Chromate reduction inStreptomyces

Summary Streptomyces species 3M grew in peptone yeast extract medium with 1000 g/ml K₂Cr₂O₇. Incubation of the chromate with different cell fractions in the presence of NADH and NADPH resulted in a decrease of Cr⁶⁺ in the reaction mixture. The level of Cr⁶⁺ was reduced by 82.7% by a particulate cell fraction obtained by centrifugation at 105,000×g for 1 h, in the presence of NADH. The reducing enzyme was associated with this cell fraction. The enzyme was constitutive and reduced Cr⁶⁺ to Cr³⁺.     

The possible involvement of protein kinase C and phospholipase A2 inHydra tentacle regeneration

The participation of protein kinase C (PKC) in the regeneration of tentacles ofHydra vulgaris was studied. Regeneration was induced by 1,2-sn-dioctanoyl-glycerol (diC₈) and the novel diterpenoidic diacylglycerol verrucosin B (VB), a potent PKC activator extracted from marine sources. VB substantially increasedHydra average tentacle number (ATN) at concentrations 10,000 times lower than those needed for diC₈ to exert an analogous effect. When both synthetic and natural VB analogues were tested, the structure/activity relationship found inHydra tentacle regeneration was identical to that known for DAG-induced activation of PKC in vitro. VB-induced increase of ATN was strongly counteracted by the PKC inhibitors sphingosine and A3, but was not synergic with a tenfold increase of extracellular Ca²⁺ concentration or with an increase of intracellular Ca²⁺ concentration obtained either with the ionophore A23187 or with thapsigargin. This suggested the involvement of a non-Ca²⁺-dependent PKC in VB-triggeredHydra tentacle regeneration. The involvement of phospholipase A₂ (PLA₂) activation inHydra regenerative processes was studied using the novel site-specific inhibitor of the enzyme, oleyloxyethylphosphorylcholine (OOPC), which brought about a striking inhibition of ATN in the low molar range. This effect was reversed by arachidonic acid (AA), while an enhancement of ATN was also observed with an inhibitor of AA uptake from membrane phospholipids, thus suggesting that PLA₂-catalysed liberation of AA is involved inHydra tentacle regeneration. OOPC also blocked verrucosin B-induced PKC-mediated enhancement of ATN, thus suggesting that this effect is also mediated by PLA₂ activation. ATN was increased also by compound 48/80, a direct activator of pertussis toxin-sensitive GTP-binding proteins, and this effect was counteracted by pertussis toxin pretreatment. None of the known AA cascade inhibitors exhibited an effect on ATN comparable to that exerted by OOPC, but, surprisingly, the cycloxygenase inhibitor indomethacin strongly enhanced ATN, thus suggesting that prostanoids might effect a negative control onHydra regenerative processes. This represents the first attempt so far reported to study the implication of more than one biochemical pathway as a signalling event in the hydroid regenerative processes.     

Hyposmotic shock-induced discharge in acontia ofCalliactis parasitica is blocked by gadolinium

On acontia ofCalliactis parasitica it was observed that mechanical stimuli applied by a gelatin probe, a method effective in tentacles of Anthozoa, do not induce the discharge of nematocytes. Hyposmotic shock, performed by treatment with NaCl solution 35% hyposmotic with respect to sea water, induces, in the presence of Ca²⁺, the discharge that spreads along the acontial filament, as previously observed following treatment with SCN^–. The hyposmotic shock-induced discharge is blocked by Gd³⁺ at a concentration of 1 M. 10 M Gd³⁺ prevents also the SCN^–-induced discharge. These results suggest the presence of stretch activated cation channels either in nematocytes and/or in supporting cells as well as a possible effect of SCN^– on this class of ion channels.     

Cl−/HCO 3 − exchanger is operative in isolated enterocytes from rat jejunum

Enterocytes isolated from rat jejunum were tested for the existence of a Cl^–/HCO ₃ ^– exchange, previously evidenced in basolateral membrane vesicles but not in brush border. Cells were found to retain functional integrity and transport capabilities long enough to allow Cl^– fluxes to be measured. Both efflux and uptake experiments indicate that a Cl^–/HCO ₃ ^– antiport, inhibited by 4,4-diisothiocyanostilbene-2-2-disulfonic acid (DIDS), is functional under resting conditions.     

