Phylogenetic diversification of immunoglobulin genes and the antibody repertoire

Immunoglobulins are encoded by a large multigene system that undergoes somatic rearrangement and additional genetic change during the development of immunoglobulin-producing cells. Inducible antibody and antibody-like responses are found in all vertebrates. However, immunoglobulin possessing disulfide-bonded heavy and light chains and domain-type organization has been described only in representatives of the jawed vertebrates. High degrees of nucleotide and predicted amino acid sequence identity are evident when the segmental elements that constitute the immunoglobulin gene loci in phylogenetically divergent vertebrates are compared. However, the organization of gene loci and the manner in which the independent elements recombine (and diversify) vary markedly among different taxa. One striking pattern of gene organization is the "cluster type" that appears to be restricted to the chondrichthyes (cartilaginous fishes) and limits segmental rearrangement to closely linked elements. This type of gene organization is associated with both heavy- and light-chain gene loci. In some cases, the clusters are "joined" or "partially joined" in the germ line, in effect predetermining or partially predetermining, respectively, the encoded specificities (the assumption being that these are expressed) of the individual loci. By relating the sequences of transcribed gene products to their respective germ-line genes, it is evident that, in some cases, joined-type genes are expressed. This raises a question about the existence and/or nature of allelic exclusion in these species. The extensive variation in gene organization found throughout the vertebrate species may relate directly to the role of intersegmental (V<==>D<==>J) distances in the commitment of the individual antibody-producing cell to a particular genetic specificity. Thus, the evolution of this locus, perhaps more so than that of others, may reflect the interrelationships between genetic organization and function.      

Measurement by electroantennogram of airborne pheromone in cotton treated for mating disruption of Pectinophora gossypiella following removal of pheromone dispensers

The presence of pheromone in cotton foliage after removal of pheromone dispensers was assessed by measuring the airborne pheromone concentration with an electroantennogram device. Plots of 0.4 ha in isolated cotton fields were treated with Shin-Etsu PBW-Rope® pheromone dispensers for mating disruption of Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae). The dispensers contained ( Z , Z )- and ( Z , E )-7,11-hexadecadienyl acetates (gossyplure) in a 49:51 ratio and were applied at a density of 1 000/ha. The 400 pheromone dispensers were removed 1–12 days later. In four experiments involving canopy heights from 30–150 cm, the decay of the pheromone concentration was recorded repeatedly in short intervals for up to 7 h. Decay to undetectable concentrations generally occurred within 1–10 h, depending on plant size and wind conditions. In all four experiments, pheromone concentration 24 h after removal was found to be near or below detection threshold of the electroantennogram. The presence of pheromone within and above the cotton after dispenser removal would be due to re-entrainment of pheromone that had been adsorbed on cotton foliage or possibly some residual airborne pheromone.     

Enhanced heterologous gene expression in novel rpoH mutants of Escherichia coli.

Extragenic temperature-resistant suppressor mutants of an rpoD800 derivative of Escherichia coli W3110 were selected at 43.5 degrees C. Two of the mutants were shown to have a phenotype of enhanced accumulation of heterologous proteins. Genetic mapping of the two mutants showed that the mutation conferring temperature resistance resided in the rpoH gene. P1-mediated transduction of the rpoD+ gene into both of the rpoD800 rpoH double mutants resulted in viable rpoH mutants, MON102 and MON105, that retained temperature resistance at 46 degrees C, the maximum growth temperature of W3110. The complete rpoH gene, including the regulatory region, from MON102, MON105, and the parental W3110 was cloned and sequenced. Sequencing results showed that a single C----T transition at nucleotide 802 was present in both MON102 and MON105, resulting in an Arg(CGC)----Cys(TGC) substitution at amino acid residue 268 (R-268-C; this gene was designated rpoH358). Heterologous protein accumulation levels in both MON102 and MON105, as well as in rpoH358 mutants constructed in previously unmanipulated W3110 and JM101, were assessed and compared with parental W3110 and JM101 levels. Expression studies utilizing the recA or araBAD promoter and the phage T7 gene 10L ribosome-binding site (g10L) showed that increased accumulation levels of a number of representative heterologous proteins (i.e., human or bovine insulin-like growth factor-1, bovine insulin-like growth factor-2, prohormone of human atrial natriuretic factor, bovine placental lactogen, and/or bovine prolactin) were obtained in the rpoH358 mutants compared with the levels in the parental W3110 and JM101. The mechanism of enhanced heterologous protein accumulation in MON102 and MON105 was unique compared with those of previously described rpoH mutants. Pulse-chase and Northern (RNA) blot analyses showed that the enhanced accumulation of heterologous proteins was not due to decreased proteolysis but was instead due to increased levels of the respective heterologous mRNAs accompanied by increased synthesis of the respective heterologous proteins. The plasmid copy number remained unaltered.     

