High degree of homology of the deduced protein structures of the tetracycline-resistance determinants between Bacillus subtilis plasmid pNS1981 and Staphylococcus aureus plasmid pTP5

The amino acid sequences of the tetracycline-resistance (Tc^r) determinants of Bacillus subtilis plasmid pNS1981 and Staphylococcus aureus plasmid pTP5 have been deduced from their nucleotide sequences and compared. The deduced Tc^r proteins (TETs) of pNS1981 (458 amino acids) and pTP5 (459 amino acids) show a considerable homology (60% identical). If homologous amino acid replacement is taken into account, the homology becomes 80%. Both TET proteins are highly hydrophobic, as expected for a membrane-binding protein, and their polarities are calculated at 32–33%. The putative secondary structures of both TET proteins have been also shown to be significantly homologous, being abundant in -sheets. The predicted positions of -sheets show a nice coincidence between both TET proteins. -Helix has a tendency to be formed at nonhomologous regions of the primary structures between both TET proteins. However, the predicted positions of -helices coincide in a frequency greater than 50%. -Helix and random coil moderately occur at the hydrophilic regions in both TET proteins.     

Characterization of a Na∶K∶2Cl cotransport system in the apical membrane of a renal epithelial cell line (LLC-PK1)

Summary ⁸⁶Rb uptake into LLC-PK₁ cells (an established renal epithelial cell line) was found to be comprised of an active ouabain-sensitive component, a loop diuretic-sensitive component which was passive and strictly dependent upon the presence of extracellular Na⁺ and Cl^– for activity, and a leak component. The diuretic-sensitive component of influx was investigated further in apical membrane vesicles derived from these cells. A large fraction of⁸⁶Rb,²²Na and³⁶Cl flux into these vesicles was sensitive to inhibition by furosemide and dependent upon the presence of the other two co-ions, in keeping with the presence of a loop diuretic-sensitive Na⁺K⁺Cl^– cotransport system. The kinetic parameters for Na⁺ and K⁺ interaction have been analyzed under initial linear zerotrans conditions. The following values were obtained:K _mNa+=0.42±0.05 mmol/liter,V _max=303±24 pmol/mg/6 sec;K _mK+=11.9±1.0 mmol/liter,V _maxK+=307±27 pmol/mg/6 sec. For Cl^– interaction evidence for two cooperative binding sites with different affinities and different specificities were obtained. Thus, a stoichiometry of 1Na⁺1K⁺2Cl^– can be calculated. It is concluded that the apical membrane of LLC-PK₁ cells contains a Na⁺K⁺2Cl^– cotransport system with properties similar to those described for the thick ascending limb of the loop of Henle.     

Toxicity of organotin compounds in primary cultures of rat cortical astrocytes

The neurotoxic organotin compounds trimethyl (TMT) and triethyltin (TET) are known to induce astrogliosis in vivo, which is indicated by an increased synthesis of glial fibrillary acidic protein (GFAP) in astrocytes. In contrast, tributyltin (TBT) does not induce astrogliosis. The aim of this study was to investigate whether trialkyltin derivatives can induce an increased GFAP synthesis in astrocyte cultures in the absence of neurons and whether differences between the action of TMT, TET, and TBT can be detected. Primary cultures of rat cortical astrocytes from 2-day-old rats were grown in 96-well plates until confluency and then exposed to various concentrations of TMT, TET, and TBT for 40 h. Effects on basal cell functions were measured by colorimetric determination of cell protein contents and by assessment of viability by means of the MTT assay. An indirect sandwich ELISA for 96-well plates was used for quantitative measurements of the GFAP content of the cells. All three compounds induced a concentration-dependent cytotoxicity indicated by parallel decreases of protein contents and MTT reduction. Half-maximum cytotoxic concentrations were 3 μmol/L (TBT), 30 μmol/L (TET), and 800 μmol/L (TMT). Cellular GFAP contents were reduced in parallel to cytotoxic action but no increase in GFAP expression at subcytotoxic concentrations could be observed. Thus, the astrocytes were not able to respond to TMT or TET exposure by an increased synthesis of GFAP in the absence of neuronal signals. This revised version was published online in July 2006 with corrections to the Cover Date.     

