Isolation and characterization of the cDNA for pulmonary surfactant-associated protein-B (SP-B) in the rabbit

Pulmonary surfactant contains phospholipids including dipalmitoyl-phosphatidylcholine and three surfactant-associated proteins designated SP-A, SP-B and SP-C. A cDNA for rabbit SP-B has been isolated from a fetal (30 days gestation) rabbit lung cDNA library constructed in lambda gt11. The cDNA and deduced amino acid sequences show strong homology with the cDNAs and predicted 40 kDa proproteins for human and canine SP-B. Strong homology is also observed with the amino acid sequences directly determined for the mature 8 kDa bovine and porcine SP-B isolated from lung lavage. SP-B is remarkable for its high cysteine and proline content and for the hydrophobic nature of the organic solvent-soluble, mature protein. In vitro translation of sense but not antisense RNA transcribed from the cDNA led to the production of 40 kDa and 32 kDa proteins. These proteins were immunoprecipitated by an antibody raised against bovine SP-B. Northern blot analysis revealed the mRNA for rabbit SP-B appears in fetal rabbit lung late in gestation and falls slightly in the neonate.     

Proteomic signatures of in vivo muscle oxidative capacity in healthy adults

Adequate support of energy for biological activities and during fluctuation of energetic demand is crucial for healthy aging; however, mechanisms for energy decline as well as compensatory mechanisms that counteract such decline remain unclear. We conducted a discovery proteomic study of skeletal muscle in 57 healthy adults (22 women and 35 men; aged 23–87 years) to identify proteins overrepresented and underrepresented with better muscle oxidative capacity, a robust measure of in vivo mitochondrial function, independent of age, sex, and physical activity. Muscle oxidative capacity was assessed by ³¹P magnetic resonance spectroscopy postexercise phosphocreatine (PCr) recovery time (τ_PCr) in the vastus lateralis muscle, with smaller τ_PCr values reflecting better oxidative capacity. Of the 4,300 proteins quantified by LC‐MS in muscle biopsies, 253 were significantly overrepresented with better muscle oxidative capacity. Enrichment analysis revealed three major protein clusters: (a) proteins involved in key energetic mitochondrial functions especially complex I of the electron transport chain, tricarboxylic acid (TCA) cycle, fatty acid oxidation, and mitochondrial ABC transporters; (b) spliceosome proteins that regulate mRNA alternative splicing machinery, and (c) proteins involved in translation within mitochondria. Our findings suggest that alternative splicing and mechanisms that modulate mitochondrial protein synthesis are central features of the molecular mechanisms aimed at maintaining mitochondrial function in the face of impairment. Whether these mechanisms are compensatory attempt to counteract the effect of aging on mitochondrial function should be further tested in longitudinal studies.     

Ancestral and neo-sex chromosomes contribute to population divergence in a dioecious plant

Empirical evidence from several animal groups suggests sex chromosomes disproportionately contribute to reproductive isolation. This effect may be enhanced when sex chromosomes are associated with turnover of sex determination systems resulting from structural rearrangements to the chromosomes. We investigated these predictions in the dioecious plant Rumex hastatulus, which is composed of populations of two different sex chromosome cytotypes caused by an X-autosome fusion. Using population genomic analyses, we investigated the demographic history of R. hastatulus and explored the contributions of ancestral and neo-sex chromosomes to population genetic divergence. Our study revealed that the cytotypes represent genetically divergent populations with evidence for historical but not contemporary gene flow between them. In agreement with classical predictions, we found that the ancestral X chromosome was disproportionately divergent compared with the rest of the genome. Excess differentiation was also observed on the Y chromosome, even when we used measures of differentiation that control for differences in effective population size. Our estimates of the timing of the origin of neo-sex chromosomes in R. hastatulus are coincident with cessation of gene flow, suggesting that the chromosomal fusion event that gave rise to the origin of the XYY cytotype may have also contributed to reproductive isolation.     

