Unfolding of trp repressor studied using fluorescence spectroscopic techniques.

The unfolding properties of the trp repressor of Escherichia coli have been studied using a number of different time-resolved and steady-state fluorescence approaches. Denaturation by urea was monitored by the average fluorescence emission energy of the intrinsic tryptophan residues of the repressor. These data were consistent with a two-state transition from dimer to unfolded monomer with a free energy of unfolding of 19.2 kcal/mol. The frequency response profiles of the fluorescence emission brought to light subtle urea-induced modifications of the intrinsic tryptophan decay parameters both preceding and following the main unfolding transition. The increase of lifetime induced by urea required higher concentrations of urea than the increase in the total intensity described by Gittelman and Matthews [(1990) Biochemistry 29, 7011]. This indicates that the intensity increase has both dynamic and static origins. To assess the effect of tryptophan binding upon repressor stability, and to determine whether repressor oligomerization would be detectable in an unfolding experiment, we examined denaturation profiles of repressor labeled with the long-lived fluorescence probe 5-(dimethylamino)naphthalene-1-sulfonyl (DNS), by monitoring the average rotational correlation time of the probe. These experiments revealed a protein concentration dependent transition at low urea concentrations. This transition was promoted by tryptophan binding. We ascribe this transition to urea-induced dissociation of repressor tetramers. The main unfolding transition of the dimer to unfolded monomer was also observable using this technique, and the free energies associated with this transition were 18.3 kcal/mol in the absence of tryptophan and 24.1 kcal/mol in its presence, demonstrating that co-repressor binding stabilizes the repressor dimer against denaturation.(ABSTRACT TRUNCATED AT 250 WORDS)     

A new species of Lernanthropus De Blainville, 1822 (Copepoda: Lernanthropidae) parasitic on Menticirrhus ophicephalus (Jenyns) (Teleostei: Sciaenidae) from the Peruvian coast

Lernanthropus huamani n. sp. (Copepoda: Lernanthropidae), a parasite of the Peruvian sciaenid fish Menticirrhus ophicephalus (Jenyns), is described and illustrated. The new species differs from all other species of Lernanthropus by a combination of characters, including the dorsal plate, legs and other appendages. L. guacoldae Villalba & Fernández, 1984 is considered a synonym of L. pacificus Oliva & Duran, 1982.     

Neuromuscular development following tetrodotoxin-induced inactivity in mouse embryos

Developmental aspects of the neuromuscular system in mouse embryos chronically paralyzed in utero with tetrodotoxin (TTX) between embryonic days 14 and 18 were studied using biochemical and histological methods. The number of lumbar spinal motoneurons (MNs) was higher in inactive embryos than in controls suggesting a decreased motoneuron cell death. In association with the increase in MN number, choline acetyltransferase activity was significantly increased in both spinal cord and peripheral synaptic sites. Paralyzed muscles exhibited a decreased number of mature myofibers and the nuclei were centrally located. Creatine kinase activity was greatly decreased and total acetylcholine receptor and receptor cluster numbers per myofiber were significantly increased in paralyzed muscles. A similar pattern of changes occurs in the neuromuscular system of the mutant mouse muscular dysgenesis (mdg). However, in contrast to the mdg mutant, tetrodotoxin-treated muscles were similar to controls in their innervation pattern, in the ultrastructural aspects of the excitation–contraction coupling system (i.e., dyads and triads) and in the extent of dihydropyridine binding. Thus, neuromuscular inactivity is not sufficient to impair the pattern of muscle innervation or the appearance of either the triadic junctions or dihydropyridine receptors. These results indicate that alterations of dihydropyridine binding sites and triads in muscular dysgenesis cannot be accounted for by inactivity but rather must reflect a more primary defect involving the structural gene(s) regulating the development of one or more aspects of muscle differentiation.     

Involvement of the Dihydropyridine Receptor and Internal CaStores in Myoblast Fusion

The process of myoblast fusion during skeletal myogenesis is calcium regulated. Both dihydropyridine receptor and ryanodine receptor are already present on muscle precursors, at the prefusional stage, before they are required for excitation–contraction coupling. Previous pharmacological studies have shown the need for a special pool of Ca²⁺associated with the membrane for the fusion process to occur. We hypothesized that this pool of Ca²⁺is mobilized via a machinery similar to that involved in excitation–contraction coupling. The process of fusion in rat L6 muscle precursors was either totally or partially abolished in the presence of the L-type calcium channel inhibitors SR33557 and nifedipine (half inhibition towards 2 μM), respectively. The inhibition was reversible and dose-dependent. Drugs able to deplete internal calcium stores (caffeine, ryanodine, and thapsigargin) were also tested on the fusion. Both caffeine and thapsigargin drastically inhibited fusion whereas ryanodine had no effect. This suggests that fusion may be controlled by internal pools of Ca²⁺but that its regulation may be insensitive to ryanodine. We presumed that an early form of the ryanodine receptor may exist, with different pharmacological properties than the adult forms. Indeed, Western blot analysis of pre- and postfusional L6 cells demonstrated the presence, at the prefusional stage, of a transient form of the ryanodine receptor protein with an apparent molecular weight slightly different from those of the classical skeletal and cardiac forms. Taken together, these results support the hypothesis that the fusion process is driven by a mechanism involving both the dihydropyridine receptor (α1 subunit of the L-type Ca²⁺channel) and the internal stores of Ca²⁺. The machinery underlying this mechanism might consist of slightly different forms of the classic molecules that in adult muscle ensure excitation–contraction coupling. It remains to be seen, however, whether the mobilization of the internal pool of Ca²⁺is triggered by the type of mechanism already described in skeletal muscle.     

