Acrylonitrile inhalation in rats: II. Excretion of thioethers and thiocyanate in urine

In male Wistar rats, the inhalation exposure to acrylonitrile (AN), 271 mg X m-3, 8 hours a day, 5 days a week, did not affect protein sulfhydryl concentration in liver and blood and decreased glutathione concentration in the liver, but not in the brain at the end of the fifth exposure. The urinary excretion of the main AN metabolites, thioethers (AN-mercapturic acids) and thiocyanate was proportional to the inhaled AN concentration (57, 125, 271 mg X m-3, respectively) in a single exposure for 12 hours, and their mutual ratio was greatly different from that after injection of AN. The results revealed that the urinary excretion of thioethers is a very sensitive and dose-related indicator of exposure to AN and extrapolation of the results indicates that the exposure to AN concentration below 10 mg X m-3 could thus be demonstrated.     

Thermal melting and circular dichroism of DNA complexes with (Lys-Ala-Ala)10 and (Lys-Ala-Ala)n: effect of polypeptide chain length

DNA complexes with polypeptides (Lys-Ala-Ala)₁)] and (Lys-Ala-Ala)₃₄ have been studied using the methods of thermal melting and circular dichroism. Derivative melting curves of (Lys-Ala-Ala)₁₀ DNA differed substantially from those of (Lys-Ala-Ala)₃₄ prepared either by salt gradient dialysis or by direct mixing. Melting curves of the former complex were unimodal or bimodal with T_m increasing continuously withn input lysin-to-DNA phosphate ratio (r); those of the latter complex consisted of three separate transitions with T_m values almost independent of r. Complete reversibility of binding in the (Lys-Ala-Ala)₁₀-DNA system but a slow redistribution of (Lys-Ala-Ala)₃₄ on DNA at low temperature were found in the redistribution experiments Much faster redistribution from denatured to native DNA occurs at the temperature of melting, contributing to the unusual trimodal melting pattern. Circular dichroism curves are very similar for both complexes and indicate little change of DNA conformation upon polypeptide binding.     

Amplification of a Cryptosporidium parvum gene fragment encoding thymidylate synthase.

Currently, there is no effective therapy for cryptosporidiosis and it is unclear why antifolate drugs which are effective treatments for infections caused by closely related parasites are not also effective against Cryptosporidium parvum. In protozoa, the target of these drugs, dihydrofolate reductase (DHFR), exists as a bifunctional enzyme also manifesting thymidylate synthase (TS) activity and is encoded by a fused DHFR-TS gene. In order to prepare a probe to isolate the C. parvum DHFR-TS gene we have used degenerate oligonucleotides whose sequences are based on strongly conserved regions of TS protein sequence to prime the polymerase chain reaction (PCR) with C. parvum DNA. The PCR amplified a 375-bp DNA fragment which was cloned and sequenced; the deduced amino acid sequence had significant identity with known TS sequences, including strict conservation of all phylogenetically invariant TS amino acid residues. The cloned PCR fragment was used as a probe to isolate a number of overlapping clones from a C. parvum genomic library which were definitively shown to be of cryptosporidial origin by genomic Southern and molecular karyotype analyses. The deduced protein sequence of C. parvum TS was most similar to the bifunctional TS enzymes of Plasmodium chabaudi and Plasmodium falciparum.     

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.      

A chromosome-level genome assembly enables the identification of the follicule stimulating hormone receptor as the master sex-determining gene in the flatfish Solea senegalensis

Sex determination (SD) shows huge variation among fish and a high evolutionary rate, as illustrated by the Pleuronectiformes (flatfishes). This order is characterized by its adaptation to demersal life, compact genomes and diversity of SD mechanisms. Here, we assembled the Solea senegalensis genome, a flatfish of great commercial value, into 82 contigs (614 Mb) combining long- and short-read sequencing, which were next scaffolded using a highly dense genetic map (28,838 markers, 21 linkage groups), representing 98.9% of the assembly. Further, we established the correspondence between the assembly and the 21 chromosomes by using BAC-FISH. Whole genome resequencing of six males and six females enabled the identification of 41 single nucleotide polymorphism variants in the follicle stimulating hormone receptor (fshr) consistent with an XX/XY SD system. The observed sex association was validated in a broader independent sample, providing a novel molecular sexing tool. The fshr gene displayed differential expression between male and female gonads from 86 days post-fertilization, when the gonad is still an undifferentiated primordium, concomitant with the activation of amh and cyp19a1a, testis and ovary marker genes, respectively, in males and females. The Y-linked fshr allele, which included 24 nonsynonymous variants and showed a highly divergent 3D protein structure, was overexpressed in males compared to the X-linked allele at all stages of gonadal differentiation. We hypothesize a mechanism hampering the action of the follicle stimulating hormone driving the undifferentiated gonad toward testis.     