Differential gliotoxicity of organotins

On the basis of reports that astrocytes play an important role in the neurotoxicity of trimethyltin (TMT), we investigated the sensitivity of astrocytes to TMT and compared it to triethyltin (TET), a neurotoxic analog with a different in vivo specificity. The gliotoxicity of these two compounds was further compared to that of tributyltin (TBT) and triphenyltin (TPT), two purportedly nonneurotoxic organotin compounds. The time and concentration components of organotin toxicity were determined by measuring lactate dehydrogenase (LDH) release and formazan production from dimethylthiazolyldiphenyltetrazolium bromide (MTT).A TMT concentration of 100 mol/L did not elevate extracellular LDH until 48 h after exposure, while signs of toxicity were not seen at 72 h for concentrations less than 10 mol/L. Extracellular LDH activity increased 24 h after exposure to concentrations of TET, TBT, and TPT as low as 2.5 mol/L.TMT was the only organotin to produce a delayed cytotoxicity, requiring both higher concentrations and more time to produce discernible toxicity. In contrast with TBT and TPT, the toxicity of the two neurotoxic organotins (TMT and TET) produced an early increase in MTT reduction. The distinct pattern of toxicity for TMT does not explain its selective in vivo toxicity, but the lack of sensitivity of astrocytes to this organotin also does not rule out more subtle changes in these cells that could disrupt normal glial/neuronal interactions.     

Pharmacology of the neuronal nicotinic acetylcholine receptor of cultured Kenyon cells of the honeybee, <Emphasis Type="Italic">Apis mellifera</Emphasis>

We investigated the pharmacology of the nicotinic acetylcholine receptor of honeybee Kenyon cells, a subset of olfactory interneurons, which are crucial for olfactory learning and memory. Whole-cell currents were recorded using patch-clamp techniques. Pressure application of agonists induced inward currents in cultured Kenyon cells at holding potentials of –110 mV. Acetylcholine or carbamylcholine were full agonists, nicotine, epibatidine and cytisine were only partial agonists. Coapplications of these partial agonists with acetylcholine reduced the current amplitude. The most efficient antagonists were dihydroxy--erythroidine (EC₅₀=0.5 pmol·l^–1) and methyllycaconitine (EC₅₀=24 pmol·l^–1). The open channel blocker mecamylamine, d-tubocurarine and hexamethonium were rather weak blockers of the honeybee nicotinic response. Bath applications of the muscarinic antagonist atropine inhibited nicotinic currents dependent on concentration (EC₅₀=24.3 mol·l^–1). Muscarine, pilocarpine or oxotremorine (1 mmol·l^–1) did not induce any measurable currents. The non-cholinergic drugs strychnine, bicuculline and picrotoxin partially and reversibly blocked the acetylcholine-induced currents. Our results indicate the expression of only one nicotinic acetylcholine receptor subtype in cultured Kenyon cells. Muscarinic as well as non-cholinergic antagonists also inhibit the receptor function, distinguishing the honeybee nicotinic receptor from the typical nicotinic receptor of vertebrates and from many described insects receptors.     

Zinc compounds inhibit osteoclast-like cell formation at the earlier stage of rat marrow culture but not osteoclast function

The effect of zinc compounds on osteoclast-like cell formation in rat marrow culture in vitro was investigated. The bone marrow cells were cultured for 7 days in -minimal essential medium containing a well-known bone resorbing hormone (1, 25-dihydroxyvitamin D₃ and parathyroid hormone [1–34]). Osteoclast-like cell formation was estimated by staining for tartrateresistant acid phosphatase (TRACP), a marker enzyme of osteoclasts. The presence of 1, 25-dihydroxyvitamin D₃ (10^–8 M) or parathyroid hormone (PTH; 10^–8 M) induced a remarkable increase in osteoclast-like multinucleated cells (MNC). These increases were clearly inhibited by the presence of zinc sulfate or zinc-chelating dipeptide (-alanyl-L-histidinato zinc; AHZ) in the concentration range of 10^–7 to 10^–5 M. The inhibitory effect was seen at the earlier stage of osteoclast-like MNC formation. However, zinc compounds (10^–6 M) did not have an effect on PTH (10^–8 M)-induced osteoclast-like cell formation in the presence of EGTA (5 × 10^–4 M), dibucaine (10^–5 M) or staurosporine (10^–9 M). Moreover, when osteoclasts isolated from rat femoraldiaphyseal tissues were cultured for 24 h in the presence of zinc compounds (10^–7 to 10^–5 M), the compounds did not have an effect on cell numbers or lysosomal enzymes activity (acid phosphatase and -glucuronidase) in the cells. The present study clearly demonstrates that zinc compounds inhibit osteoclast-like cell formation at the earlier stage with differentiation of marrow cells.     

