Structure/activity relationships in the arginine taste pathway of the channel catfish

To characterize the molecular/structural requirements for activationor antagonism of the arginine taste pathways in catfish, Ictaluruspunctatus, structure/activity studies were performed using integratedmultiunit responses and cross-adaptation. Of all the guanidinium-containingcompounds tested, only L-arginine, L--amino-ß-guanidinopropionic acid (L-AGPA) and L-arginine methyl and ethyl esterswere strong stimuli. Results of functional group substitutionsand modification of the L-arginine parent molecule indicatedthat: (i) stereospecificity was observed with D-arginine beinga much less effective stimulus than L-arginine; (ii) an L-aminogroup must be present and unblocked (-chloro-guanidino-N-valericacid and N-acetyl L-arginine were weak or inactive stimuli);(iii) a free carboxylic acid group was not necessary for activity;(iv) the distance between the anomeric carbon and the guanidiniumgroup was not critical (L-AGPA, having two methylene groupsless than L-arginine was a moderately strong stimulus as wasL-canavanine) and (v) modification or substitution of the guanidinumgroup by other basic groups including amine, methyl or dimethylamineor by an isosterc (ureido) resulted in loss of stimulatory ability.In general, those stimuli and analogs that were good cross-adaptersof L-arginine stimulation were also good competitors for L-[³H]argininebinding to a partial membrane fraction (P2) from catfish tasteepithelium. On the other hand, compounds that were poor cross-adaptingstimuli were also poor binding competitors. While D-argininewas a poor stimulus, it did cross-adapt L-arginine and competedwell with L-[³H]arginine for binding to fraction P2.     

A novel and remarkably thermostable ferredoxin from the hyperthermophilic archaebacterium Pyrococcus furiosus.

The archaebacterium Pyrococcus furiosus is a strict anaerobe that grows optimally at 100 degrees C by a fermentative-type metabolism in which H2 and CO2 are the only detectable products. A ferredoxin, which functions as the electron donor to the hydrogenase of this organism was purified under anaerobic reducing conditions. It had a molecular weight of approximately 12,000 and contained 8 iron atoms and 8 cysteine residues/mol but lacked histidine or arginine residues. Reduction and oxidation of the ferredoxin each required 2 electrons/mol, which is consistent with the presence of two [4Fe-4S] clusters. The reduced protein gave rise to a broad rhombic electronic paramagnetic resonance spectrum, with gz = 2.10, gy = 1.86, gx = 1.80, and a midpoint potential of -345 mV (at pH 8). However, this spectrum represented a minor species, since it quantitated to only approximately 0.3 spins/mol. P. furiosus ferredoxin is therefore distinct from other ferredoxins in that the bulk of its iron is not present as iron-sulfur clusters with an S = 1/2 ground state. The apoferredoxin was reconstituted with iron and sulfide to give a protein that was indistinguishable from the native ferredoxin by its iron content and electron paramagnetic resonance properties, which showed that the novel iron-sulfur clusters were not artifacts of purification. The reduced ferredoxin also functioned as an electron donor for H2 evolution catalyzed by the hydrogenase of the mesophilic eubacterium Clostridium pasteurianum. P. furiosus ferredoxin was resistant to denaturation by sodium dodecyl sulfate (20%, wt/vol) and was remarkably thermostable. Its UV-visible absorption spectrum and electron carrier activity to P. furiosus hydrogenase were unaffected by a 12-h incubation of 95 degrees C.     

Sex differences in the behaviour of immature captive lowland gorillas

Studies of the behaviour of 26 (12 males and 14 females) captive infant and juvenile lowland gorillas showed clear sex differences. Females showed greater interest in young infants and were more active in nest building as well as in solitary and social grooming. Males were more active in locomotive, dominance, and aggressive behaviour and in social play. Hand-rearing further increased aggression. Males were more aggressive when they lived with only one partner, and they rose in rank even above older females, a pattern that has not been observed in naturally reared gorillas.     

Efficiency of bacterial protein synthesis during anaerobic degradation of cattle waste

The rate of [15N]ammonia (15NH3) uptake or incorporation into bacterial cells was studied, using stirred, 3-liter benchtop digestors fed on a semicontinuous basis with cattle waste. The fermentations were carried out at 40 and 60 degrees C and at four different loading rates (3, 6, 9, and 12 g of volatile solids per liter of reactor volume per day). The rate of NH3-N incorporation for the period 1 to 5 h after feeding at the four different loading rates was 0.49, 0.83, 1.05, and 1.08 mg/liter per h in the mesophilic digestor and 0.68, 1.07, 1.17, and 1.21 mg/liter per h in the thermophilic digestor. Values were lower 7 to 21 h after feeding in both digestors and were related to the rate of fermentation or CH4 production. In the mesophilic digestors, the rate of bacterial cell production ranged from 3.97 to 8.72 mg of dry cells per liter per h, 1 to 5 h after feeding at the different loading rates. Corresponding values for the thermophilic digestors ranged from 5.46 to 9.77 mg of dry cells per liter per h. Cell yield values ranged from 2.3 to 3.1 mg of dry cells per mol of CH4 produced in the mesophilic and thermophilic digestors at the two lower loading rates. The values were higher (2.8 to 3.4) in the mesophilic digestors at the two higher loading rates because of the accumulation of propionate and a consequent reduction in CH4 production. Low cell yields such as those measured in this study are characteristic of low-specific-growth rates under energy-limited conditions.     