Developmental changes of aldehyde dehydrogenase isozymes in human livers: Mitochondrial ALDH2 isozyme is expressed in fetal livers

Summary Previous reports suggested that the major cytosolic aldehyde dehydrogenase (ALDH₁) was present in fetal and infant livers, but the major mitochondrial isozyme (ALDH₂) was absent or severely diminished. Re-examination by means of starch gel electrophoresis followed by enzyme activity staining, and by means of dot blot immuno-hybridization of liver samples with known genotypes of theALDH ₂ locus, indicated that bothALDH ₁ andALDH ₂ genes are expressed in fetal and infant livers. In addition, ALDH₄ isozyme was also observed. The results imply that a fetus with the usual homozygousALDH ₂ ¹ /ALDH ₂ ¹ genotype, but not one with the atypicalALDH ₂ ¹ /ALDH ₂ ² orALDH ₂ ² /ALDH ₂ ² , is capable of detoxifying acetaldehyde transferred from the mother.     

Complex formation betweenγ-immunoglobulin and calmodulin in calcium-free conditions

We show that -immunoglobulin (IgG) binds calmodulin (CaM) in a Ca²⁺-independent manner, with Kd value of (1.7±0.5)×10^–7M. A single IgG molecule maximally bound 10 CaM molecules. The binding is to the heavy chain or Fab portion, but not the Fc portion, of the IgG molecules. Ca²⁺ greatly diminished the interaction between IgG and CaM, with IC₅₀=8–9M. These data give a novel insight into protein-protein interactions.     

Zinc antagonizes the effect of botulinum type A toxin at the mouse neuromuscular junction

Summary Zn²⁺ (10–100 M) elevated the frequency of miniature end-plate potentials (MEPPs) in the mouse diaphragm. The effect did not depend on external Ca²⁺. Botulinum type A toxin (BTX_A, 50 ng/ml) abolished MEPPs almost completely within 30 min. Zn²⁺ (100 M) restored MEPPs and increased their frequency after they had been abolished by BTX_A in Ca²⁺-free solutions. The antagonistic effect of Zn²⁺ in the Ca²⁺-free solution was reduced by exposing the diaphragm to the toxin in the Ca²⁺-free solutions containing high K⁺. Thus, the action of BTX_A is probably enhanced by depolarization of the motor nerve terminals.     

Investigation of the effects of copper ions on protein aggregation using a model system

Protein aggregation is a notable feature of various human disorders, including Parkinsons disease, Alzheimers disease and many others systemic amyloidoses. An increasing number of observations in vitro suggest that transition metals are able to accelerate the aggregation process of several proteins found in pathological deposits, e.g. -synuclein, amyloid (A) peptide, ₂-microglobulin and fragments of the prion protein. Here we report the effects of metal ions on the aggregation rate of human muscle acylphosphatase, a suitable model system for aggregation studies in vitro. Among the different species tested, Cu²⁺ produced the most remarkable acceleration of aggregation, the rate of the process being 2.5-fold higher in the presence of 0.1 mM metal concentration. Data reported in the literature suggest the possible role played by histidine residues or negatively charged clusters present in the amino acid sequence in Cu²⁺-mediated aggregation of pathological proteins. Acylphosphatase does not contain histidine residues and is a basic protein. A number of histidine-containing mutational variants of acylphosphatase were produced to evaluate the importance of histidine in the aggregation process. The Cu²⁺-induced acceleration of aggregation was not significantly altered in the protein variants. The different aggregation rates shown by each variant were entirely explained by the changes of hydrophobicity or propensity to form a structure introduced by the point mutation. The effect of Cu²⁺ on acylphosphatase aggregation cannot therefore be attributed to the specific factors usually invoked in the aggregation of pathological proteins. The effect, rather, seems to be a general related to the chemistry of the polypeptide backbone and could represent an additional deleterious factor resulting from the alteration of the homeostasis of metal ions in cells.Received 4 December 2003; received after revision 7 January 2004; accepted 3 February 2004     

Dynamic laser light scattering studies of the effects of pyrophosphate on cyclic motions of cross-bridges in isolated thick myofilaments fromLimulus striated muscle

Pyrophosphate (PP_i) is a non-hydrolyzable ATP analogue known to affect the binding between myosin heads and actin. By using a dynamic laser light scattering method, we have shown that 1–2 mM PP_i enhances the increase in values induced by Ca²⁺ in isolated thick myofilaments fromLimulus striated muscle. However, similar treatment has no effect on the values of filaments suspended in either relaxing solution or ATP-free solution. is the average linewidth of the photoelectron count autocorrelation function of the light scattered. PP_i had no effect on the increase of values by Sr²⁺ but it substantially increased the values of the thick myofilaments suspended in Ba²⁺-substituted Ca²⁺ activating solution. The results show that PP_i also affects the energy-requiring cyclic cross-bridge motions.     

