Mutagenesis of the N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4, 5-b]pyridine DNA adduct in mammalian cells. Sequence context effects.

Site-specifically modified oligodeoxynucleotides were used to investigate the mutagenic properties of a major cooked food mutagen-derived DNA adduct, N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4, 5-b]pyridine (dG-C8-PhIP). dG-C8-PhIP-modified oligodeoxynucleotides were prepared by reacting an oligodeoxynucleotide containing a single dG (5'-TCCTCCTXGCCTCTC, where X = C, A, G, or T) with N-acetoxy-PhIP. The unmodified and dG-C8-PhIP-modified oligomers were inserted into single-stranded phagemid vectors. These single-stranded vectors were transfected into simian kidney (COS-7) cells. The progeny plasmid obtained was used to transform Escherichia coli DH10B. When dC was at the 5'-flanking position to dG-C8-PhIP, preferential incorporation of dCMP, the correct base, was observed opposite the dG-C8-PhIP. Targeted G --> T transversions were detected, along with lesser amounts of G --> A transitions and G --> C transversions. No mutations were detected for the unmodified vector. The influence of sequence context on the dG-C8-PhIP mutation frequency and spectrum was also explored. When the dC 5'-flanking base was replaced by dT, dA, or dG, the mutational spectra were similar to that observed with dC-flanking base. Higher mutational frequencies (28-30%) were observed when dC or dG was 5' to dG-C8-PhIP. A lower mutational frequency (13%) was observed when dA was at the 5' to the lesion. Single-base deletions were detected only when dG or dT flanked the adduct. We conclude that dG-C8-PhIP is mutagenic, generating primarily G --> T transversions in mammalian cells. The mutational frequency and specificity of dG-C8-PhIP vary depending on the neighboring sequence context.     

Germination and growth responses of hybridizing Carpobrotus species (Aizoaceae) from coastal California to soil salinity

Germination, growth, and physiological responses of hybridizing Carpobrotus from coastal California to soil salinity were studied. Hybrids are presumably the result of hybridization and introgression between the exotic Carpobrotus edulis, a succulent perennial invading coastal habitats, and the native or long-naturalized C. chilensis. Germination responses were investigated at 0, 10, 20, and 50% seawater. Seedling growth and physiology were compared by irrigating seedlings with solutions of the same seawater concentrations and in low and high nutrients. Germination was inhibited in the presence of salt, but recovered after transferring the seeds to fresh water. Seeds exposed to salt had higher final germination rates than control. Growth of Carpobrotus was slightly enhanced by low seawater concentrations but reduced at high salinity at both nutrient regimes. Leaf cell sap osmolarity increased with increasing soil salinity, and taxa did not differ significantly in this physiological adjustment. Leaf carbon isotope ratios (∂¹³C) ranged from −28 to −22‰ and became less negative at higher salinities, indicating an improved water use efficiency in the seedlings at high salt concentrations. In addition, ∂¹³C values were generally less negative at high than at low nutrients. Differences among taxa were generally small. The results show that salinity affects both establishment and growth of hybridizing Carpobrotus. The overall weak species differences in salt tolerance indicate that the exotic C. edulis can occupy the same sites as C. chilensis in terms of salinity. The similarity of hybrids in their response to salinity suggests that they may contribute to the invasion by Carpobrotus.     

Study of Adenosine A2 Receptors in Membrane Preparations from Optic Tectum of Chicks

Binding properties of the subtypes of adenosine A2 receptors in membrane preparations and the effects of adenosine receptor ligands on cAMP accumulation in slices from the optic tectum of neonatal chicks have been investigated. [³H]2-[4-(2-p-carboxyethyl)phenylamino]-5'-N-ethylcarboxaminoadenosine (CGS 21680), a selective ligand for adenosine A2a receptors, did not bind to optic tectal membranes, as observed with rat striatal membranes. CGS 21680 also did not induce cyclic AMP accumulation in optic tectum slices. However, 5'-N-ethylcarboxamidoadenosine (NECA), 2-chloro-adenosine or adenosine induced a 2.5- to 3-fold increase on cyclic AMP accumulation in this preparation. [³H]NECA binds to fresh non-washed-membranes obtained from optic tectum of chicks, displaying one population of binding sites, which can be displaced by NECA, 8-phenyltheophylline, 2-chloro-adenosine, but is not affected by CGS 21680. The estimated K_D value was 400.90 ± 80.50 nM and the B_max was estimated to be 2.51 ± 0.54 pmol/mg protein. Guanine nucleotides, which modulate G-proteins activity intracellularly, are also involved in the inhibition of glutamate responses by acting extracellularly. Moreover, we have previously reported that guanine nucleotides potentiate, while glutamate inhibits, adenosine-induced cyclic AMP accumulation in slices from optic tectum of chicks. However, the guanine nucleotides, GMP or GppNHp and the metabotropic glutamate receptors agonist, 1S,3R-ACPD did not alter the [³H]NECA binding observed in fresh non-washed-membranes. Therefore, the adenosine A2 receptor found in the optic tectum must be the adenosine A2b receptor which is available only in fresh membrane preparations, and its not modulated by guanine nucleotides or glutamate analogs.     

