Total and free plasma concentrations of progesterone, cortisol and oestradiol-17 beta during pregnancy, parturition and early lactation in sows

The total (bound plus free) concentrations of progesterone, 20 alpha-dihydroprogesterone, oestradiol-17 beta and cortisol were determined in the plasma of sows at three stages during pregnancy and more intensively from 5 days pre-partum to 5 days post-partum. The free fractions of progesterone, oestradiol-17 beta and cortisol were measured in the same samples by a rate dialysis method. Up to day 110 of gestation, the amounts of free hormone in plasma did not fluctuate independently of their total concentrations. During farrowing, the total and free concentrations of progesterone and cortisol varied independently of each other, whereas total and free oestradiol-17 beta declined simultaneously. The initiation of parturition was associated with a decrease in circulating total progesterone, and was accentuated by a decrease in the free fraction (P less than 0.005) so that its active free concentration was only 20% of its day 1 pre-partum value. Total and free cortisol concentrations rose rapidly during labour so that at 12-18 h after birth of the first piglet 30% of that cortisol in maternal plasma was free hormone.     

Adrenocortical suppression in workers manufacturing synthetic glucocorticoids.

A man who had worked for 16 years in the manufacture of a potent corticosteroid was found to be suffering from chronic adrenocortical insufficiency attributed to chronic absorption of the glucocorticoid. Eleven other symptom-free workers were therefore screened. Two of these workers, like the first patient, gave grossly abnormal responses to the Synacthen (tetracosactrin) test; one had been employed for only seven months. All 12 men had facial plethora, suggesting absorption of the drug in spite of their having adhered to the safety precautions. All workers manufacturing potent steroids should therfore be screened regularly by measurement of their plasma cortisol concentrations and should be moved regularly to processing other drugs.     

Coliforms from hides and meat.

Coliform tests were performed on 85 hide and 75 meat samples. IMViC reactions were determined on isolates from positive confirmed and fecal tests, and strains other than Escherichia coli were identified. Strains typed as Aerobacter aerogenes types I and II were identified as Enterobacter cloacae (51.4%), Klebsiella pneumoniae (21.5%), Enterobacter aerogenes (15%), and Enterobacter liquefaciens, Serratia, and unidentified coliforms (12.1%). K. pneumoniae appeared to be responsible for less than 1% positive fecal tests.     

The effects of specific lipogenic substrates and metabolic inhibitors on de Novo fatty acid synthesis in isolated hepatocytes from chow-fed female rats

The effects of glucose (10 mm), glycerol (3 mm), and lactate/pyruvate (10 mm) on the incorporation of ³H from ³H₂O into fatty acids were studied in isolated hepatocytes prepared from chow-fed female rats. Lactate/pyruvate markedly increased lipogenic rates, while glucose and glycerol did not significantly affect rates of lipogenesis. In cells incubated with lactate/pyruvate plus glycerol, the increase in ³H incorporation was greater than observed with lactate/pyruvate alone. In hepatocytes isolated from 24-h starved rats, lactate/pyruvate again increased de novo fatty acid synthesis to a greater extent than either glucose or glycerol. Glycerol significantly increased lipogenesis compared to the endogenous rates and when incubated with lactate/pyruvate produced an increase above lactate/pyruvate alone. (?)-Hydroxycitrate, a potent inhibitor of ATP-citrate lyase (EC 4.1.3.8), and agaric acid, an inhibitor of tricarboxylate anion translocation, were studied in hepatocytes to determine their effects on lipogenesis by measuring ³H₂O, [1-¹⁴C]acetate, and [2-¹⁴C]lactate incorporation into fatty acids. ³H incorporation into fatty acids was markedly inhibited by both inhibitors with agaric acid (60 μm) producing the greater inhibition. (?)-Hydroxycitrate (2 mm) increased acetate incorporation into fatty acids from [1-¹⁴C]acetate and agaric acid produced a strong inhibitory effect. Combined effects of (?)-hydroxycitrate and agaric acid on lipogenesis from [1-¹⁴C]acetate showed an inhibitory response to a lesser extent than with agaric acid alone. With substrate concentrations of acetate present, there was no significant increase in rates of lipogenesis from [1-¹⁴C]acetate and the increase previously observed with (?)-hydroxycitrate alone was minimized. Agaric acid significantly inhibited fatty acid synthesis from acetate in the presence of exogenous substrate, but the effect was decreased in comparison to rates with only endogenous substrate present. With [2-¹⁴C]lactate as the lipogenic precursor, agaric acid and (?)-hydroxycitrate strongly inhibited fatty acid synthesis. However, agaric acid despite its lower concentration (60 μm vs 2 mm) was twice as effective as (?)-hydroxycitrate. A similar pattern was observed when substrate concentrations of lactate/pyruvate (10 mm) were added to the incubations. When (?)-hydroxycitrate and agaric acid were simultaneously incubated in the presence of endogenous substrate, there was an additive effect of the inhibitors on decreasing fatty acid synthesis. Results are discussed in relation to the origin of substrate for hepatic lipogenesis and whether specific metabolites increase lipogenic rates.     

