized simultaneously. Thus, discrimination of these 2 systems becomes urgent for the diagnosis and treatment of the underlying diseases. Using as immunogen a 97-kd granulocyte-elastase digest of human fibrinogen, we raised an antibody IF-123 that specifically recognizes elastase digests of human fibrin(ogen). The 97-kd elastase fragment resembles plasmic fragment D(1), and the epitope of this antibody is located on the Aalpha (196-204) residue segment. This segment appears to be masked in fibrin(ogen) but exposed when the Aalpha Leu 204-Ile 205 peptide bond is cleaved by elastase. Cathepsin G concomitantly released from granulocytes failed to expose the epitope. By an enzyme immunoassay using IF-123 as the capture antibody, the elastase digests of fibrin(ogen) can be measured in plasma samples without interference by abundantly coexisting fibrinogen. Indeed, we found that the elastase digests were mostly elevated in patients with inflammation or malignant tumors, but remained in a normal range in patients with a benign gastrointestinal tract disease such as duodenal ulcer and polyps in the gallbladder or the colon. Like the plasmic D-dimer, the elastase digests predominantly consisted of the DD/E complex and DD/E-containing high-molecular weight derivatives apparently corresponding to the phase-3 plasmic digests of cross-linked fibrin. (Blood. 2000;95:1721-1728)

Microbial (1→3)- β- d-glucans, their preparation, physico-chemical characterization and immunomodulatory activity

(1→3)- β- d-glucans have the ability to stimulate the immune system and are classified as biological response modifiers (BRMs). Glucans from two technologically important species, baker's yeast Saccharomyces cerevisiae and filamentous fungus Aspergillus niger were isolated and characterized. Water-insoluble yeast glucan has a low branched structure with a ratio of the glucose units in the side chains and the backbone of 1:8. Using ultrasonic treatment and subsequent chemical derivatization, water-soluble derivatives (carboxymethyl and sulfoethyl) of yeast glucan were obtained with high yield. The glucan isolated from A. niger forms a complex with chitin and is relatively resistant to solubilization. The yield of its carboxymethylated derivative was only 30%. A method of determination of the relative ratio of α- and β-glycosidic linkages in the glucans using FTIR spectroscopy has been developed as well as a method for ultrasonic purification of the isolated glucans. Using ultrasonic treatment, glucan derivatives with decreased molecular weight (90–100 kDa) have been prepared, which show broader application possibilities in comparison with the initial high molecular-weight derivatives (300–600 kDa). It has been found that the derivatives prepared reveal high mitogenic and comitogenic activities, as well as radioprotective and antimutagenic effects.

Gas Chromatographic-Mass Spectrometric Identification and Quantitation of Benzyl Alcohol from Human Serum and Postmortem Blood after Derivatization with 4-Carbethoxy Hexafluorobutyryl Chloride: A Novel Derivative

Benzyl alcohol is commonly used as an antibacterial agent in a variety of pharmaceutical formulations. Several fatalities in neonates have been linked to benzyl alcohol poisoning. Most methods for measuring benzyl alcohol concentrations in serum utilize direct extraction followed by high performance liquid chromatography. We describe a novel derivatization of benzyl alcohol using 4-carbethoxyhexafluorobutyryl chloride after extraction from human plasma, and subsequent analysis by gas chromatography-mass spectrometry (GC/MS). The derivative was eluted at a significantly higher temperature and the method was free from interferences from more volatile components in serum and hemolyzed specimens. However, with postmortem specimens, we observed multiple peaks which were eluted at a very high temperature, long after derivatized benzyl alcohol and the internal standard. Therefore, baking the column at 310 degrees C is recommended after analysis of a postmortem specimen. Another advantage of this derivatization technique is the conversion of low molecular weight benzyl alcohol (MW 108) to a high molecular weight derivative (MW 358). The positive identification of benzyl alcohol can be easily achieved by observing a distinct molecular ion at m/z 358 as well as other characteristic ions at m/z 107 and 91. Quantitation of benzyl alcohol in human serum can easily be achieved by using 3,4-dimethylphenol as an internal standard. The within run and between run precisions (using serum standard of benzyl alcohol: 50 mg/L) were 2.2% (mean = 50.6, SD = 1.1 mg/L), and 6.9% (mean = 50.8, SD = 3.5 mg/L). The assay was linear for the serum benzyl alcohol concentrations of 5 mg/L to 200 mg/L and the detection limit was 1 mg/L. We observed no carry-over problem in our assay as when 2 microL ethyl acetate was injected into the GC/MS after analyzing serum specimens containing 200 mg/L of benzyl alcohol, we observed no peak for either benzyl alcohol or the internal standard in the total ion chromatogram.