The Proteolytic Activity and Cleavage Specificity of Fibronectin-Gelatinase and Fibronectin-Lamininase

Human plasma fibronectin contains two latent aspartic proteinases, FN-gelatinase and FN-lamininase. Both enzymes can be generated and activated in the presence of Ca²⁺ from the purified cathepsin D-produced 190-kDa fibronectin fragment. We investigated the proteolytic activity and cleavage specificity of both enzymes in a range of pH from 3.5 to 9.0 using the B chain of oxidized bovine insulin and chromogenic peptides as substrates. The inhibition of the enzymes by several natural inhibitors from human plasma was also tested. The specificities of FN-gelatinase and FN-lamininase are similar to other major acidic proteinases, including pepsin, renin, cathepsin D, and HIV-proteinases. Both enzymes mainly hydrolyze three peptide bonds in the oxidized insulin B chain, namely Glu–Ala (residues 13–14), Tyr–Leu (residues 16–17), and Phe–Phe (residues 24–25). For the peptide substrates H-Pro-Thr-Glu-Phe-p-nitro-Phe-Arg-Leu-OH and H-Phe-Gly-His-p-nitro-Phe-Phe-Val-Leu-OMe that were cleaved the respective values of k _cat/K _M were 105.1 and 11.8 mM^–1 sec^–1 for cleavage by FN-gelatinase, and 123.2 and 15.5 mM^–1 sec^–1 for cleavage by FN-lamininase. The maximal activities of both enzymes were observed in a range between pH 5.6 and 6.3 and they became inactivated at a pH value above 8.4. Both FN-gelatinase and FN-lamininase were efficiently inhibited by ₂-macroglobulin.     

Effects induced by mono- and divalent cations on protein regions responsible for thermal adaptation in β-glycosidase from<Emphasis Type="Italic"> Sulfolobus solfataricus</Emphasis>

The perturbation induced by mono- and divalent cations on the thermophilicity and thermostability of Solfolobus solfataricus -glycosidase, a hyperthermophilic tetrameric enzyme, has been investigated by spectroscopic and computational simulation methods to ascertain the Hofmeister effects on two strategic protein regions identified previously. Specifically, (1) an extra segment (83–124), present only in the sequence of hyperthermophilic glycosidases and recognized as an important thermostability determinant for the enzyme structure; and (2) a restricted area of the subunit interface responsible for the quaternary structure maintenance. Mono- and divalent cations inhibit to a different extent the -glycosidase activity, whose kinetic constants show an apparent competitive inhibition of the catalytic process that reflects the Hofmeister order. The thermostability is also affected by the nature and charge of the cations, reaching maximal effects for the case of Mg²⁺. Fourier transform infrared spectroscopy has revealed very small changes in the protein secondary structure in the presence of the investigated cations at 20 °C, while large effects on the protein melting temperatures are observed. Computational analysis of the enzyme structure has identified negative patches on the accessible surface of the two identified regions. Following the Hofmeister series, cations weaken the existing electrostatic network that links the extra segment to the remaining protein matrix. In particular, the perturbing action of cations could involve the ionic pair interactions E107–R245 and E109–R185, thus leading to a local destructuring of the extra segment as a possible starting event for thermal destabilization. A detailed investigation of the electrostatic network at the A–C intermolecular interface of Sgly after energy minimization suggests that cations could cause a strong attenuation of the ion pair interactions E474–K72 and D473–R402, with consequent partial dissociation of the tetrameric structure.Abbreviations Amide I amide I band in a ²H₂O medium - EM energy minimization - ONPG o-nitrophenyl--d-galactopyranoside - Sgly Escherichia coli expressed Sulfolobus solfataricus -glycosidase     

Natural CD8<Superscript>+</Superscript> T-cell responses against MHC class I epitopes of the HER-2/<Emphasis Type="Italic">neu</Emphasis> oncoprotein in patients with epithelial tumors