A microdeletion of less than 250 kb, including the proximal part of the FMR-1 gene and the fragile-X site, in a male with the clinical phenotype of fragile-X syndrome

A gene designated "FMR-1" has been isolated at the fragile-X locus. One exon of this gene is carried on a 5.1-kb EcoRI fragment that exhibits length variation in fragile-X patients because of amplification of or insertion into a CGG-repeat sequence. This repeat probably represents the fragile site. The EcoRI fragment also includes an HTF island that is hypermethylated in fragile-X patients showing absence of FMR-1 mRNA. In this paper, we present further evidence that the FMR-1 gene is involved in the clinical manifestation of the fragile-X syndrome and also in the expression of the cellular phenotype. A deletion including the HTF island and exons of the FMR-1 gene was detected in a fragile X-negative mentally retarded male who presented the clinical phenotype of the fragile-X syndrome. The deletion involves less than 250 kb of genomic DNA, including DXS548 and at least five exons of the FMR-1 gene. These data support the hypothesis that loss of function of the FMR-1 gene leads to the clinical phenotype of the fragile-X syndrome. In the fragile-X syndrome, there are pathogenetic mechanisms other than amplification of the CGG repeat that do have the same phenotypic consequences.     

Co-purification of 130 kD nitric oxide synthase and a 22 kD link protein from human neutrophils.

The synthesis of nitric oxide (NO) from L-arginine has been demonstrated in several cell types. Both constitutive and inducible forms of NO synthase have been described in different cells. We purified the constitutive form of NO synthase enzyme in human neutrophils using a two-column procedure. Crude 100,000g supernatant of human neutrophils was passed through a 2'-5'-ADP-agarose column followed by a DEAE-Bio-Gel A anion exchange column. NO synthase enzyme migrated as a single band (MW approximately 130,000) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Its activity was dependent upon nicotinamide adenine dinucleotide phosphate (NADPH) and (6R)-tetrahydro-L-biopterin (BH4). In addition, flavin adenine dinucleotide (FAD) was also found to be essential for its maximal activity. A second NADPH, FAD-dependent component (MW approximately 22kD) was also found consistently on the SDS-PAGE gel. These observations suggest co-regulation between NO synthase enzyme and this NADPH, FAD-dependent component, which may be associated with the superoxide radical generating system.     

Effect of nucleotide cofactor structure on recA protein-promoted DNA pairing. 2. DNA renaturation reaction.

We have examined the effects of the structurally related nucleoside triphosphates, adenosine triphosphate (ATP), purine riboside triphosphate (PTP), inosine triphosphate (ITP), and guanosine triphosphate (GTP), on the recA protein-promoted DNA renaturation reaction (phi X DNA). In the absence of nucleotide cofactor, the recA protein first converts the complementary single strands into unit-length duplex DNA and other relatively small paired DNA species; these initial products are then slowly converted into more complex multipaired network DNA products. ATP and PTP stimulate the conversion of initial product DNA into network DNA, whereas ITP and GTP completely suppress network DNA formation. The formation of network DNA is also inhibited by all four of the corresponding nucleoside diphosphates, ADP, PDP, IDP, and GDP. Those nucleotides which stimulate the formation of network DNA are found to enhance the formation of large recA-ssDNA aggregates, whereas those which inhibit network DNA formation cause the dissociation of these nucleoprotein aggregates. These results not only implicate the nucleoprotein aggregates as intermediates in the formation of network DNA, but also establish the functional equivalency of ITP and GTP with the nucleoside diphosphates. Additional experiments indicate that the net effect of ITP and GTP on the DNA renaturation reaction is dominated by the corresponding nucleoside diphosphates, IDP and GDP, that are generated by the NTP hydrolysis activity of the recA protein.     

Aggregation and Metal-Binding Properties of Mutant Forms of the Amyloid Aβ Peptide of Alzheimer's Disease

Abstract: The fibrillogenic properties of Alzheimer's Aβ peptides corresponding to residues 1–40 of the normal human sequence and to two mutant forms containing the replacement Ala²¹ to Gly or Glu²² to Gln were compared. At pH 7.4 and 37°C the Gln²² peptide was found to aggregate and precipitate from solution faster than the normal Aβ, whereas the Gly²¹ peptide aggregated much more slowly. Electron microscopy showed that the aggregates all had fibrillar structures. Circular dichroism spectra of these peptides revealed that aggregation of the normal and Gln²² sequences was associated with spectral changes consistent with a transformation from random coil to β sheet, whereas the spectrum of the Gly²¹ peptide remained almost unchanged during a period in which little or no aggregation occurred. When immobilised by spotting onto nitrocellulose membranes the peptides bound similar amounts of the radioisotope ⁶⁵Zn²⁺. Of several competing metal ions, tested at 20× the concentration of Zn²⁺, Cu²⁺ displaced >95% of the radioactivity from all three peptides and Ni²⁺ produced >50% displacement in each case. Some other metal ions tested caused lesser displacement, but Fe²⁺ and Al³⁺ were without effect. In a saturation binding assay, a value of 3.2 µM was obtained for the binding of Zn²⁺ to Aβ but our data provided no evidence for a reported higher affinity site (107 nM). The results suggest that the neuropathology associated with the Gly²¹ mutation is not due to enhanced fibrillogenic or different metal-binding properties of the peptide and that the binding of zinc to amyloid peptides is not a specific phenomenon.