Mode of action of pectic enzymes. II. Further purification of exopolygalacturonate lyase and pectinesterase from Clostridium multifermentans

Exopolygalacturonate lyase and pectinesterase from Clostridium multifermentans were purified 156-fold and 178-fold, respectively, by gel filtration chromatography on Sephadex G-200. The activities of both enzymes coincided in a single protein peak. Profiles of the two activities also coincided in diethylaminoethyl-cellulose chromatography and zonal centrifugation. These studies indicated that the esterase and the lyase were either complexed or similar molecular species. The former seems more probable because of the relatively high molecular weight. Both activities were most stable at pH 6.0. The esterase was inactivated rapidly at pH 5 or 7. Lyase preparations were freed of pectinesterase activity by heating for 30 min at 38 C and pH 7.0.     

Similarities in the Action Patterns of Exopolygalacturonate Lyase and Pectinesterase from Clostridium multifermentans

Exopolygalacturonate lyase and pectinesterase from Clostridium multifermentans were assayed simultaneously in the same reaction mixture which contained a highly esterified pectin, polymethyl polygalacturonic acid methyl glycoside. Lyase is specific for unesterified galacturonide residues and cannot degrade this substrate in the absence of the esterase. The rate for esterase was twice the rate for lyase throughout the entire course of the combined reaction. Thus, the molar ratio of the two enzyme activities was the same since the product of the lyase is an unsaturated digalacturonic acid containing two free carboxyl groups. Since clostridial exopolygalacturonate lyase is known to degrade polygalacturonate in a linear manner beginning from the reducing ends of polygalacturonate chains, it was apparent that clostridial pectinesterase must hydrolyze methyl groups in highly esterified pectins with an action pattern similar to that of the lyase. Otherwise it would be impossible for the two enzyme rates to have corresponded on the basis of a 2:1 ratio.     

Production and ecological significance of yeast cell wall-degrading enzymes from oerskovia

Motile actinomycetes capable of degrading walls of viable yeast cells were isolated from soil and identified as Oerskovia xanthineolytica. A lytic assay based on susceptibility of enzyme-treated cells to osmotic shock was developed, and 10 of 15 strains of O. xanthineolytica, Oerskovia turbata, and nonmotile Oerskovia- like organisms from other collections were found to possess yeast lytic activities. All lytic strains produced laminaranase and alpha-mannanase, but the amounts, determined by reducing group assays, were not proportional to the observed lytic activities. The Oerskovia isolates demonstrated chemotactic, predatory activity against various yeast strains and killed yeasts in mixed cultures. Of 15 carbon sources tested for production of lytic enzyme, purified yeast cell walls elicited the highest activity. Glucose repressed enzyme production and caused cells to remain in the microfilamentous and motile rod stages of the Oerskovia cell cycle. Crude lytic activity was optimal at pH 5.6 to 7.0 and inactivated by heating for 6 min at 50 degrees C. Partial purification by isoelectric focusing showed that all lytic activity was associated with four beta-(1-->3)-glucanases. The absence of protein disulfide reductase, N-acetyl-beta-d-hexosaminidase, and phosphomannanase in crude preparations indicated that the principal enzyme responsible for yeast wall lysis was a beta-(1-->3)-glucanase that produced relatively little reducing sugar from yeast glucan.     

Purification of cellobiohydrolase I fromTrichoderma reesei using monoclonal antibodies in an immunomatrix

Summary The several components of the fungal cellulase system present practical problems in devising facile and efficient schemes for their purification. We report on a new single-step affinity chromatographic method for purification of cellobiohydrolase I ofTrichoderma reesei based on its selective absorption and elution using an immunomatrix constructed with CnBr-activated Sepharose 4B and monoclonal antibody specific for the enzyme. Isoenzymes of cellobiohydrolase I were purified directly from crude culture filtrate. The method is fast, simple, and of high resolution.     

