Ultrastructure of Bovine Ovarian Follicles Induced to Extended Growth by Perioestrous Suprabasal Progesterone Levels

The present study was undertaken to determine if a short-term prolonged growth of the ovulatory follicle (12 to 18 h after expected time of ovulation), induced by progesterone implants, would cause ultrastructural changes in the follicular wall. Oestrous behaviour, follicular growth, follicular and blood plasma levels of oestradiol-17ß, progesterone and plasma luteinizing hormone (LH) were monitored in heifers oophorectomized at 9 to 12 h (controls) or 36 h after the onset of oestrus, in order to sample the pre-ovulatory follicle present. The suprabasal plasma progesterone concentrations (approximately 1.2 nmol L−1) allowed expression of oestrus at the expected time, but ovulation was delayed owing to the absence of a LH-surge. The resulting prolongation of follicle growth was associated with mild degenerative changes in the follicle wall, i.e. both granulosa and thecal cells presented increased electron density, higher amounts of secondary lysosomes and lipid droplets, increased intercellular spaces with presence of debris. No signs of luteinization were seen.     

The rumen: a unique source of enzymes for enhancing livestock production

Increasing competition in the livestock industry has forced producers to cut costs by adopting new technologies aimed at increasing production efficiency. One particularly promising technology is feeding enzymes as supplements for animal diets. Supplementation of diets for non-ruminants (e.g., swine and poultry) with fibrolytic enzymes, such as cellulases, xylanases and beta-glucanases, increases the feed conversion efficiency and growth rate of the animals. Enzymatic hydrolysis of plant cell wall polymers (e.g., cellulose, xylan, beta-glucans) releases glucose and xylose and eliminates the antinutritional effects of beta-glucans and arabinoxylans. Enzyme supplementation of diets for ruminants has also been shown to improve growth performance, even though the rumen itself represents the most potent fibrolytic fermentation system known. Implementation of this technology in the livestock industry has been limited largely because of the cost of development and production of enzymes. Over the last decade, however, developments in recombinant DNA technology have increased the efficiency of existing microbial production systems and facilitated exploitation of alternative sources of industrial enzymes. The ruminal ecosystem is among the novel enzyme sources currently being explored. Understanding the role of enzymes in feed digestion through characterization of the enzymology and genetics involved in digestion of feedstuffs by ruminants will provide insight required to improve the products currently available to producers. Characterization of genes encoding a variety of hydrolytic enzymes, such as cellulases, xylanases, beta-glucanases, amylases, pectinases, proteases, phytases and tannases, will foster the development of more efficacious enzyme supplements and enzyme expression systems for enhancing nutrient utilization by domestic animals. Characteristics of the original source organism need no longer restrict the production of a useful enzyme. Recent reports of transgenic plants expressing fibrolytic or phytase activity and of transgenic mice able to produce endoglucanase in the pancreas speak to the feasibility of improving feed digestion through genetic modification of the feedstuffs and the animals.     

Methanogenic Activity and Structural Characteristics of the Microbial Biofilm On a Needle-Punched Polyester Support

In a downflow stationary fixed-film anaerobic reactor receiving a swine waste influent, few bacteria were observed to be tightly adherent to the surfaces of the needle-punched polyester support material. However, there was a morphologically complex, dense population of bacteria trapped within the matrix. Frequently large microcolonies of a uniform morphological type of bacteria were observed. These were particularly evident for methanosarcina-like bacteria which grew forming large aggregates of unseparated cells. Leafy deposits of electron-dense, calcium- and phosphorus-enriched material coated the polyester matrix and some cells. As the biofilm matured there was more extensive mineral deposition which completely entrapped cells. The entrapped cells appeared to autolyze, and many were partially degraded. Further impregnation of the matrix with minerals and apparent cell death may eventually have a deleterious effect on the methanogenic activity of the biofilm.     

