Characterization of the Butyrivibrio fibrisolvens glgB gene, which encodes a glycogen-branching enzyme with starch-clearing activity.

A Butyrivibrio fibrisolvens H17c glgB gene, was isolated by direct selection for colonies that produced clearing on starch azure plates. The gene was expressed in Escherichia coli from its own promoter. The glgB gene consisted of an open reading frame of 1,920 bp encoding a protein of 639 amino acids (calculated Mr, 73,875) with 46 to 50% sequence homology with other branching enzymes. A limited region of 12 amino acids showed sequence similarity to amylases and glucanotransferases. The B. fibrisolvens branching enzyme was not able to hydrolyze starch but stimulated phosphorylase alpha-mediated incorporation of glucose into alpha-1,4-glucan polymer 13.4-fold. The branching enzyme was purified to homogeneity by a simple two-step procedure; N-terminal sequence and amino acid composition determinations confirmed the deduced translational start and amino acid sequence of the open reading frame. The enzymatic properties of the purified enzyme were investigated. The enzyme transferred chains of 5 to 10 (optimum, 7) glucose units, using amylose and amylopetin as substrates, to produce a highly branched polymer.     

The assessment of cellular proliferation by immunohistochemistry: A review of currently available methods and their applications

Summary Immunohistochemical methods using antibodies to cell cycle-related antigens may be used as a means of assessing various aspects of proliferation in tissue, and have the important advantage of preserving the spatial orientation of proliferating cells in histological sections. Currently, the most widely available antibodies for this purpose are antibodies to bromodeoxyuridine (BrdU), Ki67 and antibodies to proliferating cell nuclear antigen (PCNA). BrdU is a thymidine analogue incorporated during the S phase of the cell cycle, which can be introduced by in vivo administration or by in vitro incubation, and monoclonal antibodies are available to display its localization. Ki67 demonstrates a nuclear antigen expressed in all phases of the cell cycle, except G₀ and early G₁, but can only be applied to frozen tissue. PCNA is a nuclear antigen which is essential for DNA synthesis, two commercially available antibodies to PCNA work in paraffin-embedded tissue, but may have different staining characteristics under different conditions of fixation. The main advantages and disadvantages of these different techniques are discussed, together with their main applications to date.     

Role of the embryonic vesicle and progesterone in embryonic loss in mares.

Characteristics of spontaneous embryonic loss in 21 mares were compared with those of 52 contemporary mares that maintained pregnancy. Embryonic losses were, in retrospect, grouped according to day of loss and length of the interovulatory interval, respectively, as follows: group 1, less than or equal to day 20 and less than or equal to 30 days (n = 10); group 2, less than or equal to day 20 and greater than 30 days (n = 3); and group 3, greater than day 20 and greater than 30 days (n = 8); ovulation was day 0. Mean diameter of the embryonic vesicle in group 1 was smaller (P less than 0.05) on days 12-18 than in the pregnancy-maintained group, but among the pregnancy-maintained group and the embryonic-loss groups, the mean individual growth rates of vesicles was similar (no significant difference). A more frequent (P less than 0.05) location of the vesicles in the uterine body on day 13 in group 1 was due to a greater proportion of small vesicles and for day 18 was due to a greater incidence of fixation failure. Luteal regression occurred at the expected time in 77% of the mares with loss sooner than day 20. Low concentration of progesterone on days 12, 15 and 18, a detected decrease in diameter of the corpus luteum on days 15 and 18, and an interovulatory interval of less than or equal to 30 days indicated that luteolysis was not prevented by the embryonic vesicle in group 1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prostaglandin E2-specific binding to the equine oviduct.

Prostaglandin E2 (PGE2) bound specifically (P less than 0.001) to ampullary and isthmic tissue on Day 2 and Day 5 after ovulation. No significant differences (P greater than 0.8) were detected between Day 2 and Day 5 in the specific binding of ampullary or isthmic tissue. Significantly more (P less than 0.05) PGE2 bound specifically to ampullary versus isthmic tissue on both days. Detection of PGE2-specific binding in the oviductal isthmus on Day 2 and Day 5 indicates that the oviduct is responsive to PGE2 when it is capable of transporting equine embryos.     

Prostaglandin E2 secretion by day-6 to day-9 equine embryos

Prostaglandin E₂ (PGE₂) secreted by Day-6, Day-7, Day-8 and Day-9 equine embryos (ovulation = Day 0) during in vitro incubation was measured by radioimmunoassay. Embryonic PGE₂ secretion (ng/embryo/24 hr) was detectable on Day 6 (0.27±0.39), tended to increase (P <0.1) on Day 7 (0.57±0.88), and increased significantly (P <0.05) on Day 8 (2.23±0.86) and Day 9 (4.13±0.71). Embryo diameter at the start of the incubation period was linearly correlated (P <0.01) to embryonic PGE₂ secretion.     

Purification and Characterization of an Autolysin from Clostridium acetobutylicum

A proteinaceous substance with antibiotic-like activity, resembling that of a bacteriocin, was isolated from an industrial-scale acetone-butanol fermentation of Clostridium acetobutylicum. The substance, purified by acetone precipitation, diethylaminoethyl cellulose chromatography, and polyacrylamide gel electrophoresis, was characterized as a glycoprotein with a molecular weight of 28,000. The glycoprotein was partially inactivated by certain protease enzymes. It had no effect on deoxyribonucleic acid, ribonucleic acid, or protein synthesis, and it did not result in the loss of intracellular adenosine triphosphate. The glycoprotein lysed sodium dodecyl sulfate-treated cells and cell wall preparations, and therefore it is referred to as an autolysin. The autolysin gene appeared to be chromosomal since plasmid deoxyribonucleic acid was not detected in the C. acetobutylicum strain.     

Antibacterial effect of the scandium and indium complexes of enterochelin on Escherichia coli

Enterochelin, the iron chelator produced by a number of pathogenic enterobacteria, appears to be an essential metabolite for multiplication within the host, where it transports iron from the host iron-binding proteins to the bacteria. Previous work showed that complexes of enterochelin containing either scandium (Sc3+) or indium (In3+) exerted a bacteriostatic effect on Klebsiella pneumoniae in serum, whilst the Sc3+ complex exerted a significant therapeutic effect on mice infected with K. pneumoniae. These observations have now been extended to a number of pathogenic serotypes of Escherichia coli including those carrying either the K1 antigen or the ColV plasmid. The Sc3+ and In3+ complexes each exert a bacteriostatic effect on these organisms growing in either whole serum or media containing an iron-binding protein. Evidence is presented that the Sc3+ complex may act as a competitive inhibitor of the Fe3+ complex. In contrast to their effects on K. pneumoniae, sideramines other than enterochelin fail to reverse the bacteriostatic effect of the Sc3+ complex of enterochelin in E. coli, suggesting that the complex produces a more profound derangement of metabolism in this organism. The Sc3+ complex exerts a significant therapeutic effect on E. coli infections in mice although the In3+ complex is less active.