Embryo transfer in the White-tailed deer: A reproductive model for endangered deer species of the world

A model for artificial propagation of wild, seasonal breeding deer was developed using the White-tailed deer, Odocoileus , virginianus . Preseason estrus induction following removal of vaginal pessaries containing medroxyprogesterone acetate (MPA) was successful in four of seven animals. Synchronization of estrus was achieved in seven of nine attempts when does were treated with prostaglandin F(2) alpha (PGF(2)alpha) 7 to 14 d following observed estrus. Superovulation was achieved in two of five does treated with 1000 IU of pregnant mare's serum gonadotropin (PMSG) at the time of vaginal pessary MPA withdrawal. Superovulation was achieved in two of three does treated with 1000 IU of PMSG at the time of estrus induction with PGF(2)alpha. Surgical embryo recoveries attempted on six does resulted in a 68% embryo recovery rate based on numbers of corpora lutea observed. Surgical embryo transfers from two donors to three recipient does resulted in one pregnancy maintained to full term.     

Effects of sepiapterin and 6-acetyldihydrohomopterin on the guanosine triphosphate cyclohydrolase I of mouse, rat and the fruit-fly Drosophila.

The regulation of GTP cyclohydrolase I would lead to the regulation of tetrahydrobiopterin, an important cofactor for synthesis of neurotransmitters. In an attempt to extend a previous finding [Bellahsene, Dhondt, & Farriaux (1984) Biochem. J. 217, 59-65] that GTP cyclohydrolase I of rat liver is inhibited by subnanomolar concentrations of reduced biopterin and sepiapterin, we found that this could not be verified with the enzyme from mouse liver, fruit-fly (Drosophila) heads or, indeed, from rat liver. It was shown, however, that 12 microM-sepiapterin inhibited mouse liver GTP cyclohydrolase I. Another compound, namely 6-acetyldihydrohomopterin, was also employed in the present study to explore its effect on enzymes that lead to its synthesis in Drosophila and for effects on mammalian systems; at 2-5 microM this compound was shown to stimulate one form of mouse liver GTP cyclohydrolase I and then to inhibit at higher concentrations (40 microM). Neither sepiapterin nor 6-acetyldihydrohomopterin caused any effect on the Drosophila head enzyme. On the other hand, the sigmoid GTP concentration curve for the Drosophila enzyme may indicate a regulatory characteristic of this enzyme. Another report, on the lower level of GTP cyclohydrolase I in mutant mouse liver [McDonald, Cotton, Jennings, Ledley, Woo & Bode (1988) J. Neurochem. 50, 655-657], was confirmed and extended. Instead of having 10% activity, we find that the hph-1 mouse mutant has less than 2% activity in the liver. These studies demonstrate that micromolar levels of reduced pterins may have regulatory effects on GTP cyclohydrolase I and that a mouse mutant is available that has low enough activity to be considered as a model for human atypical phenylketonuria.     

The effect of low- and high-density lipoprotein cholesterol on steroid hormone production and ACTH-induced differentiation of rat adrenocortical cells in primary culture

Summary The choroid plexus consists of the choroidal epithelium, a derivative of the neural tube, and the choroidal stroma, which originates from the embryonic head mesenchyme. This study deals with epithelio-mesenchymal interactions of these two components leading to the formation of the organ. Grafting experiments of the prospective components have been performed using the quail-chicken marker technique. Prospective choroidal epithelium of quail embryos, forced to interact with mesenchyme of the body wall of chicken embryos, gives rise to a choroid plexus showing normal morphogenesis and differentiation. The choroidal epithelium induces the differentiation of organtypical fenestrated capillaries, which are highly permeable to intravenously injected horseradish peroxidase. The choroidal epithelium of the grafts constitutes a blood-cerebrospinal fluid barrier. On top of the choroidal epithelium, there are epiplexus cells displaying a typical ultrastructure. The experimental results show that these cells do not originate from the transplanted neural epithelium. Prospective choroidal stroma of chicken embryos does not exert a choroid plexus-inducing influence upon a quail embryo's neural epithelium isolated from parts of the brain that normally do not develop a choroid plexus. The experiments show that the choroidal epithelial cells are determined at least three days before the first organ anlage is detectable.This work was supported by the Deutsche Forschungsgemeinschaft (grant Ch 44/7-1)     

Plasmids in the bacterial assemblage of a dystrophic Lake: Evidence for plasmid-encoded nickel resistance

