Diosmetin alleviates neuropathic pain by regulating the Keap1/Nrf2/NF-κB signaling pathway.

BACKGROUND: Neuropathic pain, a chronic condition with a high incidence, imposes psychological burdens on both patients and society. It is urgent to improve pain management and develop new analgesic drugs. Traditional Chinese medicine has gained popularity as a method for pain relief. Diosmetin (Dio) is mainly found in Chinese herbal medicines with effective antioxidant, anti-cancer, and anti-inflammatory properties. There are few known mechanisms underlying the effectiveness of Dio in treating neuropathic pain. However, the complete understanding of its therapeutic effect is missing. PURPOSE: This study aimed to evaluate Dio's therapeutic effects on neuropathic pain models and determine its possible mechanism of action. We hypothesized that Dio may activate antioxidants and reduce inflammation, inhibit the activation of Kelch-like epichlorohydrin-associated protein 1 (Keap1) and nuclear factor-k-gene binding (NF-κB), promote the metastasis of nuclear factor erythroid 2-related factor 2 (Nrf2) and the expression of heme oxygenase 1 (HO-1), thus alleviating the neuropathic pain caused by spinal nerve ligation. METHODS: Chronic nociceptive pain mouse models were established in vivo by L4 spinal nerve ligation (SNL). Different dosages of Dio (10, 50, 100 mg/kg) were intragastrically administered daily from the third day after the establishment of the SNL model. Allodynia, caused by mechanical stimuli, and hyperalgesia, caused by heat, were assessed using the paw withdrawal response frequency (PWF) and paw withdrawal latency (PWL), respectively. Cold allodynia were assessd by acetone test. RT-PCR was used to detect the content of interleukin-(IL)- 1ß, IL-6 and tumor necrosis factor (TNF)-a. Immunofluorescence and western blotting were employed to assess the expression levels of Glial fibrillary acidic protein (GFAP), ionized calcium-binding adapter molecule (Iba1), Keap1, Nrf2, HO-1, and NF-κB p-p65 protein. RESULTS: Dio administration relieved SNL-induced transient mechanical and thermal allodynia in mice. The protective effect of Dio in the SNL model was associated with its anti-inflammatory and anti-glial responses in the spinal cord. Dio inhibited both inflammatory factors and macrophage activation in the DRG. Furthermore, Dio regulated the Keap1/Nrf2/NF-κB signaling pathway. HO-1 and Nrf2 were upregulated following Dio administration, which also decreased the levels of Keap1 and NF-κB p65 protein. CONCLUSION: Mice with SNL-induced neuropathic pain were therapeutically treated with Dio. Dio may protect against pain by inhibiting inflammatory responses and improved Keap1/Nrf2/NF-κB pathway. These results highlight the potential therapeutic effect of Dio for the development of new analgesic drugs.

A new Coffee Brewing Control Chart relating sensory properties and consumer liking to brew strength, extraction yield, and brew ratio.

The classic Coffee Brewing Control Chart (BCC) was originally developed in the 1950s. It relates coffee quality to brew strength and extraction yield, and it is still widely used today by coffee industry professionals around the world to provide guidance on the brewing of coffee. Despite its popularity, recent experimental studies have revealed that sensory attributes and consumer preferences actually follow much more complicated trends than those indicated by the classic BCC. Here, we present a methodology to synthesize the results of these recent studies on drip-brewed coffee to generate new versions of the BCC: a new Sensory BCC that displays a broad array of statistically significant sensory attributes across typical total dissolved solids and percent extraction ranges, a new Consumer BCC that highlights the existence of two preference clusters with different likes and dislikes across those ranges, a new Sensory and Consumer BCC that combines both sensory descriptive and consumer preferences on the same chart, and a more streamlined BCC that omits consumer preferences and focuses on the overarching sensory descriptive trends. The new BCCs provide more accurate insight on how best to brew coffee to achieve desired sensory profiles. PRACTICAL APPLICATION: Through the manipulation of yield and extraction parameters, the new Sensory and Consumer Coffee Brewing Control Chart presented here can be used by brewers of drip coffee to design coffees with specific sensory profiles and match the preferences of different consumer types.

