The stabilizing effect of an oligomeric proanthocyanidin on red blood cell membrane structure of poorly controlled Type II diabetes.

Type II diabetes (T2D) is a pandemic characterized by pathological circulating inflammatory markers, high-glucose levels and oxidative stress. The hematological system is especially vulnerable to these aberrant circulating molecules, and erythrocytes (RBCs) show aberrant rheology properties, owing to the direct contact with these molecules. Pathological levels of circulating inflammatory markers in T2D therefore have a direct effect on the molecular and cellular structure of RBCs. Previous research has suggested that antioxidants may reduce oxidative stress that results from the pathological inflammatory markers. Particularly, polyphenol antioxidants like oligomeric proanthocyanidins (OPCs) may act as a hydroxyl mopping agent, and may have a positive effect on the deformability and membrane protein structure of RBCs from T2D. In this paper, we look at the effect of one such agent, Pinus massoniana bark extract (standardized to 95% oligomeric proanthicyanidins), on the RBC membrane structures and RBC shape changes of T2D, after laboratory exposure at physiological levels. Our methods of choice were atomic force microscopy and scanning electron microscopy to study RBC elasticity and ultrastructure. Results showed that in our hands, this OPC could change both the eryptotic nature of the RBCs, as viewed with scanning electron microscopy, as well as the elasticity. We found a significant difference in variation between the elasticity measurement values between the RBCs before and after OPC exposure (P-value <0.0001). In conclusion, the data from both these techniques therefore suggest that OPC usage might contribute to the improvement of RBC functioning.

The prevalence of dietary-related complementary and alternative therapies and their perceived usefulness among cancer patients.

BACKGROUND: The present study aimed to directly assess and compare the usage, benefits and side-effects of dietary-related complementary and alternative medicine (CAM) use among adult cancer patients and non-cancer adults in Norwich, UK. METHODS: Self-administered questionnaires were completed by 98 cancer patients and 92 non-cancer adults to compare demographics, types of CAM usage with reasons, benefits, side-effects and CAM information sources. The groups were matched for gender, age, marital status, education and household income. The mean ages were 62.7 and 59.7 years, respectively, with slightly more female than male participants. RESULTS: CAM use was high in both groups (47% in cancer and 53% in non-cancer respondents, P > 0.05). The most widely-used diet-related CAM among both groups was the large intake of fruit, vegetables and juice, multivitamins, fish oils and glucosamine. Fish oil intake was significantly higher in the non-cancer group (P < 0.05), whereas selenium and beta-carotene supplements were significantly higher in the cancer group (P < 0.01 and P = 0.02, respectively). The main reasons for using CAM were to boost the immune system and to improve quality of life (P > 0.05). Reported benefits included increased optimism and hope for the cancer group and increased optimism and pain relief for the non-cancer group. CONCLUSIONS: Diet-related CAM is used frequently by both cancer patients and non-cancer adults, with many reported benefits and few reported side-effects. Significant differences between the groups included a higher prevalence of fish oil used by the non-cancer group, and a higher use of selenium and beta-carotene supplements in the cancer group.

Hormonal versus non-hormonal contraceptives in women with diabetes mellitus type 1 and 2.