HER-2/neu is an immunogenic protein eliciting both humoral and cellular immune responses in patients with HER-2/neu-positive (⁺) tumors. Preexisting cytotoxic T lymphocyte (CTL) immunity to HER-2/neu has so far been mainly evaluated in terms of detection of CTL precursor (CTLp) frequencies to the immunogenic HLA-A2–binding nona-peptide 369-377 (HER-2(9₃₆₉)). In the present study, we examined patients with HER-2/neu⁺ breast, ovarian, lung, colorectal, and prostate cancers for preexisting CTL immunity to four recently described HER-2/neu–derived and HLA-A2–restricted "cytotoxic" peptides and to a novel one spanning amino acids 777–785 also with HLA-A2–binding motif. We utilized enzyme-linked immunosorbent spot (ELISpot) assay, which allows a quantitative and functional assessment of T cells directed against specific peptides after only brief in vitro incubation. CTL reactivity was determined with an interferon (IFN-) ELISpot assay detecting T cells at the single cell level secreting IFN-. CTLp were defined as peptide-specific precursors per 10⁶ peripheral blood mononuclear cells (PBMCs). Patients' PBMCs with increased CTLp were also tested against autologous tumor targets and peptide-pulsed dendritic cells (DCs) in cytotoxicity assays. We also studied patients with HER-2/neu-negative (^-) tumors and healthy individuals. Of the HER-2/neu⁺ patients examined, 31% had increased CTLp to HER-2(9₉₅₂), 19% to HER-2(9₆₆₅), 16% to HER-2(9₆₈₉), and 12.5% HER-2(9₄₃₅), whereas only 2 of 32 patients (6%) responded to HER-2(9₇₇₇). The CTLp recognizing HER-2(9₉₅₂) were extremely high in two patients with breast cancer, one with lung cancer, and one with prostate cancer. None of the HER-2/neu^- patients or healthy donors exhibited increased CTLp to any of these peptides. Besides IFN- production, preexisting CTL immunity to all five HER-2/neu peptides was also shown in cytotoxicity assays where patients' PBMCs with increased CTLp specifically lysed autologous tumor targets and autologous peptide-pulsed DCs. Our results demonstrate for the first time that (1) preexisting immunity to peptides HER-2(9₄₃₅), HER-2(9₉₅₂), HER-2(9₆₈₉), HER-2(9₆₆₅), and HER-2(9₇₇₇) is present in patients with HER-2/neu⁺ tumors of distinct histology, (2) HER-2(9₇₇₇) is a naturally processed peptide expressed on the surface of HER-2/neu⁺ tumors, as are the other four peptides, and (3) HER-2/neu⁺ prostate tumor cells can be recognized and lysed by autologous HER-2 peptide-specific CTL. Our findings broaden the potential application of HER-2/neu-based immunotherapy.     

The Effect of γ-Radiation and Desiccation on the Viability of the Soil Bacteria Isolated from the Alienated Zone around the Chernobyl Nuclear Power Plant

Methylobacterium extorquens, M. mesophilicum, and Bacillus subtilis strains were found to be resistant to -radiation, irrespective of whether they were isolated from the alienated zone around the Chernobyl Nuclear Power Plant or outside this zone. The LD₉₀ of Methylobacterium and B. subtilis strains with respect to -radiation was 2.0–3.4 and 3.7–4.4 kGy, respectively, whereas their LD_99.99 values were 4.5–6.9 and more than 10 kGy, respectively. The high threshold levels of -radiation for Methylobacterium and B. subtilis imply the efficient functioning of DNA repair systems in these bacteria. Unlike Bacillus polymyxa cells, the cells of M. extorquens, M. mesophilicum, and B. subtilis were also resistant to desiccation. Pseudomonas sp., Nocardiasp., and nocardioform actinomycetes were sensitive to both -radiation and desiccation. Similar results were obtained when the bacteria studied were exposed to hydrogen peroxide and ultraviolet radiation. The results obtained indicate that the bacteria that are resistant to -radiation are also resistant to desiccation, UV radiation, and hydrogen peroxide. The possibility of using common laboratory tests (such as the determination of bacterial resistance to UV light and desiccation) for the evaluation of bacterial resistance to -radiation is discussed.     