Capillary zone electrophoresis in a hydrodynamically closed separation system with enhanced sample loadability

Some phenomena linked with capillary zone electrophoresis (CZE) performed in a hydrodynamically closed separation system with enhanced sample load capacity via the use of capillary tubes of larger I.D.s were studied. Calculations of the plate heights for varying amounts of the analytes loaded onto 50 and 300 μm I.D. columns under identical CZE separating conditions revealed that the analytical advantages of using columns of larger I.D. include significantly reduced contributions of electromigration dispersion. At higher concentrations of the carrier electrolytes this gain, however, can be partially lost due to increased thermal dispersive effects. Calculated resolutions for a varying ratio of a pair of the analytes loaded onto 50 and 300 μm I.D. columns favoured the use of the latter I.D. in situations when the ratio of the analytes was higher than ca. 102:1. CZE experiments were carried out in a 300 μm I.D. capillary tube made of fluorinated ethylene–propylene copolymer (FEP) with a porous cellophane membrane serving as a hydrodynamic barrier to prevent a flow of the solution in the separation compartment due to a pressure difference between the electrode vessels. Movement of the membrane was found to be a source of undesired flows in the separation compartment, which adversely affected both the reproducibilities of migration times of the analytes and their separation efficiencies. Mechanical support eliminated these problems. Dispersive phenomena associated with electroosmosis in the closed separation compartment were effectively suppressed by using high molecular weight derivatives of water soluble polymers (methylhydroxyethylcellulose and polyethyleneglycol) in the carrier electrolyte solutions. Examples from the separations of various groups of analytes (synthetic food colourants, some inorganic anions, alkali and alkaline earth metal cations and glycoforms of erythropoietin) are given to illustrate a practical utility of the studied CZE approach.

High Ethoxylated Alcohols with Narrow Distribution of Homologues

Homologue distributions of alcohol ethoxylates obtained with an unconventional calcium-based catalyst and NaOH as the conventional catalyst are studied in the range of average polyaddition degree from 8 to 15. The fractional compositions of those high molecular weight derivatives of dodecanol and other technical alcohols, Lial 125 and Radianol 1724, are determined as peak area percentages from HPLC chromatograms. Some physico-chemical characteristics of the products as well as by-product contents are also presented. It is shown that the differences in fractional contents between the broad and narrow homologue distributed equivalents remain very significant in that range of higher ethoxylation grades. Consequently, the influence of homologue distributions on some properties of the products can be expected in their practical applications.

D-dimer specific monoclonal antibodies react with fibrinogen aggregates

Human fibrinogen exposed to 46.5°C was subjected to gel permeation chromatography. The protein eluted in two distinct peaks. The first peak appeared in the void volume containing soluble fibrinogen aggregates, while the other peak represented monomeric fibrinogen. In contrast to the monomeric peak material, the aggregate fraction reacted with a panel of monoclonal antibodies specific for fragment D-dimer using an ELISA system. Edman degradation showed that both the aggregate and the monomeric fractions were devoid of soluble fibrin, and immunoblots of SDS-PAG electrophoretic profiles disclosed no sign of stabilized high molecular weight derivatives. We have previously shown that the aggregate fraction of similarly treated fibrinogen, in contrast to the monomeric fraction, stimulates the t-PA catalyzed conversion of plasminogen to plasmin and concomitantly exposes the sequences Aα-(148–160) and γ-(312–324) involved in t-PA stimulation. Our present and previous findings suggest that soluble fibrinogen aggregates possess a fibrin-like structure, and that fibrin or fibrinogen polymer formation is a prerequisite for the enhancing effect on t-PA-mediated plasminogen to plasmin conversion which is seen even with the polymers in the soluble state.

Regulatory subunits of cAMP-dependent protein kinases are degraded after conjugation to ubiquitin: a molecular mechanism underlying long-term synaptic plasticity.

In Aplysia, behavioral sensitization of defensive reflexes and the underlying presynaptic facilitation of sensory-to-motor neuron synapses lasts for several minutes (short term) or days to weeks (long term). Short-term sensitization has been explained by modulation of ion-channel function through cAMP-dependent protein phosphorylation. Long-term facilitation requires additional molecular changes including protein synthesis. A key event is the persistent activation of the cAMP-dependent protein kinase at baseline concentrations of cAMP. This activation is due to selective loss of regulatory (R) subunits of PKA without any change in catalytic (C) subunits. To understand the molecular mechanisms that produce the loss of R subunits in long-term facilitation, we investigated how R subunits are degraded in extracts of Aplysia nervous tissue and in rabbit reticulocyte lysates. Degradation of Aplysia R subunits requires ATP, ubiquitin, and a particulate component that appears to be the proteasome complex. Degradation is blocked by hemin, which causes the accumulation of high molecular weight derivatives of R subunits that are likely to be ubiquitin conjugates of R subunits and intermediates in the degradative pathway. We also show that vertebrate RI and RII subunits can be degraded through the ubiquitin pathway. We suggest that degradation is initiated by cAMP, which causes the holoenzyme to dissociate and, further, that the altered R-to-C ratio in Aplysia sensory neurons is maintained in long-term facilitation by newly synthesized proteins that help target R subunits for accelerated degradation.