Protection of probiotic bacteria in a synbiotic matrix following aerobic storage at 4 °C.

The survival of single strains of Bifidobacterium breve, Bifidobacterium longum, Lactobacillus acidophilus, and Lactobacillus reuteri was investigated in synbiotics that included 10 mg/ml of fructo-oligosaccharides, inulin and pectic-oligosaccharides in an alginate matrix under refrigerated (4 °C) aerobic storage conditions. When the matrices were cross-linked with calcium (45 mM), 102-103 cfu/ml of L. acidophilus and L. reuteri, and 0-103 cfu/ml of B. breve and B. longum survived refrigerated aerobic storage for 28 days. Following refrigerated storage, acetic (3-9 mM), butyric (0-2 mM), propionic (5-16 mM) and lactic acids (1-48 mM) were produced during the growth of probiotics in BHI broth at 37 °C, suggesting their metabolic activity after storage was stressed. When calcium cross-linking was not used in synbiotics, the matrix remained more gel-like after inoculation when compared to the calcium cross-linked matrix. At least 107 cfu/ml of probiotic bacteria survived after 21 days of storage within these gel-like alginate matrices. Significantly higher levels of B. breve, L. acidophilus and L. reuteri were obtained from the synbiotic matrices supplemented with fructo-oligosaccharides, inulin and pectic-oligosaccharides compared to alginate alone. B. longum survival was the same (~7 logs) in all gel-like synbiotic matrices. These results show that synbiotics protected probiotic bacteria and extended their shelf-life under refrigerated aerobic conditions. Synbiotics represent a viable delivery vehicle for health-promoting bacteria.

In utero exposure to an AR antagonist plus an inhibitor of fetal testosterone synthesis induces cumulative effects on F1 male rats.

Risk assessments are typically conducted on a chemical-by-chemical basis; however, many regulatory bodies are developing frameworks for assessing the cumulative risk of chemical mixtures of chemicals. The current investigation examined how chemicals that disrupt rat sex differentiation via two diverse mechanisms disrupt F1 male rat reproductive development, when administered together orally on days 14-18 of gestation. Experiment 1 used a mixture of 50 mg/kg-d procymidone and 500 mg/kg-d dibutyl phthalate (DBP), whereas experiment 2 used 150 mg/kg-d procymidone and 1125 mg/kg-d DBP (top dose), or 0, 4.17, 8.33, 16.7, 33.3, 50, 66.7, and 83.3% of the top dose. When we compared the dose and response addition predictions to the observed effects we found that dose addition models were more accurate than response addition models, indicating that compounds that act by different mechanisms of toxicity produce cumulative dose-additive effects.

Inhibition by pectic oligosaccharides of the invasion of undifferentiated and differentiated Caco-2 cells by Campylobacter jejuni.

The ability of pectic oligosaccharides (POS) to inhibit adherence to and invasion of undifferentiated (UC) and differentiated (DC) Caco-2 cells by Campylobacter jejuni (C. jejuni) was investigated. It was observed that both adherence and invasion were significantly higher in UC than in DC. POS (2.5mg/ml) had no significant effect on the number of bacteria which can adhere to cells, but they significantly inhibited cell invasion. The extent of the anti-invasive effect of POS was dependent on the concentration, although the entire range tested (from 2.5mg/ml to 0.05 mg/ml) was capable of inhibiting the invasion of Caco-2 cells by Campylobacter to some degree. The pre-incubation or not of C jejuni with POS did not influence the behaviour observed. The results obtained in this work suggest that POS could be potentially useful as alternatives to antibiotics in the control of C. jejuni.

Oligosaccharide-mediated inhibition of the adhesion of pathogenic Escherichia coli strains to human gut epithelial cells in vitro.

