Synthesis and oxidative insolubilization of cell-wall proteins during osmotic stress

The cell walls in the new white roots of jack pine (Pinus banksiana Lamb.) were observed to constrict around the shrinking protoplast of osmotically stressed roots, and pressure was maintained via an apparent adjustment of cell-wall size and elasticity. These elastic alterations of the cell wall permitted the root cells to maintain full turgor despite the loss of most of the water in the tissue. The constriction of the root cell wall around the dehydrating protoplasts to maintain turgor may reflect changes in cell wall structure. We found that these shrinking root cells synthesize and secrete into the intercellular fluid a set of proteins. These proteins become tightly associated (i.e. guanidine HCl- and sodium dodecyl sulfate-insoluble) with the cell wall but can be released from the matrix, after briefly boiling in 0.1% sodium dodecyl sulfate, by the combination of guanidine HCl, CaCl₂ and dithiothreitol. However, these cell-wall proteins became insoluble with time. The proteins could subsequently be destructively extracted from the wall with acid NaClO₂ treatments. After these proteins were incorporated into the cell walls, the roots adopted a new, smaller maximal tissue volume and elastic coefficients returned to normal levels. Received: 8 July 1998 / Accepted: 19 November 1998     

A study of prostaglandin F2α as the luteolysin in swine: II characterization and comparison of prostaglandin F, estrogens and progestin concentrations in utero-ovarian vein plasma of nonpregnant and pregnant gilts

Polyvinyl catheters were inserted into the right and left utero-ovarian veins (UOV) and saphenous vein (SV) and artery (SA)_of six nonpregnant (O) and five pregnant (P) gilts on day 11 after onset of estrus. Beginning on day 12, UOV blood samples were collected at 15-min intervals from 0800 to 1100 hr and 2000 to 2300 hr, and single samples were taken at 1200 and 2400 hrs. Peripheral blood (SA or SV) was sampled at 0800, 1200, 2000 and 2400 hr until gilts returned to estrus ( ) or day 24 or pregnancy. UOV plasma PGF concentrations (ng/ml; n = 1929) were measured by RIA. Status (P O) by day interactions were detected (P<.01) but variances among treatments were heterogenous (P<.01). Cuvilinear day trends were detected for PGF in 0 gilts (P<.01) but not P gilts. PGF peaks, defined as concentrations greater than two SD above the mean concentration for each gilt, occurred with greater frequency ((ifχ² = 16.4; P>.01)) in 0 than P gilts; and mean peak levels ( ) were 5.0 ± .27 and 3.84 ± .13 ng/ml, respectively.Progesterone concentrations were maintaiend in pregnant pigs and were indicative of luteal maintenance. Systematic differences in day trends of utero-ovarian venous plasma estradiol were detected between O and P pigs. These differences may be of paramount physiological importance and are discussed.     

Selective inhibition of Escherichia coli in the presence of Salmonella typhimurium by phenethyl beta-D-galactopyranoside

Phenethyl beta-d-galactopyranoside (PEG) was hydrolyzed by the beta-galactosidase of Escherichia coli to form the toxic product phenethyl alcohol. Salmonella typhimurium did not hydrolyze PEG. In mixed culture, the ratio of S. typhimurium to E. coli was increased by growing the organisms in lactose broth containing 2.5% PEG. The high concentration of PEG required for inhibition of E. coli can be attributed to inadequate cell permeability rather than to prevention of beta-galactosidase induction.     

The Schistosoma mansoni Cytochrome P450 (CYP3050A1) Is Essential for Worm Survival and Egg Development

Schistosomiasis affects millions of people in developing countries and is responsible for more than 200,000 deaths annually. Because of toxicity and limited spectrum of activity of alternatives, there is effectively only one drug, praziquantel, available for its treatment. Recent data suggest that drug resistance could soon be a problem. There is therefore the need to identify new drug targets and develop drugs for the treatment of schistosomiasis. Analysis of the Schistosoma mansoni genome sequence for proteins involved in detoxification processes found that it encodes a single cytochrome P450 (CYP450) gene. Here we report that the 1452 bp open reading frame has a characteristic heme-binding region in its catalytic domain with a conserved heme ligating cysteine, a hydrophobic leader sequence present as the membrane interacting region, and overall structural conservation. The highest sequence identity to human CYP450s is 22%. Double stranded RNA (dsRNA) silencing of S. mansoni (Sm)CYP450 in schistosomula results in worm death. Treating larval or adult worms with antifungal azole CYP450 inhibitors results in worm death at low micromolar concentrations. In addition, combinations of SmCYP450-specific dsRNA and miconazole show additive schistosomicidal effects supporting the hypothesis that SmCYP450 is the target of miconazole. Treatment of developing S. mansoni eggs with miconazole results in a dose dependent arrest in embryonic development. Our results indicate that SmCYP450 is essential for worm survival and egg development and validates it as a novel drug target. Preliminary structure-activity relationship suggests that the 1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethan-1-ol moiety of miconazole is necessary for activity and that miconazole activity and selectivity could be improved by rational drug design.     