BACKGROUND: Adequate contraceptive advice is important in women with diabetes mellitus type 1 and 2 to reduce the risk of maternal and infant morbidity and mortality in unplanned pregnancies. A wide variety of contraceptives are available for these women. However hormonal contraceptives might influence carbohydrate and lipid metabolism and increase micro- and macrovascular complications. So caution in selecting a contraceptive method is required. OBJECTIVES: To investigate whether progestogen-only, combined estrogen/progestogen or non-hormonal contraceptives differ in terms of effectiveness in preventing pregnancy, in their side effects on carbohydrate and lipid metabolism and in long-term complications such as micro- and macrovascular disease, when used in women with diabetes mellitus. SEARCH STRATEGY: The search was performed in MEDLINE, EMBASE, CENTRAL/CCTR, POPLINE, CINAHL, WorldCat, ECO, ArticleFirst, the Science Citation Index, the British Library Inside, and reference lists of relevant articles. Last search was performed in May 2005. In addition, experts in the field and pharmaceutical companies marketing contraceptives were contacted to identify published, unpublished or ongoing studies. SELECTION CRITERIA: Randomised and quasi-randomised controlled trials that studied women with diabetes mellitus comparing: 1. hormonal versus non-hormonal contraceptives. 2. progestogen-only versus estrogen/progestogen contraceptives. 3. contraceptives containing <50 microg estrogen versus contraceptives containing > or = 50 microg estrogen. 4. contraceptives containing 'first'-, 'second'- and 'third'-generation progestogens, drospirenone and cyproterone acetate. Principal outcomes were contraceptive effectiveness, diabetes control, lipid metabolism and micro- and macrovascular complications. DATA COLLECTION AND ANALYSIS: Two investigators evaluated the titles and abstracts from the literature search. Quality assessment was performed independently with discrepancies resolved by discussion or consulting a third reviewer. Because the trials differed in studied contraceptives, participant characteristics and methodological quality, we could not combine the data in a meta-analysis. The trials were therefore examined on an individual basis and narrative summaries were provided. MAIN RESULTS: Three randomised controlled trials were included. Only one was of good methodological quality. It compared the influence of levonorgestrel-releasing IUD versus copper-IUD on carbohydrate metabolism in women with type 1 diabetes mellitus. No difference was found in daily insulin requirement, glycosylated hemoglobin (HbA1c) or fasting blood sugar after twelve months. The other two trials were of limited methodological quality. Both compared progestogen-only pills with different estrogen/progestogen combinations. The trials reported blood glucose levels to remain stable during treatment with most regimens. Only high-dose combined oral contraceptives were found to slightly impair glucose homeostasis. Combined oral contraceptives also appeared to have a minor adverse effect on lipid metabolism whereas progestogen-only contraceptives slightly improved lipid-metabolism. Only one study reported on micro- and macrovascular complications. No signs or symptoms of thromboembolic incidents or visual disturbances were observed. However study duration was short. Minor adverse effects were reported in one study. The trial found progestogen-only pills to cause more bleeding irregularities when compared with combined oral contraceptives. Unintended pregnancies were not observed during any of the studies. AUTHORS' CONCLUSIONS: The three included randomised controlled trials in this systematic review provided insufficient evidence to assess whether progestogen-only and combined contraceptives differ from non-hormonal contraceptives in diabetes control, lipid metabolism and complications. Two of the three studies were of limited methodological quality, sponsored by pharmaceutical companies and described surrogate outcomes. Ideally, an adequately reported, high-quality randomised controlled trial analysing both intermediate outcomes (i.e. glucose and lipid metabolism) and true clinical endpoints (micro- and macrovascular disease) in users of combined, progestogen-only and non-hormonal contraceptives should be conducted. However, due to the low incidence of micro- and macrovascular disease and accordingly the large sample size and follow-up period needed to observe differences in risk, a randomised controlled trial might not be the ideal design.

Medico-legal investigation of suspected fatal poisonings in Pretoria: 2000-2001.

Accurate data on the incidence and spectrum of fatal poisonings in South Africa is lacking. Most studies focus on "traditional poisons". This is a retrospective study on the medico-legal investigation of suspected fatal poisonings seen at the Pretoria Medico-Legal Laboratory. Post mortem records from January 2000 to December 2001 were examined. There were 153 cases of suspected fatal poisoning or overdose. However, toxicology results were conclusively positive in only 53 (34.64%) cases with some cases testing positive for more than one type or class of "poison". The fatal "poisons" identified in these 53 cases were as follows: pharmaceuticals (47.17%), illicit drugs (41.51%), agricultural poisons (20.75%), alcohol (13.21%), traditional medicines (3.77%) and heavy metals (3.77%). The average delay in completing toxicology analyses was 10 months. This study demonstrates that the current medico-legal investigation of fatal cases of suspected poisoning does not yield adequate results for definitive judicial administration in two-thirds of suspected cases. This creates a legal dilemma, as the primary medical cause of death remains unresolved.

Botrytis cinerea endopolygalacturonase genes are differentially expressed in various plant tissues.

