Mechanisms ensuring optimal conditions of implantation and embryo development in the pig

The paper summarizes results of a series of studies concerning luteolysis and early pregnancy in pigs. The involvement of the oxytocin (OT)/OT receptor system in the mechanism of corpus luteum (CL) protection during early pregnancy as well as the implication of luteinizing hormone (LH) in the endometrial prostaglandin (PG) release and synthesis are described. In addition, the role of leptin in the regulation of ovarian steroidogenesis and the expression of leptin and its receptor (OB-Rb) genes in hypothalamus, pituitary and reproductive tissues are reported. Moreover, a strong emphasis was placed on the mechanism of PGE2 participation in the local endocrine regulations of reproductive processes occurring in the utero-ovarian area as well as on the vascular endothelial growth factor (VEGF) ligand-receptor system in the ovary and uterus.     

Synthesis of siderophores by strains of Staphylococcus cohnii isolated from various environments

Siderophore activity as the feature of microorganisms enabling colonization of human body and the survival in inanimate environment was investigated in 108 strains of Staphylococcus cohnii; S. cohnii ssp. cohnii (50 strains) and S. cohnii ssp. urealyticus (58 strains). Strains were isolated from people, hospital and non-hospital environment. Highest siderophore activity was noted in strains S. cohnii ssp. urealyticus particularly from the inanimate environments origin. In 86% analyzed strains siderophores of hydroxamate class were detected. Larger amounts of these compounds were synthesized in strains S. cohnii ssp. urealyticus. Strains belonging to both subspecies from human origin showed lower activity of siderophores (total pool) and did not produce hydroxamate class chelators or produced very small amounts of these compounds.     

Transformations of testosterone and related steroids by Botrytis cinerea

Botrytis cinerea (strain AM235) was used to investigate the transformations of testosterone and related steroids. It was found that the position and stereochemistry of the introduced hydroxyl group, as well as the yield of products, depended on the structure of the substrate. Botrytis cinerea converts the examined substrates mainly to 7 alpha-hydroxy derivatives. 1-Dehydrotestosterone was also significantly hydroxylated at a 14 alpha-position.     

Multiple forms of arylalkylamine N-acetyltransferase (NAT) from cockroach female colleterial glands and activity changes during oocyte maturation

Arylalkylamine N-acetyltransferase (NAT) from the female colleterial glands of Periplaneta americana showed activity peaks at pH 6.0 and 9.5 and the pH profile changed during oogenesis. The left gland contained higher activity than the right gland but the right gland also contained recognizable activity. The patterns in activity change depended on the substrate used, tryptamine (TN) or serotonin (5-HT). When TN was used as the substrate, the alkaline peak was higher than the acidic peak. In contrast, when 5-HT was used, the acidic peak was much higher than the alkaline peak. This suggests that at least two NATs are present in this species that are specific to pH and substrate species. Of the four combinations of the two pH ranges and two substrate indolamines, the enzyme activity that showed a similar change to the oocyte maturation was obtained in the combination of pH 6.0 and TN. TN was actually detected in the colleterial glands by fluorescent measurements according to Hess and Uderfriend [J. Pharmacol. Exp., 127 (1959) 175-177]. It peaked on the 6th day of emergence, which corresponded to the first rise of oocyte length and yolk accumulation, whereas a small peak appeared in the phase of the second rise. TN, or more likely N-acetyl TN, may therefore be involved in the regulation of oocyte maturation which could be a novel mechanism in oocyte maturation.     

Structure of formamidopyrimidine-DNA glycosylase covalently complexed to DNA

Formamidopyrimidine-DNA glycosylase (Fpg) is a DNA repair enzyme that excises oxidized purines from damaged DNA. The Schiff base intermediate formed during this reaction between Escherichia coli Fpg and DNA was trapped by reduction with sodium borohydride, and the structure of the resulting covalently cross-linked complex was determined at a 2.1-A resolution. Fpg is a bilobal protein with a wide, positively charged DNA-binding groove. It possesses a conserved zinc finger and a helix-two turn-helix motif that participate in DNA binding. The absolutely conserved residues Lys-56, His-70, Asn-168, and Arg-258 form hydrogen bonds to the phosphodiester backbone of DNA, which is sharply kinked at the lesion site. Residues Met-73, Arg-109, and Phe-110 are inserted into the DNA helix, filling the void created by nucleotide eversion. A deep hydrophobic pocket in the active site is positioned to accommodate an everted base. Structural analysis of the Fpg-DNA complex reveals essential features of damage recognition and the catalytic mechanism of Fpg.     