Analysis of biological thiols: derivatization with monobromotrimethylammoniobimane and characterization by electrophoresis and chromatography

A new method for analysis of biological thiols based upon their conversion to fluorescent derivatives by reaction with monobromotrimethylammoniobimane (qBBr) is described. The derivatives are separated by chromatography and by electrophoresis on cellulose thinlayer chromatography plates. The use of two-dimensional mapping makes it possible to differentiate between a wide variety of biological thiols including N-acetylcysteine, CoA, cysteine, cysteinylglycine, cysteamine, ergothioneine, glutathione, γ-glutamylcysteine, homocysteine, mercaptopyrimidine, pantetheine, 4′-phosphopanetheine, thiosulfate, and thiouracil. For applications to biological samples thiols were isolated from crude extracts by binding to a mercuriagarose gel. Following removal from the gel with dithiothreitol, the thiols were derivatized with qBBr. The methods were tested by showing that glutathione is the major thiol in human red blood cells, that glutathione and ergothioneine are the major thiols in Neurospora crassa conidia, and that Bacillus cereus vegetative cells lack glutathione but contain cysteine, pantetheine, and an unidentified thiol in significant amounts.     

The immune response of inbred mouse strains to DNP-BGG

The immune response of six inbred mouse strains (SJL, A, C57BL/6, CBA, BALB/c, and DBA/1) to DNP₅₆BGG was tested under three separate immunization schedules: 1 Μg DNP-BGG in 1 mg Al(OH)₃ adjuvant, 50 Μg DNP-BGG in 1 mg A1(OH)₃ adjuvant, and 1 Μg DNP-BGG in complete Freund's adjuvant. Individual serum samples were titered using a modified Farr assay. It was found that the first schedule allowed classification of the mice into responder (SJL, A) and nonresponder (C57BL/6, CBA, BALB/ c, DBA/1) strains. The second schedule produced quantitative as well as qualitative differences among the strains and allowed classification of the mice into higher-responder (SJL, A), intermediate-responder (C57BL/6, CBA, BALB/c), and low-responder (DBA/1) categories. When complete Freund's adjuvant was used in the third schedule, the differences among strains became insignificant. The sera from each strain were pooled and assayed for relative antibody affinity and IgM content. Both of these parameters were dependent largely on the dose of antigen and type of adjuvant used, rather than on the particular mouse strain being studied. The mechanism of adjuvant action, and possible cell interactions in the genetic control of the immune response, are discussed.     

