The effect of tunicamycin on development of the mammalian embryonic pancreas

The role of cell-surface glycoproteins in histogenesis of the embryonic rat pancreas was investigated by studying the effect of tunicamycin (TM) on in vitro development. TM has been shown to block glycosylation of asparagine residues in glycoproteins by inhibiting formation of dolichol oligosaccharide intermediates. Exposure of Day 15 pancreatic rudiments to 1.0 μg TM/ml for 15 or 24 hr inhibited [³H]mannose, [³H]glucosamine, and [³H]fucose incorporation by 95, 85, and 90%, respectively, while [³H]leucine incorporation was reduced by 35%. Similar results were obtained with Day 17 rudiments. These trends were confirmed using sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis and fluorography. Inhibition of [³H]monosaccharide incorporation correlated with reduced binding of RCA I-ferritin conjugates to the cell surface and both effects of TM were reversed by reculturing rudiments in medium lacking the antibiotic. Morphologically, TM treatment resulted in a delay in pancreatic histogenesis and this delay correlated with an inhibition of the normal increase in specific activity of amylase, an acinar cell secretory protein. These effects were not mimicked by treatment with cycloheximide at a concentration which inhibited [³H]leucine incorporation to the same degree observed with TM. The percentage of delayed rudiments decreased as reculturing in the absence of TM was extended.     

Temperature-sensitive periods and autonomy of pleiotropic effects of l(1)Nts1, a conditional notch lethal in Drosophila.

Analysis of temperature-shift experiments using strains homo- and/or hemizygous for a temperature-sensitive (ts) mutation of the Notch locus, l(1)N^ts1, has permitted us to localize temperature-sensitive periods (TSPs) both for lethality and for adult ectodermal morphology defects. Discrete TSPs for lethality are localized to the first half of the embryonic period, to the second larval instar, to the third larval instar, and to a 15 hr period immediately after pupation. TSPs for adult morphology defects are localized to the second and third larval instars for eyeless-headless and duplicated antenna, to the third larval instar for small and rough (spl-like) eye, eye scar, fused leg segments, shortened tarsal leg segments, Notch wings, and extra macrochaetae, and to the early pupal period for extra and missing microchaetae, fa^g-like rough eye and thick wing vein defects. Within the third larval instar, distinct patterns of eye, wing, and leg defects are observed. There is a striking similarity between the adult morphology defects and TSPs of l(1)N^ts1 and those of the larval and adult locomotor mutant, shi^ts1 (C. A. Poodry, L. Hall, and D. T. Suzuki, 1973, Develop. Biol.32, 373–386). Expression of l(1)N^ts1 also has been studied in genetic mosaics, in which we find that the pleiotropic effects of l(1)N^ts1 are autonomously expressed.     

Protein synthesis in dorsal and ventral regions of Xenopus laevis embryos in relation to dorsal and ventral differentiation

Two-dimensional gel electrophoresis has been used to analyze protein synthesis in dorsal and ventral regions in embryonic stages of Xenopus laevis. Proteins specific either to dorsal or to ventral regions are synthesized for the first time at gastrulation, concomitant with morphological differentiation. The reliability of these proteins as markers of dorsal and ventral differentiation was tested by examining their synthesis in Uv-irradiated embryos, which have severely reduced capacity for dorsal development, reflected in reduced levels of the neuromuscular-specific enzyme acetylcholinesterase, but which continue to synthesize the great majority of proteins at normal rates. Synthesis of dorsal indicator proteins should be reduced or absent in these embryos, whereas ventral indicators should be synthesized at least to the same extent as in control embryos. Some of the putative dorsal and ventral indicators failed this test, but the majority were confirmed as reliable markers of dorsal and ventral differentiation, thus providing a connection between morphology and gene expression in the establishment of the dorsal-ventral axis in X. laevis.     

