Both linked and unlinked mutations can alter the intracellular site of synthesis of exported proteins of Escherichia coli.

It previously has been demonstrated that synthesis of the periplasmic maltose-binding protein (MBP) and alkaline phosphatase (AP) of Eschericha coli predominantly occurs on membrane-bound polysomes. In this study, signal sequence alterations that adversely affect export of MBP and AP, resulting in their cytoplasmic accumulation as unprocessed precursors, were investigated to determine whether they have an effect on the intracellular site of synthesis of these proteins. Our findings indicate that export-defective MBP and AP are not synthesized or are synthesized in greatly reduced levels on membrane-bound polysomes. In some instances, a concomitant increase in the amount of these proteins synthesized on free polysomes was clearly discerned. We also determined the site of synthesis of MBP and AP in strains harboring mutations thought to alter the cellular secretion machinery. It was found that the presence of a prlA suppressor allele partially restored synthesis of export-defective MBP on membrane-bound polysomes. On the other hand, the absence of a functional SecA protein resulted in the synthesis of wild-type MBP and AP predominantly on free polysomes.     

Characteristics of angiotensin II-, K+- and ACTH-induced calcium influx in adrenal glomerulosa cells. Evidence that angiotensin II, K+, and ACTH may open a common calcium channel

The characteristics of angiotensin II-, K+-, and adrenocorticotropin (ACTH)-induced calcium influx were studied in isolated adrenal glomerulosa cells. Basal calcium influx rate is 0.64 +/- 0.09 nmol/min/mg of protein. Addition of angiotensin II (1 nM) causes a rapid 230% increase in calcium influx rate. This angiotensin II-induced calcium influx is sustained and is rapidly reversed by angiotensin II antagonist, [Sar1,Ala8]angiotensin II. Addition of either K+ or ACTH (1 nM) causes a 340 or 160% increase, respectively, in the rate of calcium influx. The effect of either angiotensin II, K+, or ACTH on calcium influx is dependent on extracellular calcium. The apparent Km for calcium is 0.46, 0.35, and 0.32 mM, respectively. When the extracellular concentration of K+ is 2 mM, neither angiotensin II nor ACTH stimulates calcium influx. Conversely, when extracellular K+ is increased to 6 mM, both angiotensin II and ACTH cause a greater stimulation of calcium influx than at 4 mM K+. When extracellular K+ is increased to 10 mM, calcium influx is 360% of the basal influx seen at 4 mM K+, and neither angiotensin II nor ACTH further stimulates the influx rate. Nitrendipine (1 microM) blocks both angiotensin II- and K+-induced calcium influx completely. In contrast, 10 microM nitrendipine does not completely block ACTH-induced calcium influx. The calcium channel agonist, BAY K 8644, also stimulates calcium influx; 10 nM BAY K 8644 leads to a rate of calcium influx which is 185% of basal. This BAY K 8644-induced increase in calcium influx and that caused by either angiotensin II or ACTH are additive. In contrast, BAY K 8644 has more than an additive effect on the calcium influx when paired with 6 mM K+. These results suggest that angiotensin II, K+, and ACTH stimulate calcium influx via a common calcium channel but act by different mechanisms to alter its function.     

Viral polypeptides detected by a complement-dependent neutralizing murine monoclonal antibody to human cytomegalovirus.

Murine monoclonal antibodies were produced which coimmunoprecipitated, under reducing conditions, 130,000- and 55,000-dalton (Da) polypeptides from cells infected with human cytomegalovirus (CMV) strain AD169. A 92,000-Da species, possibly a biosynthetic intermediate, was also detectable. One of the monoclonal antibodies, 15D8, neutralized CMV AD169 only in the presence of guinea pig complement. A second monoclonal antibody, 14E10, coimmunoprecipitated the 130,000- and 55,000-Da polypeptides but did not neutralize viral infectivity. By sequential immunoprecipitation, both monoclonal antibodies have been shown to recognize the same polypeptides. Monoclonal antibody 15D8 detected the 130,000- and 55,000-Da polypeptides in five of six clinical strains and three laboratory strains tested. The 14E10 monoclonal antibody detected the 130,000-Da protein in four of six CMV clinical isolates and in strain AD169 but did not immunoprecipitate any polypeptides from extracts of cells infected with either Towne or Davis laboratory strains. In kinetic studies, the synthesis of the 130,000-Da polypeptide preceded the appearance of the 55,000-Da polypeptide. In infected cells radiolabeled with a pulse of L-[35S]methionine, the isotope was initially detected in the 130,000-Da polypeptide but could be chased into the 55,000-Da polypeptide. These polypeptides exist in the intracellular and extracellular virus as disulfide-linked multimers. Extracellular virus contained a high-molecular-weight (greater than 200,000 Da) multimer composed entirely of 55,000-Da polypeptides. In extracts from infected cells an additional high-molecular-weight multimer was detected consisting of disulfide-linked 130,000-Da polypeptides.     

