Analysis of the regulation of adenosine 5′-phosphosulfate sulfotransferase activity inLemna minor L. using15N-density labeling

The role of de novo synthesis in the regulation of adenosine 5-phosphosulfate sulfotransferase activity by H₂S inLemna minor L. was investigate using density labeling with¹⁵N applied as¹⁵NO ₃ ^– in the culture medium. While adenosine 5-phosphosulfate sulfotransferase activity was rapidly reduced by H₂S and rapidly recovered upon removal of H₂S, O-acetyl-L-serine sulfhydrylase (EC 4.2.99.8) did not show changes in extractable activity in response to H₂S and could therefore be used as an internal marker enzyme for density labeling. The incorporation of¹⁵N into adenosine 5-phosphosulfate sulfotransferase was strongly reduced upon transfer of plants into a H₂S-containing atmosphere. Half-maximal labeling was reached only after 70–80 h compared to 40–50 h in the control. After removal of H₂S, adenosine 5-phosphosulfate sulfotransferase activity increased to the initial level within 20 h, and the enzyme reached halfmaximal labeling after only 15 h. The time course of the density increase of O-acetyl-L-serine sulfhydrylase was not affected very significantly by H₂S. These results provide evidence that de novo synthesis of enzyme protein is involved in the regulation of adenosine 5-phosphosulfate sulfotransferase activity by H₂S.Abbreviations APS adenosine 5-phosphosulfate - APSSTase adenosine 5-phosphosulfate sulfotransferase - BSA Bovine serum albumine - DTE dithioerythritol - OAS O-acetyl-L-serine - OASSase O-acetyl-L-serine sulfhydrylase - POPOP 1,4-bis-(5-phenyl-2-oxazolyl)-benzene - PPO 2,5-diphenyloxazole This is no. 9 in the series Regulation of Sulfate Assimilation in Plants     

The fine structure of the neural lobe of the mouse after transplantation under the kidney capsule

Summary The neurohypophysis of donor mice was implanted under the renal capsule of the recipients. The pituicytes survived while the neurosecretory axons disappeared. The ultrastructure of the glial cells was observed seven and nine weeks after transplantation. There were no signs of phagocytotic activity although remnants of axons were still present at seven weeks. The numerous processes of the pituicytes form a network with intercellular spaces wide in younger and narrower in older implants. The cells are connected by desmosomes and gap junctions. Pituicytes as well as blood vessels preserve their organotypic appearance. The transplant thus represents an experimental model for investigations on pituicytes in vivo in the absence of neurosecretory axons.Fellow of the Alexander von Humboldt-Stiftung. On leave from Department of Histology and Embryology, Institute of Biostructure, Academy of Medicine, Pozna, Poland     

Linkage between low-density lipoprotein and igk light-chain genes in rabbits

The rabbit geneLpq, which codes for a low-density serum lipoprotein², is linked (34.6 ± 5.3 centimorgans) to the Ig kappa light-chain gene (Ab). There is no evidence thatLpq is linked to another gene,Prt, that was previously found to be linked to theAb gene. This suggests that the gene order for the three genes isPrt- Ab- Lpq. Abbreviations used in this paper Ig immunoglobulin - a the heavy-chain variable-region geneAa - b the kappa light-chain geneAb - q the low-density serum lipoprotein geneLpq     

Tomato alcohol dehydrogenase: Purification and substrate specificity

Alcohol dehydrogenase of tomato (Lycopersicon esculentum) has been purified to homogeneity, using affinity chromatography on Cibacron F3GA-agarose. The enzyme is a dimer, M_r 90,000–100,000. The coenzyme is NAD⁺; no NADP⁺-dependent activity was detected even in crude extracts. Among saturated substrates, ethanol and acetaldehyde show the lowest apparent K_m values (2.67 and 0.174 mm, respectively) and highest V values, supporting a primary role in acetaldehyde metabolism, with action also on “flavor aldehydes”; 2-unsaturated alcohols show still lower K_m values, probably due to a more favorable K_eq. This enzyme and other plant alcohol dehydrogenases form a definite class, intermediate in specificity between liver and yeast alcohol dehydrogenases: they differ from the former in being essentially inactive on secondary and aromatic substrates, from the latter in showing only a mild decrease in V with increasing chain length of alkyl substrates, and from both in showing the lowest K_m as well as highest V on ethanol and acetaldehyde. The tomato enzyme differs from other reported plant enzymes in showing substantial activity on geraniol. Kinetic studies are in agreement with an ordered sequential mechanism. The enzyme is inhibited slowly by iodoacetamide, and reversibly by acetamide and zinc-chelating compounds.     