Temporary suppression of pulsatile LH release following a single injection of a GnRH agonist (deslorelin) in ovariectomised Holstein dairy cows

The objective of the experiment was to investigate the potential for using a single injection of the GnRH agonist [D-Trp(6), Pro(9)-des-Gly(10)-NH(2)] GnRH-ethylamide (deslorelin) to suppress LH secretion in ovariectomised Holstein cows. Each dose of 10, 100 and 1000 microg deslorelin was injected intravenously into each of four ovariectomised cows on day 0. Blood samples were collected hourly on day 0 to profile the induced LH release. Frequent serial blood samples were collected at 10min intervals over 4h on days -3, -1, +2, +4 and +6. The injection of deslorelin induced a surge-like release of LH that begun within 1h in all cows. There was no difference between deslorelin doses in terms of maximum LH concentration, area under the LH curve (AUC) or log(10)(AUC). The average interval from injection to maximum LH concentration was longer for cows receiving 1000 microg than in those receiving 10 microg (3.5 versus 1.5h; P<0.01), though no different to 100 microg (2.8h; P>0.1). This relationship was described by a logarithmic function of deslorelin dose in micrograms (R(2)=73.3%, P<0.01). Pre-treatment smoothed mean LH concentration was significantly correlated with peak LH concentration of the induced surge: max_LH=5.37+9.57 x pre-amplitude (R(2)=33.2%, P=0.05). Similarly, LH pulse amplitude pre-deslorelin was also correlated with peak LH of the induced surge max_LH=0.07+12.9 x pre-amplitude (R(2)=53.7%, P=0.07). Pulsatile release of LH was suppressed only with the 1000 microg dose on day +2. Suppression was characterised by a reduction in mean LH, smoothed mean LH and LH pulse amplitude. By day +4, LH parameters were no different to pre-treatment ones. Pulse frequency was not affected by the treatment, although a small non-significant reduction at day +2 for 1000 microg dose was observed (3.9 versus 2.8, P=0.14). In conclusion, temporary suppression of LH output for at least 48h occurred following a single intravenous injection of 1000 microg of deslorelin, even though there were similar peak LH concentrations were for the three doses.     

Population Changes in Enteric Bacteria and Other Microorganisms During Aerobic Thermophilic Windrow Composting

Composting of wastes from swine feeding operations was studied. The effects of the frequency of turning the wastes and addition of straw to improve the physical structure were studied to determine the most effective technique to rapidly increase the temperature and, consequently, destroy coliforms and Salmonella. Four different treatments were studied; the results showed that, with addition of 5% (wt/wt) straw and mechanical turning of the compost 20 times per week, the temperature reached 60 C within 3 days and enteric bacteria were destroyed within 14 days.     

A Highlights from MBoC Selection: Quantitative Analysis of the Mechanism of Endocytic Actin Patch Assembly and Disassembly in Fission Yeast

We used quantitative confocal microscopy to measure the numbers of 16 proteins tagged with fluorescent proteins during assembly and disassembly of endocytic actin patches in fission yeast. The peak numbers of each molecule that accumulate in patches varied <30–50% between individual patches. The pathway begins with accumulation of 30–40 clathrin molecules, sufficient to build a hemisphere at the tip of a plasma membrane invagination. Thereafter precisely timed waves of proteins reach characteristic peak numbers: endocytic adaptor proteins (∼120 End4p and ∼230 Pan1p), activators of Arp2/3 complex (∼200 Wsp1p and ∼340 Myo1p) and ∼300 Arp2/3 complexes just ahead of a burst of actin assembly into short, capped and highly cross-linked filaments (∼7000 actins, ∼200 capping proteins, and ∼900 fimbrins). Coronin arrives last as all other components disperse upon patch internalization and movement over ∼10 s. Patch internalization occurs without recruitment of dynamins. Mathematical modeling, described in the accompanying paper (Berro et al., 2010, MBoC 21: 2803–2813), shows that the dendritic nucleation hypothesis can account for the time course of actin assembly into a branched network of several hundred filaments 100–200 nm long and that patch disassembly requires actin filament fragmentation in addition to depolymerization from the ends.     

