The gene for trypsin inhibitor CMe is regulated in trans by the lys 3a locus in the endosperm of barley (Hordeum vulgare L.)

Summary A cDNA encoding trypsin inhibitor CMe from barley endosperm has been cloned and characterized. The longest open reading frame of the cloned cDNA codes for a typical signal peptide of 24 residues followed by a sequence which is identical to the known amino acid sequence of the inhibitor, except for an Ile/Leu substitution at position 59. Southern blot analysis of wheat-barley addition lines has shown that chromosome 3H of barley carries the gene for CMe. This protein is present at less than 2%–3% of the wild-type amount in the mature endosperm of the mutant Risø 1508 with respect to Bomi barley, from which it has been derived, and the corresponding steady state levels of the CMe mRNA are about I%. One or two copies of the CMe gene (synonym Itc1) per haploid genome have been estimated both in the wild type and in the mutant, and DNA restriction patterns are identical in both stocks, so neither a change in copy number nor a major rearrangement of the structural gene account for the markedly decreased expression. The mutation at the lys 3a locus in Risø 1508 has been previously mapped in chromosome 7 (synonym 5H). A single dose of the wild-type allele at this locus (Lys 3a) restores the expression of gene CMe (allele CMe-1) in chromosome 3H to normal levels.     

Sucrose transport into the phloem of Ricinus communis L. seedlings as measured by the analysis of sieve-tube sap

Careful cutting of the hypocotyl of Ricinus communis L. seedlings led to the exudation of pure sieve-tube sap for 2–3 h. This offered the possibility of testing the phloem-loading system qualitatively and quantitatively by incubating the cotyledons with different solutes of various concentrations to determine whether or not these solutes were loaded into the sieve tubes. The concentration which was achieved by loading and the time course could also be documented. This study concentrated on the loading of sucrose because it is the major naturally translocated sieve-tube compound. The sucrose concentration of sieve-tube sap was approx. 300 mM when the cotyledons were buried in the endosperm. When the cotyledons were excised from the endosperm and incubated in buffer, the sucrose concentration decreased gradually to 80–100 mM. This sucrose level was maintained for several hours by starch breakdown. Incubation of the excised cotyledons in sucrose caused the sucrose concentration in the sieve tubes to rise from 80 to 400 mM, depending on the sucrose concentration in the medium. Thus the sucrose concentration in the sieve tubes could be manipulated over a wide range. The transfer of labelled sucrose to the sieve-tube sap took 10 min; full isotope equilibration was finally reached after 2 h. An increase of K⁺ in the medium or in the sieve tubes did not change the sucrose concentration in the sievetube sap. Similarly the experimentally induced change of sucrose concentration in the sieve tubes did not affect the K⁺ concentration in the exudate. High concentrations of K⁺, however, strongly reduced the flow rate of exudation. Similar results were obtained with Na⁺ (data not shown). The minimum translocation speed in the sieve tubes in vivo was calculated from the growth increment of the seedling to be 1.03 m·h^-1, a value, which on average was also obtained for the exudation system with the endosperm attached. This comparison of the in-vivo rate of phloem transport and the exudation rate from cut hypocotyls indicates that sink control of phloem transport in the seedlings of that particular age was small, if there was any at all, and that the results from the experimental exudation system were probably not falsified by removal of the sink tissues.Abbreviations PTS 3-hydroxy-5,8, 10-pyrenetrisulfonate     

A similar protein portion for two exoglucanases secreted by Saccharomyces cerevisiae

Exoglucanase (exo-1,3-β-D-glucan glycohydrolase, EC 3.2.1.56) activity secreted by Saccharomyces cerevisiae into the culture medium was separated by ion exchange chromatography into two glycoprotein isoenzymes which contributed 10% (exoglucanase I) and 90% (exoglucanase II) towards the total activity. Analysis of the “in vitro” deglycosylated products by polyacrylamide gel electrophoresis under native or denaturing conditions indicated that the protein portions of both exoglucanases exhibited identical mobility, each one consisting of two polypeptides with M _r of 47000 and 48000. The same profile was shown by the exoglucanase secreted in the presence of tunicamycin. Antibodies raised against the protein portion of exoglucanase II did react with both native exoglucanases and their deglycosylated products with a pattern indicative of immunological identity. Digestion of the “in vitro” deglycosylated products of both exoglucanases with Staphylococcus aureus V-8 protease or trypsin generated the same proteolytic fragments in each case. Only exoglucanase II was secreted by protoplasts. These and previously reported results indicate that the protein portions of both isoenzymes may be the product of the same gene (or a family of related genes), and that exoglucanase I is a product of enzyme II, modified by a process occurring beyond the permeability barrier of the cell.     