Tissue-specific synthesis of yolk proteins in Caenorhabditis elegans

The primary site of yolk protein synthesis in the nematode, Caenorhabditis elegans, has been determined. In animals containing no gonadal cells (obtained by laser ablation of the gonadal precursor cells early in development), yolk proteins are present in abundance. This demonstrates that yolk proteins are made outside the gonad. An examination of proteins present in tissues isolated by dissection, and a comparison of proteins synthesized by isolated tissues incubated in vitro have identified the intestine as the major site of yolk protein synthesis. We propose that yolk proteins are synthesized in the intestine, secreted from the intestine into the body cavity, and taken up from the body cavity by the gonad to reach oocytes. The site of yolk protein synthesis has also been examined in four mutants that have largely male somatic tissues, but a hermaphrodite germ line. Here again, yolk proteins are produced by intestines in a hermaphrodite-specific manner. This suggests that sex determination is coordinately regulated in intestinal and germ line tissues.     

Asymmetric distribution of nitrate reductase subunits in the cytoplasmic membrane of Escherichia coli: evidence derived from surface labeling studies with transglutaminase.

The enzyme transglutaminase has been used to label surface proteins of Escherichia coli cytoplasmic membranes by covalently attaching to them a small fluorescent primary amine, dansyl cadaverine. Spheroplasts lacking outer membrane, osmotically lysed vesicles from the spheroplasts, and vesicles made by breaking cells in a French pressure cell were each labeled with transglutaminase and dansyl cadaverine. When the total cytoplasmic membrane proteins of each were examined on sodium dodecyl sulfate gels, three rather different labeling patterns were obtained. Labeling of the respiratory enzyme, nitrate reductase, in the membranes of each of these preparations was also examined. Membrane-bound nitrate reductase contains three subunits: A, B, and C. Dansyl cadaverine labeling of nitrate reductase in the presence of Triton X-100 indicated that subunits A and C could be labeled. When nitrate reductase was isolated from dansyl cadaverine-labeled spheroplasts, none of the subunits was labeled. When nitrate reductase was isolated from French press vesicles, subunit A was labeled and labeling was enhanced by the presence of nitrate during labeling. When nitrate reductase from osmotic vesicles was examined, subunit A was labeled in the presence of nitrate but no labeled subunits appeared when the vesicles were labeled in the absence of nitrate. It was concluded that (i) nitrate reductase is buried in the membrane with subunit A exposed only on the inner surface of the membrane, (ii) subunit C is sufficiently buried within the membrane so that it is inaccessible to transglutaminase, (iii) subunit B is not labeled under any condition, so its location is not known, and (iv) large osmotic vesicles are probably mosaics in which some protein components have been reoriented.     

Effect of N,N,N',N'-tetramethylethylenediamine on the migration of proteins in SDS polyacrylamide gels

Changing the concentration of TEMED in SDS polyacrylamide gels was found to affect the migration of proteins. Elevation of TEMED levels caused a generalized decrease in mobility with some proteins being affected more than others. With various brain protein preparations this differential effect could be used to improve the separation of adjacent protein bands. In additon, it was found that a change in the TEMED concentration affected the results of molecular weight determinations. The effect of TEMED was also observed in one non-SDS system.     

Temporal changes in embryonic nerve cell recognition: correlate with cholinergic development in aggregate cultures

Chick embryo retina and optic tectum cells can be dissociated into single cells and then reaggregated in suspension cultures to give highly organized and differentiated aggregates. These aggregates show a degree of cholinergic differentiation that is characteristic of each cell type; the low activity of choline acetyltransferase in the optic tectum aggregates probably reflects the condition of natural deafferentation inherent in the culture situation. It is possible, in this respect, to study the retina-tectum interaction in vitro by preparing coaggregates including both types of cells. When coaggregates are prepared from tectum and retina cells of the same developmental age, the activity of choline acetyltransferase measured in the coaggregates is consistently higher than would be expected from the simple addition of the activities of the component cells, pointing to some kind of metabolic synergism between retinal and tectal cells. As for acetylcholinesterase, this synergism occurs only under special circumstances, and it is generally less marked. No synergism was observed when retina and tectum cells of different developmental age were coaggregated, suggesting the existence of a temporal control of neuronal interaction specificity. On the other hand, the synergism is only observed between neuronal systems that are known to establish synaptic connections during normal in vivo development: No interaction could be detected when either retinal or tectal cells were combined with telencephalon, cerebellum, or liver cells. Experimental evidence is presented suggesting that the retina-tectum interaction depends on intimate cell-cell contact, and it is not mediated by freely diffusible molecules. Neurotransmission-related metabolic studies in coaggregates seem to offer a promising tool to study recognition-interaction phenomena in groups of neurons establishing synaptic links during development.     