Biotinidase is a Novel Marker for Papillary Thyroid Cancer Aggressiveness

Biotinidase was identified in secretome analysis of thyroid cancer cell lines using proteomics. The goal of the current study was to analyze the expression of biotinidase in thyroid cancer tissues and fine needle aspiration (FNA) samples to evaluate its diagnostic and prognostic potential in thyroid cancer. Immunohistochemical analysis of biotinidase was carried out in 129 papillary thyroid cancer (PTC, 34 benign thyroid tissues and 43 FNA samples and correlated with patients’ prognosis. Overall biotinidase expression was decreased in PTC compared to benign nodules (p = 0.001). Comparison of aggressive and non-aggressive PTC showed decrease in overall biotinidase expression in the former (p = 0.001). Loss of overall biotinidase expression was associated with poor disease free survival (p = 0.019, Hazards ratio (HR) = 3.1). We examined the effect of subcellular compartmentalization of nuclear and cytoplasmic biotinidase on patient survival. Decreased nuclear expression of biotinidase was observed in PTC as compared to benign tissues (p<0.001). Upon stratification within PTC, nuclear expression was reduced in aggressive as compared to non-aggressive tumors (p<0.001). Kaplan-Meier survival analysis showed significant association of loss of nuclear biotinidase expression with reduced disease free survival (p = 0.014, HR = 5.4). Cytoplasmic biotinidase expression was reduced in aggressive thyroid cancers in comparison with non-aggressive tumors (p = 0.002, Odds ratio (OR) = 0.29) which was evident by its significant association with advanced T stage (p = 0.003, OR = 0.28), nodal metastasis (p<0.001, OR = 0.16), advanced TNM stage (p<0.001, OR = 0.21) and extrathyroidal extension (p = 0.001, OR = 0.23). However, in multivariate analysis extrathyroidal extension emerged as the most significant prognostic marker for aggressive thyroid carcinomas (p = 0.015, HR = 12.8). In conclusion, loss of overall biotinidase expression is a novel marker for thyroid cancer aggressiveness.     

Effect of treatment with progesterone and oestradiol benzoate on ovarian follicular turnover in postpartum anoestrous cows and cows which have resumed oestrous cycles.

Two experiments were carried out to determine the effect of a low dose of progesterone (P) with and without the addition of an injection of oestradiol benzoate (ODB) on ovarian follicle dynamics, oestradiol production and LH pulsatility in postpartum anoestrous cows, compared with cows which had resumed oestrous cycles (cycling cows). In the first experiment, anoestrous Jersey cows were treated with (AN+P, n=8) or without (AN-3, n=3) a previously used intravaginal progesterone releasing (CIDR) device for 10 days, commencing 3 or 4 days after emergence of a new dominant follicle (DF1) as determined by transrectal ultrasonography. Contemporary cycling cows (CYC+P, n=8) were similarly treated with used CIDR devices and injected with prostaglandin F(2alpha) (PGF) at the time of device insertion. Follicle turnover was monitored by daily ultrasonography and pulsatile release of LH was measured on the ninth day after device insertion. During the period of CIDR device insertion, a second dominant follicle emerged in 4/8 of the CYC+P group and 7/8 of the AN+P group (P=0.14). Maximum diameter of DF1 was greater in cows in the CYC+P compared with the AN+P group (P=0.02), but did not differ between cows in the AN+P and AN-P groups (P>0.1). Frequency of LH pulses was greater in cows in the CYC+P than AN+P group (P=0.06), and in cows in the AN+P than AN-P group (P=0.02).In the second experiment, anoestrous (n=20) and cycling (n=11) Friesian cows were treated with a new CIDR device for 6 days commencing 3 days after emergence of a new dominant follicle (DF1). Cycling cows were also injected with PGF on the day of device insertion. Half of the cows in each group were injected with 2mg ODB on the day of device insertion. Daily ultrasonography was used to monitor follicular dynamics throughout the experimental period. Follicular turnover was increased by ODB in cycling (5/5 versus 1/6; P<0.05), but not anoestrous cows (5/9 versus 4/11). Persistence of DF1 was reduced by ODB treatment in both cycling and anoestrous cows (P<0.001). Maximum diameter of DF1 was influenced by ODB treatment and reproductive status (P<0.05). In anoestrous cows in which a second dominant follicle did not emerge during the period of device insertion, the interval from emergence of DF1 to emergence of a second dominant follicle was significantly delayed by treatment with ODB (P=0.04).In conclusion, P treatment of anoestrous cows increased pulsatile release of LH, but did not induce the development of persistent follicles. Injection of ODB in association with P treatment reduced the persistence of dominant follicles in both cycling and anoestrous cows, but delayed subsequent follicular development in a proportion of anoestrous cows.