Helix–coil transition in poly(γ-benzyl-L-glutamate) and poly-ε-carbobenzoxy-L-lysine

In this paper two points are considered: the methods of evaluating the helical content θ and the calculation of the parameters of the transition from experimental data and its interpretation. The parameter ΔH obtained is in good agreement with the calorimetric one and v is found to be independent of temperature and solvent and in agreement with the ordinarily accepted value for poly(γ-benzyl-L -glutamate). The different methods of estimating θ are discussed for both polypeptides.     

Mikropinozytose im Zentralnervensystem

Zusammenfassung An durch Perfusion mit Glutaraldehyd fixierten Rattengehirnen wurde das Erscheinungsbild der Mikropinozytose in Elementen der Meso- und Neuroglia sowie an den Perikarya und synaptischen Endformationen der Nervenzellen elektronenmikroskopisch dargestellt.Die bei der Mikropinozytose von der Zellmembran invaginierten Caveolen und Tubuli können einfache Verzweigungen zeigen. Ihre Oberfläche und die der mikropinozytotischen Bläschen zeigen an der gegen das Zytoplasma gerichteten Membranseite einen Stachelsaum. Diese Membrandifferenzierung dürfte mit der Resorption besonderer, zum Teil makromolekularer Substanzen zusammenhängen.Im Bereich großer Synapsen, z.B. in den Moosfasertelodendren der Glomerula cerebellaria oder in der Zona glomerulosa des Bulbus olfactorius sind mikropinozytotische Invaginationen und Bläschen sehr häufig. Möglicherweise übernehmen sie von den postsynaptischen Dendriten, die dünne Zytoplasmaprotrusionen in die Invaginationen hineinsenden, Stoffe. Es wird vermutet, daß es sich hierbei um inaktivierte Transmittersubstanz handelt, die auf diesem Wege dem präsynaptischen Abschnitt wieder zugeführt wird. Die zurückresorbierten Abbauprodukte der Transmittersubstanz werden in einem präsynaptischen Golgi-Komplex resynthetisiert und in synaptischen Bläschen angereichert. Dieses morphologische Bild ergänzt die biochemische Hypothese eines Acetylcholin-Kreislaufes im Bereich von Nervenendigungen.Entsprechende mikropinozytotische Erscheinungen wurden in caudalen Abdominalganglien von Leucophaea maderae beobachtet. Es wird angenommen, daß die Mikropinozytose ein allgemein verbreiteter Resorptionsmechanismus im Zentralnervensystem ist.Herrn Prof. Dr. F. Wassermann zum 80. Geburtstag gewidmet.Mit dankenswerter Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Serotypes and antibiotic resistance of Verotoxigenic (VTEC) and Necrotizing (NTEC) Escherichia coli strains isolated from calves with diarrhoea

Serotypes and antibiotic resistance of 51 Verotoxigenic (VTEC) and 33 Necrotizing (NTEC) bovine Escherichia coli strains were determined and compared with those shown by 205 non-VTEC non-NTEC strains isolated from the same batch of calves. E. coli untypable for O-antigen represented 47% of the VTEC, 12% of the NTEC and 8.8% of the non-VTEC non-NTEC. Typable VTEC belonged to serotypes 02:K?, 0103:K-, 0104:K?, 0128:K?, 0153:K- and O157:K-:H7, whereas typable NTEC were of serotypes 08:K87, 015:K14, 015:K-, 054:K?, 076:K-, 078:K(80), 088:K?, 0123:K-, 0139:K- and 0153:K-. Non-VTEC non-NTEC showed a wide variety of serotypes which were generally unrelated to those found in VTEC and NTEC. VTEC were resistant to antibiotics at higher rates than NTEC and non-VTEC non-NTEC, and showed also the highest multidrug-resistant pattern. Our results show that bovine VTEC strains belonged to O-groups usually found in human VTEC causing sporadic diarrhoea, haemorrhagic colitis and/or haemolytic uraemic syndrome, such as 02, 0103, 0104, 0153 and especially 0128 and O157. In contrast, bovine NTEC strains belonged to serotypes different from those previously found in necrotizing E. coli strains of human origin.     