Spectral shift supported epichlorohydrin toxicity and the protective role of sage.

In this study, the toxicity of epichlorohydrin, a chemical intermediate, was investigated by using Allium cepa L. test material as a bio-indicator. In addition, the protective role of sage leaf extract (Slex) against this toxicity was investigated. Toxicity was handled with the help of physiological (germination percentage, root elongation, and weight gain), cytogenetic (mitotic index = MI, micronucleus = MN, and chromosomal abnormalities = CAs), biochemical (malondialdehyde = MDA, superoxide dismutase = SOD, and catalase = CAT), and anatomical (root meristem cell damages) parameters. A. cepa bulbs were divided into 6 groups (1 control, 5 applications). The bulbs in the control group were treated with tap water, and the bulbs in the application group were treated with epichlorohydrin at a dose of 100 mg/L and Slex at two different doses (190 mg/L and 380 mg/L) and germinated. Germination process was continued uninterruptedly for 72 h in all groups. At the end of the period, physiological parameter measurements were carried out in the bulbs. In addition, root tips were collected and made ready for cytogenetic, biochemical, and anatomical measurements and microscopic observations. As a result, exposure to epichlorohydrin caused statistically significant (p < 0.05) decreases in germination percentage, root length, weight gain, and MI, and statistically significant (p<0.05) increases in MN frequency, CA numbers, MDA level, SOD, and CAT enzyme activities. Epichlorohydrin exposure induced CAs such as fragment, sticky chromosome, unequal distribution of chromatin, reverse polarization, and disordered mitosis in root meristem cells. The toxicity of epichlorohydrin was due to its interaction with DNA, and this interaction was confirmed by the spectral shift in the DNA spectrum. In addition, epichlorohydrin caused anatomical damages such as epidermis cell damage, cortex cell damage, thickening of the cortex cell wall, and flattened cell nuclei in root meristem cells. The application of Slex together with epichlorohydrin decreased the toxicity of epichlorohydrin and again caused statistically significant (p < 0.05) improvements in the values of all the parameters examined. In other words, germination percentage, root length, weight gain, and MI increased again and MN frequency, CAs numbers, MDA level, SOD, and CAT enzyme activities decreased. It was determined that this improvement was even more pronounced at 380 mg/L dose of Slex. As a result, it was determined that epichlorohydrin caused multiple-toxicity for the investigated indicator organism, and Slex had a reducing role in this toxicity. For this reason, Slex should be included in the daily diet as an antioxidant beverage in order to protect from the toxicity of chemical agents exposed in daily life or to reduce their effects.

Interconnected macropores cryogel with nano-thin crosslinked network regenerated cellulose.

Dissolved oil palm empty fruit bunch cellulose (EFBC) and sodium carboxymethylcellulose (NaCMC) were chemically crosslinked with epichlorohydrin (ECH) to generate designated hydrogel. After swelling process in distilled water, the swollen hydrogel was frozen and freeze-dried to form cryogel. The swelling phenomenon of hydrogel during the absorption process gave substantial effects on thinning of crosslinked network wall, pore size and volume, steadiness of cryogel skeletal structure, and re-swelling of cryogel. The swelling effects on hydrogel were confirmed via microscopic study using variable pressure scanning electron microscope (VPSEM). From the retrieved VPSEM images, nano-thin crosslinked network wall of 24.31 ± 1.97 nm and interconnected pores were observed. As a result, the amount of water, the swelling degree, and the freeze-drying process indirectly affected the VPSEM images that indicated pore size and volume, formation of interconnected pores, and re-swelling of cryogel. This study determined the intertwined factors that affected both hydrogel and cryogel properties by investigating the swelling phenomenon and its ensuing effects.

