Changes in [3H]inositol-labelled phosphoinositides of pig platelets in response to thrombin

Pig platelet phosphoinositides have been labelled with [³H]inositol and then treated with thrombin in the absence of Ca²⁺. There was a loss of labelled phosphatidylinositol 4,5-bisphosphate between 30 and 60 s after the addition of thrombin but the general picture was of increased labelling over a 4-min period. Labelling of phosphatidylinositol 4-phosphate showed no period of loss but there was an early loss of phosphatidylinositol and no increased labelling during the 4-min incubation. The small amount of lysophosphatidyl[³H]inositol in the platelets was not affected by thrombin treatment. Thrombin caused loss of [¹⁴C]arachidonate-labelled phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol.     

The study of differentiative potential of the lactating mouse mammary gland in organ culture

Summary The organ culture of the mammary gland of lactating mice was used to examine the response of the differentiated gland to lactogenic stimuli, insulin, cortisol, and prolactin. Time course studies showed that casein synthesis in cultured tissue decreased rapidly during the first 2 d despite the presence of the three hormones, but on the 3rd d tissue cultured with either insulin and prolactin or all three hormones regained the ability to synthesize milk proteins, casein, and α-lactalbumin: a greater increase occurred in the three hormone system. The delayed addition of prolactin on Day 2 to the culture system containing insulin and cortisol also stimulated casein synthesis. The addition of cytarabine, which inhibited insulin-dependent cell proliferation in cultured explants, did not block the rebound of milk protein synthesis. The results indicate that in the presence of insulin, cortisol, and prolactin mammary epithelial cells in culture first lose and then regain the ability of synthesizing milk protein without requiring the formation of new daughter cells.     

Regulation of the synthesis of mucin glycoproteins in swine trachea explants

Summary Swine tracheal epithelium has been cultured as explants in a chemically defined medium for periods of up to 2 wk. The viability of the explants was shown by the preservation of the ultrastructural features of cells in the epithelial layer and by the active incorporation of radioactive glucosamine and sulfate into secreted mucin glycoproteins. The rate of secretion of mucin glycoprotein was about 0.035 mg per cm² per d. After initial 24 h lag period was shown to be due to the equilibration of intracellular mucin glycoprotein pools with radioactive precursors. The rate of secretion of glycoprotein showed a linear dependence on the area of the explant, and maximal incorporation was observed at 200 μM glucosamine. A higher concentration of³⁵SO₄, 1000 μM, was required for maximal incorporation of the precursor. Insulin at 0.1 to 1 μg/ml increased the rate of secretion twofold, whereas 0.1 to 100 μg/ml of hydrocortisone and 0.1 to 100 μg/ml of epinephrine significantly decreased the rate of secretion. Vitamin A had little or no effect of normal trachea explants at low concentrations, and, at higher concentrations, 10⁻⁵ M, it decreased the secretion of mucin glycoproteins. Vitamin A, at a concentration of 10⁻⁹ M, increased the rate of synthesis of glycoprotein at least fourfold in trachea explants from vitamin A-deficient rats. Mucus secretions collected from the surface of swine trachea and from the culture medium of trachea explants were purified. The mucus was solubilized by reduction and carboxymethylation, and the high molecular weight mucin glycoproteins were purified by chromatography on Sepharose CL-6B columns under dissociating conditions in 2M guanidine HCl. The mucin glycoproteins purified from swine trachea and from the culture medium of trachea explants were virtually indistingushable. They showed the same properties when examined by gel electrophoresis and immunoprecipitation. The purified glycoproteins contained about 25% protein, and serine, threonine, and proline were the principal amino acids present. More than 80% of the carbohydride chains in both samples were released by treatment with alkaline borohydride. Nearly the same molar ratio ofN-acetylgalactosamine,N-acetylglucosamine, galactose, fucose, sulfate, and sialic acid was found in both preparations. This investigation was supported by U.S. Public Health Service Grants HL 20868, HL 24688, and HL 24718 from the National Heart, Lung and Blood Institute, Bethesda, MD, and AM 28187 from the National Institute of Arthritis, Diabetes and Digestive and Kidney Diseases, Bethesda, MD.     