UV-microscopic studies on the vacuolar changes caused by the flavone “aglycone” isovitexin inSilene pratensis plants

Summary UV-microscopic and chromatographic studies have been performed on the variation in contents and configuration of the flavones present in epidermal cells of the petals, stem leaves, rosette leaves and cotyledons ofSilene pratensis plants. Most of the flavone contents is located in the vacuole of the upper epidermis cells, the concentration depending on the light intensity at which the plants were grown. In plants able to glycosylate isovitexin in the petals (genotypegG/. gl/gl fg/fg, accumulating isovitexin 7-O-glucoside) the vacuole is completely filled with the UV absorbing flavone. In plants which are unable to glycosylate isovitexin in their petals (genotypeg/g gl/gl fg/fg, accumulating only isovitexin) the upper epidermal cells of stem leaves and petals contain droplet like structures in their vacuoles. At high light intensities these structures increase in mass and become detectable in the visible light. These denser structures often condense to structures with radiating threads.As compared with the accumulation of isovitexin in upper epidermal cells of stem leaves and petals in genotypeg/g gl/gl fg/fg, the cotyledons and the rosette leaves contain two isovitexin glycosides. In the latter organs the upper epidermal cells are very similar to the upper epidermal cells fromgG/. gl/gl fg/fg plants, having a vacuole filled with UV absorbing material. It appears therefore that isovitexin itself causes the formation of the structurés in the cells. It was shown by varying the light intensity that a relative high concentration of isovitexin is necessary for the droplet like structures to appear. Still higher concentrations are needed for the formation of the structures with radiating threads. It is hypothesized that isovitexin interferes with the energy supply of the cells, which therefore are not able to maintain their turgor.     

A high-affinity folate binding protein in normal human leukocytes: Ligand binding characteristics,ionic charge and molecular size

High-affinity binding of [³H]folate to supernatant from homogenized human leukocytes containing large amounts of binding protein displayed apparent positive cooperativity. The DEAE-Sepharose^® CL-6B chromatographic profile of the supernatant at pH 6.3 contained a major peak of folate binding (M_r approx. 25 000) in the front effluent and a smaller more acidic peak (M_r approx. 25 000) that emerged after a rise in NaCl from 30 mmol/l to 1 mol/l. Triton X-100 solubilized ceil sediment from the leukocyte homogenate contained some high-affinity folate binding activity (M_r approx 25 000), typically 5–10% of the total binding activity.     

The influence of proteases on the activity of glucoamylase from Aspergillus niger C

Summary Two proteases from Aspergillus niger C post-culture medium were isolated by fractionation on a DEAE-sepharose column and ultrafiltration. The four fractions of glucoamylase activity (GA1, GA2, GA3 and GA4) present in the medium showed different susceptibility to the influence of proteases. The effects of proteases on the different glucoamylase fractions during the growth of the fungus are demonstrated. The activity was found to decrease at the beginning of the culture, but by its end there was a stimulation of GA4 glucoamylase. After treating GA2 and GA3 with protease II, a new additional fraction of glucoamylase was detected.     