Botrytis cinerea, the causal agent of blight, rot, and gray mold on many plant species, secretes various endopolygalacturonases during all stages of infection. The expression pattern of the encoding genes (Bcpg 1-6) was studied on four hosts: tomato, broad bean, apple, and courgette (also known as zucchini). All gene family members are differentially expressed, depending on the stage of infection and the host. Bcpg1 is expressed in all tissues tested although differences in transcript levels occur. Bcpg2 expression is detected early in the infection of three of four plant tissues tested. Bcpg3 and Bcpg5 are expressed in apple fruit tissue, although probably as a result of different regulatory mechanisms. The expression patterns of Bcpg4 and 6 are in agreement with their inducibility by monogalacturonic acid. The pattern of Bcpg gene expression indicates that B. cinerea is equipped with a flexible enzymatic pectate degradation machinery. The studies pinpoint new targets for gene disruption studies.

Synergy between enzymes from Aspergillus involved in the degradation of plant cell wall polysaccharides.

Synergy in the degradation of two plant cell wall polysaccharides, water insoluble pentosan from wheat flour (an arabinoxylan) and sugar beet pectin, was studied using several main-chain cleaving and accessory enzymes. Synergy was observed between most enzymes tested, although not always to the same extent. Degradation of the xylan backbone by endo-xylanase and beta-xylosidase was influenced most strongly by the action of alpha-L-arabinofuranosidase and arabinoxylan arabinofuranohydrolase resulting in a 2.5-fold and twofold increase in release of xylose, respectively. Ferulic acid release by feruloyl esterase A and 4-O-methyl glucuronic acid release by alpha-glucuronidase depended largely on the degradation of the xylan backbone by endo-xylanase but were also influenced by other enzymes. Degradation of the backbone of the pectin hairy regions resulted in a twofold increase in the release of galactose by beta-galactosidase and endo-galactanase but did not significantly influence the arabinose release by arabinofuranosidase and endo-arabinase. Ferulic acid release from sugar beet pectin by feruloyl esterase A was affected most strongly by the presence of other accessory enzymes.

Analysis of pectic epitopes recognised by hybridoma and phage display monoclonal antibodies using defined oligosaccharides, polysaccharides, and enzymatic degradation.

The structure of epitopes recognised by anti-pectin monoclonal antibodies (mAbs) has been investigated using a series of model lime-pectin samples with defined degrees and patterns of methyl esterification, a range of defined oligogalacturonides and enzymatic degradation of pectic polysaccharides. In immuno-dot-assays, the anti-homogalacturonan (HG) mAbs JIM5 and JIM7 both bound to samples with a wide range of degrees of methyl esterification in preference to fully de-esterified samples. In contrast, the anti-HG phage display mAb PAM1 bound most effectively to fully de-esterified pectin. In competitive inhibition ELISAs using fully methyl-esterified or fully de-esterified oligogalacturonides with 3-9 galacturonic acid residues, JIM5 bound weakly to a fully de-esterified nonagalacturonide but JIM7 did not bind to any of the oligogalacturonides tested. Therefore, optimal JIM5 and JIM7 binding occurs where specific but undefined methyl-esterification patterns are present on HG domains, although fully de-esterified HG samples contain sub-optimal JIM5 epitopes. The persistence of mAb binding to epitopes in pectic antigens, with 41% blockwise esterification (P41) and 43% random esterification (F43) subject to fragmentation by endo-polygalacturonase II (PG II) and endo-pectin lyase (PL), was also studied. Time course analysis of PG II digestion of P41 revealed that JIM5 epitopes were rapidly degraded, but a low level of PAM1 and JIM7 epitopes existed even after extensive digestion, indicating that some HG domains were more resistant to cleavage by PG II. The chromatographic separation of fragments produced by the complete digestion of P41 by pectin lyase indicated that a very restricted population of fragments contained the PAM1 epitope while a (1-->4)-beta-D-galactan epitope occurring on the side chains of pectic polysaccharides was recovered in a broad range of fractions.

Regulation of endopolygalacturonase gene expression in Botrytis cinerea by galacturonic acid, ambient pH and carbon catabolite repression.