Mechanism of inhibition of IgE-dependent histamine release from rat mast cells by xestobergsterol A from the Okinawan marine spongeXestospongia bergquistia

Histamine release from rat peritoneal mast cells induced by anti-IgE was essentially complete within 4–5 min. Xestobergsterol A and B, which are constituents of the Okinawan marine spongeXestospongia bergquistia Fromont, dose-dependently inhibited anti-IgE-induced histamine release from rat mast cells. The IC₅₀ values of xestobergsterol A and B for histamine release in mast cells activated by anti-IgE were 0.07 and 0.11 M, respectively. Anti-IgE stimulated PI-PLC activity in a mast cell membrane preparation. Xestobergsterol A dose-dependently inhibited the generation of IP3 and membrane-bound PI-PLC activity. Moreover, xestobergsterol A inhibited Ca²⁺-mobilization from intracellular Ca²⁺-stores as well as histamine release in mast cells activated by anti-IgE. On the other hand, xestobergsterol B did not inhibit the membrane-bound and cytosolic PI-PLC activity, IP3 generation or the initial rise in [Ca²⁺]_i in mast cells activated by anti-IgE. These results suggest that the mechanism of inhibition by xestobergsterol A of the initial rise in [Ca²⁺]_i, of the generation of IP3, and of histamine release induced by anti-IgE, was through the inhibition of PI-PLC activity.     

HAb18G/CD147-mediated calcium mobilization and hepatoma metastasis require both C-terminal and N-terminal domains

HAb18G/CD147 is a heavily glycosylated protein containing two immunoglobulin superfamily domains. Our previous studies have indicated that overexpression of HAb18G/CD147 enhances metastatic potentials in human hepatoma cells by disrupting the regulation of store-operated Ca²⁺ entry by nitric oxide (NO)/cGMP. In the present study, we investigated the structure-function of HAb18G/CD147 by transfecting truncated HAb18G/CD147 fragments into human 7721 hepatoma cells. The inhibitory effect of HAb18G/CD147 on 8-bromo-cGMP-regulated thapsigargin-induced Ca²⁺ entry was reversed by the expression of either C or N terminus truncated HAb18G/CD147 in T7721C and T7721N cells, respectively. The potential effect of HAb18G/CD147 on metastatic potentials, both adhesion and invasion capacities, of hepatoma cells was abolished in T7721C cells, but not affected in T7721N cells. Release and activation of matrix metalloproteinases (MMPs), MMP-2 and MMP-9, were found to be enhanced by the expression of HAb18G/CD147, and this effect was abolished by both truncations. Thapsigargin significantly enhanced release and activation of MMPs (MMP-2 and MMP-9) in non-transfected 7721 cells, and this effect was negatively regulated by SNAP. However, no effects of thapsigargin or SNAP were observed in T7721 cells, and expression of HAb18G/CD147 enhanced secretion and activation of MMPs at a stable and high level. Taken together, these results suggest that both ectodomain and intracellular domains of HAb18G/CD147 are required to mediate the effect of HAb18G/CD147 on the secretion and activation of MMPs and metastasis-related processes in human hepatoma cells by disrupting the regulation of NO/cGMP-sensitive intracellular Ca²⁺ mobilization although each domain may play different roles.Received 1 April 2004; received after revision 15 June 2004; accepted 22 June 2004     

Effects of a new cholecystokinin antagonist (GE 410) on the smooth muscle of the guinea pig ileum

Summary Suc-Tyr-(SE)-Met-Gly-Trp-Met-Asp--phenethylamide (GE 410) competitively antagonized the contractions of smooth muscle strips from guinea pig ileum (pA₂=7.6, n=0.95) induced by cholecystokinin-octapeptide (CCK8). GE 410 inhibited the electrically-induced cholinergically mediated contractile responses and the [³H]ACh release in the ileum, as well as the CCK-stimulated electrical contractile responses and the [³H]ACh release in the cholinergic nerve terminals. The results suggest the existence of CCK-receptors not only in the smooth muscles but also on the neurons.     

Dissociation-constants of metal-ion-complexes with alkaline phosphatase from pig kidney

Summary Using metal-ion buffers it was possible to remove Zn²⁺, Mg²⁺ and Mn²⁺ ions of pig kidney alkaline phosphatase reversibly. The dissociation constants obtained are K_EMg:4·10^–7 M, K_EMn:4·10^–8 M and K_EZn:8·10^–13 M (22°C, pH:9.6, :0.07).Acknowledgement: The authors thank Dr.H. U. Wolf for helpful suggestions and valuable discussion and MissH. Köth for technical assistance.     