The aim of the study was to investigate the ability of pectic oligosaccharides (POS) to inhibit adhesion of three strains of verotoxigenic Escherichia coli, three strains of enteropathogenic E. coli, and one nonclinical strain of Desulfovibrio desulfuricans to human intestinal epithelial cell cultures. Lactobacillus acidophilus and Lactobacillus gasseri were included for comparison. Attachment was determined in the human HT29 cell line by viable count of adherent bacteria. POS in buffer at pH 7.2 were antiadhesive at a dose of 2.5 mg ml(-1), reducing adhesion of enteropathogenic E. coil and verotoxigenic E. coli strains to less than 30% of control values. Concentrations resulting in 50% inhibition ranged from 0.15 to 0.46 mg ml(-1). L. acidophilus was not significantly affected, but adhesion of L. gasseri was reduced to 29% of the control value. POS reduced the adhesion of D. desulfuricans to 0.33% of the control value. POS also had a protective effect against E. coli verocytotoxins VT1 and VT2 at concentrations of 0.01 and 1 microg ml(-1), respectively.

In utero exposure to the environmental androgen trenbolone masculinizes female Sprague-Dawley rats.

Recently, the occurrence of environmental contaminants with androgenic activity has been described from pulp and paper mill effluents and beef feedlot discharges. A synthetic androgen associated with beef production is trenbolone acetate, which is used to promote growth in cattle. A primary metabolite, 17beta Trenbolone (TB), has been characterized as a potent androgen in both in vitro and in vivo studies with rats. The current study was designed to characterize the permanent morphological and functional consequences of prenatal TB exposure on female rats compared with those produced in an earlier study with testosterone propionate (TP). Female rat offspring were exposed to 0mg/day, 0.1mg/day, 0.5mg/day, 1.0mg/day, or 2.0mg/day TB on gestational days 14-19. The 0.5mg/day, 1.0mg/day, or 2.0mg/day TB groups displayed increases in neonatal anogenital distance (AGD) which persisted in the high dose group. Puberty was delayed in the high dose group and there were increased incidences of external genital malformations and the presence of male prostatic tissue in the 0.5mg/day, 1.0mg/day, or 2.0mg/day groups. These changes were associated with amniotic fluid concentrations of TB that compare favorably with concentrations known to be active in both in vitro systems and in fish.

Refractoriness to short day lengths augments tonic and gonadotrophin-releasing hormone-stimulated lutenising hormone secretion.

Siberian hamsters (Phodopus sungorus) undergo reproductive involution following exposure to short winter day lengths. Following approximately 20 weeks of exposure to short day (SD) lengths, hamsters become refractory to the inhibitory effects of SD, and reproductive competence is restored in anticipation of spring. The extent to which changes in gonadal steroid-dependent and -independent regulation of gonadotrophin secretion participate in this vernal reactivation of the gonads is not known. This experiment tested whether tonic and gonadotrophin-releasing hormone (GnRH)-stimulated regulation of lutenising hormone (LH) secretion differs between photoresponsive and photorefractory Siberian hamsters. Male hamsters born into long day (LD) lengths were castrated or subjected to a sham-castration surgery at 17 days of age, implanted s.c. with blank or testosterone-filled capsules, and housed in LD or SD thereafter. Baseline LH and LH responses to GnRH (200 ng/kg, s.c) were measured at 14 (photoresponsive) and 40 (photorefractory) weeks of age. Despite lower circulating testosterone concentrations in gonadally regressed SD hamsters on week 14, tonic LH concentrations were comparable among all groups of gonad-intact hamsters on weeks 14 and 40; however, week 14 SD hamsters exhibited significantly higher GnRH-stimulated LH responses. Tonic LH concentrations were indistinguishable among all groups of castrated hamsters bearing empty implants on week 14, but prolonged exposure to LD led to a decrease in resting LH, whereas prolonged exposure to SD resulted in an increase in LH. In castrated hamsters bearing testosterone implants, baseline LH concentrations were comparable in all groups, but GnRH treatment resulted in significantly higher LH concentrations in photorefractory (week 40, SD) hamsters relative to all other groups. The data suggest that the development of photorefractoriness in Siberian hamsters is characterised by enhanced gonadal hormone-independent stimulation of LH secretion, and diminished sensitivity to inhibitory negative-feedback effects of testosterone on LH secretion. Decreases in responsiveness of gonadotrophin secretion to gonadal hormone negative feedback may contribute to the process of photorefractoriness and assist in maintaining the growth of reproductive organs during the process of gonadal recrudescence.

In vitro determination of prebiotic properties of oligosaccharides derived from an orange juice manufacturing by-product stream.