Effects of double ligation of Stensen's duct on the rabbit parotid gland

Salivary gland duct ligation is an alternative to gland excision for treating sialorrhea or reducing salivary gland size prior to tumor excision. Duct ligation also is used as an approach to study salivary gland aging, regeneration, radiotherapy, sialolithiasis and sialadenitis. Reports conflict about the contribution of each salivary cell population to gland size reduction after ductal ligation. Certain cell populations, especially acini, reportedly undergo atrophy, apoptosis and proliferation during reduction of gland size. Acini also have been reported to de-differentiate into ducts. These contradictory results have been attributed to different animal or salivary gland models, or to methods of ligation. We report here a bilateral double ligature technique for rabbit parotid glands with histologic observations at 1, 7, 14, 30, 60 days after ligation. A large battery of special stains and immunohistochemical procedures was employed to define the cell populations. Four stages with overlapping features were observed that led to progressive shutdown of gland activities: 1) marked atrophy of the acinar cells occurred by 14 days, 2) response to and removal of the secretory material trapped in the acinar and ductal lumens mainly between 30 and 60 days, 3) reduction in the number of parenchymal (mostly acinar) cells by apoptosis that occurred mainly between 14–30 days, and 4) maintenance of steady-state at 60 days with a low rate of fluid, protein, and glycoprotein secretion, which greatly decreased the number of leukocytes engaged in the removal of the luminal contents. The main post- ligation characteristics were dilation of ductal and acinar lumens, massive transient infiltration of mostly heterophils (rabbit polymorphonuclear leukocytes), acinar atrophy, and apoptosis of both acinar and ductal cells. Proliferation was uncommon except in the larger ducts. By 30 days, the distribution of myoepithelial cells had spread from exclusively investing the intercalated ducts pre-ligation to surrounding a majority of the residual duct-like structures, many of which clearly were atrophic acini. Thus, both atrophy and apoptosis made major contributions to the post-ligation reduction in gland size. Structures also occurred with both ductal and acinar markers that suggested acini differentiating into ducts. Overall, the reaction to duct ligation proceeded at a considerably slower pace in the rabbit parotid glands than has been reported for the salivary glands of the rat.     

The development of enantiostyly

Enantiostyly, the deflection of the style either to the left (left-styled) or right (right-styled) side of the floral axis, has evolved in at least ten angiosperm families. Two types of enantiostyly occur: monomorphic enantiostyly, in which individuals exhibit both stylar orientations, and dimorphic enantiostyly, in which the two stylar orientations occur on separate plants. To evaluate architectural or developmental constraints on the evolution of both forms of enantiostyly, we examined inflorescence structure and floral development among unrelated enantiostylous species. We investigated relations between the position of left- and right-styled flowers and inflorescence architecture in four monomorphic enantiostylous species, and we examined the development of enantiostyly in nine monomorphic and dimorphic enantiostylous species from five unrelated lineages. The location of left- and right-styled flowers within inflorescences ranged from highly predictable (in Solanum rostratum) to random (in Heteranthera mexicana). There were striking differences among taxa in the timing of stylar bending. In Wachendorfia paniculata, Dilatris corymbosa, and Philydrum lanuginosum, the style deflected in the bud, whereas in Heteranthera spp., Monochoria australasica, Cyanella lutea, and Solanum rostratum, stylar bending occurred at the beginning of anthesis. Comparisons of organ initiation and development indicated that asymmetries along the left-right axis were expressed very late in development, despite the early initiation of a dorsiventral asymmetry. We suggest that the evolution of dimorphic enantiostyly from monomorphic enantiostyly may be constrained by a lack of left-right positional information in the bud.     

