Structural organization of the beta-globin locus of B-haplotype sheep

Goats and some sheep synthesize a juvenile hemoglobin, Hb C (alpha 2 beta C2), at birth and produce this hemoglobin exclusively during severe anemia. Sheep that synthesize this juvenile hemoglobin are of the A haplotype. Other sheep, belonging to a separate group, the B haplotype, do not synthesize hemoglobin C and during anemia continue to produce their adult hemoglobin. To understand the basis for this difference we have determined the structural organization of the beta- globin locus of B-type sheep by constructing and isolating overlapping genomic clones. These clones have allowed us to establish the linkage map 5' epsilon I-epsilon II-psi beta I-beta B-epsilon III-epsilon IV- psi beta II-beta F3' in this haplotype. Thus, B sheep lack four genes, including the BC gene, and have only eight genes, compared with the 12 found in the goat globin locus. The goat beta-globin locus is as follows: 5' epsilon I-epsilon II-psi beta X-beta C-epsilon III-epsilon IV-psi beta Z-beta A-epsilon V-epsilon VI-psi beta Y-beta F3'. Southern blot analysis of A-type sheep reveals that these animals have a beta- globin locus similar to that of goat, i.e., 12 globin genes. Thus, the beta-globin locus of B-haplotype sheep resembles that of cows and may have retained the duplicated locus of the ancestor of cows and sheep. Alternatively, the B-sheep locus arrangement may be the result of a deletion of a four-gene set from the triplicated locus.      

Developmental toxicity of soman in rats and rabbits

Soman (GD; phosphonofluoridic acid, methyl-,1,2,2-trimethylpropyl ester) is an organophosphate compound with potent anticholinesterase activity. To determine developmental toxicity, soman was administered orally to CD rats on days 6 through 15 of gestation at dose levels of 0, 37.5, 75, 150, or 165 micrograms/kg/day and to New Zealand White (NZW) rabbits on days 6 through 19 of gestation at dose levels of 0, 2.5, 5, 10, or 15 micrograms/kg/day. At sacrifice, gravid uteri were weighed and examined for number and status of implants. Individual fetal body weights and external, visceral, and skeletal malformations were recorded. Mean maternal weight changes, fetal implantation status/litter, fetal weight, and fetal malformations/litter were compared between dose groups. Monitors for maternal toxicity were net body weight change, treatment weight change, mortality, and clinical signs of toxicity such as lethargy, ataxia, and tremors. Maternal rats and rabbits in the high-dose groups exhibited statistically significant increases in toxicity and mortality when compared to controls. There were no significant dose-related effects among dose groups in the prevalence of postimplantation loss, malformations, or in average body weight of live fetuses per litter. There was no evidence of increased prenatal mortality or fetal toxicity in the CD rat or NZW rabbit following exposure to soman, even at a dose that produced significant maternal toxicity.     

Binding and fluorescence studies of the relationship between neurophysin-peptide interaction and neurophysin self-association: an allosteric system exhibiting minimal cooperativity

The mechanism of peptide-enhanced neurophysin self-association was investigated to address questions raised by the crystal structure of a neurophysin-dipeptide complex. The dependence on protein concentration of the binding of a broad range of peptides to the principal hormone-binding site confirmed that occupancy of this site alone, and not a site that bridges the monomer-monomer interface, is the trigger for enhanced dimerization. For the binding of most peptides to the principal hormone-binding site on bovine neurophysin I, the affinity of each dimer site was at least 10 times that of monomer under the conditions used. No interactions between the two sites of the dimer were evident. Fluorescence polarization studies of pressure-induced dimer dissociation indicated that the volume change for this reaction was almost 4 times greater in the liganded than in the unliganded state, pointing to a significant alteration of the monomer-monomer interface upon peptide binding. Novel conformational changes in the vicinity of the single neurophysin tyrosine, Tyr-49, induced by pressures lower than required for subunit dissociation, were also observed. The bovine neurophysin I dimer therefore appears to represent an allosteric system in which there is thermodynamic and functional communication between each binding site and the monomer-monomer interface, but no communication across the interface to the binding site of the other subunit. A model for the peptide-enhanced dimerization is proposed in which intersubunit contacts between monomers reduce the large unfavorable free energy associated with binding-induced intrasubunit conformational change. Structural origins of the lack of communication across the interface are suggested on the basis of the low volume change associated with dimerization in the unliganded state and monomer-monomer contacts in the crystal structure. Potential roles for the peptide alpha-amino group and position 2 phenyl ring in triggering conformational change are discussed.     