A human melanoma-derived cell line (IGR39) with a very high number of vasoactive-intestinal-peptide (VIP) receptors. 2. Effect of VIP on cAMP production and on cell-surface VIP-binding sites

Vasoactive intestinal peptide (VIP) stimulated in a dose-dependent manner the accumulation of cAMP in human melanoma-derived cell line IGR39. The maximal effect (about 100 times the basal level) was observed with 10 nM VIP. Half-maximum cAMP production was obtained at 0.78 nM VIP. VIP-related peptides were also potent in stimulating the cAMP production in IGR39 cells. The order of potency was VIP much greater than peptide histidine-methioninamide greater than human growth-hormone-releasing factor(1-44) greater than secretin greater than glucagon. Using the same conditions, IGR37 cells, a metastasic counterpart of IGR39 cells, displayed a weak stimulation of cAMP production. After exposure of IGR39 cells to 10 nM VIP, the cAMP response to a new stimulation by VIP was strongly reduced. This desensitization of IGR39 cells to VIP was rapid (t1/2 less than 2 min) and homologous. Preincubation of IGR39 cells in the presence of native VIP induced disappearance of the VIP-binding sites at the cell surface. This phenomenon was dependent on time and VIP concentration. Maximum effect (loss of 80% of binding capacity) was obtained after exposure of the cells at 37 degrees C with a VIP concentration of 1 microM. The t1/2 of maximum disappearance was less than 2 min and the concentration of VIP giving half-maximum decrease in binding of mono[125I]iodinated VIP (125I-VIP) was 8 nM. This phenomenon was also reversible since 85% of the VIP-binding capacity could be restored in less than 1 h by incubating IGR39 cells in a VIP-free medium. The IGR39 cell line should be a useful model for further study of the structure and function of the human VIP receptor.     

A human melanoma-derived cell line (IGR39) with a very high number of vasoactive-intestinal-peptide (VIP) receptors. 1. Molecular characterization of the binding site

Using mono[125I]iodinated vasoactive intestinal peptide (125I-VIP), a very high number of specific binding sites for VIP were identified at the surface of the human melanoma cell line IGR39. The Scatchard analysis of competitive displacement experiments between native VIP and 125I-VIP was consistent with the existence of two classes of VIP-binding sites. IGR39 cells possess 0.54 x 10(6) high-affinity sites with a dissociation constant (Kd) of 0.66 nM and 1.3 x 10(6) sites of moderate affinity with a Kd of 4.7 nM. Pharmacological studies indicated that the order of potency in inhibiting 125I-VIP binding of the VIP/secretin family peptides was VIP much greater than peptide histidine methioninamide greater than human growth-hormone-releasing factor(1-44) greater than secretin. Glucagon has no effect on the binding of the labelled peptide. By means of photoaffinity labelling a polypeptide of Mr 63,000 was characterized. The labelling of this species was completely abolished by native VIP. The order of potency of VIP-related peptides in inhibiting 125I-VIP cross-linking to its receptor was the same as in the competition experiments. The glycoprotein nature of the VIP-binding sites of IGR39 cells has been investigated by affinity chromatography on wheat-germ-agglutinin-Sepharose.     

Localization of the nerve growth factor receptor on fetal human schwann cells in culture

Previous studies have established that Schwann cells (SC) in culture express an NGF receptor. In this study, cultures of fetal human SC were established from fetal nerves and various light microscopic (LM) and electron microscopic (EM) techniques were used to localize the NGF receptor on the SC. Results indicate that NGF receptor is localized to the plasma membrane of the SC. Quantitative digital analysis determined that the distal portion of the SC process had high concentrations of NGF receptor. The possible functional significance of this latter observation is discussed in terms of SC migration and ensheathment of axons.     