Stimulation of the cyclooxygenase activity of prostaglandin H synthase by salicylate-derived quinhydrones

Two novel salicylate-derived quinhydrones were evaluated for their effect on the kinetics of cyclooxygenase activity of sheep seminal vesicle prostaglandin H synthase. These quinhydrones, which form semiquinone radicals in solution, were designed to resemble oxidized intermediates of salicylic acid metabolism. Although initially investigated for their potential role in irreversible inactivation of cyclooxygenase, these derivatives were found to give three-fold stimulation of this activity. In the absence of arachidonic acid, preincubation of the enzyme with these quinhydrones did not lead to inactivation of the cyclooxygenase activity. These compounds thus resemble the phenolic antioxidants in their effects on the cyclooxygenase activity of the synthase.     

Isolation and amino-terminal sequences of subunits from the photosynthetic reaction center of Rhodopseudomonas capsulata

The amino-terminal sequences have been determined by Edman degradation for the reaction center polypeptides from a carotenoidless mutant of Rhodopseudomonas capsulata. Individual polypeptides were isolated by preparative electrophoresis and electroelution. By comparison with the sequences deduced from the DNA (Youvan, D.C., Alberti, M., Begush, H., Bylina, E.J. and Hearst, J.E. (1984) Proc. Natl. Acad. Sci. USA 81, 189–192) we conclude that the M and L subunits are processed so as to remove the amino-terminal methionine, whereas the H subunit is not processed at the amino-terminus after translation. None of the subunits is synthesized with a significant amino-terminal extension peptide.     

EPR detection of a cryogenically photogenerated intermediate in photosynthetic oxygen evolution

In Photosystem II preparations at low temperature we were able to generate and trap an intermediate state between the S₁ and S₂ states of the Kok scheme for photosynthetic oxygen evolution. Illumination of dark-adapted, oxygen-evolving Photosystem II preparations at 140 K produces a 320-G-wide EPR signal centered near g = 4.1 when observed at 10 K. This signal is superimposed on a 5-fold larger and somewhat narrower background signal; hence, it is best observed in difference spectra. Warming of illuminated samples to 190 K in the dark results in the disappearance of the light-induced g = 4.1 feature and the appearance of the multiline EPR signal associated with the S₂ state. Low-temperature illumination of samples prepared in the S₂ state does not produce the g = 4.1 signal. Inhibition of oxygen evolution by incubation of PS II preparations in 0.8 M NaCl buffer or by the addition of 400 μM NH₂OH prevents the formation of the g = 4.1 signal. Samples in which oxygen evolution is inhibited by replacement of Cl^? with F^? exhibit the g = 4.1 signal when illuminated at 140 K, but subsequent warming to 190 K neither depletes the amplitude of this signal nor produces the multiline signal. The broad signal at g = 4.1 is typical for a $\text{S =}\text{5}\text{2}$ spin system in a rhombic environment, suggesting the involvement of non-heme Fe in photosynthetic oxygen evolution.     

A thin quartz cell suitable for vacuum ultraviolet absorption and circular dichroism measurements

The design of a thin quartz cell suitable for absorption and circular dichroism measurements in the vacuum ultraviolet is described. Important features of the cell are (1) that it can be disassembled for cleaning and reproducibly reassembled with path lengths up to 0.3 mm, and (2) that strain in the windows from the compressed sample can be relieved by a sample overflow port. The latter feature allows the cell to be used for circular dichroism as well as absorption measurements.     

Muralytic Activities of Ruminococcus albus 8

Ruminococcus albus 8 was cultured with isolated alfalfa cell walls as the carbon source. The culture broth was assayed for muralytic enzyme activities. The effect, with respect to the production of such muralytic enzymes, of growing the microorganism on different carbon sources was also investigated. Also, the rates of solubilization and utilization by R. albus of individual alfalfa cell wall sugars during a 96-h growth period were examined.