Enzymic synthesis,chemical characterisation and Sda activity of GalNAcß1-4[NeuAcα2-3]Galß1-4GlcNAc and GalNAcß1-4[NeuAcα2-3]Galß1-4Glc

The tetrasaccharides GalNAcß1-4[NeuAc2-3]Galß1-4Glc and GalNAcß1-4[NeuAc2-3]Galß1-4GlcNAc were synthesised by enzymic transfer of GalNAc from UDP-GalNAc to 3-sialyllactose (NeuAc2-3Galß1-4Glc) and 3-sialyl-N-acetyllactosamine (NeuAc2-3Galß1-4GlcNAc). The structures of the products were established by methylation and¹H-500 MHz NMR spectroscopy. In Sd^a serological tests the product formed with 3-sialyl-N-acetyllactosamine was highly active whereas that formed with 3-sialyllactose had only weak activity.     

Occurrence of the hepatotoxic cyanobacterium Nodularia spumigena in the Baltic Sea and structure of the toxin

Water blooms formed by potentially toxic species of cyanobacteria are a common phenomenon in the Baltic Sea in late summer. Twenty-five cyanobacterial bloom samples were collected from open and coastal waters of the Baltic Sea during 1985 to 1987, and their toxicity was determined by mouse bioassay. All of 5 bloom samples from the southern Baltic Sea, 6 of 6 from the open northern Baltic Sea (Gulf of Finland), and 7 of 14 Finnish coastal samples were found to contain hepatotoxic cyanobacteria. Nodularia spumigena and Aphanizomenon flos-aquae occurred together in high amounts in blooms from the open-sea areas. In addition, coastal samples contained the species Anabaena lemmermannii, Microcystis aeruginosa, and Oscillatoria agardhii. Eighteen hepatotoxic N. spumigena cultures were isolated from water bloom and open-sea water samples. High-pressure liquid chromatographic analysis of both hepatotoxic bloom samples and Nodularia strains showed a single toxic fraction. The toxin concentrations of the blooms were less than or equal to 2.4 mg/g of freeze-dried material, and those of laboratory-grown cultures were 2.5 to 8.0 mg/g of freeze-dried cells. A single toxin was isolated from three N. spumigena-containing bloom samples and three N. spumigena laboratory isolates. Amino acid analysis and low- and high-resolution fast-atom bombardment mass spectroscopy indicated that the toxin from all of the sources was a cyclic pentapeptide (molecular weight, 824) containing glutamic acid, beta-methylaspartic acid, arginine, N-methyldehydrobutyrine, and 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl-4,6-decadienoic acid.(ABSTRACT TRUNCATED AT 250 WORDS)     

Occurrence of the hepatotoxic cyanobacterium Nodularia spumigena in the Baltic Sea and structure of the toxin.

Water blooms formed by potentially toxic species of cyanobacteria are a common phenomenon in the Baltic Sea in late summer. Twenty-five cyanobacterial bloom samples were collected from open and coastal waters of the Baltic Sea during 1985 to 1987, and their toxicity was determined by mouse bioassay. All of 5 bloom samples from the southern Baltic Sea, 6 of 6 from the open northern Baltic Sea (Gulf of Finland), and 7 of 14 Finnish coastal samples were found to contain hepatotoxic cyanobacteria. Nodularia spumigena and Aphanizomenon flos-aquae occurred together in high amounts in blooms from the open-sea areas. In addition, coastal samples contained the species Anabaena lemmermannii, Microcystis aeruginosa, and Oscillatoria agardhii. Eighteen hepatotoxic N. spumigena cultures were isolated from water bloom and open-sea water samples. High-pressure liquid chromatographic analysis of both hepatotoxic bloom samples and Nodularia strains showed a single toxic fraction. The toxin concentrations of the blooms were less than or equal to 2.4 mg/g of freeze-dried material, and those of laboratory-grown cultures were 2.5 to 8.0 mg/g of freeze-dried cells. A single toxin was isolated from three N. spumigena-containing bloom samples and three N. spumigena laboratory isolates. Amino acid analysis and low- and high-resolution fast-atom bombardment mass spectroscopy indicated that the toxin from all of the sources was a cyclic pentapeptide (molecular weight, 824) containing glutamic acid, beta-methylaspartic acid, arginine, N-methyldehydrobutyrine, and 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl-4,6-decadienoic acid.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ouabain-insensitive salt and water movements in duck red cells. II. Norepinephrine stimulation of sodium plus potassium cotransport

