Molecular characteristics of subfractions of laminarin

Pacific Ocean Institute of Bioorganic Chemistry, Far Eastern Scientific Center of the Russian Academy of Sciences, Vladivostok. Translated from Khimiya Prirodnykh Soedinenii, No. 2, pp. 296–298, March–April, 1994.     

Characterization of Hyaluronidase 4 Involved in the Catabolism of Chondroitin Sulfate

Hyaluronidases (HYALs) are endo-beta-N-acetylhexosaminidases that depolymerize not only hyaluronan but also chondroitin sulfate (CS) at the initial step of their catabolism. Although HYAL1 hydrolyzes both CS and HA, HYAL4 is a CS-specific endoglycosidase. The substrate specificity of HYAL4 and identification of amino acid residues required for its enzymatic activity have been reported. In this study, we characterized the properties of HYAL4 including the expression levels in various tissues, cellular localization, and effects of its overexpression on intracellular CS catabolism, using cultured cells as well as mouse tissues. Hyal4 mRNA and HYAL4 protein were demonstrated to be ubiquitously expressed in various organs in the mouse. HYAL4 protein was shown to be present both on cell surfaces as well as in lysosomes of rat skeletal muscle myoblasts, L6 cells. Overexpression of HYAL4 in Chinese hamster ovary cells decreased in the total amount of CS, suggesting its involvement in the cellular catabolism of CS. In conclusion, HYAL4 may be widely distributed and play various biological roles, including the intracellular depolymerization of CS.     

Aggregation and solubility behavior of asphaltenes and their subfractions

Asphaltenes from four different crude oils (Arab Heavy, B6, Canadon Seco, and Hondo) were fractionated in mixtures of heptane and toluene and analyzed chemically, by vapor pressure osmometry (VPO), and by small angle neutron scattering (SANS). Solubility profiles of the asphaltenes and their subfractions indicated strong cooperative asphaltene interactions of a particular subfraction that is polar and hydrogen bonding. This subfraction had lower H/C ratios and modestly higher N, V, Ni, and Fe contents than the less polar and more soluble subfraction of asphaltenes. VPO and SANS studies indicated that the less soluble subfractions formed aggregates that were considerably larger than the more soluble subfractions. In general, asphaltene aggregate size increased with decreasing solvent aromaticity up to the solubility limit, beyond which the aggregate size decreased with heptane addition. The presence of a low wavevector Q feature in the scattering curves at 25 degrees C indicated that the individual aggregates were flocculating; however, the intensity of the feature was diminished upon heating of the samples to 80 degrees C. The solubility mechanism for Canadon Seco asphaltenes, the largest aggregate formers, appears to be dominated by aromatic pi-bonding interactions due to their low H/C ratio and low nitrogen content. B6 and Hondo asphaltenes formed similar-sized aggregates in heptol and the solubility mechanism is most likely driven by polar interactions due to their relatively high H/C ratios and high nitrogen contents. Arab Heavy, the least polar asphaltene, had a H/C ratio similar to Canadon Seco but formed the smallest aggregates in heptol. The enhancement in polar and pi-bonding interactions for the less soluble subfraction indicated by elemental analysis is reflected by the aggregate size from SANS. The less soluble asphaltenes contribute the majority of species responsible for aggregation and likely cause many petroleum production problems such as pipeline deposition and water-in-oil emulsion stabilization.     

The determination of Se, Mg and Mn concentrations in leukocyte subfractions

This continuing study explores the feasibility of determination of elemental concentrations in a number of leukocyte subfractions. Establishing a reliable separation method using blood of normal subjects prior to the separation and elemental analysis of blood from heart bypass patients is the first step. In addition, three blood collection vessels were compared and assessed. Instrumental Neutron Activation Analysis (INAA) using short irradiations was the technique employed. Results indicated that determination of elemental concentration was possible, in some of the leukocyte subfractions although these are in relatively small amounts. These results highlight the need for further investigation. This revised version was published online in August 2006 with corrections to the Cover Date.     