Tyrosine phosphorylation of Munc18‐1 inhibits synaptic transmission by preventing SNARE assembly

Tyrosine kinases are important regulators of synaptic strength. Here, we describe a key component of the synaptic vesicle release machinery, Munc18‐1, as a phosphorylation target for neuronal Src family kinases (SFKs). Phosphomimetic Y473D mutation of a SFK phosphorylation site previously identified by brain phospho‐proteomics abolished the stimulatory effect of Munc18‐1 on SNARE complex formation (“SNARE‐templating”) and membrane fusion in vitro. Furthermore, priming but not docking of synaptic vesicles was disrupted in hippocampal munc18‐1‐null neurons expressing Munc18‐1_Y473D. Synaptic transmission was temporarily restored by high‐frequency stimulation, as well as by a Munc18‐1 mutation that results in helix 12 extension, a critical conformational step in vesicle priming. On the other hand, expression of non‐phosphorylatable Munc18‐1 supported normal synaptic transmission. We propose that SFK‐dependent Munc18‐1 phosphorylation may constitute a potent, previously unknown mechanism to shut down synaptic transmission, via direct occlusion of a Synaptobrevin/VAMP2 binding groove and subsequent hindrance of conformational changes in domain 3a responsible for vesicle priming. This would strongly interfere with the essential post‐docking SNARE‐templating role of Munc18‐1, resulting in a largely abolished pool of releasable synaptic vesicles.     

Agar Concentration in Counting Clostridium Colonies

Decreasing the agar concentration of a counting medium from the usual 1.5% resulted in larger colonies with less interference from gas in Clostridium botulinum 115B and C. sporogenes PA 3679. Optimal agar concentration was 0.65% for C. botulinum with 24-hr incubation and 0.50% for C. sporogenes with 48-hr incubation. Lower concentrations yielded growth too diffuse for counting. Motility was considered the explanation for increased colony size in softer agar. The greater the degree of motility, the greater would be the diffusibility expected, and thus the higher the agar concentration required to insure discrete colonies. For quantitating motility, evaluations were made by use of microscopic examination of liquid cultures and rate of diffusion in a semisolid medium. With both criteria, the degree of motility of C. botulinum 115B clearly exceeded that of C. sporogenes PA 3679. Small-colony variants of C. botulinum in 0.65% agar yielded only small colonies on subculture, with a corresponding decrease in degree of motility of the cells by both criteria. Colony size of the nonmotile C. perfringens ATCC 3624 was unaffected by lowered agar concentrations.     

