Autophosphorylation is required for high kinase activity and efficient transformation ability of proteins encoded by host range alleles of v-src.

pp60v-src is a nonreceptor protein tyrosine kinase that can transform both chicken and rodent fibroblasts. The src homology 2 (SH2) domain of this protein serves a critical role in the regulation of protein tyrosine kinase activity. The host range proteins pp60v-src-L, which contains a deletion of a highly conserved residue (Phe-172) in the SH2 domain, and pp60v-src-PPP, which contains a change from a Leu to a Phe at amino acid 186 in the SH2 domain, transform chicken but not rat cells and have slightly reduced kinase activity measured in vitro. The data presented here show that these altered proteins require autophosphorylation on Tyr-416 for high kinase activity and transforming ability. In the absence of autophosphorylation, there is a further decrease of at least threefold in in vitro kinase activity relative to the phosphorylated host range parental protein, no morphological transformation, a reduction in anchorage independent growth, and no disruption of the actin cytoskeleton. In addition, these SH2 mutations abolish the ability of the SH2 domain to bind a phosphorylated peptide that corresponds to the autophosphorylation site of pp60src. Thus, like mutant alleles of c-src encoding transformation competent proteins, and unlike v-src, transformation by pp60v-src-F172 delta and pp60v-src-L186F is dependent on phosphorylation of Y-416 for high kinase activity and transformation ability. The dependence of transformation on phosphotyrosine is not a reflection of an intramolecular interaction between the autophosphorylation site and the SH2 domains since purified SH2 domains are incapable of binding phosphorylated autophosphorylation site peptides in vitro.     

Corpus luteal function in nonpregnant mares following intrauterine administration of prostaglandin E(2) or estradiol-17beta

The objective of this study was to test the hypothesis that intrauterine administration of prostaglandin E(2) (PGE(2)) or estradiol-17beta (E-17beta) would prolong CL function in nonpregnant mares. Nonpregnant mares were continuously infused with 240 mug/d of PGE(2), 6 mug/d of E-17beta, or vehicle (sham-treated) on Days 10 to 16 post ovulation (ovulation = Day 0), using osmotic minipumps surgically placed into the uterine lumen on Day 10 (n = 11 per group). Nonpregnant and pregnant mares served as negative and positive controls, respectively (n = 11 per group). Mares were defined as having prolonged CL function if plasma progesterone remained > 2.5 ng/ml and if ovulation did not occur on Days 9 to 30. Corpus luteal function was prolonged until Day 30 in 1 11 nonpregnant mares, 4 11 sham-treated mares, 6 11 E-17beta-treated mares, 8 11 PGE(2)-treated mares, and 11 11 pregnant mares. The incidence of prolonged CL function was similar (P=0.16) in the sham-treated and nonpregnant mares. The hypothesis that PGE(2) would prolong CL function in nonpregnant mares was supported, since the incidence of prolonged CL function was higher (P=0.003) in PGE(2)-treated versus nonpregnant mares, tended to be higher (P=0.09) in PGE(2)-versus sham-treated mares, and was not lower (P=0.11) in PGE(2)-treated versus pregnant mares. The hypothesis that E-17beta would prolong CL function in nonpregnant mares was not supported, since the incidence of prolonged CL function was not higher (P=0.34) in E-17beta-versus sham-treated mares, and was lower (P=0.02) in E-17beta-treated versus pregnant mares. These results demonstrate that intrauterine administration of a pharmacologic dose of PGE(2) initiated prolonged CL function in nonpregnant mares. Further experiments are needed to confirm the role of conceptus secretion of PGE(2) in CL maintenance, and to determine the mechanism of action of PGE(2) within the equine reproductive tract.     

Comparison of reactors for oxygen-sensitive reactions: reductive dechlorination of chlorophenols by vitamin b(12s)

Serum bottles are frequently used for studies of reductive dechlorination by vitamin B(12), but reducing conditions can be maintained only for several days. This time period is inadequate for evaluating the reductive dechlorination of some slow-reacting aromatic compounds. Sealed glass ampoules maintain reducing conditions for many months, but this method has the disadvantage of disallowing subsampling of the reaction mixture. A glass serum tube was modified for these experiments which not only maintained anoxic conditions for several days but also allowed subsamples to be removed during experiments. The modification was a restriction placed in the middle of the tube by heating in a flame, creating two chambers separated by a narrow neck. The lower chamber contained the oxygen-sensitive reaction mixture. The upper chamber, sealed with a septum and screw cap, was purged with purified nitrogen or argon introduced and vented through fused silica capillaries. Reductive dechlorination of chlorophenols by vitamin B(12) reduced with Ti(III) citrate was monitored in all three reactor types. Sealed ampoules maintained reducing conditions for up to 12 months. The two-chambered reactor maintained reducing conditions longer than the serum vials when frequent samples were taken.     

Role of the rfe gene in the synthesis of the O8 antigen in Escherichia coli K-12.