The main features of Bacillus thuringiensis δ-endotoxin molecular structure

The crystal-forming proteins (-endotoxins) produced by various serotypes of Bacillus thuringiensis and toxic for Lepidoptera reveal the same pattern of molecular organisation. These proteins (M _r of ca. 145,000–130,000) contain an N-terminal domain (M _r of 85,000–65,000) resistant to proteolysis whereas their C-terminal moieties (M _r of 65,000) undergo an extensive degradation by trypsin that leads to stepwise cleavage off the fragments with M _r of 15,000–35,000.The N-terminal domain from serotype V -endotoxin is active when introduced into the hemocoel of Galleria mellonella larvae. Hence, it correponds to the true toxin normally formed by larva proteases action on the crystalforming protein (protoxin). Some differences were found in the properties of the N-terminal domains isolated from the crystal-forming proteins of III, V and IX serotypes, e.g., in their solubility, digestion by subtilisin, molecular weights and the distribution of methionine residues along the polypeptide chains.Abbreviations SDS sodium dodecyl sulphate - PAGE polyacryl amide gel electrophoresis - CFP crystal-forming protein - DNS 5-dimethylamino-1-naphthalene-sulphonyl     

Optimization of cell density and dilution rate in <Emphasis Type="Italic">Pichia pastoris</Emphasis> continuous fermentations for production of recombinant proteins

This paper provides an approach for optimizing the cell density (X_c) and dilution rate (D) in a chemostat for a Pichia pastoris continuous fermentation for the extracellular production of a recombinant protein, interferon (INF-). The objective was to maximize the volumetric productivity (Q, mg INF- l^–1 h^–1), which was accomplished using response surface methodology (RSM) to model the response of Q as a function of X_c and D within the ranges 150 X_c 450 g cells (wet weight) l^–1 and 0.1 _mD0.9 _m (_m=0.0678 h^–1, the maximum specific growth rate obtained from a fed-batch phase controlled with a methanol sensor). The methanol and medium feed rates that resulted in the desired X_c and D were determined based on the mass balance. From the RSM model, the optimal X_c and D were 328.9 g l^–1 and 0.0333 h^–1 for a maximum Q of 2.73 mg l^–1 h^–1. The model of specific production rate (, mg INF- g^–1 cells h^–1) was also established and showed the optimal X_c=287.7 g l^–1 and D=0.0361 h^–1 for the maximum (predicted to be 8.92×10^–3 mg^–1 g^–1 h^–1). The methanol specific consumption rate (, g methanol g^–1 cells h^–1) was calculated and shown to be independent of the cell density. The relationship between and (specific growth rate) was the same as that discovered from fed-batch fermentations of the same strain. The approach developed in this study is expected to be applicable to the optimization of continuous fermentations by other microorganisms.     

Activation of JNK/SAPK in Primary Glial Cultures: II. Differential Activation of Kinase Isoforms Corresponds to Their Differential Expression

Recently, we reported on the activation of c-Jun N-terminal kinase (JNK) in primary glial cells noting certain differences in the patterns of kinase activation in astrocytes and oligodendrocytes (Zhang et al., J Neurosci Res 46:114–121;1996). In this extended study, we have examined the activation and expression levels of JNK1 and JNK2 isoforms in different glial cell types including the two in vitro-defined astroglial subtypes (type-1 and type-2), oligodendrocytes and microglia. An in-gel kinase assay of cell extracts and JNK-immunoprecipitates revealed the activation of both JNK1 and JNK2 in type-1 astrocytes in response to TNF, and in microglia, in response to TNF and bacterial lipopolysaccharide. The strong activation of the two JNK isoforms in type-1 astrocytes and microglia contrasted with a predominant activation of JNK1 over JNK2 in type-2 astrocytes and oligodendrocytes, the two glial subtypes sharing a common lineage. Immunoblot and immunocytochemical analyses using isoform-speciflc antibodies showed a differential expression of the two isoforms in different glial cells thereby accounting for their observed differential activation.     

