Properties,synthesis, and accumulation of storage proteins in Pieris rapae L.

Two kinds of storage proteins (SP-1, SP-2) were confirmed in hemolymph and fat body of Pieris rapae during metamorphosis. Both proteins were present in high concentrations in the hemolymph during the last larval instar. Hemolymph concentrations of SP-1 and SP-2 dropped after pupation as the proteins were being deposited in fat bodies. SP-2 is present in a larger amount than SP-1. Detailed studies on storage proteins determined their properties, mode of synthesis, and accumulation in the fat body. SP-1 has a molecular weight of 500,000 and consists of one type of subunit (M_r 77,000), while SP-2 has a molecular weight of 460,000 and is composed of two types of subunits (M_r 80,000 and 69,000). The pl values of SP-1 and SP-2 were determined to be 6.97 and 7.06, respectively. Fat body cells from 1-day-old fifth instar larvae synthesized storage proteins in large amounts, whereas those from late prepupae exhibited high protein sequestration. Proteins taken up in fat body accumulated in dense granules during the pupal stage but sharply decreased at the adult stage. Morphological changes in the fat body tissues were observed during the larval-pupal transformation; the nuclei of fat body cells became irregularly shaped, and the boundaries between cells seemed to be obscure. Synthesis, storage, or degradation of storage proteins in fat body during development is closely associated with morphological changes in the tissues.     

The atokous-epitokous border is determined before the onset of heteronereid development in Platynereis dumerilii (Annelida,Polychaeta)

Summary In most nereids sexual maturation is accompanied by a dramatic reorganization of the body that enables swarming of the formerly benthic worms. However, a border exists between unchanged anterior (atokous) and metamorphosed posterior (epitokous) segments. The site of this atokous-epitokous border (a/e border) is different in sexually mature males and females of Platynereis dumerilii. There is no correlation between the total number of setigerous segments of a specimen and the location of the a/e border. The location of the a/e border and sexual development are affected neither by cutting off caudal segments of juveniles (including the prospective a/e border) nor by transecting the ventral nerve cord. When parapodia are transplanted from prospective epitokous regions to prospective atokous regions and vice versa, they maintain their original character during metamorphosis. The results presented here suggest that prospective atokous as well as epitokous characters are determined at or only very shortly after formation of the respective segments. Thus the a/e border is established well in advance of the onset of epitokous metamorphosis.     

Modulation of secretion of human chorionic gonadotropin by biologic response modifiers on term placenta and choriocarcinoma cells

The effects of biologic response modifiers such as interferon-gamma, tumor necrosis factor alpha (TNF), and retinoic acid on the human chorionic gonadotropin (hCG) secretion of cultured choriocarcinoma cells (JAR) and term placenta have been studied. Although the proliferation of JAR cells was not inhibited by these agents, retinoic acid and TNF markedly increased both the intracellular levels as well as the secreted amounts of hCG. In the case of the term placenta, only retinoic acid increased the hCG secretion into the culture medium, whereas interferon-gamma and TNF both markedly reduced secretion. The cytostatic agent etoposide (VP-16) was able to augment the hCG secretion on the choriocarcinoma cells but did not alter its production on term placenta. The The data presented indicate different mechanisms of regulation of hCG secretion in the normal and malignant trophoblast.     

Cu(I)-thionein release from copper-loaded yeast cells

Summary The release of intact CU(I)₈-thionein from copper-resistant copper-loaded yeast cells, strain X 2180-1Aa, has been shown. This copper(I)-thiolate-rich protein was characterized and compared with the chemical and physicochemical properties of intracellular yeast Cu-thionein. The same molecular mass and stoichiometry of 8 mol copper atoms/mol protein was found. No detectable difference between the Cu-thioneins was seen in luminescence emission, electronic absorption in the ultraviolet region, chiroptical data or amino acid composition. The importance of stable Cu(I)-thiolates in Cu-thionein as a safe vehicle for transporting copper in a non-reactive manner is confirmed.     

Identification of a new V kappa gene family that is highly expressed in hybridomas from an autoimmune mouse strain

We have identified a new murine V kappa family that contains five to seven members, one member of which encodes the L chain V region of an anti-dsDNA antibody produced by a BALB/c hybridoma, C8.5. The cloned C8.5 V kappa gene exhibits highest homology with a human V kappa gene that was cloned from a nonproductive rearrangement but has never been seen in an expressed repertoire. Because this family was first identified in an autoantibody, we studied its expression in an autoimmune mouse strain. This V kappa family is expressed in 20% of hybridomas from NZB mice.     

Two substrate sites in the renal Na+-d-glucose cotransporter studied by model analysis of phlorizin binding and-d-glucose transport measurements

Summary Time courses of phlorizin binding to the outside of membrane vesicles from porcine renal outer cortex and outer medulla were measured and the obtained families of binding curves were fitted to different binding models. To fit the experimental data a model with two binding sites was required. Optimal fits were obtained if a ratio of low and high affinity phlorizin binding sites of 1:1 was assumed. Na⁺ increased the affinity of both binding sites. By an inside-negative membrane potential the affinity of the high affinity binding site (measured in the presence of 3 mM Na⁺) and of the low affinity binding site (measured in the presence of 3 or 90 mM Na⁺) was increased. Optimal fits were obtained when the rate constants of dissociation were not changed by the membrane potential. In the presence of 90 mM Na⁺ on both membrane sides and with a clamped membrane potential,K _D values of 0.4 and 7.9 M were calculated for the low and high affinity phlorizin binding sites which were observed in outer cortex and in outer medulla. Apparent low and high affinity transport sites were detected by measuring the substrate dependence ofd-glucose uptake in membrane vesicles from outer cortex and outer medulla which is stimulated by an initial gradient of 90 mM Na⁺(out>in). Low and high affinity transport could be fitted with identicalK _m values in outer cortex and outer medulla. An inside-negative membrane potential decreased the apparentK _m ofhigh affinity transport whereas the apparentK _m of low affinity transport was not changed. The data show that in outer cortex and outer medulla of pighigh and low affinity Na⁺-d-glucose cotransporters are present which containlow and high affinity phlorizin binding sites, respectively. It has to be elucidated from future experiments whether equal amounts of low and high affinity transporters are expressed in both kidney regions or whether the low and high affinity transporter are parts of the same glucose transport moleculc.     