Characterization of modified low density lipoprotein subfractions by capillary isotachophoresis

Oxidative modification of low density lipoproteins (LDLs) is an important pathogenetic factor in atherosclerosis. The various steps in oxidative modifications of LDL can be monitored using different methodologies with varying degrees of complexity. In this study, we propose capillary isotachophoresis (CITP) as a suitable tool to detect and measure the degree of oxidation of LDL. LDL was isolated from pooled plasma of healthy volunteers by sequential ultracentrifugation, and oxidation was performed in vitro as well as in cell culture experiments. Native LDL and oxidatively modified LDL were characterized by apo B-100 fluorescence and conjugated diene formation. Samples were separated by CITP combined with sudan black B staining. To underline the inherent advantages of this approach, CITP was compared with classical lipoprotein electrophoresis using agarose gel. We demonstrate the CITP method to be highly sensitive, as changes in peak area of the separated LDL subfractions were detected after only 2 h of oxidation. The leading LDL peaks increased, while the terminating LDL peaks decreased in parallel throughout the duration of oxidation. The LDL samples, oxidized for 4-24 h, also exhibited an increased migration velocity of the fractions. In summary, we present the first study investigating LDL-subfractions separated by CITP and the alterations of these LDL-subfractions after gradual in vitro oxidation and after oxidative modification by monocyte-derived macrophages and vascular smooth muscle cells.     

Quantitation of methanol formed in cell culture cytotoxicity assays and as a metabolite in microsome suspensions

Two methods have been used to isolate Hb F from red cells with low levels of Hb F (less than 2% FAD), namely an alkali denaturation procedure, as described by Tsuchiya et al. [Dokkyo J. Med. Sci., 10 (1983) 13], and anion-exchange chromatography. Analyses of these Hb F enriched hemoglobin solutions by high-performance liquid chromatography allowed quantitation of the different gamma chains in the Hb F. Although the data showed considerable variation, particularly for samples with low levels of Hb F, the final results obtained with the two approaches were comparable, suggesting that the much simpler and more economical alkali denaturation procedure can be used for this purpose.     

The Biosynthesis and Catabolism of the Maleic Anhydride Moiety of Stipitatonic Acid

A series of directed knockout experiments, combined with an in vitro assay of pathway components, has elucidated for the first time the chemical steps involved in the biosynthesis of the tropolone class of fungal maleic anhydrides. The pathway involves the stepwise oxidation of aldehyde and methyl carbon atoms to form a 1,2‐dicarboxylate. A hydrolase‐catalyzed interconversion of this and the corresponding maleic anhydride, followed by decarboxylation of the diacid leads to the pathway’s final product of stipitatic acid.     

Protein-protein interactions in complex cosolvent solutions.

The effects of various kosmotropic and chaotropic cosolvents and salts on the intermolecular interaction potential of positively charged lysozyme is evaluated at varying protein concentrations by using synchrotron small-angle X-ray scattering in combination with liquid-state theoretical approaches. The experimentally derived static structure factors S(Q) obtained without and with added cosolvents and salts are analysed with a statistical mechanical model based on the Derjaguin-Landau-Verwey-Overbeek (DLVO) potential, which accounts for repulsive and attractive interactions between the protein molecules. Different cosolvents and salts influence the interactions between protein molecules differently as a result of changes in the hydration level or solvation, in charge screening, specific adsorption of the additives at the protein surface, or increased hydrophobic interactions. Intermolecular interaction effects are significant above protein concentrations of 1 wt %, and with increasing protein concentration, the repulsive nature of the intermolecular pair potential V(r) increases markedly. Kosmotropic cosolvents like glycerol and sucrose exhibit strong concentration-dependent effects on the interaction potential, leading to an increase of repulsive forces between the protein molecules at low to medium high osmolyte concentrations. Addition of trifluoroethanol exhibits a multiphasic effect on V(r) when changing its concentration. Salts like sodium chloride and potassium sulfate exhibit strong concentration-dependent changes of the interaction potential due to charge screening of the positively charged protein molecules. Guanidinium chloride (GdmCl) at low concentrations exhibits a similar charge-screening effect, resulting in increased attractive interactions between the protein molecules. At higher GdmCl concentrations, V(r) becomes more repulsive in nature due to the presence of high concentrations of Gdm(+) ions binding to the protein molecules. Our findings also imply that in calculations of thermodynamic properties of proteins in solution and cosolvent mixtures, activity coefficients may not generally be neglected in the concentration range above 1 wt % protein.     