Interference of thiol-compounds with dextrinizing activity assay of α-amylase by starch-iodine colour reaction: Modification of the method to eliminate this interference

Interference of Luria broth and other bacteriological media with the starch–iodine colour assay of the dextrinizing activity of -amylase (Fuwa's method) was observed, complete bleaching occurring with 0.4ml of the Luria broth. The interference was found to be due to the thiol groups present in the medium which compete with starch for iodine. Among the various metal salts tested for counteracting the interference, ZnSO₄ was found to be the best which reverted the colour to about 73–85% of that of the blank. A combination of hydrogen peroxide (10 l of 30% solution) and CuSO₄ · 5H₂O (50 l of 0.1 M solution) completely protected the starch–iodine reaction in the presence of even 0.5 ml of Luria broth and a modified assay was developed based on this finding. The colour intensity, however, was almost double than that obtained for the same amount of starch and iodine in the absence of these protective agents. Nevertheless, with different concentrations of starch as well as with varying amounts of enzymes, the modified method showed perfect linearity and could be effectively used for estimation of dextrinizing activity of -amylase in the presence of thiol groups.     

Polyclonal activation of human lymphocytes and induction of cytotoxic lymphocytes by streptococcal preparations

Polyclonal activation of human peripheral blood lymphocytes (PBLs)in vitro by preparations ofStreptococcus pyogenes Su strain (OK-432) and other heat-killed strains was investigated. The streptococcal preparations tested induce a proliferative response of PBLs via interleukin-2 (IL-2)-independent pathways. The proliferative response is accompanied by the generation of lymphoblastic cells (LBCs), which consist of heterologous lymphocyte populations: CD4⁺ helper type of T cells, and CD4^–CD8^– double-negative (DN) lymphocytes, including both CD3⁺ TcR ⁺ T cells and CD2⁺CD3^– immature type of T or non-T cell type of lymphocytes. Almost all the LBCs express Leu19, TfR (transferrin receptor), LFA-1 and CD38 (OKT10) antigens, which are expressed on activated T cells, NK cells and some other lymphocytes. The proliferative response of human PBLs is also accompanied by the generation of potent cytotoxic activity against NK-sensitive and -resistant targets. C-dependent cytolysis and cell sorting experiments of OK-432-activated LBCs revealed that both CD3⁺ and CD3^– types of CD4^–CD8^– DN lymphocytes, but not CD4⁺ helper T cells, may be major populations responsible for the cytotoxicity induced. On the other hand, CD4^–CD8 T cells may be required for the proliferation of PBLs and generation of cytotoxic effector cells. These results suggest that the OK-432 and other streptococcal preparations stimulate the human PBLsin vitro to induce the proliferation/activation of CD4⁺ T cells, mediating the following generation of DN cytotoxic effector lymphocytes.     

δ15N as an indicator of N2-fixation by cyanobacterial mats in tropical marshes

Cyanobacterial mats (CBM) are important components of wetland ecosystems in limestone-based regions of the Caribbean. During two sampling periods (July 1999 and January 2000) we measured N₂-fixation in samples from 23 different marshes simultaneously with measurements of relevant environmental factors. Samples were evaluated for abundance of five groups of cyanobacteria: (1) Leptolyngbya, (2) Oscillatoria, (3) Chroococcales, (4) Nostoc-& Stigonematales, and (5) dead sheaths. Differences in nitrogen fixation, expressed as nitrogenase activity in nmol C₂H₄ cm^–2 h^–1, were best explained by the proportion of heterocyst-forming cyanobacteria. The samples were analyzed for the natural abundance of ¹⁵N. ¹⁵N values ranged from –1.99 to 11.44 and were strongly negatively correlated with N₂-fixation. With all data included, ¹⁵N was also strongly correlated with nitrates in water. With the samples from Little Belize (high nitrate content marshes) excluded, the effect of nitrate became insignificant. N₂-fixation predicted from ¹⁵N measured on an independent data set from September 2000 was moderately accurate (r² = 0.68, 0.52 and 0.54 for predictions based on July 1999, January 2000 and combined data sets, respectively). When individual sample sets were divided into two groups with ¹⁵N < 2 and ¹⁵N > 2, the two groups were always highly significantly different in terms of their N₂-fixation. The presented evidence suggests that ¹⁵N can be used as a reliable indicator of N₂-fixation by CBM.     