Regenerating adult chicken skeletal muscle and satellite cell cultures express embryonic patterns of myosin and tropomyosin isoforms

Regenerating areas of adult chicken fast muscle (pectoralis major) and slow muscle (anterior latissimus dorsi) were examined in order to determine synthesis patterns of myosin light chains, heavy chains and tropomyosin. In addition, these patterns were also examined in muscle cultures derived from satellite cells of adult fast and slow muscle. One week after cold-injury the regenerating fast muscle showed a pattern of synthesis that was predominately embryonic. These muscles synthesized the embryonic myosin heavy chain, beta-tropomyosin and reduced amounts of myosin fast light chain-3 which are characteristic of embryonic fast muscle but synthesized very little myosin slow light chains. The regenerating slow muscle, however, showed a nearly complete array of embryonic peptides including embryonic myosin heavy chain, fast and slow myosin light chains and both alpha-fast and slow tropomyosins. Peptide map analysis of the embryonic myosin heavy chains synthesized by regenerating fast and slow muscles showed them to be identical. Thus, in both muscles there is a return to embryonic patterns during regeneration but this return appears to be incomplete in the pectoralis major. By 4 weeks postinjury both regenerating fast and slow muscles had stopped synthesizing embryonic isoforms of myosin and tropomyosin and had returned to a normal adult pattern of synthesis. Adult fast and slow muscles yielded a satellite cell population that formed muscle fibers in culture. Fibers derived from either population synthesized the embryonic myosin heavy chain in addition to alpha-fast and beta-tropomyosin. Thus, muscle fibers derived in culture from satellite cells of fast and slow muscles synthesized a predominately embryonic pattern of myosin heavy chains and tropomyosin. In addition, however, the satellite cell-derived myotubes from fast muscle synthesized only fast myosin light chains while the myotubes derived from slow muscle satellite cells synthesized both fast and slow myosin light chains. Thus, while both kinds of satellite cells produced embryonic type myotubes in culture the overall patterns were not identical. Satellite cells of fast and slow muscle appear therefore to have diverged from each other in their commitment during maturation in vivo.     

Kinetic identification of component X as P430: a primary electron acceptor of Photosystem I

Kinetics of dark recoveries of Component X, Center A, and Center B at 20 and 0 °C after a 30-s illumination were studied in membrane fragments from a blue-green alga by using low temperature electron paramagnetic resonance spectroscopy in combination with a quick-freeze method. These kinetics were compared with those obtained by spectrophotometry under the same conditions. Contrary to the currently popular view, the result strongly suggests that Component X, rather than Center A or Center B, is P430.     

The use of potassium iodide equilibrium density gradient centrifugation in the purification of RNA for hybridization with nonreiterated DNA sequences.

A new instrument has been developed that is able to monitor enzyme activities in chromatography column eluents automatically. The instrument consists of a simple stopped-flow instrument, equipped with a multishutoff valve to direct reagents and eluent flow. Assays of the eluate may be done at present intervals. The instrument may also be used for monitoring enzyme activities in perfasates as well as for monitoring the elution of metabolites.     

Binding of active and inactive hemolytic factor of sea anemone nematocyst venom to red blood cells

Sea anemone nematocyst venom, in the presence of Ca²⁺, induced the lysis of red blood cells after an induction period. In the absence of Ca²⁺, however, no lysis occurred, but the hemolytic factor was shown to bind to the cells. This binding was shown to be requisite for the Ca²⁺ dependent lysis to ensue. After freeze thawing, the venom proteins responsible for lysis lost their hemolytic activity, yet still bound to the cells. The freezethawed inactivated venom competitively blocked hemolysis by active venom.     

