Determination of the relative and absolute stereochemistry of a potent and α1A-selective adrenoceptor antagonist

The binding affinities and selectivities of antagonists Figure 1 and Scheme 2 for the α_1A-adrenoceptor are dependent on the stereochemical orientation of the groups at the C-4 and C-5 positions of the oxazolidinone ring. The unambiguous assignment of the relative and absolute configurations of the diastereomers of SNAP 7915 (Figure 1 and Scheme 2) is reported.     

A proposed neural pathway for vocalization in South African clawed frogs,Xenopus laevis

Vocalizations of South African clawed frogs are produced by contractions of laryngeal muscles innervated by motor neurons of the caudal medulla (within cranial nerve nucleus IX-X). We have traced afferents to laryngeal motor neurons in male and female frogs using retrograde axonal transport of horseradish peroxidase conjugated to wheat germ agglutinin (HRP-WGA). After iontophoretic injection of HRP-WGA into n. IX-X, retrogradely labelled neurons were seen in the contralateral n. IX-X, in rhombencephalic reticular nuclei, and in the pre-trigeminal nucleus of the dorsal tegmental area (DTAM) of both males and females.     

Buchbesprechungen

Ohne Zusammenfassung     

A general method for highly selective cross-linking of unprotected polypeptides via pH-controlled modification of N-terminal alpha-amino groups

A method is described for the highly selective modification of the alpha-amino groups at the N-termini of unprotected peptides to form stable, modified peptide intermediates which can be covalently coupled to other molecules or to a solid support. Acylation with iodoacetic anhydride at pH 6.0 occurs with 90-98% selectivity for the alpha-amino group, depending on the N-terminal residue (as shown with a series of model hexapeptides containing a competing Lys residue). Although Cys residues must be protected (reversibly or irreversibly) before the anhydride reaction, there are no detectable side reactions of the alpha-amino moiety--of the reagent or of modified peptide--with the side chains of His, Met, or Lys. The reaction works well in denaturants, so that inhibitory effects of noncovalent structure can be minimized. In a second step the iodoacetyl-peptide can be reacted with a thiol group on a protein, on a solid chromatography matrix, on a spectroscopic probe, etc. This is illustrated by reaction of a series of N alpha-iodoacetyl-peptides with murine interferon-gamma, which contains a C-terminal Cys residue. Data are presented which suggest that this iodoacetic anhydride scheme is superior in selectivity for alpha-amino groups to conventional chemical approaches to cross-linking such as use of 2-iminothiolane or N-hydroxysuccinimide-activated carboxylic acid esters. The reaction is ideally suited for modifying peptide fragments, as pure species or as mixtures, derived from proteolytic or chemical fragmentation of proteins. Furthermore, polypeptides synthesized biosynthetically, for example via recombinant DNA techniques, can be cross-linked in this way.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nutrient additions by waterfowl to lakes and reservoirs: predicting their effects on productivity and water quality

Lakes and reservoirs provide water for human needs and habitat for aquatic birds. Managers of such waters may ask whether nutrients added by waterfowl degrade water quality. For lakes and reservoirs where primary productivity is limited by phosphorus (P), we developed a procedure that integrates annual P loads from waterfowl and other external sources, applies a nutrient load-response model, and determines whether waterfowl that used the lake or reservoir degraded water quality. Annual P loading by waterfowl can be derived from a figure in this report, using the days per year that each kind spent on any lake or reservoir. In our example, over 6500 Canada geese (Branta canadensis) and 4200 ducks (mostly mallards, Anas platyrhynchos) added 4462 kg of carbon (C), 280 kg of nitrogen (N), and 88 kg of P y^–1 to Wintergreen Lake in southwestern Michigan, mostly during their migration. These amounts were 69% of all C, 27% of all N, and 70% of all P that entered the lake from external sources. Loads from all external sources totaled 840 mg P m^–2 y^–1. Application of a nutrient load-response model to this concentration, the hydraulic load (0.25 m y^–1), and the water residence time (9.7 y) of Wintergreen Lake yielded an average annual concentration of total P in the lake of 818 mg m^–3 that classified the lake as hypertrophic. This trophic classification agreed with independent measures of primary productivity, chlorophyll-a, total P, total N, and Secchi disk transparency made in Wintergreen Lake. Our procedure showed that waterfowl caused low water quality in Wintergreen Lake.Contribution 824 of the National Fisheries Research Center-Great Lakes, 1451 Green Road, Ann Arbor, Michigan 48105, U.S.A. and 722 of the Kellogg Biological Station, Michigan State University.     