Lipase Immobilization on Silica Xerogel Treated with Protic Ionic Liquid and its Application in Biodiesel Production from Different Oils.

Treated silica xerogel with protic ionic liquid (PIL) and bifunctional agents (glutaraldehyde and epichlorohydrin) is a novel support strategy used in the effective immobilization of lipase from Burkholderia cepacia (LBC) by covalent binding. As biocatalysts with the highest activity recovery yields, LBC immobilized by covalent binding with epichlorohydrin without (203%) and with PIL (250%), was assessed by the following the hydrolysis reaction of olive oil and characterized biochemically (Michaelis⁻Menten constant, optimum pH and temperature, and operational stability). Further, the potential transesterification activity for three substrates: sunflower, soybean, and colza oils, was also determined, achieving a conversion of ethyl esters between 70 and 98%. The supports and the immobilized lipase systems were characterized using Fourier transform infrared spectra (FTIR), scanning electron microscopy (SEM), elemental analysis, and thermogravimetric (TG) analysis.

Efficient removal of toxic phosphate anions from aqueous environment using pectin based quaternary amino anion exchanger.

Pectin based quaternary amino anion exchanger (Pc-QAE) was prepared using simple crosslinking polymerization method. This anion exchanger was characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR). Pc-QAE was applied for the removal of phosphate anion from the aqueous solution. The adsorption process which was pH dependent showed maximum adsorption of phosphate anions at pH 7. Pc-QAE showed good monolayer adsorption capacity for phosphate anions which demonstrated its good capability towards Langmuir isotherm model. Moreover, the adsorption was evaluated thermodynamically and the negative value of Gibbs free energy (-1.791KJ/mol) revealed the spontaneity of adsorption process. The value of ΔH° and ΔS° were found to be 15.28 and 49.48KJ/mol, respectively representing the endothermic nature and enhancement in degree of freedom due to the adsorption process.

Enhancing adsorption of U(VI) onto EDTA modified L. cylindrica using epichlorohydrin and ethylenediamine as a bridge.

Benefiting from strong coordination ability and unique vascular structure, EDTA modified L. cylindrica opens up an alternative way for uranium recovery from seawater. However, limitations, such as poor adsorption capacity and slow adsorption rate due to low graft ratio of EDTA via one-step esterification block its practical application. Here, a strategy for increasing the graft ratio is proposed in order to improve the adsorption performance. The strategy initially involves immobilization of epichlorohydrin (EPI) onto L. cylindrica and then ethylenediamine (EDA) is introduced via facile ring-opening reaction. EPI and EDA serve as a bridge between L. cylindrica and EDTA. The graft ratio is promoted (15.01 to 21.44%) contributing to the smaller steric hindrance of EPI and EDA than EDTA and improvement in adsorption performance. In addition, the adsorbent prepared by the new strategy exhibits excellent adsorption properties in simulated seawater.

Dual crosslinked pectin-alginate network as sustained release hydrophilic matrix for repaglinide.

Repaglinide, an oral antidiabetic agent, has a rapid onset of action and short half-life of approximately 1h. Developing a controlled and prolonged release delivery system is required to maintain its therapeutic plasma concentration and to eliminate its adverse effects particularly hypoglycemia. The present study aimed to develop controlled release repaglinide loaded beads using sodium alginate and pectin with dual cross-linking for effective control of drug release. The prepared beads were characterized for size, percentage drug entrapment efficiency, in vitro drug release and the morphological examination using scanning electron microscope. For the comparative study, the release profile of a marketed conventional tablet of repaglinide (Prandin® tablets 2mg, Novo Nordisk) was determined by the same procedure as followed for beads. The particle size of beads was in the range of 698±2.34-769±1.43µm. The drug entrapment efficiency varied between 55.24±4.61 to 82.29±3.42%. The FTIR results suggest that there was no interaction between repaglinide and excipients. The XRD and DSC results suggest partial molecular dispersion and amorphization of the drug throughout the system. These results suggest that repaglinide did not dissolve completely in the polymer composition and seems not to be involved in the cross-linking reaction. The percent drug release was decreased with higher polymer concentrations. In conclusion, the developed beads could enhance drug entrapment efficiency, prolong the drug release and enhance bioavailability for better control of diabetes.