Analysis of inbreeding in a colonizing population of Drosophila subobscura

An analysis of the effects of inbreeding on the genetic structure of a colonizing population of Drosophila subobscura has been carried out. Species of Drosophila, particularly D. subobscura, may have lethal alleles associated with chromosomal inversions and our aim was to assess the extent to which the genome is balanced in this way. The frequencies of chromosomal inversions were compared between a large population and a set of 72 lines that were maintained by brother-sister mating for 10 generations. Fisher's matrix method was used to calculate the expected homozygosity in these inbred lines for 5 allozyme loci (Aph, Hk-1, Lap, Odh, and Pept-1) used as markers of large chromosomal segments. Furthermore, the expected rates of fixation corresponding to these allozyme loci were also calculated. The results show that the amount of homozygosis observed did not differ significantly from expectations (with the corresponding loss of lines as a consequence of the reduction in viability). However, two deviations from strict neutrality were observed: there was a heterozygote excess at the Lap locus, and the frequency of the O ₅ inversion (always associated with a lethal gene in colonizing populations) was higher than expected.     

Trinuclear iridium and rhodium complexes: the solution to a puzzle involving the multiple coordination possibilities of 1,8-naphthyridine-2-one ligands

The multiple coordination possibilities of 1,8-naphthyridine-2-one (HOnapy) and 5,7-dimethyl-1,8-napthyridine-2-one (HOMe₂napy) ligands allow the synthesis of a variety of tri- di- and mononuclear complexes, showing fluxional behaviour and frequent exchange of the coordinated ML₂ fragments. Thus, reactions of [M₂(μ-OMe)₂(cod)₂] (cod = 1,5-cyclooctadiene) with HOnapy and HOMe₂napy yield the compounds of the general formula [M(μ-OR₂napy) (cod)]_n (M = Ir, R = Me (1a, 1b, H (2); M = Rh, R = Me (3a, 3b). They crystallise as inconvertible yellow (a) and purple/orange (b) forms and also show a puzzling behaviour in solution. X-ray diffraction studies on both forms (3a, 3b) and spectroscopic data reveal that the yellow forms are mononuclear complexes whilst the dark-coloured crystals contain dinuclear complexes. In solution, the nuclearity of the complexes depends on the solvent. In addition both types of complexes are fluxional. The mixed-ligand complexes [M₂(μ-OMe₂napy)₂(CO)₂(cod)] M = Ir (5), Rh (6) have been isolated and characterised; they are found to be intermediates in the synthesis of the trinuclear complexes [M₃(μ₃-OMe₂napy)₂(CO)₂(cod)₂]⁺ M = Rh (8), Ir (9). Reactions of [IrCl(CO)₂(NH₂-p-tolyl] with the complexes [Rh(μ-OR₂napy)(diolefin)]_n followed by addition of a poor donor anion is a general one-pot synthesis for the hetertrinuclear complexes [Rh₂Ir(μ₃-OR₂napy)₂(CO)₂(diolefin)₂]⁺ (R=Me, DIOLEFIN = cod (10), tetrafluorobenzo-barrelene (tfbb) (11), 2,5-norbornadiene (nbd) (12); R=H, DIOLEFIN=cod (13)). This synthesis follows a stepwise mechanism from the mononuclear to the trinuclear complexes in which mixed-ligand heterodinuclear complexes are involved as intermediates of the type [(diolefin)Rh(μ-OMe₂napy)₂Ir(CO)₂]. Heteronuclear complexes which possess the core [RhIr₂]³⁺, such as [RhIr₂(μ₃-OR₂napy)₂(CO)₂(cod)₂]BF₄ (R=Me (14), H (15)), result from the reaction of 1 or 2 with [Rh(CO)₂S_x]⁺ (S = solvent). The trinuclear complexes undergo two chemically reversible one-electron oxidation processes. The chemical oxidation of 10, 14 and 9 with silver salts gives the mixed-valence trinuclear radicals [Rh₂Ir(μ₃-OMe₂napy)₂(CO)₂(cod)₂]²⁺ (16), [RhIr₂(μ₃-OMe₂napy)₂(CO)₂(cod)₂]²⁺ (17) and [Ir₃(μ₃-OMe₂napy)₂(CO)₂(cod)₂]²⁺ (18), which have been isolated as the perchlorate and tetrafluoroborate salts. The EPR spectrum of 16 indicates that the unpaired electron is essentially in an orbital delocalised on the metals. The molecular structures of the complexes 3a, 3b, 6, 10b and 16a are described. Crystals of 3a are triclinic, P-1, with a = 9.7393(2), b = 14.0148(4), c = 16.0607(4) Å, α = 88.122(3), β = 83.924(3), γ = 87.038(3)°, Z = 4; 3b crystallises in the Pna2_i orthorhhombic space group, with a = 16.7541(3), B = 11.7500(8), c = 17.7508(7) Å, Z = 4; complex 6 is packed in the monoclinic space group P2_i/c, a = 9.6371(1), b = 11.8054(4), c = 27.2010(9) Å, β = 90.556(4)°, Z = 4; crystals of 10b are monoclinic, P2₁/n, with a = 17.546(7), b = 13.232(6), c = 17.437(8) Å, β = 106.18(1)°, Z = 4; crystals of 16a are triclinic, P-1, with a = 10.318(4), b = 12.562(6), C = 19.308(8) Å, α = 92.12(8), β = 97.65(9), γ = 90.68(5)°, Z = 2. The five different structures show the coordination versatility of the OMe₂napy molecule as ligand, which behaves as a N,N′-chelating (3a), bidentate N,O-donor (3b, 6), or as a tridentate N,N′,O-donor bridging ligand (10b, 16a).     