Catecholamines induce net salt and water movements in duck red cells incubated in isotonic solutions. The rate of this response is approximately three times greater than a comparable effect observed in 400 mosmol hypertonic solutions in the absence of hormone (W.F. Schmidt and T. J. McManus. 1977 a.J. Gen. Physiol. 70:59-79. Otherwise, these two systems share a great many similarities. In both cases, net water and salt movements have a marked dependence on external cation concentrations, are sensitive to furosemide and insensitive to ouabain, and allow the substitution of rubidium for external potassium. In the presence of ouabain, but the absence of external potassium (or rubidium), a furosemide-sensitive net extrusion of sodium against a large electrochemical gradient can be demonstrated. When norepinephrine-treated cells are incubated with ouabain and sufficient external sodium, the furosemide-sensitive, unidirectional influxes of both sodium and rubidium are half- maximally saturated at similar rubidium concentrations; with saturating external rubidium, the same fluxes are half-maximal at comparable levels of external sodium. In the absence of sodium, a catecholamine-stimulated, furosemide-sensitive influx of rubidium persists. In the absence of rubidium, a similar but smaller component of sodium influx can be seen. We interpret these results in terms of a cotransport model for sodium plus potassium which is activated by hypertonicity or norepinephrine. When either ion is absent from the incubation medium, the system promotes an exchange-diffusion type of movement of the co-ion into the cells. In the absence of external potassium, net movement of potassium out of the cell leads to a coupled extrusion of sodium against its electrochemical gradient.     

Isolation and characterization of hepatotoxic microcystin homologs from the filamentous freshwater cyanobacterium Nostoc sp. strain 152.

A strain of the filamentous cyanobacterium Nostoc sp. isolated from a lake in Finland was found to produce at least nine hepatotoxic peptides with chemical and toxicological properties similar to those of the hepatotoxic hepta- and pentapeptides produced by other cyanobacteria. Toxins were isolated and purified by high-performance liquid chromatography. Amounts available for five of the purified toxins (P6, P14, P15, P16, and P18) were adequate for high-performance liquid chromatography amino acid analysis and determination of molecular weight by fast-atom bombardment-mass spectrometry (FAB-MS). Quantities of three toxins (P14, P15, and P16) were adequate for further analysis by high-resolution FAB-MS, FAB-MS/MS, and 1H-nuclear magnetic resonance. Analysis showed that the toxins are new types of microcystin-LR homologs. Microcystin-LR contains equimolar amounts of D-alanine, L-leucine, D-erythro-beta-methylaspartic acid, L-arginine, ADDA (3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl-4,6-decadienoic acid), D-glutamic acid, and N-methyldehydroalanine (molecular weight [MW], 994). Nostoc sp. strain 152 was found to produce the following microcystin-LR homologs: (i) P6 contains an extra methylene group most probably due to the presence of N-methyldehydrobutyrine instead of N-methyldehydroalanine (MW, 1,008); (ii) P14 is O-acetyl-O-demethyl ADDA-microcystin-LR (MW, 1,022); (iii) P15 is 3-demethyl-O-acetylADDA-homoarginine-microcystin-LR (MW, 1,036); (iv) P16 is 3-demethyl-O-acetyl-O-demethylADDA-microcystin-LR (MW, 1,008); (v) P18 is 3-demethyl-O-acetyl-O-demethylADDA-homoarginine-microcystin- LR (MW, 1,022). The toxicities of the new microcystin homologs were not significantly different from those of microcystin-LR or demethylmicrocystin-LR.     