Patterns of population genetic variation in sympatric chiltoniid amphipods within a calcrete aquifer reveal a dynamic subterranean environment

Calcrete aquifers from the Yilgarn region of arid central Western Australia contain an assemblage of obligate groundwater invertebrate species that are each endemic to single aquifers. Fine-scale phylogeographic and population genetic analyses of three sympatric and independently derived species of amphipod (Chiltoniidae) were carried out to determine whether there were common patterns of population genetic structure or evidence for past geographic isolation of populations within a single calcrete aquifer. Genetic diversity in amphipod mitochondrial DNA (cytochrome c oxidase subunit I gene) and allozymes were examined across a 3.5 km² region of the Sturt Meadows calcrete, which contains a grid of 115 bore holes (=wells). Stygobiont amphipods were found to have high levels of mitochondrial haplotype diversity coupled with low nucleotide diversity. Mitochondrial phylogeographic structuring was found between haplogroups for one of the chiltoniid species, which also showed population structuring for nuclear markers. Signatures of population expansion in two of the three species, match previous findings for diving beetles at the same site, indicating that the system is dynamic. We propose isolation of populations in refugia within the calcrete, followed by expansion events, as the most likely source of intraspecific genetic diversity, due to changes in water level influencing gene flow across the calcrete.     

Beverage specific alcohol intake in a population-based study: Evidence for a positive association between pulmonary function and wine intake

Background  

Lung function is a strong predictor of cardiovascular and all-cause mortality. Previous studies suggest that alcohol exposure may be linked to impaired pulmonary function through oxidant-antioxidant mechanisms. Alcohol may be an important source of oxidants; however, wine contains several antioxidants. In this study we analyzed the relation of beverage specific alcohol intake with forced expiratory volume in one second (FEV₁) and forced vital capacity (FVC) in a random sample of 1555 residents of Western New York, USA.     

DNA integrity and transgene expression after passage through the NOGA needle catheter used for therapeutic myocardial angiogenesis

BACKGROUND: The NOGA (Biosense Webster, Markham, ON, Canada) injection catheter is an innovative navigational device that provides an ideal platform for intra-myocardial injection material. However, injection through a long (1.91 m), narrow (27G) nitinol needle could result in deterioration in the integrity and functionality of DNA. METHODS: To test this possibility, DNA in plasmid form (pcDNA3.1) containing the Lac Z transgene (250 micro l) was passed through the NOGA needle using a hand-held 1 cc syringe at a gentle hand injection pressure (43 +/- 3 PSI, 3.0 +/- 0.2 kg/cm(2)) or at maximal manual pressure (90 +/- 6 PSI, 6.3 +/- 0.4 kg/cm(2)), either once or 20 times. This DNA, compared to DNA not passed through the NOGA needle (control), was then used to transfect primary cultures of rat skin fibroblasts (FB) from Fisher 344 rats and the cells were subsequently stained for beta galactosidase (betagal). RESULTS: Transfection efficiency was significantly reduced by passing the DNA through the needle at both 43 +/- 3 PSI (78 +/- 4% of control, n = 10, P < 0.05 versus control) and 90 +/- 6 PSI (66 +/- 4 % of control, n = 10, P < 0.01 versus control, P < 0.02 versus 43 +/- 3 PSI). Passage of the DNA through the NOGA needle 20 times resulted in a transfection efficiency of only 5 +/- 1% of control (n = 20, P < 0.1 x 10(-11) versus control). Capillary Electrophoresis revealed that the reduction in transfection efficiency was due to a conformational change in the DNA from predominantly supercoiled to nicked and linearized DNA. Transfection efficiency as compared with control decreased as the concentration of the DNA solution which was passed through the needle was increased from 0.3 micro g/ micro l to 2.4 micro g/ micro l. Recovery experiments confirmed that the reduction in transfection efficiency was not due to loss of DNA by binding to the NOGA needle. CONCLUSION: These results suggest that DNA is susceptible to shear forces when injected through the NOGA needle even at nominal clinical injection pressures, suggesting that careful and controlled injections will be required to achieve optimal gene integrity and expression.     