Use of Adenosine 5′-Triphosphate as an Indicator of the Microbiota Biomass in Rumen Contents

A number of techniques were tested for their efficiency in extracting adenosine 5′-triphosphate (ATP) from strained rumen fluid (SRF). Extraction with 0.6 N H₂SO₄, using a modification of the procedure described by Lee et al. (1971), was the most efficient and was better suited for extracting particulate samples. Neutralized extracts could not be stored frozen before assaying for ATP because large losses were incurred. The inclusion of internal standards was necessary to correct for incomplete recovery of ATP. The ATP concentration in rumen contents from a cow receiving a ration of dried roughage (mainly alfalfa hay) ranged from 31 to 56 μg of ATP per g of contents. Approximately 75% of the ATP was associated with the particulate material. The ATP was primarily of microbial origin, since only traces of ATP were present in the feed and none was found in “cell-free” rumen fluid. Fractionation of the bacterial and protozoal populations in SRF resulted in the isolation of an enriched protozoal fraction with a 10-fold higher ATP concentration than that of the separated rumen bacteria. The ATP pool sizes of nine functionally important rumen bacteria during the exponential phase of growth ranged from 1.1 to 17.6 μg of ATP per mg of dry weight. This information indicates that using ATP as a measure of microbial biomass in rumen contents must be done with caution because of possible variations in the efficiency of extraction of ATP from rumen contents and differences in the concentration of ATP in rumen microbes.     

Hyaluronan-binding proteins on cultured J 774 macrophages.

Cultivated macrophages of murine cell-line J 774 were found to bind high-molecular-weight (molecular weight average approx. 5.10(6) [3H]hyaluronan (HA) by a saturable mechanism at 4 degrees C. Half-maximal binding was observed at 7-8 microgram/ml (1.4-1.6 nM) and the maximal binding was reached at 30-40 microgram/ml. Scatchard plot analysis revealed that approx. 20,000 molecules could bind to each cell with a Kd of 1.5 nM. The binding could be effectively inhibited by unlabeled HA. Also chondroitin sulphate inhibited the binding, but only to about 50%. At 37 degrees C the J 774 cells took up and degraded the polysaccharide effectively. Affinity chromatography on HA coupled to agarose of solubilized surface-iodinated J 774 cells, revealed that a protein of approx. 60 kDa, when analyzed by sodium dodecylsulfate polyacrylamide gel electrophoresis and autoradiography, could be specifically eluted with HA-oligosaccharides. Our results suggest that J 774 macrophages can bind HA by a mechanism compatible with receptor-binding, and carry a 60 kDa HA-binding protein on their surface. This receptor-binding may mediate uptake and degradation of the polysaccharide and influence the levels and turnover of HA in interstitial fluid as well as the release of HA into the bloodstream.     

Effects of neonatal exposure to diethylstilbestrol on early mouse embryo development in vivo and in vitro

Eight-week-old female mice of the NMRI strain that had been treated neonatally with diethylstilbestrol (DES, 5 micrograms/day for five days) or not (controls) were treated with gonadotropins to induce ovulation and then were artificially inseminated. Ova or young embryos were recovered from the oviducts on the morning after insemination and on Days 2, 3, and 4. In other experiments, ova were obtained from inseminated females on the morning after ovulation and cultured in vitro. In DES-treated females, a few zygotes developed to the 4-cell stage, but no more advanced stages were seen. Under in vitro conditions, zygotes from DES-treated females developed into blastocysts and to the implantation stage, but the incidence of these stages was lower than with zygotes from controls. Our results point to an abnormal oviductal function in DES-treated females that is not compatible with early embryo survival, even though an additional zygote factor contributing to degeneration of early cleavage stages cannot be excluded.     

Genetic analysis of the yopE region of Yersinia spp.: identification of a novel conserved locus, yerA, regulating yopE expression.

The yopE gene of Yersinia pseudotuberculosis was recently sequenced, and YopE was identified as an indispensable virulence determinant when tested in a mouse model (A. Forsberg and H. Wolf-Watz, Mol. Microbiol. 2:121-133, 1988). In the study described here, the DNA sequences of the yopE genes of Yersinia pestis EV76 and Yersinia enterocolitica 8081 were determined and compared with that of the Y. pseudotuberculosis gene. Only two codons were found to differ, both leading to amino acid replacements, when the gene from Y. pestis was compared. These two replacements were also present in the gene from Y. enterocolitica; in addition, 18 other codons were found to differ. Thirteen of these substitutions led to amino acid replacements. Downstream of the yopE gene, the plasmid partition locus par was found to be conserved in all three species. In Y. enterocolitica 8081, the sequence homology was interrupted by a putative insertion sequence element inserted between the yopE gene and the par region at a position only 5 base pairs downstream of the yopE stop codon. Upstream of the yopE gene, 620 base pairs were conserved in the three species. This region contained a 130-amino-acid-long open reading frame reading in the opposite direction to the yopE gene and expressed a 14-kilodalton protein in minicells. An insertion mutation in this region constructed in Y. pseudotuberculosis expressed significantly lower amounts of YopE protein in vitro than did the corresponding wild type. The expression level could be restored by transcomplementation. This new locus was designated yerA, for yopE-regulating gene A. The yerA mutant was avirulent when mice were challenged by oral infection.     