Fermentation properties of oligosaccharides derived from orange peel pectin were assessed in mixed fecal bacterial culture. The orange peel oligosaccharide fraction contained glucose in addition to rhamnogalacturonan and xylogalacturonan pectic oligosaccharides. Twenty-four-hour, temperature- and pH-controlled, stirred anaerobic fecal batch cultures were used to determine the effects that oligosaccharides derived from orange products had on the composition of the fecal microbiota. The effects were measured through fluorescent in situ hybridization to determine changes in bacterial populations, fermentation end products were analyzed by high-performance liquid chromatography to assess short-chain fatty acid concentrations, and subsequently, a prebiotic index (PI) was determined. Pectic oligosaccharides (POS) were able to increase the bifidobacterial and Eubacterium rectale numbers, albeit resulting in a lower prebiotic index than that from fructo-oligosaccharide metabolism. Orange albedo maintained the growth of most bacterial populations and gave a PI similar to that of soluble starch. Fermentation of POS resulted in an increase in the Eubacterium rectale numbers and concomitantly increased butyrate production. In conclusion, this study has shown that POS can have a beneficial effect on the fecal microflora; however, a classical prebiotic effect was not found. An increase in the Eubacterium rectale population was found, and butyrate levels increased, which is of potential benefit to the host.

Skeleton photoperiods alter delayed-type hypersensitivity responses and reproductive function of Siberian hamsters (Phodopus sungorus).

Photoperiod (day length) can modulate immune function. Whether these photoperiodic effects on immune function are mediated directly by a circadian photoperiodic time measurement system or indirectly by nonspecific (e.g. stressful) effects of light is unknown. To discriminate between these two possibilities, Siberian hamsters (Phodopus sungorus) were housed in either long or short photoperiods (LD 16 : 8 h or LD 8 : 16 h) or in 'skeleton' long or short photoperiods (LD 1 : 14 h: LD 1 : 8 h or LD 1 : 6 h: LD 1 : 16 h). In the skeleton photoperiods, both long- and short-day animals received 2 h of light per day. After 10 weeks in their respective photoperiods, hamsters were tested for an antigen specific immune response using a delayed type hypersensitivity (DTH) model. Reproductive and endocrine responses of hamsters in each of the skeleton photoperiods were equivalent to those in standard long or short days, respectively. Hamsters in skeleton short days and LD 8 : 16 increased DTH responses compared to hamsters in both long-day groups. DTH responses were equivalent in both long-day groups. These results suggest that the influences of day length on immune function potentially are due to circadian photoperiodic time measurement systems.

Photoperiod affects neuronal nitric oxide synthase and aggressive behaviour in male Siberian hamsters (Phodopus sungorus).

Many nontropical animals display physiological and behavioural changes in response to seasonal environmental cues including photoperiod (day length). Male Siberian hamsters (Phodopus sungorus) housed in short photoperiod undergo testicular regression accompanied by reduced circulating testosterone and decreased reproductive behaviour. By contrast to the majority of small mammals studied, aggressive behaviour is elevated in short-day Siberian hamsters when blood testosterone concentrations are not detectable. Because gonadal steroid hormones influence neuronal nitric oxide synthase (nNOS), and this enzyme has been implicated in aggressive behaviour, we hypothesized that nNOS expression would be decreased in short-day male Siberian hamsters and negatively correlated with the display of territorial aggression. Adult male Siberian hamsters were individually housed in either long (LD 16:8 h) or short (LD 8:16 h) photoperiods for 10 weeks. Hamsters were assigned to one of two categories by assessing testicular volume and plasma testosterone values: (i) photoperiodic responsive (i.e. regressed testes and low testosterone concentrations) or (ii) photoperiodic nonresponsive (i.e. testes size and circulating testosterone concentrations equivalent to hamsters maintained in long days). At week 10, aggression was assessed using a resident-intruder test. Latency to initial attack, frequency of attacks and duration of total attacks were recorded during a 10-min aggression trial. Brains were collected immediately after behavioural testing and stained for nNOS expression using immunohistochemistry. All short day-housed hamsters were significantly more aggressive than long-day animals, regardless of gonadal size or testosterone concentrations. Short-day animals, both reproductively responsive and nonresponsive morphs, also had significantly less nNOS-immunoreactive cells in the anterior and basolateral amygdaloid areas and paraventricular nuclei compared to long-day hamsters. Together, these results suggest that seasonal aggression in male Siberian hamsters is regulated by photoperiod, through mechanisms that are likely independent from gonadal steroid hormones.