The cost of incubation in relation to clutch-size in the Collared Flycatcher Ficedula albicollis

This study examines the importance of avian incubation costs as determinants of clutch-size variation by performing clutch-size and brood-size manipulations in the same population of Collared Flycatchers Ficedula albicollis during the same breeding season. In 2 5 cases when three or more clutches of the same size were completed on the same day, we moved two eggs on the day after the last egg had been laid from one randomly selected clutch (C) to another (C) and moved two other eggs from this to a third clutch (C⁺). In 20 other cases of simultaneously completed clutches of the same size, we moved two randomly selected young from one brood to a second and from that moved two other young to a third (B, B and B⁺groups). Most females were weighed the day after completion of the clutch and 1–4 days before hatching of the young, and some of them also 10–14 days after hatching of the young. We measured the daily energy expenditure of females incubating manipulated clutches of 4, 6 and 8 eggs by means of the doubly-labelled water (D₂¹⁸O) technique and also recorded their nest attendance. Hatching success of fertilized eggs was reduced in the enlarged clutches compared with control and reduced clutches. Females expired on average 3142.6 ml CO₂ and expended 78.6 kJ per day while incubating, which corresponds to a metabolic intensity of 3.3 times BMR. Daily energy expenditure increased with clutch-size due to higher costs while incubating, and not because of changed activity patterns. There were no significant differences in length of incubation, female mass or mass changes between phases for the C, C and C⁺groups. In both the C and B groups, enlarged broods produced significantly more fledged young than control broods, and those significantly more than reduced broods. Fledgling tarsus-length and mass did not differ significantly between treatments in either the C or B groups. There was no significant difference in breeding success between clutch and brood manipulations. In this season, incubation costs did not entail significant fitness losses, expressed either as fledgling production or female condition. Also, control females could have raised more young to fledging age than they did with no apparent costs.     

Insulin action and secretion in endurance-trained and untrained humans

To evaluate insulin sensitivity and responsiveness, a two-stage hyperinsulinemic euglycemic clamp procedure (insulin infusions of 40 and 400 mU.m-2.min-1) was performed on 11 endurance-trained and 11 untrained volunteers. A 3-h hyperglycemic clamp procedure (plasma glucose approximately 180 mg/dl) was used to study the insulin response to a fixed glycemic stimulus in 15 trained and 12 untrained subjects. During the 40-mU.m-2.min-1 insulin infusion, the glucose disposal rate was 10.2 +/- 0.5 mg.kg fat-free mass (FFM)-1.min-1 in the trained group compared with 8.0 +/- 0.6 mg.kg FFM-1.min-1 in the untrained group (P less than 0.01). In contrast, there was no significant difference in maximally stimulated glucose disposal: 17.7 +/- 0.6 in the trained vs. 16.7 +/- 0.7 mg.kg FFM-1.min-1 in the untrained group. During the hyperglycemic clamp procedure, the incremental area for plasma insulin was lower in the trained subjects for both early (0-10 min: 140 +/- 18 vs. 223 +/- 23 microU.ml-1.min; P less than 0.005) and late (10-180 min: 4,582 +/- 689 vs. 8,895 +/- 1,316 microU.ml-1.min; P less than 0.005) insulin secretory phases. These data demonstrate that 1) the improved insulin action in healthy trained subjects is due to increased sensitivity to insulin, with no change in responsiveness to insulin, and 2) trained subjects have a smaller plasma insulin response to an identical glucose stimulus than untrained individuals.     

Effect of resistance to Bacillus thuringiensis cotton on pink bollworm (Lepidoptera: Gelechiidae) response to sex pheromone

Fitness costs associated with resistance to transgenic crops producing toxins from Bacillus thuringiensis (Bt) could reduce male response to pheromone traps. Such costs would cause underestimation of resistance frequency if monitoring was based on analysis of males caught in pheromone traps. To develop a DNA-based resistance monitoring program for pink bollworm, Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae), we compared the response to pheromone traps of males with and without cadherin alleles associated with resistance to Bt cotton (Gossypium hirsutum L.). When irradiated males from two hybrid laboratory strains with an intermediate frequency of resistance alleles were released in large field cages, the probability of capture in pheromone traps was not lower for males with resistance alleles than for males without resistance alleles. These results suggest that analysis of trapped males would not underestimate the frequency of resistance. As the time males spent in traps in the field increased from 3 to 15 d, the success of DNA amplification declined from 100 to 30%. Thus, the efficiency of a DNA-based resistance monitoring program would be improved by analyzing males remaining in traps for 3 d or less.