Purification and properties of glutamine synthetase from the non-N2-fixing cyanobacterium Phormidium laminosum.

Soluble glutamine synthetase activity (L-glutamate:ammonia ligase, ADP forming, EC 6.3.1.2) was purified to electrophoretic homogeneity from the filamentous non-N2-fixing cyanobacterium Phormidium laminosum (OH-1-p.Cl1) by using conventional purification procedures in the absence of stabilizing ligands. The pure enzyme showed a specific activity of 152 mumol of gamma-glutamylhydroxamate formed.min-1 (transferase activity), which corresponded to 4.4 mumol of Pi released.min-1 (biosynthetic activity). The relative molecular mass of the native enzyme was 602 kilodaltons and was composed of 12 identically sized subunits of 52 kilodaltons. Biosynthetic activity required the presence of Mg2+ as an essential activator, although Co2+ and Zn2+ were partially effective. The kinetics of activation by Mg2+, Co2+, and Zn2+ were sigmoidal, and concentrations required for half-maximal activity were 18 mM (h = 2.2), 6.3 mM (h = 5.6), and 6.3 mM (h = 2.45), respectively. However, transferase activity required Mn2+ (Ka = 3.5 microM), Cu2+, Co2+, or Mg2+ being less effective. The substrate affinities calculated for L-Glu, ammonium, ATP, L-Gln, and hydroxylamine were 15, 0.4, 1.9 (h = 0.75), 14, and 4.1 mM, respectively. Optimal pH and temperature were 7.2 and 55 degrees C for biosynthetic activity and 7.5 and 45 degrees C for transferase activity. The biosynthetic reaction mechanism proceeded according to an ordered three-reactant system, the binding order being ammonium, L-Glu, and ATP. The presence of Mn2+ or Mg2+ drastically affected the thermostability of transferase and biosynthetic activities. Heat inactivation of biosynthetic activity in the presence of Mn2+ obeyed first-order kinetics, with an Ea of 76.8 kcal (ca. 321 kJ) mol-1. Gly, L-Asp, L-Ala, L-Ser and, with lower efficiency, L-Lys and L-Met, L-Lys, and L-Glu inhibited only transferase activity. No cumulative inhibition was observed when mixtures of amino acids were used. Biosynthetic activity was inhibited by AMP (Ki= 7 mM), ADP (Ki= 2.3 mM), p-hydroxymercuribenzoate (Ki= 25 microM), and L-methionine-D, L-sulfoximine (Ki= 2 microM). The enzyme was not activated in vitro by chemically reduced Anabaena thioredoxin. This is the first report of glutamine synthetase activity purified from a filamentous non-N2-fixing cyanobacterium.     

Surface and virulence properties of environmental Vibrio cholerae non-O1 from Albufera Lake (Valencia, Spain).

A total of 140 environmental Vibrio cholerae non-O1 isolates, together with several culture collection strains from both environmental and clinical sources, were studied in relation to hemagglutination, surface hydrophobicity, and the enzymatic, hemolytic, cytotoxic, and enterotoxic activities of their extracellular products. A total of 78 and 62% of the strains produced hemagglutinins and exohemagglutinins, respectively. Four different hemagglutinating and two exohemagglutinating activities were found by using eight sugars in the inhibition assays. Cell-bound mannose-sensitive hemagglutination was detected mainly in chicken blood, whereas fucose-sensitive hemagglutination was recorded only in human blood. Cell-bound hemagglutinin resistant to all sugars tested was the only one related to surface hydrophobicity. The surface properties varied along the growth curves. The non-O1 strains displayed strong enzymatic and hemolytic activities, except for esculin hydrolysis. Of 26 non-O1 isolates selected for cytotoxin and enterotoxin production, 23 showed a wide spectrum of cytotoxic effects on cell lines of poikilothermic and homoiothermic species, but they were weakly enterotoxigenic in the infant mouse test. All extracellular products of cytotoxic strains were proteolytic, lipolytic, and hemolytic, and a high percentage produced hemagglutination of chicken blood. The cytotoxic factors in the non-O1 strains analyzed were not R plasmid mediated.     