A mixture of the "antiandrogens" linuron and butyl benzyl phthalate alters sexual differentiation of the male rat in a cumulative fashion.

Prenatal exposure to environmental chemicals that interfere with the androgen signaling pathway can cause permanent adverse effects on reproductive development in male rats. The objectives of this study were to 1) determine whether a documented antiandrogen butyl benzyl phthalate (BBP) and/or linuron (an androgen receptor antagonist) would decrease fetal testosterone (T) production, 2) describe reproductive developmental effects of linuron and BBP in the male, 3) examine the potential cumulative effects of linuron and BBP, and 4) investigate whether treatment-induced changes to neonatal anogenital distance (AGD) and juvenile areola number were predictive of adult reproductive alterations. Pregnant rats were treated with either corn oil, 75 mg/kg/day of linuron, 500 mg/kg/day of BBP, or a combination of 75 mg/kg/day linuron and 500 mg/kg/day BBP from gestational Day 14 to 18. A cohort of fetuses was removed to assess male testicular T and progesterone production, testicular T concentrations, and whole-body T concentrations. Male offspring from the remaining litters were assessed for AGD and number of areolae and then examined for alterations as young adults. Prenatal exposure to either linuron or BBP or BBP + linuron decreased T production and caused alterations to androgen-organized tissues in a dose-additive manner. Furthermore, treatment-related changes to neonatal AGD and infant areolae significantly correlated with adult AGD, nipple retention, reproductive malformations, and reproductive organ and tissue weights. In general, consideration of the dose-response curves for the antiandrogenic effects suggests that these responses were dose additive rather than synergistic responses. Taken together, these data provide additional evidence of cumulative effects of antiandrogen mixtures on male reproductive development.

An environmental antiandrogen, vinclozolin, alters the organization of play behavior.

During mammalian sexual differentiation, the androgens, testosterone and dihydrotestosterone are critical for the organization of the male phenotype. In rats, play behavior is sexually dimorphic. Administration of exogenous androgens during the perinatal period results in masculine-like play behavior of juveniles. Recently, there has been increasing concern about the potential for environmental endocrine-disrupting chemicals (EDCs) to alter sexual differentiation in mammals. One such EDC is the fungicide and androgen receptor (AR) antagonist, vinclozolin. We tested whether developmental exposure to an EDC could alter androgen-dependent behaviors such as play. To examine this possibility, neonatal male rats were injected from Postnatal Days (PND) 2 to 3 with corn oil, pharmacological antiandrogen flutamide (50 mg/kg/day) or vinclozolin (200 mg/kg/day); whereas neonatal females were treated either with corn oil or testosterone propionate (TP, 250 microg/kg/day). At PNDs 36-37, animals were observed for social play. Behaviors associated with general social activity, such as sniffing and dorsal contact, were unaffected by treatment or sex. However, play behavior in males treated with flutamide or vinclozolin was significantly reduced to near-female levels when compared to control males. Play behavior in females exposed to TP during the neonatal period was significantly increased when compared with control females. Hence, this study suggests that perinatal exposure to vinclozolin, an environmental antiandrogen, can alter androgen-dependent behavior, such as play, in the male rat.

Xenoendocrine disrupters-tiered screening and testing: filling key data gaps.