Phosphate uptake studies of cross-linked chitosan bead materials.

A systematic experimental study is reported that provides a molecular based understanding of cross-linked chitosan beads and their adsorption properties in aqueous solution containing phosphate dianion (HPO42-) species. Synthetically modified chitosan using epichlorohydrin and glutaraldehyde cross-linkers result in surface modified beads with variable hydrophile-lipophile character and tunable HPO42- uptake properties. The kinetic and thermodynamic adsorption properties of cross-linked chitosan beads with HPO42- species were studied in aqueous solution. Complementary structure and physicochemical characterization of chitosan beads via potentiometry, Raman spectroscopy, DSC, and dye adsorption measurements was carried out to establish structure-property relationships. The maximum uptake (Qm) of bead systems with HPO42- at equilibrium was 52.1mgg-1; whereas, kinetic uptake results for chitosan bead/phosphate systems are relatively rapid (0.111-0.113min-1) with an intraparticle diffusion rate-limiting step. The adsorption process follows a multi-step pathway involving inner- and outer-sphere complexes with significant changes in hydration. Phosphate uptake strongly depends on the composition and type of cross-linker used for preparation of chitosan beads. The adsorption isotherms and structural characterization of bead systems illustrate the role of surface charge, hydrophile-lipophile balance, adsorption site accessibility, and hydration properties of the chitosan bead surface.

New agar microspheres for the separation and purification of natural products.

A new type of agar chromatography media has been prepared with a yield over 80% using a water-in-oil emulsion technique. These microspheres have regular spherical shapes and particle diameters in the range 40-165 µm (average ∼90 µm). Cross-linking of the resulting agar microspheres with epichlorohydrin and 1,4-butanediol diglycidyl ether enhanced their mechanical and thermal stability. The alkaline conditions used during the cross-linking reaction also decreased the content of ionized sulfate groups of the polysaccharide, thus reducing the nonspecific adsorption of positively charged molecules. The cross-linked agar microspheres were functionalized with (i) branched poly(ethyleneimine) to obtain a stationary phase useful for the separation of proteins in an anion-exchange mode and (ii) with poly-ß-cyclodextrin enabling direct isolation and purification of puerarin from a crude extract of Radix puerariae. Using a 23.5 mL column loaded with 20 mg extract (0.85 mg/mL gel), puerarin with a purity of 96% was recovered with a yield of 86%.

Two-photon fluorescence imaging and bimodal phototherapy of epidermal cancer cells with biocompatible self-assembled polymer nanoparticles.

We have developed herein an engineered polymer-based nanoplatform showing the convergence of two-photon fluorescence imaging and bimodal phototherapeutic activity in a single nanostructure. It was achieved through the appropriate choice of three different components: a ß-cyclodextrin-based polymer acting as a suitable carrier, a zinc phthalocyanine emitting red fluorescence simultaneously as being a singlet oxygen ((1)O2) photosensitizer, and a tailored nitroaniline derivative, functioning as a nitric oxide (NO) photodonor. The self-assembly of these components results in photoactivable nanoparticles, approximately 35 nm in diameter, coencapsulating a multifunctional cargo, which can be delivered to carcinoma cells. The combination of steady-state and time-resolved spectroscopic and photochemical techniques shows that the two photoresponsive guests do not interfere with each other while being enclosed in their supramolecular container and can thus be operated in parallel under control of light stimuli. Specifically, two-photon fluorescence microscopy allows mapping of the nanoassembly, here applied to epidermal cancer cells. By detecting the red emission from the phthalocyanine fluorophore it was also possible to investigate the tissue distribution after topical delivery onto human skin ex vivo. Irradiation of the nanoassembly with visible light triggers the simultaneous delivery of cytotoxic (1)O2 and NO, resulting in an amplified cell photomortality due to a combinatory effect of the two cytotoxic agents. The potential of dual therapeutic photodynamic action and two-photon fluorescence imaging capability in a single nanostructure make this system an appealing candidate for further studies in biomedical research.