Effect of distance,aggregation, and habitat on levels of seed predation for two mammal — dispersed neotropical rain forest tree species

The effect of seed aggregation and distance from conspecific trees on seed predation was experimentally examined for two neotropical tree species, Macoubea guianensis (Apocynaceae) and Pouteria sp. (Sapotaceae) in a lowland tropical rain forest in northeastern Peru. Results of these experiments are discussed in the context of the Janzen-Connell model (Janzen 1970; Connell 1971), which predicts decreased seed survival near parent trees due to either density-or distance-responsive mortality, and Howe's model (Howe 1989) which predicts that trees with seeds dispersed in clumps (aggregated) will not suffer density-dependent predation, and will have higher survival of seeds near the parent tree than other trees. We also examined whether predation on seeds of these species was affected by seed placement in or near 30-m-wide strips regenerating after clear-cutting. Both species appeared to be mammal-dispersed but differed in how frugivores handled seeds, seed size, overall fruit crop size, and gemination time. Neither of the two species studied appeared to suffer seed predation in a manner predicted by the Janzen-Connell model, and patterns of seed predation for only one of the species was similar to predictions of Howe's model. For neither species did seed predation along the edge of, or in the center of, regenerating clear cuts differ from predation 15 m into the primary forest. For Pouteria, seed predation in and near regnerating strips was significantly greater than around forest trees, but the opposite pattern held for Macoubea. Overall, seed predation was much greater on Macoubea. The difference in seed predation for these two species was most likely a result of differences in the types of seed predators that attacked these two species.     

Xp-duplications with and without sex reversal

Duplications in Xp including the DSS (dosage sensitive sex reversal) region cause male to female sex reversal. We investigated two patients from families with Xp duplications. The first case was one of two sisters with karyotype 46,XY, der(22), t(X;22)(p11.3;p11)mat and unambiguous female genitalia. The living sister was developmentally retarded, and showed multiple dysmorphic features and an acrocallosal syndrome. The second case was a boy with a maternally inherited direct duplication of Xp21.3-pter with the breakpoint close to the DSS locus. He had multiple abnormalities and micropenis, but otherwise unambiguous male genitalia. We performed quantitative Southern blot analysis with probes from Xp22.13 to p21.2 to define the duplicated region. Clinical, cytogenetic, and molecular data from both patients were compared with those of previously reported related cases. A comparison of the extragenital symptoms revealed no differences between patients with or without sex reversal. In both cases, the symptoms were non-specific. Among 22 patients with a duplication in Xp, nine had unambiguous female genitalia and a well-documented duplication of the DSS region. Two patients with duplication of DSS showed ambiguous external genitalia. From these data, we conclude that induction of testicular tissue may start in these patients, but that the type of genitalia depends on the degree of subsequent degeneration by a gene in DSS.     

Effect of abscisic acid, osmolarity and partial desiccation on the development of recalcitrant mango somatic embryos

Inhibition of mango somatic embryo growth was inducedin vitro by treatments for 4 or more weeks with abscisic acid (0–100 M ABA) with and without high osmolarity provided by mannitol (0–10%). High osmolarity and ABA significantly affected somatic embryo length, precocious germination and the production of good quality secondary somatic embryos. High osmolarity also affected root elongation. Abscisic acid was more effective in suppressing growth and development of 0.5 cm-length somatic embryos than smaller somatic embryos. Development beyond the heart stage was significantly inhibited by both ABA and mannitol treatments. The recovery of good quality somatic embryos was enhanced by high levels of ABA (100 M) with and without mannitol (0–5%). Somatic embryos that had been pulsed with ABA were partially desiccated at different relative humidities. Weight loss was affected only by relative humidity; and ABA did not enhance desiccation tolerance.Abbreviations ABA Abscisic acid - 2,4-D 2,4-Dichlorophenoxyacetic acid - MM1 Mango maturation medium - RH Relative humidity