The phytopathogenic fungus Botrytis cinerea produces a set of endopolygalacturonases (endoPGs) which are involved in the enzymatic degradation of pectin in plant cell walls. The endoPG-encoding genes of B. cinerea are differentially expressed when the fungus is grown in liquid culture on different carbon sources. A basic constitutive expression level was observed for two genes, Bcpg1 and Bcpg2, which encode basic isozymes. Galacturonic acid was shown to induce the expression of Bcpg4 and Bcpg6. Low pH of the culture medium resulted in induced expression of the Bcpg3 gene. Expression of the Bcpg5 gene was inducible; however the inducing factors could not be identified. Finally, galacturonic acid-induced expression of the Bcpg4 gene was repressed by the presence of more-favourable carbon sources, such as glucose.

Tandem mass spectrometric analysis of aspergillus niger pectin methylesterase: mode of action on fully methyl-esterified oligogalacturonates.

The substrate specificity and the mode of action of Aspergillus niger pectin methylesterase (PME) was determined using both fully methyl-esterified oligogalacturonates with degrees of polymerization (DP) 2-6 and chemically synthesized monomethyl trigalacturonates. The enzymic activity on the different substrates and a preliminary characterization of the reaction products were performed by using high-performance anion-exchange chromatography at neutral pH. Electrospray ionization tandem MS (ESI-MS/MS) was used to localize the methyl esters on the (18)O-labelled reaction products during the course of the enzymic reaction. A. niger PME is able to hydrolyse the methyl esters of fully methyl-esterified oligogalacturonates with DP 2, and preferentially hydrolyses the methyl esters located on the internal galacturonate residues, followed by hydrolysis of the methyl esters towards the reducing end. This PME is unable to hydrolyse the methyl ester of the galacturonate moiety at the non-reducing end.

The exopolygalacturonase from Aspergillus tubingensis is also active on xylogalacturonan.

Apple-pectin hairy regions were prepared from apple pectin by combined action of the recombinant Aspergillus niger enzymes endopolygalacturonase II and pectin methylesterase and the A. tubigensis exopolygalacturonase. Using this enzymically prepared pectin fraction, an additional activity of the A. tubigensis exopolygalacturonase was discovered only when the substrate was chemically saponified and when D-galacturonate, a potent inhibitor of the enzyme, was removed from the incubation mixture. The new reaction product was purified and could be hydrolysed by A. niger beta-xylosidase into D-galacturonate and beta-D-xylose in a 1:1 ratio, which identified it as xylogalacturonate. The results demonstrate that exopolygalacturonase is not only active on galacturonan but also on xylogalacturonan. The enzyme thus accomodates a substrate in which the terminal galacturonic acid residue carries a single xylose substitution. The well-defined substrate specificity of exopolygalacturonase opens the possibility for use of this enzyme in biotechnological applications, such as preparing pectins that are methylated at the non-reducing end, and for studying the fine structure of xylogalacturonan in pectin.

Cloning and partial characterization of endopolygalacturonase genes from Botrytis cinerea.

Botrytis cinerea is a plant-pathogenic fungus infecting over 200 different plant species. We use a molecular genetic approach to study the process of pectin degradation by the fungus. Recently, we described the cloning and characterization of an endopolygalacturonase (endoPG) gene from B. cinerea (Bcpg1) which is required for full virulence. Here we describe the cloning and characterization of five additional endoPG-encoding genes from B. cinerea SAS56. The identity at the amino acid level between the six endoPGs of B. cinerea varied from 34 to 73%. Phylogenetic analysis, by using a group of 35 related fungal endoPGs and as an outgroup one plant PG, resulted in the identification of five monophyletic groups of closely related proteins. The endoPG proteins from B. cinerea SAS56 could be assigned to three different monophyletic groups. DNA blot analysis revealed the presence of the complete endoPG gene family in other strains of B. cinerea, as well as in other Botrytis species. Differential gene expression of the gene family members was found in mycelium grown in liquid culture with either glucose or polygalacturonic acid as the carbon source.

Kinetic characterization of Aspergillus niger N400 endopolygalacturonases I, II and C.