The US Environmental Protection Agency (EPA) is developing a screening and testing program for endocrine disrupting chemicals (EDCs) to detect alterations of hypothalamic-pituitary-gonadal (HPG) function, estrogen (ER), androgen (AR) and thyroid hormone synthesis and AR and ER receptor-mediated effects in mammals and other animals. High priority chemicals would be evaluated in the Tier 1 Screening (T1S) battery and chemicals positive in T1S would then be tested (Tier 2). T1S includes in vitro ER and AR receptor binding and/or gene expression, an assessment of steroidogenesis and mammalian (rat) and nonmammalian in vivo assays (Table 1). In vivo, the uterotropic assay detects estrogens and antiestrogens, while steroidogenesis, antithyroid activity, (anti)estrogenicity and HPG function are assessed in a 'Pubertal Female Assay'. (Anti-) androgens are detected in the Hershberger Assay (weight of AR-dependent tissues in castrate-immature-male rats). Fish and amphibian assays also are being developed. The fathead minnow assay can identify EDCs displaying several mechanisms of concern, including AR and ER receptor agonists and antagonists and inhibitors of steroid hormone synthesis. An amphibian metamorphosis assay is being developed to detect thyroid-active substances. Several alternative mammalian in vivo assays have been proposed. Of these, a short-term pubertal male rat assay appears most promising. An in utero-lactational screening protocol also is being evaluated. For Tier 2, the numbers of endocrine sensitive endpoints and offspring (F1) examined in multigenerational tests need to be expanded for EDCs. Consideration should be given to tailoring T2, based on the results of T1S. Tier 1 and 2 also should examine relevant mixtures of EDCs. Toxicants that induce malformations in AR-dependent tissues produce cumulative effects even when two chemicals act via different mechanisms of action.

Androgens and environmental antiandrogens affect reproductive development and play behavior in the Sprague-Dawley rat.

In mammals, exposure to androgens early in development is essential for masculinization of the male reproductive phenotype. Male fetuses exposed to antiandrogens during perinatal life are permanently demasculinized in their morphology and physiology, whereas exposure to exogenous androgens permanently masculinizes females. In some litter-bearing species, proximity(italic) in utero(/italic) of females to males can partially masculinize female siblings and alter their responsiveness to endocrine-disrupting compounds. However, in our strain of rat (CD-SD Charles River), intrauterine position does not significantly influence testosterone concentrations and anogenital distance of fetuses. In comparison, administration of testosterone propionate to pregnant females, at doses that doubled fetal female testosterone levels, did masculinize the reproductive system. Discovery of androgen-active chemicals in the environment has placed increased emphasis on describing the reproductive and behavioral effects of both natural and environmental androgens and antiandrogens. Recently, the effects of an antiandrogen, vinclozolin, on the brain and behavior were cited as a special concern by the U.S. Environmental Protection Agency in its risk assessment of this pesticide. In rats, one such behavior that is perinatally organized by androgens is social play. Males play more than females, and administration of exogenous androgens during the neonatal period alters the juvenile expression of this sexually dimorphic behavior. Vinclozolin is an androgen receptor antagonist that inhibits androgen-dependent tissue growth in vivo. We were interested in whether developmental exposure to vinclozolin could also alter androgen-dependent behaviors such as play. Neonatal male rats were injected on postnatal days (PNDs) 2 and 3 with corn oil, the pharmacologic antiandrogen flutamide (50 mg/kg), or vinclozolin (200 mg/kg). On PNDs 36-37 animals were observed for social play. Behaviors associated with general social activity such as sniffing and dorsal contact were unaffected by treatment. However, play behavior in males treated with flutamide or vinclozolin was significantly reduced, resembling levels of play characteristic of females rather than untreated males. Therefore, this study demonstrates that perinatal exposure to vinclozolin, an environmental antiandrogen, can alter androgen-dependent play behavior in the male rat.

Isolation of oligogalacturonic acids up to DP 20 by preparative high-performance anion-exchange chromatography and pulsed amperometric detection.

Milligram quantities of oligogalacturonic acids up to a degree of polymerization (DP) of 20 were purified by high-performance anion-exchange chromatography utilizing a preparative-scale (21-mm i.d.) CarboPac PA1 column and a nonlinear potassium acetate (pH 7.5) gradient. Detection was accomplished by pulsed amperometry without post-column addition of hydroxide. Pulsed amperometry at near-neutral pH is an excellent detection method for preparative separations of carbohydrates because it avoids base-catalyzed degradation reactions that can occur at high pH. This method was simpler, faster, had higher sample loading capacity and allowed for the isolation of higher DP oligogalacturonic acids than other methods reported previously. With this improved method, multi-milligram quantities of valuable oligogalacturonic acids (up to DP 20) can be readily isolated.

Effects of environmental antiandrogens on reproductive development in experimental animals.