Comparison of sevelamer hydrochloride with colestimide, administered alone or in combination with calcium carbonate, in patients on hemodialysis.

Since hyperphosphatemia in hemodialysis patients can cause secondary hyperparathyroidism and promotes vascular calcification, serum phosphate (Pi) levels must be controlled by phosphate binders. Although sevelamer and colestimide are known as similar non-calcium, non-aluminum phosphate binders in hemodialysis patients, there are no studies that compare the effects of the two agents as either a monotherapy or in combination with calcium carbonate (CaCO3). We randomly allocated 62 hemodialysis patients with hyperphosphatemia to treatment with sevelamer (3.0 g/day) and colestimide (3.0 g/day). During the study, 35 subjects dropped out, leaving 13 in the sevelamer group and 14 in the colestimide group. After a 2-week CaCO3 washout, all subjects received the monotherapy for 4 weeks and then CaCO3 (3.0 g/day) was added for another 4 weeks. Serum corrected calcium levels tended to decrease in both groups during the washout period and monotherapy, but there was no significant difference between the two groups after the addition of CaCO3. Although the calcium x phosphorus product (Ca x P) in the two groups increased during the washout period, there was no significant change or difference between the two groups during monotherapy. However, the addition of CaCO3 significantly reduced serum Pi at Week 8 compared to that at Week 0 in both groups, and significantly lowered Ca x P only in the sevelamer group, but not in the colestimide group(.) In this short-term study, sevelamer and colestimide similarly ameliorated hyperphosphatemia, but the combination of sevelamer and CaCO3 was more effective than colestimide with CaCO3 in controlling the Ca x P product, and it may improve cardiovascular mortality in hemodialysis patients.

Blood glucose-lowering activity of colestimide in patients with type 2 diabetes and hypercholesterolemia: a case-control study comparing colestimide with acarbose.

BACKGROUND: An anion exchange resin has been reported to lower blood glucose levels in patients with type 2 diabetes. AIM: To examine, in comparison with an alpha-glucosidase inhibitor, the usefulness of colestimide in lowering blood glucose levels in patients with type 2 diabetes and hypercholesterolemia. METHODS: Thirty-three patients with type 2 diabetes and hypercholesterolemia were more or less randomly assigned to receive either colestimide (17 patients) or acarbose (16 patients). At 10 time points before and after administration, plasma glucose levels and serum lipid concentrations were measured in all subjects, and the J-index and M-value were calculated. RESULTS: Patients receiving colestimide showed significant decreases in glucose levels 2 hours after breakfast (from 216.9 +/- 37.2 mg/dl before treatment to 191.1 +/- 40.9 mg/dl after treatment; p=0.008), in the J-index (from 42.6 +/- 14.5 to 32.6 +/- 9.8; p<0.001), and in the M-value (from 23.1 +/- 12.1 to 14.6 +/- 7.1; p<0.001). CONCLUSION: In patients with type 2 diabetes and hyperlipidemia, colestimide was suggested to have blood glucose-lowering activity as does acarbose.

Development of a biosensor based on gilo peroxidase immobilized on chitosan chemically crosslinked with epichlorohydrin for determination of rutin.