In-depth qualitative and quantitative analysis of composite glycosylation profiles and other micro-heterogeneity on intact monoclonal antibodies by high-resolution native mass spectrometry using a modified Orbitrap

Here, we describe a fast, easy-to-use, and sensitive method to profile in-depth structural micro-heterogeneity, including intricate N-glycosylation profiles, of monoclonal antibodies at the native intact protein level by means of mass spectrometry using a recently introduced modified Orbitrap Exactive Plus mass spectrometer. We demonstrate the versatility of our method to probe structural micro-heterogeneity by describing the analysis of three types of molecules: (1) a non-covalently bound IgG4 hinge deleted full-antibody in equilibrium with its half-antibody, (2) IgG4 mutants exhibiting highly complex glycosylation profiles, and (3) antibody-drug conjugates. Using the modified instrument, we obtain baseline separation and accurate mass determination of all different proteoforms that may be induced, for example, by glycosylation, drug loading and partial peptide backbone-truncation. We show that our method can handle highly complex glycosylation profiles, identifying more than 20 different glycoforms per monoclonal antibody preparation and more than 30 proteoforms on a single highly purified antibody. In analyzing antibody-drug conjugates, our method also easily identifies and quantifies more than 15 structurally different proteoforms that may result from the collective differences in drug loading and glycosylation. The method presented here will aid in the comprehensive analytical and functional characterization of protein micro-heterogeneity, which is crucial for successful development and manufacturing of therapeutic antibodies     

The diageotropica mutation alters auxin induction of a subset of the Aux/IAA gene family in tomato

The diageotropica (dgt) mutation has been proposed to affect either auxin perception or responsiveness in tomato plants. It has previously been demonstrated that the expression of one member of the Aux/IAA family of auxin-regulated genes is reduced in dgt plants. Here, we report the cloning of ten new members of the tomato Aux/IAA family by PCR amplification based on conserved protein domains. All of the gene family members except one (LeIAA7) are expressed in etiolated tomato seedlings, although they demonstrate tissue specificity (e.g. increased expression in hypocotyls vs. roots) within the seedling. The wild-type auxin-response characteristics of the expression of these tomato LeIAA genes are similar to those previously described for Aux/IAA family members in Arabidopsis. In dgt seedlings, auxin stimulation of gene expression was reduced in only a subset of LeIAA genes (LeIAA5, 8, 10, and 11), with the greatest reduction associated with those genes with the strongest wild-type response to auxin. The remaining LeIAA genes tested exhibited essentially the same induction levels in response to the hormone in both dgt and wild-type hypocotyls. These results confirm that dgt plants can perceive auxin and suggest that a specific step in early auxin signal transduction is disrupted by the dgt mutation.     

Biochemical isolation and purification of ovulation-inducing factor (OIF) in seminal plasma of llamas

Background  

The objective of the present study was to isolate and purify the protein fraction(s) of llama seminal plasma responsible for the ovulation-inducing effect of the ejaculate.     

Combined modulation of the mitochondrial ATP-dependent potassium channel and the permeability transition pore causes prolongation of the biphasic calcium dynamics

The permeability transition pore (PTP) and the ATP-dependent potassium (mtK-ATP) channel of mitochondria are known to play key roles in mitochondrially mediated apoptosis. We investigated how modulation of the permeability transition pore (PTP) and the ATP-dependent potassium (mtK-ATP) channel, either as single elements or in combination, affects the proapoptotic intracellular calcium ([Ca(2+)](i)) transients and the mitochondrial membrane potential (psi(m)). For this purpose a model was established exploring the [Ca(2+)](i) transients in N2A cells using continuous application of ATP that causes a biphasic [Ca(2+)](i) response. This response was sensitive to endoplasmatic reticulum (ER) Ca(2+) depletion and a smooth ER Ca(2+)-ATPase (SERCA) antagonist. PTP inhibition by cyclosporine A (CsA) or its non-immunosuppressive derivative NIM811 caused an amplification of the secondary [Ca(2+)](i) peak and induced a hyperpolarization of psi(m). Both the putative mtK-ATP channel inhibitor 5-hydroxydecanoate (5-HD) and the opener diazoxide ameliorated the ATP-induced secondary [Ca(2+)](i) peak. The effect of diazoxide was accompanied by a depolarization of psi(m) whereas 5-HD had no effect on psi(m). When diazoxide and CsA or NIM811 were applied together the secondary [Ca(2+)](i) rise did not return to baseline and a not significant hyperpolarization of psi(m) was observed. So, simultaneous inhibition of PTP and activation of the mtK-ATP channel prevents the increased slope of the secondary [Ca(2+)](i) peak induced by CsA (or NIM811) and also the depolarization after diazoxide application. Hence, we propose that modulation of one of these channels leads to functional changes of the other channel by means of Delta[Ca(2+)](i) and Deltapsi(m).