Isolation and characterization of the mitochondrial ATP synthase fromChlamydomonas reinhardtii. cDNA sequence and deduced protein sequence of the α subunit

We have isolated the F₀F₁-ATP synthase complex from oligomycin-sensitive mitochondria of the green algaChlamydomonas reinhardtii. A pure and active ATP synthase was obtained by eans of sonication, extraction with dodecyl maltoside and ion exchange and gel permeation chromatography in the presence of glycerol, DTT, ATP and-21. The enzyme consists of 14 subunits as judged by SDS-PAGE. A cDNA clone encoding the ATP synthase subunit has been sequenced. The deduced protein sequence contains a presequence of 45 amino acids which is not present in the mature protein. The mature protein is 58–70% identical to corresponding mitochondrial proteins from other organisms. In contrast to the ATP synthase subunit fromC. reinhardtii (Franzen and Falk, Plant Mol Biol 19 (1992) 771–780), the protein does not have a C-terminal extension. However, the N-terminal domain of the mature protein is 15–18 residues longer than in ATP synthase subunits from other organisms. Southern blot analysis indicates that the protein is encoded by a single-copy gene.Abbreviations DM dodecyl--D-maltoside - OSCP oligomycin sensitivity conferring protein - PMSF phenyl-methylsulfonylfluoride - DTT dithiothreitol - EDTA ethylenediaminotetraacetic disodium salt     

Leaf 15N abundance of subarctic plants provides field evidence that ericoid,ectomycorrhizal and non-and arbuscular mycorrhizal species access different sources of soil nitrogen

The natural abundance of the nitrogen isotope 15, ¹⁵N, was analysed in leaves of 23 subarctic vascular plant species and two lichens from a tree-line heath at 450 m altitude and a fellfield at 1150 m altitude close to Abisko in N. Sweden, as well as in soil, rain and snow. The aim was to reveal if plant species with different types of mycorrhizal fungi also differ in their use of the various soil N sources. The dwarf shrubs and the shrubs, which in combination formed more than 65% of the total above-ground biomass at both sites, were colonized by ericoid or ectomycorrhizal fungi. Their leaf ¹⁵N was between–8.8 and–5.5 at the heath and between–6.1 and –3.3 at the fellfield. The leaf ¹⁵N of non- or arbuscular mycorrhizal species was markedly different, ranging from –4.1 to –0.4 at the heath, and from –3.4 to+2.2 at the fellfield. We conclude that ericoid and ectomycorrhizal dwarf shrubs and shrubs utilize a distinct N source, most likely a fraction of the organic N in fresh litter, and not complexed N in recalcitrant organic matter. The latter is the largest component of soil total N, which had a ¹⁵N of –0.7 at the heath and +0.5 at the fellfield. Our field-based data thus support earlier controlled-environment studies and studies on the N uptake of excised roots, which have demonstrated protease activity and amino acid uptake by ericoid and ectomycorrhizal tundra species. The leaves of ectomycorrhizal plants had slightly higher ¹⁵N (fellfield) and N concentration than leaves of the ericoids, and Betula nana, Dryas octopetala and Salix spp. also showed NO _inf3 ^sup- reductase activity. These species may depend more on soil inorganic N than the ericoids. The ¹⁵N of non- or arbuscular mycorrhizal species indicates that the ¹⁵N of inorganic N available to these plants was higher than that of average fresh litter, probably due to high microbial immobilization of inorganic N. The ¹⁵N of NH _inf4 ^sup+ -N was +12.3 in winter snow and +1.9 in summer rain. Precipitation N might be a major contributer in species with poorly developed root systems, e.g. Lycopodium selago. Our results show that coexisting plant species under severe nutrient limitation may tap several different N sources: NH _inf4 ^sup+ , NO _inf3 ^sup- and organic N from the soil, atmospheric N₂, and N in precipitation. Ericoid and ectomycorrhizal fungi are of major importance for plant N uptake in tundra ecosystems, and mycorrhizal fungi probably exert a major control on plant ¹⁵N in organic soils.     

Production of poly-γ-glutamic acid by fed-batch culture of Bacillus licheniformis

Fed-batch cultures of Bacillus licheniformis produced poly--glutamic acid (PGA), a water-soluble biodegradable polymer. PGA reached 35 g l^–1 with a productivity of 1 g l^–1 h^–1 by pulsed-feeding of citric acid (1.44 g h^–1) and l-glutamic acid (2.4 g h^–1) when citric acid was depleted from the culture medium.