Assessment of interleukin 1 and interleukin 2 effects on cycling and noncycling murine thymocytes

When mouse thymocytes are stimulated with PHA, the proliferative response is very low, unless the culture medium is enriched with interleukin 1 (IL-1)- or interleukin 2 (IL-2)-containing supernatants. Cytofluorometric analyses show, however, that PHA stimulation generates a significant number of cells with increased RNA content (transition from the G₀ to G₁ phase of the cell cycle). If IL-2 is added to such cultures, the activated cells complete their process of RNA synthesis and then enter the S phase. The use of IL-2-containing culture medium thus permits one to obtain a high correlation between the number of g₁ cells and [³H]thymidine incorporation (r = 0.97). Enrichment with IL-1-containing supernatants also results in a statistically significant correlation (r = 0.68), but the regression lines are markedly different for the two interleukins (s = 20.3 for IL-2 and s = 9.2 for IL-1), when analyzed after 48 hr of incubation. These observations suggest that the G₁ phase must be divided into two subcompartments, G_1a and G_1b, the G_1a-G_1b transition being an IL-2-dependent event. If the number of G_1b cells is used to establish correlations with [³H]thymidine incorporation, all values fall on the same regression line, regardless of culture conditions and of the addition of interleukins. It is concluded that IL-2 regulates lymphocyte proliferation at the level of RNA synthesis (G_1a-G_1b transition) rather than that of DNA synthesis (G₁-S transition).     

Characterization of a variant of carcinoembryonic antigen (CEA) using monoclonal and polyclonal anti-CEA antibodies

A variant of the carcinoembryonic antigen (CEA) with lower molecular weight than a CEA reference preparation has been separated from CEA. Using a polyclonal, spleen absorbed anti-CEA antiserum, the variant crossreacts with reference CEA in immunodiffusion. The CEA-activity of the variant has been demonstrated using an enzyme-immunoassay with monoclonal CEA specific antibodies. There is sufficient immunological evidence that this variant is a distinct antigen different from the crossreactive antigens described so far. The reactivity of the polyclonal anti-CEA antiserum with the CEA variant was abolished by absorption against the immobilized variant.     

Two distinct regulatory steps in cartilage differentiation

The effect of developmental stage on chondrogenic capacity in high-density cell cultures of chick embryonic wing bud mesenchyme is examined. Mesenchyme from stage 19 embryos forms aggregates of closely associated cells which do not form cartilage matrix, nor contain significant levels of type II collagen that are detectable by immunofluorescence, unless they are treated with dibutyryl cyclic AMP. Mesenchyme from stage 24 embryonic wing buds in high-density cell cultures will spontaneously form cartilage, as defined by electron microscopy and immunofluorescence with antibody to type II collagen. Cultures prepared from stage 26 wings form numerous aggregates which fail to accumulate an Alcian blue-staining matrix and which resemble mesenchyme cells morphologically. However, because these cells show considerable intracellular immunofluorescence for type II collagen, they are actually unexpressed cartilage cells. Several treatments, including exposure to dibutyryl cyclic AMP, ascorbic acid and an atmosphere of 5% oxygen, or mixture with small numbers of stage 24 wing mesenchyme cells, stimulate expression, as determined by the accumulation of an Alcian blue-staining matrix and an ultrastructurally recognizable cartilage matrix. Since the addition of similar numbers of differentiated cartilage cells does not stimulate expression of stage 26 cells, it is proposed that initial cartilage expression is dependent on a mesenchyme-specific influence which might be removed by cell dissociation. These studies demonstrate that there are at least two distinct transitions in cartilage differentiation: one involves the conversion of mesenchyme to unexpressed chondrocytes and the second involves mesenchyme-dependent expression of chondrogenic differentiation.