A new reagentless biosensor for the square-wave voltammetric determination of rutin in pharmaceutical formulations was developed by immobilization of gilo (Solanum gilo) crude extract in chitosan matrix. The gilo tissue acts as a source of peroxidase. The highest biosensor performance was obtained after immobilization of the peroxidase in chemically crosslinked chitosan with epichlorohydrin and glutaraldehyde that was incorporated in a carbon paste electrode. In the presence of hydrogen peroxide this enzyme catalyses the oxidation of rutin to quinone and the electrochemical reduction of the product was obtained at a fixed potential of +124 mV versus Ag/AgCl (3.0 M KCl). The performance and factors influencing the resulting biosensor were studied in detail. The bioelectrode exhibited a linear response for rutin concentrations from 3.4x10(-7) to 7.2x10(-6) M (r=0.9998) and the recovery of rutin from the samples ranged from 96.2 to 102.4%. The detection and quantification limits were 2.0x10(-8) and 6.3x10(-8) M, respectively. The relative standard deviation was less than 1.0% for solutions containing 3.4x10(-7) to 7.2x10(-6) M rutin in 0.1 M phosphate buffer solution at pH 7.0 (n=10). The lifetime of this biosensor was 8 months (at least 500 determinations).

Effects of colestimide and/or Bofu-tsusho-san on plasma and liver lipids in mice fed a high-fat diet.

Hypercholesterolemia is known to enhance the risk of coronary heart disease and fatty liver. Colestimide is an anion-exchange resin, which is not absorbed in the small intestine, decreases the intestinal reabsorption of bile acids synthesized from cholesterol in the liver and consequently increases bile acid excretion into the feces. Bofu-tsusho-san, a traditional Japanese herbal remedy, contains 18 components and has long been used as an anti-obesity agent. In the present study, we investigated the effects of colestimide and/or Bohu-tsusho-san in young male mice fed a high-fat diet. The high-fat diet supplemented with both colestimide and Bofu-tsusho-san markedly reduced the plasma levels of lipids, the liver weight and number of fatty droplets in the liver cytoplasm, and the body growth, compared with animals fed a high-fat diet alone. Neither medicine affected the blood biochemistry. Thus, the hypocholesterolemic action of colestimide, sometimes bringing light flatulence, which is improved by simultaneous administration of Bofu-tsusho-san, which activates the thermogenesis of brown adipose tissue, is suggested to reduce body mass and liver lipids, lowering the plasma levels of lipids.

Synthesis and enzymatic degradation of epichlorohydrin cross-linked pectins.

The water solubility of pectin was successfully decreased by cross-linking with increasing amounts of epichlorohydrin in the reaction media. The initial molar ratios of epichlorohydrin/ galacturonic acid monomer in the reaction mixtures were 0, 0.37, 0.56, 0.74, 1.00, 1.47, and 2.44. The resulting epichlorohydrin cross-linked pectins were thus referred to as C-LP0, C-LP37, C-LP56, C-LP75, C-LP100, C-LP150, and C-LP250, respectively. Methoxylation degrees ranged from 60.5 +/- 0.9% to 68.0 +/- 0.6%, and the effective cross-linking degrees, determined by quantification of the hydroxyl anions consumed during the reaction, were 0, 17.8, 26.0, 38.3, 46.5, 53.5, and 58.7%. respectively. After incubating the different cross-linked pectins (0.5% w/v) in 25 mL of 0.05 M acetate-phosphate buffer (pH 4.5), containing 50 microL of Pectinex Ultra SP-L (pectinolytic enzymes), between 60 and 80% of the pectin osidic bounds were broken in less than 1 hr. Moreover, increasing the cross-linking degree only resulted in a weak slowing on the enzymatic degradation velocity.

Affinity-based in situ product removal coupled with co-immobilization of oily substrate and filamentous fungus.