Use of the point-score system for breeding soundness examination in yearling Dorset, Hampshire and Suffolk rams

Performance tests were conducted on 583 purebred Dorset, Hampshire and Suffolk yearling rams at the Virginia Ram Test Station from 1986 to 1989. Birth dates at entry and weights (lbs) at entry and end-of-test were recorded for each ram. Entry and exit scrotal circumference (SC; cm) data were recorded for each year of the study. Breeding soundness examination (BSE) data at entry were obtained for only the last two years (1988-1989). The BSE followed the basic format recommended by the Society for Theriogenology. The number of seminal white blood cells per (100x) microscope field (WBC/LPF) were also recorded for each ram's ejaculate. Classification of rams into breeding groups (satisfactory, questionable and unsatisfactory) were made using a point-scale system based upon values obtained from SC, sperm motility and morphology assessments. Between-breed differences were noted for age at entry to the test station, weight per day of age, final weight at the end of the test period and average daily gain. Suffolk rams were younger in age (P0.05). Overall the percentage of rams classified as unsatisfactory, questionable and satisfactory was 11.8, 16.5 and 71.7, respectively. Rams with more than 10 WBC/LPF had significantly smaller SC at entry (P<0.01) than rams with less than 10 WBC/LPF. Most of the differences (75%) in BSE scores in this study were contributed by differences in semen quality (spermatozoal motility and morphology) not by differences in SC.     

Effect of estradiol-17beta on PGF and total protein content in bovine uterine flushings and peripheral plasma concentration of 13, 14-dihydro-15-keto-PGF(2alpha)

Two experiments were conducted to examine the effect of estradiol-17beta (E(2)-17beta) on content of immunoreactive prostagladin F(2)alpha (PGF, ng) and total protein (TUP, mg) in uterine flushings, as well as concentrations of 13, 14-dihydro-15-keto-PGF(2)alpha (PGFM) in plasma (Pg/ml). In experiment 1, Holstein heifers were utilized in a single reversal trial in which either E(2)-17beta (3 mg in 2 ml saline/ethanol 50:50; n=5) or vehicle alone (n=6) were given intravenously on day 14 or 15 of the estrous cycle (Period 1) following an induced estrus (day of estrus = day 0). Treatment (Trt) groups were reversed in Period 2 (Day 14 or 15 of the second estrous cycle). Jugular venous plasma was obtained before treatment (Oh), and at 5, 6, and 9h posttreatment (PT). Uterine flushings were collected nonsurgically in vivo , per cervix, via Foley catheter at 6h PT (20 ml of .9% saline per uterine horn). E(2)-17beta did not significantly alter (E(2)-17beta vs vehicle; x(-) +/- S.E.M.) PGF (1674 +/- .11 +/- 338.39 vs 1889.91 +/- 400.24 ng; P> .10) or TUP (33.25 +/- 2.57 vs 39.16 +/- 3.04 mg; P > .10). However, E(2)-17beta increased (P < .05) plasma PGFM (E(2)-17beta vs vehicle) after treatment (0h, 113.2 vs 163.8; 5h, 312.5 vs 203.9; 6h, 324.5 vs 198.0; 9h, 323.2 vs 246.8, pg/ml). In experiment 2, crossbred beef cattle received comparable treatments of either E(2)-17beta (n=5) or vehicle (n=5) on day 14 or 15 postestrus. Jugular venous plasma was obtained at 0h PT, and at 6h PT. Uterine flushings (1.9% saline, 20 ml per uterine horn) and peripheral plasma were collected at slaughter. Estradiol-17beta increased PGF (30.07 +/- 5.94 vs 8.46 +/- 2.01 ng; P> <.05) in uterine flushings as well as PGFM in plasma (E(2)-17beta : 55.82 +/- 19.13 pg/ml, at 0h and 89.31 +/- 14.02 pg/ml, at 6h, vs saline: 103.46 +/- 50.73 pg/ml, at 0h and 17.78 +/- 14.22, at 6h). Estradiol-17beta stimulated uterine production and release of PGF and protein as measured in flushings (experiment 2) as well as plasma PGFM responses (experiments 1 and 2). Uterine and/or cervical stimulation of experiment 1 may have masked uterine response to E(2)-17beta.