CS-A therapy in MRL-lpr/lpr mice: amelioration of immunopathology despite autoantibody production

MRL-lpr/lpr mice spontaneously develop massive T cell lymphadenopathy, autoantibodies, and immune-mediated pathology. These mice are thought to be models of various human autoimmune diseases, including systemic lupus, Sjogren's syndrome, and rheumatoid arthritis. We have used cyclosporin A (CS-A) treatment as a tool by which the mechanisms of immune-mediated pathology might be dissected. CS-A was used because of its known preferential inhibition of T cell function and the marked expansion in MRL-lpr/lpr mice of an unusual L3T4-, Lyt-2-, 6B2+ T cell population. CS-A prevented lymphadenopathy and expansion of L3T4-, Lyt-2-, 6B2+ T cells in the peripheral lymph nodes, and also in the thymus. The increased expression of the c-myb and T cell receptor beta-chain genes associated with these unusual cells was also corrected. The finding of increased numbers of L3T4-, Lyt-2-, 6B2+ thymocytes in untreated mice suggests abnormal intrathymic differentiation in lpr/lpr mice, a defect that was corrected by CS-A. Treated mice had a marked decrease in arthritis and glomerulonephritis and significantly prolonged survival. These beneficial effects of CS-A occurred despite a lack of reduction in antibodies reactive with DNA, circulating immune complexes, rheumatoid factor titers, or immunoglobulin concentrations. These results demonstrate that the B cell hyperactivity of MRL-lpr/lpr mice can proceed without the T cell proliferative disease.     

Cloning and nucleotide sequence of the Salmonella typhimurium LT2 gnd gene and its homology with the corresponding sequence of Escherichia coli K12

Summary The complete nucleotide sequence of the Salmonella strain LT2 gnd gene for 6-phosphogluconate dehydrogenase was determined. The gene contains 1404 bases and encodes a 468 amino acid polypeptide, which is the same as for Escherichia coli K12. The DNA sequence shows 14.8% difference between the two and the amino acid sequence 3.6% difference. Changes are mostly in the third codon base and most of the amino acid changes are conservative.     

Liming and deep ripping responses for a range of field crops

Grain yields were measured over 2 seasons from a range of field crops following liming and deep ripping an acid and compacted soil in north-eastern Victoria. Lime (2.5 t ha^–1) substantially reduced the level of exchangeable Al and exchangeable Mn whilst raising soil pH by about 1.0 unit. The crops grown were 7 cultivars of wheat and one cultivar each of triticale, oats, barley, rapeseed, safflower, field pea, chick pea and lupins. With the exception of lupin, liming the soil increased (p=0.05) the grain yield of all crops and cultivars. With the wheat cultivars there were 2 distinct groups with different tolerance to soil acidity. Wheat, oats, triticale and lupins had higher absolute yields than the other crops. Safflower and chick pea had very low yields without soil amendment. The magnitude of the lime response did not differ between the wheat cultivars (17%) or between any of the crop species (range 9–29%). Deep ripping the soil to break a hard compacted layer resulted in more yield for all the cereals and safflower. The results demonstrate the importance of using crops with tolerance to acid soil conditions as well as gains that can be obtained with ameliorating identifiable soil problems.     

Phosphorylation of Escherichia coli enolase

In vivo labeling of Escherichia coli JA200 pLC 11-8 resulted in 32P incorporation into enolase as demonstrated by immunoaffinity chromatography and electrophoresis followed by autoradiography. Complete acid hydrolysis, followed by thin layer chromatography was employed for determination of the phosphoamino acid residue. Comparison with phosphoamino acid standards resulted in the identification of a labeled residue corresponding to phosphoserine. In vitro labeling of cell extracts from glucose and acetate grown cells resulted in differential labeling of enolase. When specific radioactivities of in vivo labeled enolase were compared, 7 times more label was incorporated at late log phase in glucose grown cells than in late log acetate grown cells. At stationary phase, only 2.5 times more label was incorporated into glucose compared to acetate. When 32P-labeled enolase from glucose grown cells was subjected to treatment with potato acid phosphatase, dephosphorylation of the enzyme could be observed. Monitoring enzyme activity during the acid phosphatase treatment revealed a 70% decrease for the forward enzyme reaction, and a 3-fold increase, followed by a gradual decrease to almost zero, for the reverse enzyme reaction. Complete reversal of the changes in activity was possible by adding an aliquot of partially purified enolase kinase plus ATP.     

Use of a synthetic dodecapeptide (malantide) to measure the cyclic AMP-dependent protein kinase activity ratio in a variety of tissues.

1. The cyclic AMP-dependent protein kinase activity-ratio assay was investigated by comparing histone and a synthetic peptide, malantide [Malencik & Anderson (1983) Anal. Biochem. 132, 32-40], as substrates. 2. In several tissues the activity ratio was higher when assayed with histone as the substrate; this result was obtained in control tissues and also in those incubated with agents known to increase cyclic AMP. The effect of these agents to increase the activity ratio was more clearly demonstrated with malantide. 3. The higher activity ratios observed with histone are due to: (a) measurement of phosphorylation not catalysed by cyclic AMP-dependent protein kinase; (b) activation of cyclic AMP-dependent protein kinase by histone during the assay. 4. When tissues were homogenized in buffers without NACl, lower activity ratios were found, owing to the catalytic subunit being artifactually removed from the supernatant. 5. We conclude that the measured activity ratio more faithfully reflects that in the tissue when NaCl is included in the homogenization buffer and malantide is used in the assay. This was confirmed in experiments where cyclic AMP-dependent protein kinase was added to the tissue before homogenization, and no dissociation of the exogenous enzyme was observed.     