The Escherichia coli O8 antigen is a mannan composed of the trisaccharide repeat unit -->3)-alpha-Man-(1-->2)-alpha-Man-(1-->2)-alpha-Man-(1--> (K. Reske and K. Jann, Eur. J. Biochem. 67:53-56, 1972), and synthesis of the O8 antigen is rfe dependent (G. Schmidt, H. Mayer, and P. H. Mäkelä, J. Bacteriol. 127:755-762, 1976). The rfe gene has recently been identified as encoding a tunicamycin-sensitive UDP-GlcNAc:undecaprenylphosphate GlcNAc-1-phosphate transferase (U. Meier-Dieter, K. Barr, R. Starman, L. Hatch, and P. D. Rick, J. Biol. Chem. 267:746-753, 1992). However, the role of rfe in O8 side chain synthesis is not understood. Thus, the role of the rfe gene in the synthesis of the O8 antigen was investigated in an rfbO8+ (rfb genes encoding O8 antigen) derivative of E. coli K-12 mutant possessing a defective phosphoglucose isomerase (pgi). The in vivo synthesis of O8 side chains was inhibited by the antibiotic tunicamycin. In addition, putative lipid carrier-linked O8 side chains accumulated in vivo when lipopolysaccharide outer core synthesis was precluded by growing cells in the absence of exogenously supplied glucose. The lipid carrier-linked O8 antigen was extracted from cells and treated with mild acid in order to release free O8 side chains. The water-soluble O8 side chains were then purified by affinity chromatography using Sepharose-bound concanavalin A. Characterization of the affinity-purified O8 side chains revealed the occurrence of glucosamine in the reducing terminal position of the polysaccharide chains. The data presented suggest that GlcNAc-pyrophosphorylundecaprenol functions as the acceptor of mannose residues for the in vivo synthesis of O8 side chains in E. coli K-12.     

Differential cellular effects in the toxicity of haloalkene and haloalkane cysteine conjugates to rabbit renal proximal tubules

The relationship between the covalent binding, uptake, and toxicity produced by S-(1,2-dichlorovinyl)-L-cysteine (DCVC) and S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (TFEC) was investigated in suspensions of rabbit renal proximal tubules (RPT). The DCVC and TFEC at concentrations of 25 μM produced a time-dependent (1–6 hours) loss of RPT viability. The TFEC was bio-transformed rapidly by β-lyase to a reactive metabolite which bound covalently to tubular protein. Approximately 63% of the TFEC-equivalents inside the cell were bound to protein. Covalent binding of TFEC-equivalents was associated with a 30% decrease in tubular basal and state 3 respiration, a sevenfold increase in lipid peroxidation, and, ultimately, cell death. The DCVC was biotransformed rapidly to a reactive metabolite which bound covalently to tubular protein. Approximately 90% of the DCVC-equivalents inside the cell were bound covalently to tubular protein. Following exposure to 25 μM DCVC, the binding of DCVC-equivalents was associated with a 17-fold increase in lipid peroxidation but, in contrast to TFEC, had no effect on tubular respiration. However, exposure of RPT to 100 μM DCVC resulted in a ninefold increase in the binding of DCVC- equivalents and a 30% decrease in tubular state 3 respiration. The β-lyase inhibitor aminooxyacetic acid (AOAA) blocked the covalent binding, mitochondrial dysfunction, lipid peroxidation, and cell death produced by TFEC. The AOAA decreased the covalent binding and the lipid peroxidation produced by DCVC by approximately 60–70% but had no effect on cell death. These results suggest that mitochondria! bioactivation of TFEC by β-lyase is critical for TFEC-induced mitochondrial dysfunction and the resulting cell death. These results also suggest that cytosolic bioactivation and binding, but not mitochondrial bioactivation and dysfunction, are important in the toxicity produced by DCVC to rabbit RPT. The lack of protection against DCVC toxicity by AOAA may be related to incomplete inhibition of DCVC metabolism or bioactivation of DCVC by pathways other than β-lyase.     

Uterine transport of prostaglandin E(2)-releasing simulated embryonic vesicles in mares

Transrectal ultrasonography was used to test the hypothesis that prostaglandin E(2) (PGE(2)) would increase the uterine transport of simulated embryonic vesicles in mares. Uterine transport of PGE(2)-releasing (PGE) vesicles, vehicle-releasing (sham) vesicles, and equine embryos was contrasted on Day 12 or Day 13 post ovulation. In Experiment 1, there was no difference (P>0.10) in transport of PGE vesicles, sham vesicles, Day-12 embryos, and Day-12 embryos after cervical manipulation (n = 3 per group). In Experiments 2 and 3, respectively, transport of PGE and sham vesicles was contrasted with transport of Day-13 embryos after the vesicles (1 vesicle per mare) were placed into the uterine lumen with the embryo, (n = 7 per group). In Experiment 2, PGE vesicles were transported less often (P<0.05) from horn to body and from segment to segment than Day-13 embryos before vesicle insertion. In Experiment 3, sham vesicles were transported less often from horn to body (P<0.10) and from segment to segment (P<0.01) than Day-13 embryos before vesicle insertion. However, there was no difference (P>0.10) in the transport of PGE vesicles and embryos (Experiment 2) or sham vesicles and embryos (Experiment 3) together in the uterine lumen. In Experiment 4, transport of PGE and sham vesicles was contrasted by placing them together into the uterine lumen of nonpregnant mares on Day 13 (n = 7). There was no difference (P>0.10) in the transport of PGE and sham vesicles together in the uterine lumen. These results do not support the hypothesis that PGE(2) increases uterine transport of simulated embryonic vesicles. In addition, these results do not support the hypothesis that equine embryos stimulate uterine transport.     

Alkaptonuria in a cynomolgus monkey (Macaca fascicularis)

An eight year old wild caught Cynomolgus monkey was diagnosed as having alkaptonuria, a condition characterized by the passage of a normal-colored urine which darkens upon standing. The underlying cause is the congenital lack of homogentisic acid oxidase with subsequent passage of homogentisic acid in the urine. No other clinical manifestations, such as deposition of pigment in the skin or mucous membranes or development of an ochronitic arthritis, were observed in this animal.     

Analytical applications of guided neutron beams

Biological Trace Element Research - Guided beams of thermal and cold neutrons have become available to analysts at several reactors during the past decade. The very pure beams from these guides...