Isolation, characterization and transposition of an (IS2)2 intermediate

We have isolated and characterized a dimer derivative of the extensively studiedEscherichia coli insertion sequence IS2. The dimer structure — called (IS2)₂ — consists of two IS2 elements arranged as a direct repeat, separated by 1 bp. The junction between the (IS2)₂ dimer and target sequences is located at various positions in independent isolates; however, one position was preferred. The transposition of (IS2)₂ into a target plasmid resulted in cointegrate-type structures. The transposition frequency of the (IS2)₂ dimer itself was significantly higher than that of the isogenic monomer IS2 insertion. The poor stability and high activity of (IS2)₂ indicates that this is an active transposition intermediate. The mode of transposition of (IS2)₂ is analogous to the joined dimer model described in the case of (IS21)₂ and (IS30)₂.     

Correlation of iron content, spectral forms of chlorophyll and chlorophyll-proteins in iron deficient cucumber (Cucumis sativus)

Leaves and chloroplast suspensions of severely and slightly iron deficient cucumber ( Cucumis sativus L.) plants were characterized by low-temperature fluorescence emission spectroscopy and Deriphat polyacrylamide gel electrophoresis. The emission spectra of the chloroplast suspensions were resolved into Gaussian components and those changes induced by iron deficiency were related to the variations in the chlorophyll-protein pattern. The symptoms described with these methods were also correlated with the iron content of the leaves. It was concluded that the lack of physiologically active iron caused a relative decrease of photosystem I (PSI) and light harvesting complex I (LHCI), together with the long wavelength fluorescence, especially the 740 nm Gaussian component, and. to a much lesser extent, of the photosystem II (PSII) core complexes (relative increase of 685, 695 nm components). However, the relative decrease in the amount of light harvesting complex II (LHCII) was followed by a relative increase in its fluorescence band at 680 nm, showing that energy transfer from LHCII to core complex II (CCII) was partly disturbed. Thus iron deficiency affected the photosynthetic apparatus in a complex way: it decreased the synthesis of chlorophylls (Chls) and influenced the expression and assembly of Chl-binding proteins.     

Biomass dependent interactions in pond ecosystems: responses of lower trophic levels to fish manipulations

The effects of mature benthivorous cyprinid fish and theirrecruitment on sediment resuspension, turbidity, phyto- andzooplankton, and benthic macroinvertebrates were studied in fourexperimental ponds. The ponds were stocked with bream (Abramisbrama L.), white bream (Blicca bjoerkna L.), roach (Rutilus rutilus L.) and wild carp (Cyprinus carpio L.) of3+–5+ age classes at standing crop biomass varying from 0 to500 kg ha-1. Cyprinids caused an increase in sedimentresuspension and in turbidity, in proportion to their biomass. Meancrustacean biomass did not significantly affect phytoplanktonbiomass due to intense grazing by fish during spring. Ponds withhigh fish stocks showed reduced midge biomass and vegetation coverand increased biomass of predatory invertebrates.     

Methacholine-induced bronchoconstriction in rats: effects of intravenous vs. aerosol delivery

Peták, Ferenc, Zoltán Hantos, ÁgnesAdamicza, Tibor Asztalos, and Peter D. Sly. Methacholine-inducedbronchoconstriction in rats: effects of intravenous vs. aerosoldelivery. J. Appl. Physiol. 82(5):1479-1487, 1997.To determine the predominant site of action ofmethacholine (MCh) on lung mechanics, two groups of open-chestSprague-Dawley rats were studied. Five rats were measured duringintravenous infusion of MCh (iv group), with doubling of concentrationsfrom 1 to 16 µg · kg¹ · min¹.Seven rats were measured after aerosol administration of MCh with dosesdoubled from 1 to 16 mg/ml (ae group). Pulmonary input impedance(ZL) between 0.5 and 21 Hz wasdetermined by using a wave-tube technique. A model containing airwayresistance (Raw) and inertance (Iaw) and parenchymal damping (G) andelastance (H) was fitted to theZL spectra. In the iv group, MChinduced dose-dependent increases in Raw [peak response 270 ± 9 (SE) % of the control level; P < 0.05] and in G (340 ± 150%;P < 0.05), with no increase inIaw (30 ± 59%) orH (111 ± 9%). In the ae group, thedose-dependent increases in Raw (191 ± 14%;P < 0.05) andG (385 ± 35%; P < 0.05) were associated with a significant increase in H (202 ± 8%; P < 0.05).Measurements with different resident gases [air vs. neon-oxygenmixture, as suggested (K. R. Lutchen, Z. Hantos, F. Peták,Á. Adamicza, and B. Suki. J. Appl.Physiol. 80: 1841-1849, 1996)] in thecontrol and constricted states in another group of rats suggested thatthe entire increase seen in G during the ivchallenge was due to ventilation inhomogeneity, whereas the aechallenge might also have involved real tissue contractions viaselective stimulation of the muscarinic receptors.