Effects of castration and home cage residency on aggressive behavior in rats

The effects of castration of both resident and intruder rats on territorial aggressive behavior were studied. The results suggest that residence in a home cage is more important than gonadal status in determining the outcome of an aggressive encounter. Resident rats were more likely to be dominant especially if they were intact. Intact residents directed less aggressive behavior toward castrated intruders than toward intact intruders. Intruder rats generally showed low levels of aggressive behavior and were only dominant when the resident had been castrated. Thus, the aggressive behavior of a male rat depends upon both his gonadal status and that of his opponent.     

Effects of protease inhibitors on sperm-related events in sea urchin fertilization

Evidence for sperm-borne proteolytic enzymes exposed during the acrosome reaction in sea urchin sperm has been accumulating. To investigate the possible role(s) such enzymes have in fertilization, we studied the effects of several protease inhibitors on sperm-related events. Soybean trypsin inhibitor, Nα-p-tosyl-l-lysine, chloromethyl ketone, phenylmethylsulfonyl fluoride, and chymostatin neither reduced the number of acrosome reactions nor interfered with gamete binding. p-Nitrophenyl-p′-guanidinobenzoate caused sperm to fuse into irregular clumps, rendering them unable to fertilize eggs. However, l-1-tosylamide-2-phenylethyl chloromethyl ketone (TPCK), an inhibitor of chymotrypsin, prevented the acrosome reaction in Strongylocentrotus purpuratus, S. droebachiensis, and Lytechinus pictus. The effects of TPCK on sperm in subsequent steps of fertilization were also investigated. First, gamete binding assays were performed on fixed eggs. This precluded any effects TPCK might have had on egg-derived secretions (e.g., proteases). Binding of prereacted sperm occurred with both fixed and living eggs. However, fertilization of living eggs in the presence of TPCK was greatly reduced, even though sperm had been prereacted with egg jelly. Vitelline coats were then removed from eggs by trypsin treatment. Eggs in TPCK fertilized and developed normally after the above treatment. These observations are consistent with the hypothesis of a sperm protease participating in the acrosome reaction and the penetration of the egg vitelline coat in the sea urchin.     

Effect of a bifunctional imidoester on dissociation of 70S ribosomes in Escherichia coli

Treatment of the 70S ribosome from $\text{Escherichia coli}$ with diethyl malonimidate dihydrochloride, a bifunctional imidoester, was found to result in the formation of crosslinkage between the two subunits. The 70S complex thus obtained no longer dissociates into 50S and 30S particles at 0.5mM Mg²⁺ concentration, but do so at lower concentrations (0.1mM), suggesting the release of protein(s) involved in the inter-particle cross-linkage from one or both ribosomal subunits.     

Incorporation of 5-bromodeoxyuridine into DNA in newborn rat tissues

The incorporation of bromodeoxyuridine (BUdR) into newborn rat tissue DNA has been determined after i.p. injection of 5-bromo-2′-deoxy[6-³H]uridine. Incorporation of the unchanged nucleoside was shown by hydrolysis and ion exchange chromatography of extracted DNA. In all tissues examined, more than 90% of the radioactivity incorporated was in the form of bromodeoxyuridine.     

Adenylate kinase bound to the envelope membranes of spinach chloroplasts.

The activity of adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3) in both the forward (2ADP → ATP + AMP) and backward (ATP + AMP → 2ADP) reactions was found to be associated with the envelope membranes which were isolated from spinach chloroplasts. Sonication and repeated washing in a medium of high ionic strength were unable to release the enzymes from the envelope membranes. Adenylate kinase bound to the envelope is stable in the cold and inactivated by heat and acid treatments. The enzyme requires magnesium ion as an activator. The pH-activity profile of the forward reaction catalyzed by membrane-bound adenylate kinase gave a maximal activity at pH 8.5. The apparent Michaelis constant, K_m, value for ADP in the forward reaction was estimated to be 1.3 ± 0.2 × 10^?4m. A Lineweaver-Burk plot of the forward reaction gave a straight line when the reciprocal of the reaction rate was plotted versus the reciprocal, and not the square of the reciprocal, of the concentration of substrate ADP. This favors the view that the adenylate kinase bound to the chloroplast envelope has a single or equivalent binding site of Mg-ADP^?. The probable involvement of adenylate kinase bound to the chloroplast envelope in controlling the energy pool and adenylate translocation in chloroplasts is suggested.     