Identification and characterization of flavonoids in the root exudate of Robinia pseudoacacia

 Eight compounds exuded from young roots of black locust (Robinia pseudoacacia) were separated by two-dimensional HPTLC, by HPLC and GC, and were identified by spectroscopic methods (ultraviolet/visible spectroscopy and mass spectrometry) as 4′,7-dihydroxyflavone, apigenin, naringenin, chrysoeriol and isoliquiritigenin. Structural assignments were confirmed by comparison with authentic standards. The capacity to induce β-galactosidase activity in Rhizobium sp. NGR234 containing a nod box::lacZ fusion on plasmid pA27 identified these flavonoids and the chalcone as nod gene inducers. This indicates the important role of these compounds in nodulation of this legume tree. Received: 26 July 1996 / Accepted: 9 September 1996     

Reovirus variants selected during persistent infections of L cells contain mutations in the viral S1 and S4 genes and are altered in viral disassembly.

Reoviruses isolated from persistently infected cultures (PI viruses) can grow in the presence of ammonium chloride, a weak base that blocks acid-dependent proteolysis of viral outer-capsid proteins during viral entry into cells. We used reassortant viruses isolated from crosses of wild-type (wt) reovirus strain, type 1 Lang, and three independent PI viruses, L/C, PI 2A1, and PI 3-1, to identify viral genes that segregate with the capacity of PI viruses to grow in cells treated with ammonium chloride. Growth of reassortant viruses in ammonium chloride-treated cells segregated with the S1 gene of L/C and the S4 gene of PI 2A1 and PI 3-1. The S1 gene encodes viral attachment protein sigma1, and the S4 gene encodes outer-capsid protein sigma3. To identify mutations in sigma3 selected during persistent reovirus infection, we determined the S4 gene nucleotide sequences of L/C, PI 2A1, PI 3-1, and four additional PI viruses. The deduced amino acid sequences of sigma3 protein of six of these PI viruses contained a tyrosine-to-histidine substitution at residue 354. To determine whether mutations selected during persistent infection alter cleavage of the viral outer capsid, the fate of viral structural proteins was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after treatment of virions of wt and PI viruses with chymotrypsin in vitro. Proteolysis of PI virus outer-capsid proteins sigma3 and mu1C occurred with faster kinetics than proteolysis of wt virus outer-capsid proteins. These results demonstrate that mutations in either the S1 or S4 gene alter acid-dependent disassembly of the reovirus outer capsid and suggest that increased efficiency of proteolysis of viral outer-capsid proteins is important for maintenance of persistent reovirus infections of cultured cells.     

Analogs of methionyl-tRNA synthetase substrates containing photolabile groups.

Three photolabile analogs of substrates of methionyl-tRNA synthetase were synthesized. In one, the 4-thiouridine at the 8 position of E. coli tRNAfMet was alkylated with [14C]p-azidobromoacetanilide. In the second, [14C]p-azidobenzoic acid hydrazide was condensed with the 3'-terminal dialdehyde of periodate-oxidized Escherichia coli tRNAfMet. The modified tRNAs could be purified by chromatography on benzoylated DEAE-cellulose. The third photolabile compound was [3H]methioninyl-8-azido-adenosine 5'-phosphate, an analog of the methionyl adenylate intermediate in the aminoacylation reaction. Irradiation of each of these compounds in the presence of equimolar amounts of E. coli methionyl-tRNA synthetase of micrometer concentrations gave 5-15% crosslinking.     

Induction of interleukin-5 responsiveness in resting B cells by engagement of the antigen receptor and perception of a second polyclonal activation signal.

Experiments were performed to examine the nature of agents which could induce IL-5 responsiveness in small, resting splenic B lymphocytes. First, IL-5 increased plaque forming cell responses to the TI-1 antigen TNP-LPS. A second set of experiments using anti-IgM + LPS which allowed limiting dilution analysis showed induction of IL-5 responsiveness in about 20% of the resting B cell population. In the same system, IL-4 increased the percentage of proliferating cells by about 40%. A third system using the TI-2 analog conjugate anti-IgD-dextran (anti-delta-dextran) also rendered small, resting B cells responsive to IL-5. An additional system employing anti-IgM plus dextran sulfate, which also allowed limiting dilution analysis, induced IL-5 responsiveness in at least 10% of resting B cells. The features common to all four systems inducing B cell IL-5 responsiveness are at least twofold. Each system directly accesses the B cell antigen receptor and causes crosslinking. Second, each system also provides an additional polyclonal activating moiety, some of which may be similar to those in thymus independent antigens. These results suggest that some resting B cells may become IL-5 receptive after perception of at least two kinds of signals one of which perturbs sIg and the second being nonspecific and polyclonally activating.