Studies on 9-amino-6-chloro-2-methoxy acridine fluorescence quenching and enhancement in relation to energy transduction in spheroplasts and intact cells of the cyanobacterium Plectonema boryanum

The fluorescent probe 9-amino-6-chloro-2-methoxy acridine was used to study the energy transduction in the thylakoid and cell membranes of the cyanobacterium Plectonema boryanum. Apart from light-driven electron transfer, the dark endogenous respiration also leads to energization resulting in an ACMA fluorescence response, that is sensitive to the electron flow inhibitor 2, 5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, to the energy transfer inhibitors dicyclohexylcarbodiimide and venturicidine and to the uncoupler 5-chloro-3-t-butyl-2-chloro-4-nitrosalicylanilide.In spheroplasts, in which the cell membranes have lost their capacity to maintain a proton gradient, the respiration-and light-induced ACMA fluorescence changes (quenching) are similar to those in chloroplasts. In intact cells a combination of reversible quenching and enhancement of ACMA fluorescence was found. This dualistic behaviour is supposedly caused by an opposite orientation of the thylakoid and cell membranes. ACMA quenching at the level of the thylakoids was obtained either by respiratory or photosynthetic electron transfer and gave similar responses to those obtained in the spheroplasts. The slower ACMA fluorescence enhancement, only observed in cells with intact cell membranes, also evoked by both respiration and light-induced energization is sensitive to the compounds mentioned above and in addition to KCN.Our results support the view [8] that dark oxidation of substrates by O₂ proceeds via the thylakoid membrane and terminates at a CN^- sensitive oxidase located in the cell membrane which requires the involvement of a mobile cytoplasmic redox mediator.Abbreviations ACMA 9-amino-6-chloro-2-methoxy acridine - chl a chlorophyll a - DBMIB 2, 5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCCD dicyclohexylcarbodiimide - DNP dinitrophenol - DNP-INT dinitrophenyl ether of 2-iodo-4-nitrothymol - FCCP carbonylcyanide-p-trifluoro-methoxy phenylhydrazone - S-13 5-chloro-3-t-butyl-2-chloro-4-nitrosalicylanilide - tricine N-2 (2-Hydroxy-1, 1-bis (hydroxymethyl) ethyl)-glycine - Tris Tris (hydroxymethyl) amino methane     

Effects of alternating temperatures onAcarus siro L. (Acari: Acaridae)

Fecundity, longevity and survival to adulthood ofAcarus siro (L.) at constant and alternating temperatures were compared. Both fecundity and longevity were affected by alternating temperatures but the effect of the frequency of alternations was not significant. Significantly higher fecundity and longer life spans were recorded at constant temperatures of 14 and 21°C than at 28°C or at alternating temperature regimes. Alternating temperature regimes in comparison to constant regimes did not significantly change the number of males and females surviving to adulthood or the sex ratio. However, the lowest number of adults surviving was recorded when temperatures were changed every 12 h and the highest at a constant temperature of 14°C.     

An immunocytochemical study of the optic ganglia of the crayfish Astacus leptodactylus (Nordmann 1842) with antisera against biologically active peptides of vertebrates and invertebrates

Summary The peptidergic system in the optic ganglia of Astacus leptodactylus is characterized by the immunocytochemical application of 15 antisera raised against biologically active peptides of vertebrates and invertebrates. Positive reactions were found with anti-FMRFamide, antiMSH, anti-vasotocin, anti-gastrin, anti-CCK, anti-oxytocin, anti-secretin, anti-glucagon and anti-GIP. Based on immunochemical reaction and localization it is possible to distinguish 30 cell groups. Only part of these cell groups is found in the known classical neurosecretory cell regions. This observation demonstrates a more extensive peptidergic system than formerly recognized. The morphology of this peptidergic system suggests that one part is neurohormonal and the other part neurotransmitter-like or neuromodulatory.     

Implementation of periodic acid-thiosemicarbazide-silver proteinate staining for ultrastructural assessment of muscle glycogen utilization during exercise

Summary Distribution of glycogen particles in semithin and ultrathin sections of biopsy samples from human muscles subjected to either short- or long-term running were investigated using PAS and Periodic Acid-ThioSemiCarbazide-Silver Proteinate (PA-TSC-SP) staining methods. Glycogen particles were predominantly found immediately under the sarcolemma or aligned along the myofibrillar Iband. After long-term exhaustive exercise type-1 fibers with a few or no glycogen particles in the core of the fibers were frequently observed. The subsarcolemmal glycogen stores of these depleted type-1 fibers were about three times as large as after exhaustive short-time exercise. Another indication of utilization of subsarcolemmal glycogen stores during anaerobic exercise was that many particles displayed a pale, rudimentary shape. This observation suggests fragmental metabolization of glycogen. Thus, depending on type of exercise and type of fiber differential and sequential glycogen utilization patterns can be observed.