     

A plant in vitro system for the nuclear import of proteins

This paper reports the development of an in vitro system that allows the direct assay of protein import into plant nuclei. In this assay the import of fluorescently labelled karyophilic protein substrates into nuclei isolated from evacuolated tobacco BY-2 suspension cells is monitored. It is demonstrated that import of the fluorescently labelled peptide conjugates is rapid, saturable and nuclear localization signal (NLS)-dependent. Exclusion of high molecular weight (70 kDa) dextran and substrates carrying mutated NLS sequences further underline the specificity of this system. Nuclear translocation of karyophilic import substrates in tobacco, similar to mammalian systems, is inhibited by the non-hydrolysable GTP analogue GTP-γ-S. In contrast, protein uptake is not blocked by wheat germ agglutinin, N-ethyl-maleinimide and iodoacetic acid. Furthermore, it is shown that nuclear import of proteins is only partially inhibited by low temperature (0–4°C). The in vitro nuclear import assay does not depend on exogenously added ATP or cytosolic factors. However, a block of nuclear import with GTP-γ-S could be overcome by the addition of cytosolic extract, suggesting the dependence on cytosolic factors or proteins. These data indicate that the characteristics of nuclear protein import in plant and mammalian cells are similar, but may be, at least in some respects, also different from each other.     

Identifying Monoaminergic,GABAergic, and Cholinergic Characteristics in Immortalized Neuronal Cell Lines

We measured the concentration of neurotransmitters in immortalized neural cell lines of hippo-campal, septal, brainstem and cerebellar origin. While in most of the cell lines, concentrations of monoamines, -aminobutyric acid (GABA) and acetylcholine were low, in some they were markedly higher. This made it quite easy to identify possible monoaminergic, GABAergic or cholinergic cell lines. However all the cell lines contained glutamate and aspartate and there were no outstanding differences in levels of these amino acids differences between the cell lines. Deprivation of serum, which made the cells acquire a more differentiated morphology, caused an increase in the intracellular concentrations of some compounds and a switch from multiple to a single transmitter in the case of some cell lines. It suggested that measurement of transmitter concentrations combined with serum deprivation studies, may provide an indication of the neurochemical characteristics of immortalised neuronal cell lines.     

Isolation and characterization of a protease inhibitor from commercial stem bromelain acetone powder

Seven closely related protease inhibitors were isolated from commercial bromelain acetone powder in electrophoretically pure form by gel filtration on Sephadex G-75, followed by ion exchange chromatography on DEAE Sephadex at pH 7.55. The inhibitors are proteins of MW 5000-6000, which inhibit competitively the bromelaincatalyzed hydrolysis of CLN (K_i ≈ 10^?7 M). This inhibition is optimal at pH 3 to 4,. and it depends upon the ionization of two acidic residues of pK = 4.5 and 5.0. In the acidic pH range the inhibitors are also effective toward papain, ficin and trypsin.     

Multiple forms of porcine pancreatic phospholipase A2: Isolation and specificity

Porcine pancreatic phospholipase A₂ was purified from commercial pancreatin by a method involving heat denaturation, trichloroacetic acid precipitation, and DEAE-cellulose chromatography. Assaying the eluate of the chromatography step by a new titrimetric method using vegetable lecithin-albumin emulsion as the substrate, several species of phospholipase A were found. Some of these went undetected when the conventional egg yolk emulsion assay was used. Two phospholipases A₂ were isolated in a homogeneous form and shown to have similar chemical and physical properties. Catalytic specificity of the two enzymes differs remarkably toward lecithins in different emulsified states.     

Neurochemistry of GABAergic System in Cerebral Cortex Chronically Exposed to Bromide In Vivo

Neurochemical correlates of GABAergic synaptic transmission [binding, uptake, metabolism, and tissue content of gamma-aminobutyric acid (GABA)] were investigated in the cortex of rats that had been given 27 mM bromide in drinking water for periods of time ranging from 1 day to 1 month. No effect of bromide on any of the parameters was found and it is concluded that chronic administration of bromide has no profound effect on GABAergic inhibitory system in the rat cortex.