Endopolygalacturonases I, II and C isolated from recombinant Aspergillus niger strains were characterized with respect to pH optimum, activity on polygalacturonic acid and mode of action and kinetics on oligogalacturonates of different chain length (n = 3-7). Apparent Vmax values using polygalacturonate as a substrate at the pH optimum, pH 4.1, were calculated as 13.8 mukat.mg-1, 36.5 mukat.mg-1 and 415 nkat.mg-1 for endopolygalacturonases I, II and C, respectively. K(m) values were < 0.15 mg.mL-1 for all three enzymes. Product progression analysis using polygalacturonate as a substrate revealed a random cleavage pattern for all three enzymes and suggested processive behavior for endopolygalacturonases I and C. This result was confirmed by analysis of the mode of action using oligogalacturonates. Processivity was observed when the degree of polymerization of the substrate exceeded 5 or 6 for endopolygalacturonase I and endopolygalacturonase C, respectively. The bond-cleavage frequencies obtained for the hydrolysis of the oligogalacturonates were used to assess subsite maps. The maps indicate that the minimum number of subsites is seven for all three enzymes. Using pectins of various degrees of esterification, it was shown that endopolygalacturonase II is the most sensitive to the presence of methyl esters. Like endopolygalacturonase II, endopolygalacturonases I, C and E, which was also included in this part of the study, preferred the non-esterified pectate. Additional differences in substrate specificity were revealed by analysis of the reaction products of hydrolysis of a mixture of pectate lyase-generated delta 4,5-unsaturated oligogalacturonates of degree of polymerization 4-8. Whereas endopolygalacturonase I showed a strong preference for generating the delta 4,5-unsaturated dimer, with endopolygalacturonase II the delta 4,5-unsaturated trimer accumulated, indicating further differences in substrate specificity. For endopolygalacturonases C and E both the delta 4,5-unsaturated dimer and trimer were observed, although in different ratios.

Characterization of the exopolygalacturonate lyase PelX of Erwinia chrysanthemi 3937.

Erwinia chrysanthemi 3937 secretes several pectinolytic enzymes, among which eight isoenzymes of pectate lyases with an endo-cleaving mode (PelA, PelB, PelC, PelD, PelE, PelI, PelL, and PelZ) have been identified. Two exo-cleaving enzymes, the exopolygalacturonate lyase, PelX, and an exo-poly-alpha-D-galacturonosidase, PehX, have been previously identified in other E. chrysanthemi strains. Using a genomic bank of a 3937 mutant with the major pel genes deleted, we cloned a pectinase gene identified as pelX, encoding the exopolygalacturonate lyase. The deduced amino acid sequence of the 3937 PelX is very similar to the PelX of another E. chrysanthemi strain, EC16, except in the 43 C-terminal amino acids. PelX also has homology to the endo-pectate lyase PelL of E. chrysanthemi but has a N-terminal extension of 324 residues. The transcription of pelX, analyzed by gene fusions, is dependent on several environmental conditions. It is induced by pectic catabolic products and affected by growth phase, oxygen limitation, nitrogen starvation, and catabolite repression. Regulation of pelX expression is dependent on the KdgR repressor, which controls almost all the steps of pectin catabolism, and on the global activator of sugar catabolism, cyclic AMP receptor protein. In contrast, PecS and PecT, two repressors of the transcription of most pectate lyase genes, are not involved in pelX expression. The pelX mutant displayed reduced pathogenicity on chicory leaves, but its virulence on potato tubers or Saintpaulia ionantha plants did not appear to be affected. The purified PelX protein has no maceration activity on plant tissues. Tetragalacturonate is the best substrate of PelX, but PelX also has good activity on longer oligomers. Therefore, the estimated number of binding subsites for PelX is 4, extending from subsites -2 to +2. PelX and PehX were shown to be localized in the periplasm of E. chrysanthemi 3937. PelX catalyzed the formation of unsaturated digalacturonates by attack from the reducing end of the substrate, while PehX released digalacturonates by attack from the nonreducing end of the substrate. Thus, the two types of exo-degrading enzymes appeared complementary in the degradation of pectic polymers, since they act on both extremities of the polymeric chain.