Use of recombinant cellulose-binding domains of Trichoderma reesei cellulase as a selective immunocytochemical marker for cellulose in protozoa

Some unicellular organisms are able to encyst as a protective response to a harmful environment. The cyst wall usually contains chitin as its main structural constituent, but in some cases, as in Acanthamoeba, it consists of cellulose instead. Specific cytochemical differentiation between cellulose and chitin by microscopy has not been possible, due to the similarity of their constituent beta-1,4-linked hexose backbones. Thus, various fluorescent brightening agents and lectins bind to both cellulose and chitin. We have used a recombinant cellulose-binding protein consisting of two cellulose-binding domains (CBDs) from Trichoderma reesei cellulases linked together in combination with monoclonal anticellulase antibodies and anti-mouse immunoglobulin fluorescein conjugate to specifically stain cellulose in the cysts of Acanthamoeba strains for fluorescence microscopy imaging. Staining was observed in ruptured cysts and frozen sections of cysts but not in intact mature cysts. No staining reaction was observed with the chitin-containing cyst walls of Giardia intestinalis, Entamoeba dispar, or Pneumocystis carinii. Thus, the recombinant CBD can be used as a marker to distinguish between cellulose and chitin. Thirteen of 25 environmental or clinical isolates of amoebae reacted in the CBD binding assay. All 13 isolates were identified as Acanthamoeba spp. Five isolates of Hartmannella and seven isolates of Naegleria tested negative in the CBD binding assay. Whether cyst wall cellulose really is a unique property of Acanthamoeba spp. among free-living amoebae, as suggested by our findings, remains to be shown in more extensive studies.     

The phenylketonuria G272X haplotype 7 mutation in European populations

We have compiled data on the frequencies of the phenylketonuria G272X mutation in European populations. This mutation occurs north of the Alps. It has a particularly high frequency in the Oslo Fjord region of Norway with the adjacent Bohuslän region of Sweden. An intermediate frequency was noted in a separate area, the eastern part of Germany with the adjacent western part of Czechoslovakia. The G272X mutation was associated with phenylalanine hydroxylase haplotype 7, except for one case with haplotype 3. Genealogical studies going back eight to nine generations revealed no common source for this mutation, but there was some geographical convergence to the Bohuslän region. These findings suggest a single origin for this mutation, with at least one founding population in south-eastern Norway/adjacent Sweden.     

Selective isolation and purification of tat protein via affinity membrane separation

This work deals with the separation of Tat protein from a complex fermentation broth using an affinity membrane system. Tat is a regulatory protein that is critical for HIV-1 replication and thus a potential candidate for vaccine and drug development. Furthermore, Tat can facilitate transport of exogenous molecules across cell membranes and is implicated in pathogenesis of HIV dementia. Affinity membranes were prepared through coupling of avidin within a 4-stack membrane construct. Tat (naturally biotinylated) accessibility in the bacterial lysate feed was influenced by the presence of RNAse, protein concentration, and ionic strength. Enhanced accessibility translated to a marked increase in the overall product yield per pass. The purity of the membrane-isolated Tat was compared to that prepared via packed column chromatography through SDS-PAGE, Western blot, activity assay, and neurotoxicity studies. Tat protein produced via membrane separation yielded primarily monomeric forms of the oligopeptide sequence, whereas column chromatography produced predominately polymeric forms of Tat. These differences resulted in changes in the neurotoxicity and cellular uptake of the two preparations.     

Rearrangement of 23-oxospirostanes to the 22-oxo-23-spiroketal isomers promoted by Lewis acids--X-ray crystal structure of (23R,25S)-3beta-acetoxy-16beta,23:23,26-diepoxy-5beta-cholestan-22-one

Both 25R and 25S 23-oxospirostanes undergo rearrangement to the 22-oxo-23-spiroketal isomers promoted by Lewis acids. An X-ray crystal structure analysis of the rearranged product of 23-oxosarsasapogenin acetate confirmed the R configuration at the new spiro carbon atom.