Anticancer Cytotoxic Activity of Bispidine Derivatives Associated with the Increasing Catabolism of Polyamines

Polyamine (PA) catabolism is often reduced in cancer cells. The activation of this metabolic pathway produces cytotoxic substances that might cause apoptosis in cancer cells. Chemical compounds able to restore the level of PA catabolism in tumors could become potential antineoplastic agents. The search for activators of PA catabolism among bicyclononan-9-ones is a promising strategy for drug development. The aim of the study was to evaluate the biological activity of new 3,7-diazabicyclo[3.3.1]nonan-9-one derivatives that have antiproliferative properties by accelerating PA catabolism. Eight bispidine derivatives were synthetized and demonstrated the ability to activate PA catabolism in regenerating rat liver homogenates. However, only three of them demonstrated a potent ability to decrease the viability of cancer cells in the MTT assay. Compounds 4c and 4e could induce apoptosis more effectively in cancer HepG2 cells rather than in normal WI-38 fibroblasts. The lead compound 4e could significantly enhance cancer cell death, but not the death of normal cells if PAs were added to the cell culture media. Thus, the bispidine derivative 4e 3-(3-methoxypropyl)-7-[3-(1H-piperazin-1-yl)ethyl]-3,7-diazabicyclo[3.3.1]nonane could become a potential anticancer drug substance whose mechanism relies on the induction of PA catabolism in cancer cells.     

First Chemical Synthesis of Three Natural Depsides Involved in Flavonol Catabolism and Related to Quercetinase Catalysis

We report here the first chemical synthesis of three depsides related to quercetinase‐catalyzed degradation of kaempferol, quercetin, and myricetin. The three depsides were constructed through the coupling of suitably protected phloroglucinol carboxylic acid and hydroxy‐perbenzylated, derivatives of gallic, protocatechuic, and 4‐hydroxy benzoic acids. The three synthesized target compounds proved to be identical to their natural counterparts, arising from quercetinase action on corresponding flavonols.     

Chlorophyll Breakdown in Senescent Banana Leaves: Catabolism Reprogrammed for Biosynthesis of Persistent Blue Fluorescent Tetrapyrroles

Chlorophyll breakdown is a visual phenomenon of leaf senescence and fruit ripening. It leads to the formation of colorless chlorophyll catabolites, a group of (chlorophyll‐derived bilin‐type) linear tetrapyrroles. Here, analysis and structure elucidation of the chlorophyll breakdown products in leaves of banana (Musa acuminata) is reported. In senescent leaves of this monocot all chlorophyll catabolites identified were hypermodified fluorescent chlorophyll catabolites (hmFCCs). Surprisingly, nonfluorescent chlorophyll catabolites (NCCs) were not found, the often abundant and apparently typical final chlorophyll breakdown products in senescent leaves. As a rule, FCCs exist only fleetingly, and they isomerize rapidly to NCCs in the senescent plant cell. Amazingly, in the leaves of banana plants, persistent hmFCCs were identified that accounted for about 80 % of the chlorophyll broken down, and yellow leaves of M. acuminata display a strong blue luminescence. The structures of eight hmFCCs from banana leaves were analyzed by spectroscopic means. The massive accumulation of the hmFCCs in banana leaves, and their functional group characteristics, indicate a chlorophyll breakdown path, the downstream transformations of which are entirely reprogrammed towards the generation of persistent and blue fluorescent FCCs. As expressed earlier in related studies, the present findings call for attention, as to still elusive biological roles of these linear tetrapyrroles.     

Photophysical processes in the NADH-alcohol dehydrogenase complex.

Fluorescent analysis showed the high sensitivity of the NADH-alcohol dehydrogenase complex to UV irradiation. The complex reacts to UV illumination with "photoresponse," a rapid decrease in NADH fluorescence intensity to some stationary level. No such decrease was found in the NADH solution without the protein. The maximum rate of photoresponse was observed at pH 8.0, i.e, the biochemical optimum for the enzyme. It is suggested that the main reason for the photoresponse is desorption of NADH from the protein due to vibrational and conformational relaxation after photoexcitation. The data obtained point to the possibility of controlling "dark" enzymatic processes with light.     

DNA-histidine complex formation in isoelectric histidine buffers.

The free solution electrophoretic mobility of two DNA molecules of different molecular masses, 18 base pairs and 2686 base pairs, has been measured in isoelectric histidine buffers with and without added low-molecular-mass electrolytes. Extensive DNA-histidine complex formation is observed in isoelectric histidine buffer, as evidenced by distortion and splitting of the peaks in the electropherograms. Peak distortion and splitting can be decreased or eliminated by adding low-molecular-mass neutral salts to the solution, suggesting that the DNA-histidine complexes are stabilized by electrostatic interactions. The ability of various neutral salts to disrupt the DNA-histidine complexes depends on the molecular mass of the DNA and the concentration and type of added salt.