Polyadenylic acid synthesis activity of purified DNA-dependent RNA polymerase from Caulobacter

Characterization of purified DNA-dependent RNA polymerase (EC 2.7.7.6) of Caulobacter crescentus, strain CB15 has led to the conclusion that this enzyme catalyzes poly(A) synthesis in the absence of template. Poly(A) synthetase activity co-purifies with both holoenzyme and core polymerase on DNA-cellulose columns, and core polymerase purified to 98% homogeneity by glycerol gradient centrifugation is still capable of catalyzing poly(A) polymerization. Both RNA synthesis and poly(A) polymerization activities are sensitive to rifampicin. In addition, RNA polymerase purified from partially rifampicin-sensitive mutants exhibits the same partial sensitivity in vitro to the drug in the synthesis of RNA and poly(A). The enzyme used in these studies was prepared by a simple method which allows a high yield of pure RNA polymerase from large batches of exponential cells. The procedure includes high speed centrifugation of cell extracts, DEAE-cellulose column, DNA-affinity chromatography, and low salt glycerol gradient centrifugation. Holoenzyme can be resolved into core and sigma subunit by either DNA-cellulose chromatography or glycerol gradient centrifugation, and the latter step allows recovery of pure sigma factor.     

Micromanipulation of adhesion of a Jurkat cell to a planar bilayer membrane containing lymphocyte function-associated antigen 3 molecules

Cell adhesion plays a fundamental role in the organization of cells in differentiated organs, cell motility, and immune response. A novel micromanipulation method is employed to quantify the direct contribution of surface adhesion receptors to the physical strength of cell adhesion. In this technique, a cell is brought into contact with a glass-supported planar membrane reconstituted with a known concentration of a given type of adhesion molecules. After a period of incubation (5-10 min), the cell is detached from the planar bilayer by pulling away the pipette holding the cell in the direction perpendicular to the glass-supported planar bilayer. In particular, we investigated the adhesion between a Jurkat cell expressing CD2 and a glass-supported planar bilayer containing either the glycosyl-phosphatidylinositol (GPI) or the transmembrane (TM) isoform of the counter-receptor lymphocyte function-associated antigen 3 (LFA-3) at a concentration of 1,000 molecules/microns 2. In response to the pipette force the Jurkat cells that adhered to the planar bilayer containing the GPI isoform of LFA-3 underwent extensive elongation. When the contact radius was reduced by approximately 50%, the cell then detached quickly from its substrate. The aspiration pressure required to detach a Jurkat cell from its substrate was comparable to that required to detach a cytotoxic T cell from its target cell. Jurkat cells that had been separated from the substrate again adhered strongly to the planar bilayer when brought to proximity by micromanipulation. In experiments using the planar bilayer containing the TM isoform of LFA-3, Jurkat cells detached with little resistance to micromanipulation and without changing their round shape.     

Site-directed mutagenesis of glutathione S-transferase YaYa. Important roles of tyrosine 9 and aspartic acid 101 in catalysis.

The roles of tyrosine 9 and aspartic acid 101 in the catalytic mechanism of rat glutathione S-transferase YaYa were studied by site-directed mutagenesis. Replacement of tyrosine 9 with phenylalanine (Y9F), threonine (Y9T), histidine (Y9H), or valine (Y9V) resulted in mutant enzymes with less than 5% catalytic activity of the wild type enzymes. Kinetic studies with purified Y9F and Y9T mutants demonstrated poor catalytic efficiencies which were largely due to a drastic decrease in kcat. The estimated pK alpha values of the sulfhydryl group of glutathione bound to Y9F and Y9T mutant enzymes were 8.5 to 8.7, similar to the chemical reaction, in contrast to the estimated pK alpha value of 6.7 to 6.8 for the glutathione enzyme complex of wild type glutathione S-transferase. These results indicate that tyrosine 9 is directly responsible for the lowering of the pKa of the sulfhydryl group of glutathione, presumably due to the stabilization of the thiolate anion through hydrogen bonding with the hydroxyl group of tyrosine. To examine the role of aspartic acid in the binding of glutathione to YaYa, 4 conserved aspartic acid residues at positions 61, 93, 101, and 157 were changed to glutamic acid and asparagine. All mutant enzymes retained either full or partial activity except D157N, which was virtually inactive. Kinetic studies with four mutant enzymes (D93E, D93N, D101E, and D101N) indicate that only D101N exhibited a 5-fold increase in Km toward glutathione. Also, the binding of this mutant to the affinity column was greatly reduced. These results demonstrate that aspartic acid 101 plays an important role in glutathione interaction to YaYa. The role of aspartic acid 157 in catalysis remains to be determined.