Field observations quantifying attraction of four tortricid moths to high-dosage pheromone dispensers in untreated and pheromone-treated orchards

Orientational responses of four species of feral tortricid moths (Lepidoptera: Tortricidae) to polyethylene tube dispensers of pheromone were observed in a 0.8 ha apple orchard treated with such pheromone dispensers and in an untreated 0.8 ha orchard. Male oblique‐banded leafrollers, Choristoneura rosaceana (Walker) (mean 7.2 ± 0.4 moths/night during 21 nights), Oriental fruit moths, Grapholita molesta (Busck) (mean 10.5 ± 2.1 during 20 evenings), and the redbanded leafrollers, Argyrotaenia velutinana (Walker) (mean 2.0 ± 1.1 during 14 nights) were observed approaching within 100 cm of their respective polyethylene‐tube pheromone dispensers in the untreated orchard. Furthermore, C. rosaceana (mean 2.0 ± 0.7 during 17 nights) and G. molesta (mean 1.5 ± 0.4 over 20 evenings) came within 100 cm of their respective polyethylene‐tube pheromone dispensers in the pheromone‐treated orchard. Most visits lasted less than 10 s, after which the majority of moths departed by flying upwind. In the untreated orchard, the number of C. rosaceana observed orienting to polyethylene tube dispensers was greater than the number captured in optimized monitoring traps (1.9 ± 0.4) per night of observation. The numbers of A. velutinana (2.0 ± 1.1) or G. molesta (10.5 ± 2.1) attracted to polyethylene‐tube dispensers in the untreated orchard did not differ statistically from the numbers captured in optimized monitoring traps per night of observation. In the pheromone‐treated orchard, the number of C. rosaceana (2.0 ± 0.4) or G. molesta (1.2 ± 0.2) observed orienting to polyethylene‐tube dispensers did not differ statistically from the numbers of male moths of these species captured in optimized monitoring traps per night of observation. No codling moths, Cydia pomonella L. were observed orienting to, or landing near, their respective polyethylene‐tube dispensers in either the untreated or pheromone‐treated orchards, although substantial numbers were captured in monitoring traps per night of observation (6.0 ± 1.7) in the untreated orchard. The attraction of male moths to polyethylene tube dispensers thus occurred in three of the four species observed. These results provide support for the idea that false‐plume‐following is an important component of the mechanisms mediating communicational disruption in moths by polyethylene‐tube dispensers.     

Rotation of cytochrome P-450. II. Specific interactions of cytochrome P-450 with NADPH-cytochrome P-450 reductase in phospholipid vesicles

Purified rat liver microsomal cytochrome P-450 and NADPH-cytochrome P-450 reductase were co-reconstituted in phosphatidylcholine-phosphatidylethanolamine-phosphatidylserine vesicles using a cholate dialysis technique. The co-reconstitution of the enzymes was demonstrated in proteoliposomes fractionated by centrifugation in a glycerol gradient. The proteoliposomes catalyzed the N-demethylation of a variety of substrates. Rotational diffusion of cytochrome P-450 was measured by detecting the decay of absorption anisotropy r(t), after photolysis of the heme.CO complex by a vertically polarized laser flash. The rotational mobility of cytochrome P-450, when reconstituted alone, was found to be dependent on the lipid to protein ratio by weight (L/P450) (Kawato, S., Gut, J., Cherry, R. J., Winterhalter, K. H., and Richter, C. (1982) J. Biol. Chem. 257, 7023-7029). About 35% of cytochrome P-450 was immobilized and the rest was rotating with a mean rotational relaxation time phi 1 of about 95 mus in L/P450 = 1 vesicle. In L/P450 = 10 vesicles, about 10% of P-450 was immobile and the rest was rotating with phi 1 congruent to 55 mus. Co-reconstitution of equimolar amounts of NADPH-cytochrome P-450 reductase into the above vesicles results in completely mobile cytochrome P-450 with a phi 1 congruent to 40 mus. Only a small decrease in the immobile fraction of cytochrome P-450 is observed when the molar ratio of cytochrome P-450 to the reductase is 5. The results suggest the formation of a monomolecular 1:1 complex between cytochrome P-450 and NADPH-cytochrome P-450 reductase in the liposomes.