Dietary glutamine supplementation reduces plasma nitrate levels in rats.

It was recently shown that L-glutamine inhibits vascular nitric oxide (NO) production in vitro. The present study investigated the effect of glutamine enriched enteral diets on in vivo NO production in the rat. Nitrate, the stable end-product of NO production, was measured in plasma and 24 h urine collections in glutamine supplemented rats (6.25%, 12.5% and 25% w/w) and compared to the effect of isocaloric, nitrogenous control diets. Glutamine supplementation increased plasma levels of glutamine (up to 91%), arginine (up to 17%) and citrulline (up to 54%). After 1 week of glutamine supplementation plasma nitrate levels were significantly reduced by 50% compared to control (P < 0. 0001); irrespective of the amount of supplementation. No further decrease was observed after 2 weeks of feeding. No differences in daily urinary losses were found between the groups. These results point to an in vivo inhibitory effect of glutamine supplemented enteral feeding on NO production.

Cloning and characterization of two rhamnogalacturonan hydrolase genes from Aspergillus niger.

A rhamnogalacturonan hydrolase gene of Aspergillus aculeatus was used as a probe for the cloning of two rhamnogalacturonan hydrolase genes of Aspergillus niger. The corresponding proteins, rhamnogalacturonan hydrolases A and B, are 78 and 72% identical, respectively, with the A. aculeatus enzyme. In A. niger cultures which were shifted from growth on sucrose to growth on apple pectin as a carbon source, the expression of the rhamnogalacturonan hydrolase A gene (rhgA) was transiently induced after 3 h of growth on apple pectin. The rhamnogalacturonan hydrolase B gene was not induced by apple pectin, but the rhgB gene was derepressed after 18 h of growth on either apple pectin or sucrose. Gene fusions of the A. niger rhgA and rhgB coding regions with the strong and inducible Aspergillus awamori exlA promoter were used to obtain high-producing A. awamori transformants which were then used for the purification of the two A. niger rhamnogalacturonan hydrolases. High-performance anion-exchange chromatography of oligomeric degradation products showed that optimal degradation of an isolated highly branched pectin fraction by A. niger rhamnogalacturonan hydrolases A and B occurred at pH 3.6 and 4.1, respectively. The specific activities of rhamnogalacturonan hydrolases A and B were then 0.9 and 0.4 U/mg, respectively, which is significantly lower than the specific activity of A. aculeatus rhamnogalacturonan hydrolase (2.5 U/mg at an optimal pH of 4.5). Compared to the A enzymes, the A. niger B enzyme appears to have a different substrate specificity, since additional oligomers are formed.

Upregulation of antithrombotic ectonucleotidases by aspirin in human endothelial cells in-vitro.

Ecto ATP-diphosphohydrolase (apyrase) activity of human endothelial cells following aspirin treatment has been studied in-vitro. It was shown by HPLC analysis of supernatant samples that pre-incubation of the cultures with aspirin resulted in a significantly increased turnover of supplemented ATP into its degradation products (ADP and AMP). Enhanced expression of ectoenzyme after aspirin treatment could be observed as demonstrated by immunofluorescence-staining with monoclonal anti-apyrase antibodies. This suggests enhancement of endothelial ATP-diphosphohydrolase activity induced by aspirin. The present data obtained in human vascular cells in-vitro are in line with results from previous animal studies in-vivo, suggesting a novel cyclooxygenase-independent antithrombotic activity of aspirin.

Arginine supplementation in the prevention and treatment of osteoporosis.

Oral administration of L-arginine in pharmacological doses induces growth hormone and insulin-like growth factor-I responses and stimulates nitric oxide synthesis. Growth hormone and insulin-like growth factor-I are important mediators of bone turnover and osteoblastic bone formation, while nitric oxide is a potent inhibitor of osteoclastic bone resorption. Because of this dual effect on physiological regulators of bone remodeling, L-arginine could potentially increase bone formation over bone resorption, and, consequently, increase bone mass. It is, therefore, hypothesized that oral supplementation of L-arginine may be a novel strategy in the prevention and treatment of osteoporosis. Studies of this simple, safe, and inexpensive therapy seem warranted.

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.