Production and utilization of acetate in mammals

1. In an attempt to define the importance of acetate as a metabolic precursor, the activities of acetyl-CoA synthetase (EC 6.2.1.1) and acetyl-CoA hydrolase (Ec 3.1.2.1) were assayed in tissues from rats and sheep. In addition, the concentrations of acetate in blood and liver were measured, as well as the rates of acetate production by tissue slices and mitochondrial fractions of these tissues. 2. Acetyl-CoA synthetase occurs at high activities in heart and kidney cortex of both species as well as in rat liver and the sheep masseter muscle. The enzyme is mostly in the cytosol fraction of liver, whereas it is associated with the mitochondrial fraction in heart tissue. Both mitochondrial and cytosol activities have a K(m) for acetate of 0.3mm. Acetyl-CoA synthetase activity in liver was not altered by changes in diet, age or alloxan-diabetes. 3. Acetyl-CoA hydrolase is widely distributed in rat and sheep tissues, the highest activity being found in liver. Essentially all of the activity in liver and heart is localized in the mitochondrial fraction. Hepatic acetyl-CoA hydrolase activity is increased by starvation in rats and sheep and during the suckling period in young rats. 4. The concentrations of acetate in blood are decreased by starvation and increased by alloxan-diabetes in both species. The uptake of acetate by the sheep hind limb is proportional to the arterial concentration of acetate, except in alloxan-treated animals, where uptake is impaired. 5. Acetate is produced by liver and heart slices and also by heart mitochondrial fractions that are incubated with either pyruvate or palmitoyl-(-)-carnitine. Liver mitochondrial fractions do not form acetate from either substrate but instead convert acetate into acetoacetate. 6. We propose that acetate in the blood of rats or starved sheep is derived from the hydrolysis of acetyl-CoA. Release of acetate from tissues would occur under conditions when the function of the tricarboxylic acid cycle is restricted, so that the circulating acetate serves to redistribute oxidizable substrate throughout the body. This function is analogous to that served by ketone bodies.     

Mechanism of choline O-sulphate utilization in fungi

1. The position of the enzyme blocks in a number of parathiotrophic mutants of Aspergillus nidulans A 69 and mutants A and C of a biotinless mutant of Aspergillus nidulans were examined by nutritional and heterokaryosis experiments and by assay in vitro of enzyme systems and specific enzymes. 2. The mutants were in five groups: A and C blocked at sulphate transport; gamma at ATP sulphurylase; iota at adenosine 5′-sulphatophosphate kinase; eta at the adenosine 3′-phosphate 5′-sulphatophosphate reductase system; alpha, beta and zeta between sulphite and thiosulphate. 3. The ability of the various mutants to synthesize choline O-sulphate in vivo and in vitro and to utilize choline O-sulphate as a source of sulphur indicated that the utilization of endogenously formed choline O-sulphate involved the splitting off of inorganic sulphate, which was then reduced. 4. Choline O-sulphate acted as a source of choline for cholineless strains of Neurospora crassa, suggesting that choline O-sulphate breakdown occurred by simple hydrolysis involving a choline sulphatase. 5. After de-repression of mycelia by growing for a period on a sulphur-free medium the presence of choline sulphatase in physiologically significant amounts was demonstrated in all the A. nidulans strains tested. 6. Choline O-sulphate is transported across the mycelial wall by a mechanism different to that responsible for inorganic sulphate transport.     

Isolation of yeast with killer activity and its breeding with an industrial baking strain by protoplast fusion

Summary Wild strains of Saccharomyces cerevisiae were isolated from dairy products, bakery goods, fresh fruit and vegetables, and tested for killer activity. Four isolates out of 238 strains possessed killer activity. The best of these was converted to the petite form and hybridized with an industrial strain of Saccharomyces cerevisiae by protoplast fusion. Thirty-eight out of 104 isolates had killer activity, and some of these had good dough-raising activity as well.     

Cofilin-mediated F-actin severing is regulated by the Rap GTPase and controls the cytoskeletal dynamics that drive lymphocyte spreading and BCR microcluster formation

When lymphocytes encounter APCs bearing cognate Ag, they spread across the surface of the APC to scan for additional Ags. This is followed by membrane contraction and the formation of Ag receptor microclusters that initiate the signaling reactions that lead to lymphocyte activation. Breakdown of the submembrane cytoskeleton is likely to be required for the cytoskeleton reorganization that drives cell spreading and for removing physical barriers that limit Ag receptor mobility. In this report, we show that Ag receptor signaling via the Rap GTPases promotes the dephosphorylation and activation of the actin-severing protein cofilin and that this results in increased severing of cellular actin filaments. Moreover, we show that this cofilin-mediated actin severing is critical for the changes in actin dynamics that drive B and T cell spreading, for the formation of BCR microclusters, and for the increased mobility of BCR microclusters within the plasma membrane after BCR engagement. Finally, using a model APC, we show that activation of this Rap-cofilin signaling module controls the amount of Ag that is gathered into BCR microclusters and that this is directly related to the magnitude of the resulting BCR signaling that is initiated during B cell-APC interactions. Thus, Rap-dependent activation of cofilin is critical for the early cytoskeletal changes and BCR reorganization that are involved in APC-dependent lymphocyte activation.