The long-term effect of glucagon on pyruvate kinase activity in primary cultures of hepatocytes

Glucagon caused a marked decrease in the total L-pyruvate kinase activity of control hepatocytes maintained in monolayer culture (t1/2 = 54 h), while the addition of insulin to hepatocytes isolated from a fasted rat caused a four- to fivefold increase in the total enzyme activity. Maintenance of L-pyruvate kinase in control cultures of hepatocytes was shown to require insulin. However, when 1 microM glucagon was present in the medium, the total L-pyruvate kinase activity was not maintained even in the presence of 1 microM insulin, but rather the total L-pyruvate kinase activity of the cells steadily declined from 12.1 to 5.7 units/mg DNA by the 6th day in culture. The increase in the total L-pyruvate kinase activity of fasted hepatocytes cultured in the presence of insulin was shown to result from an increase in protein synthesis, since actinomycin D and cycloheximide blocked the insulin-induced increase in the enzyme activity. The addition of 1 microM glucagon to cultures of fasted hepatocytes also blocked the insulin-induced increase in total L-pyruvate kinase activity. Since glucagon decreased the total L-pyruvate kinase activity in control hepatocytes and blocked the increase in L-pyruvate kinase activity in fasted hepatocytes, it is suggested that, in addition to the phosphorylation of L-pyruvate kinase by a cAMP-dependent protein kinase, glucagon also acts to decrease the synthesis of L-pyruvate kinase in vitro.     

Measurement of biosynthetic rates and intracellular transit times for a cell-surface membrane glycoprotein, alkaline phosphatase in HeLa cells

An assay procedure for thyroid hormone receptor activity which used nitrocellulose membrane filters was developed. Receptor proteins, extracted from washed rat liver nuclei with a 0.4 M NaCl solution, were incubated with 125I-labeled thyroid hormone (T3), and filtered on the cellulose ester membranes under suction at 2 degrees C. The filters were subsequently washed with cold buffer and counted for 125I radioactivity. The method allowed an accurate estimation of the receptor activity, satisfying a linear relationship between the activity and the receptor protein concentrations. The usefulness of this filter-binding method became evident when it was compared with the conventional procedure that employs Sephadex G-25 columns. For practical application to routine assays, various filtration conditions were examined, and a standard procedure was established. Using this technique, the isolated receptors were determined to possess an apparent Kd of 1.38 X 10(-10) M and a pH optimum of T3 binding at 8.2-8.4.     

Heterogeneity of heparan sulfate proteoglycans synthesized by PYS-2 cells

Antibodies to the basement membrane proteoglycan produced by the EHS tumor were used to immunoprecipitate [35S]sulfate-labeled protoglycans produced by PYS-2 cells. The immunoprecipitated proteoglycans were subsequently fractionated by CsCl density gradient centrifugation and Sepharose CL-4B chromatography. The culture medium contained a low-density proteoglycan eluting from Sepharose CL-4B at Kav = 0.18, containing heparan sulfate side chains of Mr = 35-40,000. The medium also contained a high-density proteoglycan eluting from Sepharose CL-4B at Kav = 0.23, containing heparan sulfate side chains of Mr = 30,000. The corresponding proteoglycans of the cell layer were all smaller than those in the medium. Since the antibodies used to precipitate those proteoglycans were directed against the protein core, this suggests that these proteoglycans share common antigenic features, and may be derived from a common precursor which undergoes modification by the removal of protein segments and a portion of each heparan sulfate chain.