Chemicals that act as androgen receptor (AR) agonists and antagonists or inhibit fetal steroidogenesis can induce reproductive malformations in humans and laboratory animals. Several environmental chemicals disrupt development in rats and/or rabbits at fetal concentrations at, or near, exposure levels seen in some segments of the human population. In rats, fetal tissues concentrations of 10-20 p.p.m. of the DDT metabolite, p,p'-DDE, are correlated with reproductive abnormalities in male offspring. These concentrations are similar to those measured in first-trimester human fetal tissues in the late 1960s. The pesticides vinclozolin, procymidone, linuron and DDT are AR antagonists. They reduce male rat anogenital distance, and induce areolas at relatively low dosages. Hypospadias, agenesis of the sex accessory tissues and retained nipples are seen in the middle dosages, while undescended testes and epididymal agenesis are seen in the highest doses. Phthalate esters (PE) inhibit testosterone synthesis during fetal life, but do not appear to be AR antagonists. Prenatal administration of a single low dose of dioxin (50-1,000 ng TCDD/kg) alters the differentiation of androgen-dependent tissues at p.p.t. concentrations, but the mechanism of action likely involves interaction with a hormone-like nuclear transcription factor, the hormone-like receptor AhR, rather than AR. p,p'-DDT and p,p'-DDE, vinclozolin and di-n-butyl phthalate affect reproductive function in rabbits when administered during prenatal and/or neonatal life. Cryptorchidism and carcinoma in situ-like (CIS) testicular lesions were seen in male rabbits treated during development with p,p'-DDT or p,p'-DDE. Extrapolation of effects from rodents to humans would be enhanced if future studies incorporate determination of tissue concentrations of the active metabolites. Knowledge of the tissue concentrations of the active toxicants also would provide an important link to in-vitro studies, which provide more useful mechanistic information when they are executed at relevant concentrations.

Cellular and molecular mechanisms of action of linuron: an antiandrogenic herbicide that produces reproductive malformations in male rats.

Antiandrogenic chemicals alter sex differentiation by several different mechanisms. Some, like flutamide, procymidone, or vinclozolin compete with androgens for the androgen receptor (AR), inhibit AR-DNA binding, and alter androgen-dependent gene expression in vivo and in vitro. Finasteride and some phthalate esters demasculinize male rats by inhibiting fetal androgen synthesis. Linuron, which is a weak competitive inhibitor of AR binding (reported Ki of 100 microM), alters sexual differentiation in an antiandrogenic manner. However, the pattern of malformations more closely resembles that produced by the phthalate esters than by vinclozolin treatment. The present study was designed to determine if linuron acted as an AR antagonist in vitro and in vivo. In vitro, we (1) confirmed the affinity of linuron for the rat AR, and found (2) that linuron binds human AR (hAR), and (3) acts as an hAR antagonist. Linuron competed with an androgen for rat prostatic AR (EC(50) = 100-300 microM) and human AR (hAR) in a COS cell-binding assay (EC(50) = 20 microM). Linuron inhibited dihydrotestosterone (DHT)-hAR induced gene expression in CV-1 and MDA-MB-453-KB2 cells (EC(50) = 10 microM) at concentrations that were not cytotoxic. In short-term in vivo studies, linuron treatment reduced testosterone- and DHT-dependent tissue weights in the Hershberger assay (oral 100 mg/kg/d for 7 days, using castrate-immature-testosterone propionate-treated male rats; an assay used for decades to screen for AR agonists and antagonists) and altered the expression of androgen-regulated ventral prostate genes (oral 100 mg/kg/d for 4 days). Histological effects of in utero exposure to linuron (100 mg/kg/d, day 14-18) or DBP (500 mg/kg/d, day 14 to postnatal day 3) on the testes and epididymides also are shown here. Taken together, these results support the hypothesis that linuron is an AR antagonist both in vivo and in vitro, but it remains to be determined if linuron alters sexual differentiation by additional mechanisms of action.

Genetic and biochemical characterization of an exopolygalacturonase and a pectate lyase from Yersinia enterocolitica.