Binding of the J 1 Adhesion Molecules to Extracellular Matrix Constituents

The J1 glycoproteins can be obtained in multiple forms in the soluble fraction of developing and adult mouse brain tissue. They are recovered as two forms of apparent molecular weights of 160,000 and 180,000 (J1-160) from adult mouse brain and as forms of apparent molecular weights of 200,000 and 220,000 (J1-220) from developing brain. J1-160 and J1-220 share common epitopes but are considered as separate entities, with J1-220 being immunochemically closely related if not identical to tenascin. Based on the observation that J1 immunoreactivity appears on basement membrane and interstitial collagens after denervation of the neuromuscular junction in adult rodents, we became interested in investigating the binding properties of J1 glycoproteins to extracellular matrix constituents in vitro. Both J1-160 and J1-220 bound to collagens type I-VI and IX but not to laminin, fibronectin, bovine serum albumin, or gelatin under hypotonic buffer conditions. Under isotonic buffer conditions, J1-220 bound to all collagen types, whereas J1-160 bound only to collagen types V and VI with values that could be examined by Scatchard analysis. Binding of J1-220 to collagens displayed two binding constants (KD) between 1.5 and 4.4 X 10(-9) and 1.8 and 5.5 X 10(-8) M, respectively, under hypotonic buffer conditions and a single KD of 2.1-8.0 X 10(-8) M under isotonic buffer conditions. Binding of J1-160 to collagens had an apparent KD of 1.9-8.0 X 10(-9) M under hypotonic buffer conditions. Under isotonic buffer conditions, binding constants of J1-160 to collagen types V and VI were approximately 2 X 10(-8) M. Binding of J1-220 to collagen type I could be inhibited by J1-220, J1-160, and collagen type VI but not by fibronectin or gelatin. Conversely, binding of J1-160 was inhibited by J1-220, J1-160, and collagen type VI (in order of decreasing efficacy of competition). J1-160 and J1-220 were retained on a heparin-agarose column and eluted in a salt gradient at approximately 0.5 M NaCl. The formation of the J1-heparin complexes was inhibited 100-fold more efficiently by heparin than by chondroitin sulfate. These experiments show that J1 glycoproteins resemble in many respects the extracellular matrix constituents fibronectin, laminin, vitronectin, and von Willebrand factor.     

Purification, characterization, and rapid inactivation of thermolabile ubiquitin-activating enzyme from the mammalian cell cycle mutant ts85

Conjugation of ubiquitin to certain proteins can trigger their degradation in the in vitro reticulocyte system. In order to determine whether ubiquitin conjugation serves as an intermediate step in the turnover of cellular proteins in vivo, it is necessary to isolate proteolytic intermediates, i.e. ubiquitin-protein adducts of specific cellular proteins. While the steady-state level of conjugates of rapidly turning over proteins is relatively high, that of long-lived proteins is presumably extremely low, and therefore undetectable. Therefore, mutant cell lines with conditionally altered function(s) of the ubiquitin system can serve as powerful tools in studying the degradation of stable cellular proteins. We have characterized a temperature sensitive cell cycle arrest mutant cell (ts85) with a thermolabile ubiquitin-activating enzyme (E1; Finley, D., Ciechanover, A., and Varshavsky, A. (1984) Cell 37, 43-55). Following incubation at the restrictive temperature (39.5 degrees C), these cells fail to degrade short-lived proteins (Ciechanover, A., Finley, D., and Varshavsky, A. (1984) Cell 37, 57-66). However, involvement of the ubiquitin system in the turnover of long-lived proteins has not been addressed in these cells. A slow rate of inactivation of E1 in vivo, and significant rate of cell death following long incubation periods at the restrictive temperature, make this question difficult to address experimentally. In the present study we show that incubation of the cells for 1 h at 43 degrees C leads to rapid inactivation of ubiquitin conjugation in the intact mutant cell. Following heat treatment, the cells can be incubated at 39.5 degrees C for at least 6 h in order to study the possible involvement of the system in the turnover of long-lived cellular proteins. The viability of the cells is excellent at the end of the incubation. Following extraction, we have shown that inactivation occurs much more rapidly in the cell lysate in vitro than in the intact cell (t1/2 of 10 min compared to 4 h at 39.5 degrees C). The enzyme from both the mutant cell and the wild-type cell was purified to homogeneity. The molecular mass of the native enzyme from both cells is approximately 220 kDa with a subunit molecular mass of about 108 kDa. The structure of the enzyme is therefore very similar to that purified from rabbit reticulocytes. At the permissive temperature, the enzymes from both cells catalyze ATP-PPi and ATP-AMP exchange in similar kinetics. However, at the high temperature, the mutated enzyme is at least 7-fold less stable than the wild-type enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)