Evidence of histidine phosphorylation in isocitrate lyase from Escherichia coli

Escherichia coli isocitrate lyase (EC 4.1.3.1.) can be phosphorylated in vitro by an ATP-dependent reaction. The enzyme becomes phosphorylated by an endogenous kinase when partially purified sonic extracts are incubated with [gamma-32P]ATP. Treatment of isocitrate lyase with diethyl pyrocarbonate, a histidine-modifying reagent, blocked incorporation of [32P]phosphate from [gamma-32P]ATP. The isoelectric point of the enzyme was altered by treatment with phosphoramidate, a histidine phosphorylating agent, which suggests that isocitrate lyase can be phosphorylated at a histidine residue(s). Immunoprecipitated 32P-labeled isocitrate lyase was subjected to alkaline hydrolysis, mixed with chemically synthesized phosphohistidine standards, and analyzed by anion exchange chromatography. Characterization of the phosphoamino acid was based on the demonstration that the 32P-labeled product from alkali-hydrolyzed isocitrate lyase comigrated with synthetic 1-phosphohistidine. In addition, loss of catalytic activity after treatment with potato acid phosphatase indicates that catalytically active isocitrate lyase is the phosphorylated form of the enzyme.     

Properties of phosphorylated NADP+-specific glutamate dehydrogenase fromEscherichia coli

The NADP⁺-specific glutamate dehydrogenase (GDH) fromEscherichia coli strain D₅H₃G₇, an enzyme that catalyzes the interconversion of -ketoglutarate andl-glutamate, has been shown to be phosphorylated in vitro in an ATP-dependent enzymatic reaction. The phosphorylated protein is extremely acid labile and is unstable at high pH. Treatment of GDH with diethyl pyrocarbonate (DEP), a histidine-modifying reagent, blocked the incorporation of³²P from [-³²P]ATP. GDH catalytic activity was also inhibited by DEP treatment. Hydroxylamine, a reagent hydrolyzing phosphoramidates, catalyzed the removal of phosphate from phosphorylated GDH, suggesting that GDH may be phosphorylated at a histidine residue(s). A total enzymatic hydrolysis of phosphorylated GDH, which was electroeluted from a native polyacrylamide gel, was analyzed by a Dowex 1-8X anion exchange chromatography. The presence of³²P-labeled 3-phosphohistidine, characterized and identified from this hydrolysate, demonstrates that a histidine residue(s) is the site of phosphorylation.     

Comparative studies of nickel,cobalt, and copper uptake by some nickel hyperaccumulators of the genus Alyssum

The uptake of Ni, Co, and Cu by the nickel hyperaccumulator Alyssum troodii Boiss and the non-accumulator Aurinia saxatilis (L.) Desv. were studied in pot trials using artificial rooting media with varying concentrations of the metals added as soluble salts, singly and in combination. The ability of five other Ni hyperaccumulating species of Alyssum to hyperaccumulate Co was also investigated.Leaves and stems of A. troodii accumulated Ni to almost the same extent (8000–10 000 g g^-1). In roots, the highest Ni concentration was 2000 g g^-1. In leaves of Au. saxatilis, the maximum Ni concentration was only 380 g g^-1 and the level in roots was even lower.In media containing Co, the maximum concentration of this element in A. troodii (2325 g g^-1) was ten times higher than in the non-accumulator species. Slightly less Co was found in stems and roots of both species. Among the other Ni hyperaccumulators, the maximum concentration of Co in leaves ranged from about 1000–8000 g g^-1.Copper concentrations were the same in all organs of both species when they were grown in copper-rich media and were in the range 40–80 g g^-1, showing that neither plant was capable of taking up Cu at levels comparable to those of Ni and Co.When both plants were grown in media containing equal amounts of both Co and Ni, the Co concentrations in plant organs were the same as for specimens grown in media containing Co only. However, the Ni levels were lower in both species. Uptake of Co therefore appeared to suppress Ni uptake.Pot trials showed that the order of tolerance was Ni>Cu>Co for A. troodii and Ni>CoCu for Au. saxatilis, whereas the seedling tests showed the order to be Co>Ni>Cu. At metal concentrations 10 000 g g^-1, the overall tolerance of A. troodii was greater than that of Au. saxatilis which exhibited equally low tolerance to Ni and Cu.We conclude that in A. troodii, A. corsicum Duby, A. heldreichii Hausskn., A. murale Waldstein & Kitaibel, A. pintodasilvae T.R. Dudley, and A. tenium Hálácsy, Ni tolerance and hyperaccumulation conveys the same character towards Co. This behaviour should be investigated in other hyperaccumulators of Ni and/or Co.     

Quantity and quality of embryos collected from mice passively immunized against estradiol

The objective of this study was to determine the effects of passive immunization against estradiol on the occurrence and timing of estrus, ovulation and fertilization rates and on early embryonic development in mice. Swiss Webster fermale mice were randomly assigned to one of the three treatment groups to be injected with 0.1 ml saline (control; n=15), 0.1 ml anti-estradiol antisera (high dose: n=17) or 0.1 ml antiestradiol antisera diluted 1:10 with saline (low dose; n=17) at seven weeks of age. Immediately after injection mice were placed with males and observed daily for the presence of vaginal plugs for 10 d. Three days after vaginal plugs were observed, mice were terminated and the uteri were removed and flushed to determine the number and quality of unfertilized ova and embryos. No differences were observed in the timing of vaginal plug formation, the proportion of mice with vaginal plugs, or the mean number of unfertilized ova or embryos collected from each treatment group. However, the proportion of excellent or good quality embryos was reduced in the high dosage treatment. It was concluded from this study that passive immunization of mice against estradiol did not increase the number of embryos obtained from mice and that a high dosage of antisera against estradiol reduced embryo quality. These results provide evidence that alterations in levels of estradiol may adversely affect embryonic development.