Sixty-two aerobic bacterial strains isolated from the unproductive dystrophic Lake Skärshultsjön (South Sweden) were screened for plasmids. The lake is considered to be an extreme environment because of its high concentration of persistent but nontoxic humic compounds. One-third of the isolates harbored multiple plasmids usually of similar high molecular weights (>25 Mdal). The plasmid-bearing strains were members of the common aquatic taxaPseudomonas spp.,Acinetobacter sp.,Alcaligenes sp.,Aeromonas/Vibrio group, andEnterobacteriaceae (taxonomy is tentative). The majority of isolates displayed multiple resistance to antibiotics and heavy metals. Some of them were capable of degrading aromatic compounds. Three isolates were chosen for curing experiments. Only strain S-68, anAlcaligenes sp., could be cured of one of its two plasmids. It harbored the two cryptic plasmids pQQ32 and pQQ70 of 32 and ca. 70 Mdal, and the latter was segregated during ethidium bromide treatment. Parental strain S-68 was capable of degrading some of nonchlorinated phenolic compounds and displayed resistance to a broad spectrum of antibiotics and the heavy metals Co²⁺, Ni²⁺, Zn²⁺, Cd²⁺, and Hg²⁺. Derivative strain S-68-41 lost its resistance to nickel, suggesting segregated plasmid PQQ70 coded for nickel resistance. Transformation experiments to restore nickel resistance in the cured derivative strain were not successful.     

Protease activity and proteolytic modification of cellulases from a Trichoderma reesei QM 9414 selectant

Summary The secretion of multiple forms of cellulolytic enzymes by a Trichoderma reesei QM 9414 selectant exhibiting high protease activity (T. reesei QM 9414/A 30) was investigated using monoclonal, domain-specific antibodies against cellobiohydrolase (CBH) I, CBH II and -glucosidase, and a polyclonal antibody against endoglucanase I. The pattern of appearance of these proteins was followed during growth of the fungus on Avicel cellulose, using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)/Western blotting/immunostaining. Evidence was obtained that, at late cultivation stages, CBH I and II became partially modified to lower molecular weight components, whereas -glucosidase and endoglucanase I appeared to remain largely intact. Modification of CBH I appeared to commence from the carboxy-terminal AB region, whereas CBH II appeared to become modified both from the amino- (ABB') and the carboxy-terminal. Evidence for a protease activity that modifies the already truncated cellobiohydrolases in the culture filtrate was obtained. These results show that proteolysis at late culture stages may contribute to the multiplicity of cellulases found in T. reesei culture fluids. Initial proteolytic cleavage of CBH I and II may, however, involve an unusual protease not detectable by the azocasein method.Offprint requests to: C. P. Kubicek     

Human B cell lines can be triggered to secrete an interleukin 2-like molecule

To determine whether human B cells can be triggered to secrete interleukin 2 (IL-2), 19 tumor cell lines derived from patients with undifferentiated lymphomas of Burkitt's and non-Burkitt's types and 6 normal lymphoblastoid cell lines were tested. Cells were grown in the presence or absence of the new tumor promoter teleocidin, and culture supernatants were assayed for IL-2 activity using the standard CTLL-2 assay. Teleocidin (10 ng/ml) triggered IL-2 secretion in 7/8 (87%) EBV-negative lymphoma cell lines of American origin and in 6/6 (100%) normal lymphoblastoid cell lines, but in only 1/6 (16%) EBV-positive tumor cell lines of American origin. Teleocidin had no effect on 5/5 (0%) African Burkitt's cell lines. IL-2 secretion was not detected in control supernatants. IL-2 secretion correlated with the induction of IgM secretion and was linked to both EBV status and karyotype. The following similarities in the functional biological characteristics of T cell and B cell IL-2 suggest that B cell IL-2 is not a factor which mimics IL-2 activity in the CTLL-2 assay: (i) neutralization of IL-2 by anti-IL-2 monoclonal antibody (DMS-1); (ii) elution of IL-2 following its adsorption to CTLL-2 cells; (iii) determination of the MW of IL-2 by SDS-PAGE and Western blot analysis; and (iv) ability of B cell IL-2 to support T cell proliferation and blocking of this activity by anti-tac monoclonal antibody. cDNA probes for T cell IL-2, however, did not detect IL-2 mRNA in B cells. The cell lines were also found to constitutively express IL-2 receptors detected by anti-tac monoclonal antibody, and to secrete soluble IL-2 receptors measured by ELISA. Our results imply that under certain circumstances, B cells can be triggered to secrete IL-2 or an IL-2-like molecule and thus influence T cell activation and proliferation.     