A functional, discontinuous HIV-1 gp120 C3/C4 domain-derived, branched, synthetic peptide that binds to CD4 and inhibits MIP-1alpha chemokine binding.

This paper describes a branched synthetic peptide [3.7] that incorporates sequence discontinuous residues of HIV-1 gp120 constant regions. The approach was to bring together residues of gp120 known to interact with human cell membranes such that the peptide could fold to mimic the native molecule. The peptide incorporates elements of both the conserved CD4 and CCR5 binding sites. The 3.7 peptide, which cannot be produced by conventional genetic engineering methods, is recognized by antiserum raised to native gp120. The peptide also binds to CD4 and competitively inhibits binding of QS4120 an antibody directed against the CDR2 region of CD4. When preincubated with the CD4+ve MM6 macrophage cell line, which expresses mRNA for the CCR3 and CCR5 chemokine receptors, both 3.7 and gp120 inhibit binding of the chemokine MIP-1alpha. The peptide also inhibits infection of primary macrophages by M-tropic HIV-1. Thus, 3.7 is a prototype candidate peptide for a vaccine against HIV-1 and represents a novel approach to the rational design of peptides that can mimic complex sequence discontinuous ligand binding sites of clinically relevant proteins.     

Seasonal variation of behavior and brain size in a freshwater fish

1. Teleost fishes occupy a range of ecosystem, and habitat types subject to large seasonal fluctuations. Temperate fishes, in particular, survive large seasonal shifts in temperature, light availability, and access to certain habitats. Mobile species such as lake trout (Salvelinus namaycush) can behaviorally respond to seasonal variation by shifting their habitat deeper and further offshore in response to warmer surface water temperatures during the summer. During cooler seasons, the use of more structurally complex nearshore zones by lake trout could increase cognitive demands and potentially result in a larger relative brain size during those periods. Yet, there is limited understanding of how such behavioral responses to a seasonally shifting environment might shape, or be shaped by, the nervous system.
2. Here, we quantified variation in relative brain size and the size of five externally visible brain regions in lake trout, across six consecutive seasons in two different lakes. Acoustic telemetry data from one of our study lakes were collected during the study period from a different subset of individuals and used to infer relationships between brain size and seasonal behaviors (habitat use and movement rate).
3. Our results indicated that lake trout relative brain size was larger in the fall and winter compared with the spring and summer in both lakes. Larger brains coincided with increased use of nearshore habitats and increased horizontal movement rates in the fall and winter based on acoustic telemetry. The telencephalon followed the same pattern as whole brain size, while the other brain regions (cerebellum, optic tectum, olfactory bulbs, and hypothalamus) were only smaller in the spring.
4. These findings provide evidence that flexibility in brain size could underpin shifts in behavior, which could potentially subserve functions associated with differential habitat use during cold and warm seasons and allow fish to succeed in seasonally variable environments.

     

Docosahexaenoic acid in diluent for goat semen cryopreservation

The effects of docosahexaenoic acid (DHA) in the diluent for cryopreservation of goat semen on seminal quality and the optimal levels to be used were evaluated. After collection, semen was pooled and physically evaluated, then divided into four aliquots with different DHA levels in the diluent: 0, 10, 20, and 30 ng mL^-1. The semen was cryopreserved in a TK 3000® freezing machine and then thawed for assessment at 37 °C. Sperm motility and vigor, membrane integrity, acrosomal integrity, mitochondrial activity, and sperm chromatin compaction were evaluated after thawing. A completely randomized design was used. For normally distributed variables, ANOVA and regression analysis were used to test for differences between treatments, and for non-parametric data, the Kruskal Wallis test was used at the 5% significance level. There were no differences among groups in terms of membrane integrity, acrosomal integrity, or chromatin compaction. There was a decrease in class I mitochondrial activity with increasing DHA level (P<0.05), but no differences in classes II, III, and IV (P>0.05). The inclusion of 10 to 30 ng mL^-1 of DHA in the diluent did not result in improvements in seminal quality parameters after thawing, with some impairment observed in the mitochondrial activity of the sperm cells.