In vitro phosphorylation of human complement factor C3 by protein kinase A and protein kinase C. Effects on the classical and alternative pathways

Complement factor C3, recently found to contain covalently bound phosphate, was phosphorylated in vitro by cyclic AMP-dependent protein kinase (protein kinase A) and Ca2(+)-activated, phospholipid-dependent protein kinase (protein kinase C). Both protein kinases phosphorylated the same serine residue(s) located in the C3a portion of the alpha-chain. In addition, protein kinase C phosphorylated the beta-chain to a lesser extent. Protein kinase A gave a maximal incorporation of 1 mol of phosphate/mol of C3 while that value with protein kinase C was 1.5 mol of phosphate/mol of C3. The velocity in pmol of [32P]phosphate/(min x unit kinase) was 20 times higher for protein kinase C than for protein kinase A although a 10 times lower ratio of protein kinase to C3 was used in the former case. The apparent Km for C3 was 2.6 microM when protein kinase C was used. The phosphorylated C3 was found to be more resistant to partial degradation by trypsin than unphosphorylated C3. It was also found that phosphorylation of C3 in the C3a portion of the alpha-chain inhibited both the classical and alternative complement activation pathways on an approximately stoichiometric basis.     

Cellulose digestion and cellulase regulation and distribution in Fibrobacter succinogenes subsp. succinogenes S85.

Fibrobacter succinogenes subsp. succinogenes S85 initiated growth on microcrystalline cellulose without a lag whether inoculated from a glucose, cellobiose, or cellulose culture. During growth on cellulose, there was no accumulation of soluble carbohydrate. When the growth medium contained either glucose or cellobiose in combination with microcrystalline cellulose, there was a lag in cellulose digestion until all of the soluble sugar had been utilized, suggesting an end product feedback mechanism that affects cellulose digestion. Cl-stimulated cellobiosidase and periplasmic cellodextrinase were produced under all growth conditions tested, indicating constitutive synthesis. Both cellobiosidases were cell associated until the stationary phase of growth, whereas proteins antigenically related to the Cl-stimulated cellobiosidase and a proportion of the endoglucanase were released into the extracellular culture fluid during growth, irrespective of the substrate. Immunoelectron microscopy of cells with a polyclonal antibody to Cl-stimulated cellobiosidase as the primary antibody and 10-nm-diameter gold particles conjugated to goat anti-rabbit antibodies as the second antibody revealed protrusions of the outer surface which were selectively labeled with gold, suggesting that Cl-stimulated cellobiosidase was located on the protrusions. These data support the contention that the protrusions have a role in cellulose hydrolysis; however, this interpretation is complicated by reactivity of the antibodies with a large number of other proteins that possess related antigenic epitopes.     

Regulation and distribution of Fibrobacter succinogenes subsp. succinogenes S85 endoglucanases.

The distribution of endoglucanase activities in cultures of Fibrobacter succinogenes subsp. succinogenes S85 grown on different carbon sources was examined by a variety of biochemical and immunological techniques. Total culture endoglucanase activity was primarily cell associated and was expressed constitutively, although synthesis of endoglucanase 1 (EG1) was repressed by cellobiose. Western immunoblotting showed that EG1 and EG3 were released into the culture fluid during growth, while EG2 remained largely associated with the cell. Subcellular localization showed low endoglucanase activity in the periplasmic fraction and similar, high levels in the cytoplasmic and membrane fractions. Western immunoblotting showed that EG2 was absent from the periplasmic fraction. Data from immunoelectron microscopy with either polyclonal or monoclonal antibody to EG2 revealed a high density of gold labeling at sites where there was a disruption in the regular features of the cell surface, such as in blebbing or physical tearing of the membrane. When cells were grown on cellulose, there was a high density of labeling on the cellulose but not on the cells, indicating that EG2 has limited exposure at the cell surface. On the basis of these data, export of enzymes from their intracellular locations appears to occur via three different mechanisms: a specific secretory pathway independent of cellulose, a secretory mechanism which is mediated by contact with cellulose, and a generalized blebbing process that occurs irrespective of the carbon source.