Yersinia enterocolitica, an invasive foodborne human pathogen, degrades polypectate by producing two depolymerizing enzymes, pectate lyase (PL) and polygalacturonase (PG). The gene encoding the PG activity, designated pehY, was located in a 3-kb genomic fragment of Y. enterocolitica ATCC 49397. The complete nucleotide sequence of this 3-kb fragment was determined and an open reading frame consisting of 1803 bp was predicted to encode a PG protein with an estimated M(r) of 66 kDa and pI of 6.3. The amino acid sequence of prePG showed 59 and 43% identity to that of the exopolygalacturonase (exoPG) of Erwinia chrysanthemi and Ralstonia solanacearum, respectively. The Y. enterocolitica PG overproduced in Escherichia coli was purified to near homogeneity using perfusion cation exchange chromatography. Analysis of the PG depolymerization products by high performance anion-exchange chromatography and pulsed amperometric detection (HPAEC-PAD) revealed the exolytic nature of this enzyme. The Y. enterocolitica PL overproduced in E. coli was also partially purified and the M(r) and pI were estimated to be 55 kDa and 5.2, respectively. HPAEC-PAD analysis of the PL depolymerization products indicated the endolytic nature of this enzyme. Southern hybridization analyses revealed that pehY and pel genes of Y. enterocolitica are possibly encoded in the chromosome rather than in the plasmid. Purified exopolygalacturonase (over 10 activity units) was unable to macerate plant tissues.

Structure of a plant cell wall fragment complexed to pectate lyase C.

The three-dimensional structure of a complex between the pectate lyase C (PelC) R218K mutant and a plant cell wall fragment has been determined by x-ray diffraction techniques to a resolution of 2.2 A and refined to a crystallographic R factor of 18.6%. The oligosaccharide substrate, alpha-D-GalpA-([1-->4]-alpha-D-GalpA)3-(1-->4)-D-GalpA , is composed of five galacturonopyranose units (D-GalpA) linked by alpha-(1-->4) glycosidic bonds. PelC is secreted by the plant pathogen Erwinia chrysanthemi and degrades the pectate component of plant cell walls in soft rot diseases. The substrate has been trapped in crystals by using the inactive R218K mutant. Four of the five saccharide units of the substrate are well ordered and represent an atomic view of the pectate component in plant cell walls. The conformation of the pectate fragment is a mix of 21 and 31 right-handed helices. The substrate binds in a cleft, interacting primarily with positively charged groups: either lysine or arginine amino acids on PelC or the four Ca2+ ions found in the complex. The observed protein-oligosaccharide interactions provide a functional explanation for many of the invariant and conserved amino acids in the pectate lyase family of proteins. Because the R218K PelC-galacturonopentaose complex represents an intermediate in the reaction pathway, the structure also reveals important details regarding the enzymatic mechanism. Notably, the results suggest that an arginine, which is invariant in the pectate lyase superfamily, is the amino acid that initiates proton abstraction during the beta elimination cleavage of polygalacturonic acid.

Further studies of the role of cyclic beta-glucans in symbiosis. An NdvC mutant of Bradyrhizobium japonicum synthesizes cyclodecakis-(1-->3)-beta-glucosyl.

The cyclic beta-(1-->3),beta-(1-->6)-D-glucan synthesis locus of Bradyrhizobium japonicum is composed of at least two genes, ndvB and ndvC. Mutation in either gene affects glucan synthesis, as well as the ability of the bacterium to establish a successful symbiotic interaction with the legume host soybean (Glycine max). B. japonicum strain AB-14 (ndvB::Tn5) does not synthesize beta-glucans, and strain AB-1 (ndvC::Tn5) synthesizes a cyclic beta-glucan lacking beta-(1-->6)-glycosidic bonds. We determined that the structure of the glucan synthesized by strain AB-1 is cyclodecakis-(1-->3)-beta-D-glucosyl, a cyclic beta-(1-->3)-linked decasaccharide in which one of the residues is substituted in the 6 position with beta-laminaribiose. Cyclodecakis-(1-->3)-beta-D-glucosyl did not suppress the fungal beta-glucan-induced plant defense response in soybean cotyledons and had much lower affinity for the putative membrane receptor protein than cyclic beta-(1-->3),beta-(1-->6)-glucans produced by wild-type B. japonicum. This is consistent with the hypothesis presented previously that the wild-type cyclic beta-glucans may function as suppressors of a host defense response.