Partial purification and characterization of acid phosphatase from sporulated oocysts ofEimeria tenella

Summary Acid phosphatase ofEimeria tenella oocysts (Peak II) was purified 77-fold with a recovery of 26% using protamine sulfate precipitation, DEAE-cellulose chromatography and Sephadex G-200 gel filtration. This enzyme occurs in multiple forms as indicated by two peaks which can be separated by DEAE-cellulose chromatography and polyacrylamide gel electrophoresis. The partially purified enzyme has optimal activity at pH 4.5. With p-nitrophenyl phosphate the Km and V_max values for (Peak II) were 25 mM and 1.57 mol/min/mg protein, respectively. The enzyme (Peak II) ist strongly inhibited by Hg⁺⁺, Cu⁺⁺, iodoacetamide, fluoride and molybdate. Tartrate and other divalent metal ions have no effect on enzyme activity. The partially purified Peak II phosphatase is not a glycoprotein as it is not absorbed on concanavalin-A Sepharose and its treatment with bacterial neuraminidase does not alter its elution profile through DEAE cellulose.     

Guanylate cyclase stimulation by nitro-compounds is dependent on free Ca2+1

Summary The stimulatory effect of nitro-compounds on arterial and hepatic guanylate cyclase became significantly depressed at 0.2 M and higher concentration of free Ca²⁺. The basal enzyme activity proved to be Ca²⁺-independent.This study was supported by the Anton Dreher-Foundation for Medical Research.     

Nest cell lining of the solitary beeHylaeus bisinuatus (Hymenoptera: Colletidae)

The nest cell lining ofHylaeus bisinuatus (Hymenoptera: Colletidae) was shown by high-resolution solidstate [¹³C]NMR to be composed of lipid polymer and protein. The lipid polymer was shown by reduction and subsequent GC/MS analysis to be comprised of -hydroxy fatty acids (C₂₀, C₂₂, C₂₄ and C₂₆) and fatty alcohols (C₁₆ to C₃₀). The protein portion of the lining had a silk-like amino acid composition.     

Regulation ofL-valine absorption by opioids interacting withμ-receptors in rabbit ileum

In intact tissue, [d-Ala²,MePhe⁴, Gly-ol⁵] enkephalin (10^–5 M;-ligand), diminsihed short-circuit current (I_sc) and increased water, Na⁺ and Cl^– net fluxes in vitro under open circuit conditions; it also inhibitedL-valine absorption andL-valine-dependent variations of short-circuit current (I_{sc, val}). Naloxone (10^–6 M) antagonized these effects. In the absence of the muscularis and myenteric plexus this enkephalin or morphine (-ligand) reduced I_sc and I_{sc, val}. These enkephalin effects occurred at different times. Different concentrations of enkephalin were tested for their effects on I_{sc, val}. [d-Ala²,d-Leu⁵] enkephalin (mainly a -ligand) significantly decreased I_sc but not I_{sc, val}. The reduction ofL-valine absorption does not depend on the effects on basal ion transport. Interaction of opioids with-receptors located in the submucosal plexus and/or in the epithelial cell accounts for this reduction. This enkephalin effect seems to be at least partially under the control of the myenteric plexus.     

A correlation of the ionization energies of water and ethers with the polar and inductive substituent constants

Résumé On démontre que les énergies d'ionisation des ethers, R₂O, sont une fonction linéaire des constantes substituentes polaires et inductives. Les groupes alkyls qui donnent des électrons abaissent l'énergie d'ionisation du «lone-pair» de l'atome d'oxygène. L'eau est inclue dans la corrélation de même que lether le plus simple. On trouve qu'avec bonne approximationE _I =10,00+ 2,57 ^* ouE _I =12,59+28,0 _I .     

Chemical defense in the three European species ofCrematogaster ants

The composition of the Dufour gland of the antC. scutellaris has been reinvestigated by gas chromatography/mass spectrometry. The major components of the gland are (2E,5E,12Z)-4-oxoheneicosa-2,5,12-trien-1-ol acetate (1a) its ¹⁴ and ¹⁶ double bond isomers (1b and1c), and the corresponding (Z,Z)-dienes5a and5b, all containing an acetylated C₂₁ chain. The previously proposed structures1d, 1e, and5c, which are based on an homologous acetylated C₂₃ chain, correspond to minor derivatives present in the gland. Traces of acetylated C₁₉ homologs, tentatively identified as1g-1i, have also been found. The Dufour gland contents of the two other EuropeanCrematogaster species have also been studied.C. auberti is very similar toC. scutellaris in producing mainly1a, 1b and1c, together with the same higher and lower homologs, but it lacks the dienic derivatives5, whereasC. sordidula contains essentially the acetylated C₁₉ compounds1g, 1h, and1i, accompanied by acetylated C₁₇ homologs.