Synthesis of an affinity ligand ('UPHIT') for in vivo acylation of the kappa-opioid receptor

The isothiocyanate analog (1S,2S-trans-2-isothiocyanato-4,5-dichloro-N- methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide, 3a) of the highly selective kappa-opioid receptor agonist, U50,488, was prepared as a potential site-directed affinity ligand for acylation of kappa-opioid receptors in vivo. The isothiocyanate (3a) which we have designated UPHIT and its enantiomer (3b) were synthesized in 3 steps starting from optically pure (1S,2S)-(+)-trans-2-pyrrolidinyl-N-methyl-cyclohexylamine (4a) and its enantiomer (4b), respectively, thus defining their absolute stereochemistry. Binding in vitro of the 1S,2S enantiomer 3a to kappa receptors labelled by [3H]U69,593 was shown to occur with an IC50 value of 25.92 +/- 0.36 nM, whereas 827.42 +/- 5.88 and 115.10 +/- 1.23 nM were obtained for the IC50 value of the 1R,2R enantiomer (3b) and (+/-)-3 respectively. Intracerebroventricular (ICV) injection of 100 micrograms of (+/-)-3 into guinea-pig brain followed by analysis of remaining kappa-binding sites 24 h later revealed that (+/-)-3 depleted 98% of the kappa receptors that bind [3H]U69,593 and 40% of those that bind [3H]bremazocine. These preliminary data suggest exciting uses for these compounds in furthering our knowledge of the kappa-opioid receptor.     

Heritability of bone mass: a longitudinal study in aging male twins

Midshaft radial bone mass was first measured from 1970 through 1972 by photon absorptiometry in 42 pairs of monozygotic (MZ) and 38 pairs of dizygotic (DZ) male Caucasian twins (age 44-55 years). The MZ intraclass correlation (rMZ) of .70 was significantly larger (P less than .05) than the DZ correlation (rDZ) of .45, providing evidence for genetic influences (Smith et al. 1973). Radial bone mass measurements repeated 16 years later (1986-87) on 25 of the MZ pairs and on 21 of the DZ pairs revealed an rMZ of .61 and an rDZ of .44, but the difference was not significant (P greater than .05). The twins had an average radial mass loss of 0.49%/year between the two examinations. The rMZ (.52) and rDZ (.49) values for the 16-year loss in radial mass were both significantly different from zero, but their similar size indicated that the correlations were due to nongenetic factors. In a search for the source of genetic influences on adult radial mass, heritability was estimated by the formula 2(rMZ - rDZ) for radial width and was found to be .66 and .76 (P less than .05) for examinations 1 and 2, respectively. An index of radial density (mass/width) was calculated, and the differences between rMZ and rDZ were not significant at either examination. The intraclass correlations (rMZ = .35; rDZ = .43) were both significant for the loss of bone density between examinations but provided no evidence for genetic influences, results similar to the findings for the loss of mass.(ABSTRACT TRUNCATED AT 250 WORDS)     

Repetitive recycling of guanosine triphosphate cyclohydrolase I for synthesis of dihydroneopterin triphosphate

A procedure for enzymatic production of dihydroneopterin triphosphate is described that allows GTP cyclohydrolase I to be reused repetitively. The reaction takes place in an ultrafiltration cell, and the product is collected in the filtrate, whereas the enzyme remains in the cell to be reused with additional substrate. This is repeated until the enzyme activity drops below a desirable level. The purity of the dihydroneopterin triphosphate is satisfactory for utilization of this compound for studies on enzymes involved in the synthesis of tetrahydrobiopterin and drosopterin. A procedure for purification of dihydroneopterin triphosphate is described that uses C18-silica and silica cartridges.     

Secondary structures of rat lipolytic enzymes: circular dichroism studies and relation to hydrophobic moments

To explore the secondary structures of lingual and pancreatic lipases, circular dichroism measurements were performed. Maximum average ellipticities were used to calculate the percentage of alpha-helices, beta-sheets, and random coils. Lingual lipase had an ellipticity of -20235 +/- 140 deg cm2/dmol (mean +/- SE) at 220 nm suggesting 60% alpha-helix, 20% beta-sheet and 20% random coil structure, but the mean ellipticity for pancreatic lipase was -14093 +/- 82 deg cm2/dmol (mean +/- SE) at 210 nm suggesting a 34.8% alpha-helical, 25% beta-sheet and 40% random coil secondary structure. An alpha-helical stretch of residues with a large hydrophobic moment ("globular" alpha-helix by hydrophobic moment plot) from amino acids 382 through 389 at the COOH-terminal end of lingual lipase was noted. This sequence, absent in pancreatic lipase, may account for the avid binding of lingual lipase to fat emulsion particles.