Effect of Quin-2 on 45Ca2+ uptake mediated by Na+i/Ca2+o exchange and 45Ca2+ efflux in rat brain synaptosomes: a requirement for [Ca2+]i

The Na+/Ca2+ exchanger of squid axons, barnacle muscle and sarcolemma requires micromolar intracellular calcium for activation in the Na+i/Ca2+o exchange mode ('reverse' Na+/Ca2+ exchange). The requirement for [Ca2+]i has been demonstrated with the use of intracellular calcium buffers, such as Quin-2, to inhibit Na+i/Ca2+o exchange. However, the inhibition of Na+i/Ca2+o exchange in mammalian nerve terminals loaded with Quin-2 has not been observed [7], suggesting a lower sensitivity to low [Ca2+]i for this system. In contrast, the results reported herein indicate that 45Ca2+ uptake in synaptosomes through Na+i/Ca2+o exchange is inhibited by Quin-2 much in the same way as it is in the squid, provided that synaptosomes are preincubated in low Ca2+ medium to avoid saturation of Quin-2. Under these conditions, 45Ca2+ efflux via Ca2+i/Ca2+o exchange is also inhibited. Our results indicate that the Na+i/Ca2+o and Ca2+i/Ca2+o modes of the Na+/Ca2+ exchanger from rat brain synaptosomes require intracellular calcium for activation. However, because no clear relationship between the observed [Ca2+]i values and the inhibition of Na+i/Ca2+o exchange has been found, it is suggested that localised submembrane calcium concentrations not detected by the [Ca2+]i probe might regulate the exchanger.     

Effect of hyperoxia (PaO2 50-90 mmHg) on fetal breathing movements in the unanaesthetized fetal sheep

Hypoxia inhibits fetal breathing movements but after birth it stimulates breathing. These differences have long been thought to involve central nervous inhibitory mechanisms. Such mechanisms might exert a tonic inhibition of fetal breathing movements at normal fetal PaO2 and the rise in PaO2 at birth might lift this inhibitory effect. To test this hypothesis 7 fetal sheep were chronically instrumented at 125-130 days for recording electrocortical activity (ECoG), and the electromyograph (EMG) activity of the diaphragm and neck muscles. Catheters were placed in a fetal carotid and a brachial artery and in the fetal trachea. For an extracorporeal membrane oxygenation system a 12 F gauge silastic catheter was placed in the right atrium for draining fetal blood and a 9.6 F gauge catheter was placed in a carotid artery to return oxygenated blood. Three days after operation the fetuses were connected to the extracorporeal membrane oxygenation system and fetal PaO2 was raised to 65.2 +/- 4.4 mmHg (SEM) for 6 to 19 h without changing pH or PaCO2. Neither the incidence of high voltage ECoG (48.5 +/- SEM 2.0% vs 52.8 +/- 3.3%) nor of fetal breathing movements (37.3 +/- 2.6% vs 23.8 +/- 5.9%) changed during the periods of hyperoxia. Since fetal breathing movements did not become continuous, we conclude that the lower PaO2 in the fetus compared to the neonate does not exert a tonic inhibitory influence on fetal breathing movements.     

Enzyme profiles of single muscle fibers never exposed to normal neuromuscular activity.