In situ product removal (ISPR) involves actions taken for the fast removal of a product from the producing cell. ISPR is implemented to improve yield and productivity via minimization of product inhibition, minimization of product losses due to degradation or evaporation, and reduction of the number of subsequent downstream processing steps. Here we describe the implementation of affinity-based, specific ISPR as a crucial component of an integrative approach to problems associated with the biocatalytic production of a product exhibiting poor water solubility from an oily, water-insoluble precursor. Our integrative ISPR-based approach consists of co-immobilization of the oily substrate emulsion and the biocatalyst within bilayered alginate beads. A particulate-specific adsorbent, exhibiting high binding capacity of the product, is suspended in the reaction medium with periodical replacements. According to this approach, ISPR implementation is expected to shift the equilibration of product distribution between the co-immobilized oily substrate and the outer medium via specific product immobilization onto the added adsorbent. The product may subsequently be readily recovered via single-step final purification. This integrative approach was successfully demonstrated by the affinity-based ISPR of gamma-decalactone (4-decanolide). gamma-Decalactone was produced from castor oil via its beta-oxidation by the filamentous fungus Tyromyces sambuceus, co-immobilized with emulsified substrate within bilayered alginate beads. Product immobilization onto medium-suspended epichlorohydrin-crosslinked beta-cyclodextrin resulted in higher yield and easy pure product recovery.

Biomonitoring human exposure to environmental carcinogenic chemicals.

A coordinated study was carried out on the development, evaluation and application of biomonitoring procedures for populations exposed to environmental genotoxic pollutants. The procedures used involved both direct measurement of DNA or protein damage (adducts) and assessment of second biological effects (mutation and cytogenetic damage). Adduct detection at the level of DNA or protein (haemoglobin) was carried out by 32P-postlabelling, immunochemical, HPLC or mass spectrometric methods. Urinary excretion products resulting from DNA damage were also estimated (immunochemical assay, mass spectrometry). The measurement of adducts was focused on those from genotoxicants that result from petrochemical combustion or processing, e.g. low-molecular-weight alkylating agents, PAHs and compounds that cause oxidative DNA damage. Cytogenetic analysis of lymphocytes was undertaken (micronuclei, chromosome aberrations and sister chromatid exchanges) and mutation frequency was estimated at a number of loci including the hprt gene and genes involving in cancer development. Blood and urine samples from individuals exposed to urban pollution were collected. Populations exposed through occupational or medical sources to larger amounts of some of the genotoxic compounds present in the environmental samples were used as positive controls for the environmentally exposed population. Samples from rural areas were used as negative controls. The project has led to new, more sensitive and more selective approaches for detecting carcinogen-induced damage to DNA and proteins, and subsequent biological effects. These methods were validated with the occupational exposures, which showed evidence of DNA and/or protein and/or chromosome damage in workers in a coke oven plant, garage workers exposed to diesel exhaust and workers exposed to ethylene oxide in a sterilization plant. Dose reponse and adduct repair were studied for methylated adducts in patients treated with methylating cytostatic drugs. The biomonitoring methods have also demonstrated their potential for detecting environmental exposure to genotoxic compounds in nine groups of non-smoking individuals, 32P-postlabelling of DNA adducts being shown to have the greatest sensitivity.

Purification and characterization of polygalacturonases produced by the hyphal fungus Aspergillus niger.

Five endo-polygalacturonases (poly(1,4-alpha-D-galacturonide) glycanohydrolase, EC 3.2.1.15) and one exo-polygalacturonase (poly(1,4-alpha-D-galacturonide) galacturonohydrolase, EC 3.2.1.67) were isolated from a commercial pectinase preparation derived from Aspergillus niger. All five endo-enzymes could be purified to homogeneity by affinity chromatography on cross-linked alginate, ion-exchange chromatography, chromatofocusing, and gel permeation chromatography. The exo-polygalacturonase was only partially purified but free from endo-polygalacturonase activity. The two most abundant endo-polygalacturonases (endo-I and endo-II), with molecular masses of 55 and 38 kDa, respectively, are quite different with respect to their isoelectric point, specific activity, mode of action on oligomeric substrates, and amino acid composition. The physicochemical properties of the other three endo-polygalacturonases (endo-IIIA, endo-IIIB, and endo-IV), present in low amounts, are quite similar to those of the endo-I type. The pH optima of all these endo-polygalacturonases are in the range of 4.3-4.9.