Molecular evolution of P transposable elements in the genus Drosophila. III. The melanogaster species group

Phylogenetic relationships were determined for 76 partial P-element sequences from 14 species of the melanogaster species group within the Drosophila subgenus Sophophora. These results are examined in the context of the phylogeny of the species from which the sequences were isolated. Sequences from the P-element family fall into distinct subfamilies, or clades, which are often characteristic for particular species subgroups. When examined locally among closely related species, the evolution of P elements is characterized by vertical transmission, whereby the P-element phylogeny traces the species phylogeny. On a broader scale, however, the P-element phylogeny is not congruent with the species phylogeny. One feature of P-element evolution in the melanogaster group is the presence of more than one P-element subfamily, differing by as much as 36%, in the genomes of some species. Thus, P elements from several individual species are not monophyletic, and a likely explanation for the incongruence between P-element and species phylogenies is provided by the comparison of paralogous sequences. In certain instances, horizontal transfer seems to be a valid alternative explanation for lack of congruence between species and P-element phylogenies. The canonical P-element subfamily, which represents the active, autonomous transposable element, is restricted to D. melanogaster. Thus, its origin clearly lies outside of the melanogaster species group, consistent with the earlier conclusion of recent horizontal transfer.      

Aspidosperma (Apocynaceae) plant cytotoxicity and activity towards malaria parasites. Part II: experimental studies with Aspidosperma ramiflorum in vivo and in vitro

Several species of Aspidosperma plants are used to treat diseases in the tropics, including Aspidosperma ramiflorum, which acts against leishmaniasis, an activity that is experimentally confirmed. The species, known as guatambu-yellow, yellow peroba, coffee-peroba andmatiambu, grows in the Atlantic Forest of Brazil in the South to the Southeast regions. Through a guided biofractionation of A. ramiflorum extracts, the plant activity against Plasmodium falciparum was evaluated in vitro for toxicity towards human hepatoma G2 cells, normal monkey kidney cells and nonimmortalised human monocytes isolated from peripheral blood. Six of the seven extracts tested were active at low doses (half-maximal drug inhibitory concentration < 3.8 µg/mL); the aqueous extract was inactive. Overall, the plant extracts and the purified compounds displayed low toxicity in vitro. A nonsoluble extract fraction and one purified alkaloid isositsirikine (compound 5) displayed high selectivity indexes (SI) (= 56 and 113, respectively), whereas compounds 2 and 3 were toxic (SI < 10). The structure, activity and low toxicity of isositsirikine in vitro are described here for the first time in A. ramiflorum, but only the neutral and precipitate plant fractions were tested for activity, which caused up to 53% parasitaemia inhibition of Plasmodium berghei in mice with blood-induced malaria. This plant species is likely to be useful in the further development of an antimalarial drug, but its pharmacological evaluation is still required.     

Interleukin-1, tumor necrosis factor-alpha,and transforming growth factor-beta 1 and integrative meniscal repair: influences on meniscal cell proliferation and migration

Introduction  

Interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α) are up-regulated in injured and osteoarthritic knee joints. IL-1 and TNF-α inhibit integrative meniscal repair; however, the mechanisms by which this inhibition occurs are not fully understood. Transforming growth factor-β1 (TGF-β1) increases meniscal cell proliferation and accumulation, and enhances integrative meniscal repair. An improved understanding of the mechanisms modulating meniscal cell proliferation and migration will help to improve approaches for enhancing intrinsic or tissue-engineered repair of the meniscus. The goal of this study was to examine the hypothesis that IL-1 and TNF-α suppress, while TGF-β1 enhances, cellular proliferation and migration in cell and tissue models of meniscal repair.