Do muscle fiber properties commonly associated with fiber types in adult animals and the population distribution of these properties require normal activation patterns to develop? To address this issue, the activity of an oxidative [succinic dehydrogenase (SDH)] and a glycolytic [alpha-glycerophosphate dehydrogenase (GPD)] marker enzyme, the characteristics of myosin adenosinetriphosphatase (myosin ATPase, alkaline preincubation), and the cross-sectional area of single fibers were studied. The soleus and medial gastrocnemius of normal adult cats were compared with cats that 6 mo earlier had been spinally transected at T12-T13 at 2 wk of age. In control cats, SDH activity was higher in dark than light ATPase fibers in the soleus and higher in light than dark ATPase fibers in the medial gastrocnemius. After transection, SDH activity was similar to control in both muscles. GPD activity appeared to be elevated in some fibers in each fiber type in both muscles after transection. The cross-sectional areas most affected by spinal transection were light ATPase fibers of the soleus and dark ATPase fibers of the medial gastrocnemius, the predominant fiber type in each muscle. These data demonstrate that although the muscle fibers of cats spinalized at 2 wk of age presumably were never exposed to normal levels of activation, the activity of an oxidative marker enzyme was maintained or elevated 6 mo after spinal transection. Furthermore, although the absolute enzyme activities in some fibers were elevated by transection, three functional protein systems commonly associated with fiber types, i.e., hydrolysis of ATP by myosin ATPase and glycolytic (GPD) and oxidative (SHD) metabolism, developed in a coordinated manner typical of normal adult muscles.     

Characterization of the Erwinia chrysanthemi osmoprotectant transporter gene ousA.

Growth of Erwinia chrysanthemi in media of elevated osmolarity can be achieved by the uptake and accumulation of various osmoprotectants. This study deals with the cloning and sequencing of the ousA gene-encoded osmoprotectant uptake system A from E. chrysanthemi 3937. OusA belongs to the superfamily of solute ion cotransporters. This osmotically inducible system allows the uptake of glycine betaine, proline, ectoine, and pipecolic acid and presents strong similarities in nucleotide sequence and protein function with the proline/betaine porter of Escherichia coli encoded by proP. The control of ousA expression is clearly different from that of proP. It is induced by osmotic strength and repressed by osmoprotectants. Its expression in E. coli is controlled by H-NS and is rpoS dependent in the exponential phase but unaffected by the stationary phase.     

Recombinant Treponema pallidum rare outer membrane protein 1 (Tromp1) expressed in Escherichia coli has porin activity and surface antigenic exposure.

We recently reported the cloning and sequencing of the gene encoding a 31-kDa Treponema pallidum subsp. pallidum rare outer membrane porin protein, designated Tromp1 (D. R. Blanco, C. I. Champion, M. M. Exner, H. Erdjument-Bromage, R. E. W. Hancock, P. Tempst, J. N. Miller, and M. A. Lovett, J. Bacteriol. 177:3556-3562, 1995). Here, we report the stable expression of recombinant Tromp1 (rTromp1) in Escherichia coli. rTromp1 expressed without its signal peptide and containing a 22-residue N-terminal fusion resulted in high-level accumulation of a nonexported soluble protein that was purified to homogeneity by fast protein liquid chromatography (FPLC). Specific antiserum generated to the FPLC-purified rTromp1 fusion identified on immunoblots of T. pallidum the native 31-kDa Tromp1 protein and two higher-molecular-mass oligomeric forms of Tromp1 at 55 and 80 kDa. rTromp1 was also expressed with its native signal peptide by using an inducible T7 promoter. Under these conditions, rTromp1 fractionated predominantly with the E. coli soluble and outer membrane fractions, but not with the inner membrane fraction. rTromp1 isolated from the E. coli outer membrane and reconstituted into planar lipid bilayers showed porin activity based on average single-channel conductances of 0.4 and 0.8 nS in 1 M KCl. Whole-mount immunoelectron microscopy using infection-derived immune serum against T. pallidum indicated that rTromp1 was surface exposed when expressed in E. coli. These findings demonstrate that rTromp1 can be targeted to the E. coli outer membrane, where it has both porin activity and surface antigenic exposure.