Rapid postnatal changes in F1-ATPase proteins and in the uncoupling protein in brown adipose tissue mitochondria of the newborn rat

Changes in F1-ATPase and UCP protein contents and in the activity of respiratory complexes I, II and IV of brown adipose tissue mitochondria are reported during the first 0-6 hours of life in the rat. Mitochondrial UCP/F1-ATPase protein ratio is used to define the onset of thermogenic differentiation of brown adipose tissue mitochondria. It is concluded that mitochondrial differentiation occurs soon after birth and that the process is accelerated by hypothermic conditions.     

Histochemical detection of monoamine oxidase and alcohol dehydrogenase activities in the Syrian hamster Harderian glands: existence of a sexual dimorphism

Summary Monoamine oxidase (MAO) and alcohol dehydrogenase (AD) activities were studied histochemically in the Syrian hamster Harderian gland using tryptamine as substrate and Nitroblue Tetrazolium as the final electron acceptor. No dark: light-related changes were observed. Male type I secretory cells showed an intense MAO reaction. Female type I cells exhibited a moderate MAO activity. Both male and female glands showed a moderate/intense AD-positive reaction. Male type II cells were lacking MAO and AD activities. MAO activity found in the hamster Harderian glands corresponded mainly to MAO type A since treatment with chlorgyline (0.01, 0.1 and 0.5mm) totally inhibited it. The possible role of these two enzymes in Harderian gland indolalkylamine metabolism is discussed.     

Ca2+ ionophores inhibit superoxide generation by chemotactic peptide in rabbit neutrophils and the correlation with intracellular calcium

Summary Effects of Ca²⁺ ionophores, A23187 and lasalocid, on superoxide anion generation by chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine methyl ester, in rabbit peritoneal exudate neutrophils were studied. The ionophores by themselves did not activate superoxide anion generation in these neutrophils. When preincubated with the cells for 2 min, both the ionophores inhibited superoxide generation induced by chemotactic peptide. The inhibition was present even in the absence of extracellular Ca²⁺ and the inhibition was better then. Lasalocid produces a dose-dependent chlortetracycline fluorescence decrease response in neutrophils loaded with chlortetracycline. This response is independent of extracellular Ca²⁺ concentration and is related to release of Ca²⁺ from intracellular storage sites. The dose-range at which lasalocid gives this response is same as the dose-range at which it causes inhibition of superoxide response. It may be concluded that the inhibition of superoxide generation by these ionophores is correlated to intracellular Ca²⁺ modulation.Abbreviations FMLP Formyl-Methionyl-Leucyl-Phenylalanine methyl ester     

Processing of SP1 precursor in a cell-free system from poly(A+) mRNA of human placenta

Cell-free translation of polyadenylated mRNA from human term placenta in a wheat germ extract, after immunoprecipitation with antibodies directed against purified pregnant serum SP₁, yielded a single polypeptide of 31 kDa. Addition of dog pancreatic microsomal vesicles to the translation system resulted in the appearance of two polypeptides, one of them of 46 kDa and the other of 28 kDa. Both polypeptides were protected from limited proteolysis and when the assay was performed with lytic detergent concentrations in addition to proteases, this protection was abolished indicating that the polypeptides were segregated into the microsomal vesicles. The cleavage of a signal peptide of 3 kDa from the 31 kDa primary translation product gives rise to 28 kDa and accounts for the slight increase in electrophoretic mobility. The treatment of the immunoprecipitated products with Endoglycosidase H and -mannosidase, suggested that only the 46 kDa polypeptide is a glycoprotein.From the results obtained we conclude that SP₁ is synthesized and processed to a glycoprotein of 46 kDa which would be a protomeric form of the oligomers reported in pregnant serum by other authors.Abbreviations PMSF phenylmethyl sulfonylfluoride - TCA trichloroacetic acid - DTT dithiotreitol