Nerve growth factor, a differentiating agent, and epidermal growth factor, a mitogen, increase the activities of different S6 kinases in PC12 cells

Treatment of PC12 cells with either nerve growth factor, a differentiating agent, or epidermal growth factor, a mitogen, leads to an increase in the phosphorylation of the ribosomal protein S6. The soluble fraction of PC12 cells contains two S6 kinases, separable on heparin-Sepharose. Treatment of the cells with nerve growth factor results in an increase in the activity of one of these kinases; treatment of the cells with epidermal growth factor results in an increase in the activity of the other one. The data suggest that the patterns of phosphorylation and, in turn, the functional properties of S6 are different in cells instructed to differentiate from those in cells instructed to divide.     

Characterization of Escherichia coli chemotaxis receptor mutants with null phenotypes

Hydroxylamine mutagenesis was used to alter the tar gene that encodes the transmembrane Tar protein required for chemotaxis. Mutants defective in chemotaxis were selected, and the mutation was characterized by DNA sequencing. Two classes of mutations were found: nonsense and missense. The nonsense mutations were distributed throughout the gene, while the missense mutations were found to cluster in a region that includes 185 amino acids at the C-terminal end of the Tar protein. Partial characterization of mutant phenotypes suggested that some are completely defective in signaling while responding to attractants and repellents by differential methylation. Other mutants are undermethylated and constantly tumble, while yet another class of mutants is overmethylated and biased toward constant swimming with little or no tumbling. These mutants will be useful in experiments designed to understand the mechanism of chemotaxis.     

Abdominal distension alters regional pleural pressures and chest wall mechanics in pigs in vivo

Abdominal distension (AD) occurs in pregnancy and is also commonly seen in patients with ascites from various causes. Because the abdomen forms part of the "chest wall," the purpose of this study was to clarify the effects of AD on ventilatory mechanics. Airway pressure, four (vertical) regional pleural pressures, and abdominal pressure were measured in five anesthetized, paralyzed, and ventilated upright pigs. The effects of AD on the lung and chest wall were studied by inflating a liquid-filled balloon placed in the abdominal cavity. Respiratory system, chest wall, and lung pressure-volume (PV) relationships were measured on deflation from total lung capacity to residual volume, as well as in the tidal breathing range, before and 15 min after abdominal pressure was raised. Increasing abdominal pressure from 3 to 15 cmH2O decreased total lung capacity and functional residual capacity by approximately 40% and shifted the respiratory system and chest wall PV curves downward and to the right. Much smaller downward shifts in lung deflation curves were seen, with no change in the transdiaphragmatic PV relationship. All regional pleural pressures increased (became less negative) and, in the dependent region, approached 0 cmH2O at functional residual capacity. Tidal compliances of the respiratory system, chest wall, and lung were decreased 43, 42, and 48%, respectively. AD markedly alters respiratory system mechanics primarily by "stiffening" the diaphragm/abdomen part of the chest wall and secondarily by restricting lung expansion, thus shifting the lung PV curve as seen after chest strapping. The less negative pleural pressures in the dependent lung regions suggest that nonuniformities of ventilation could also be accentuated and gas exchange impaired by AD.     

Molecular cloning and characterization of the platelet-activating factor receptor gene expressed in the human heart.

PAF decreases cardiac contractility and blood pressure. To characterize the cardiac PAF receptor, we screened a human ventricular cDNA library in a low stringency condition, using a PCR product derived from guinea pig lung PAF receptor as a probe. Four clones were obtained and named HV1-4. In Xenopus oocytes injected with cRNA derived from HV3 or 4 but not from HV1 or 2, PAF elicited a Ca(2+)-activated Cl- current. HV3 and HV4 were duplicate clones, encoding a 342 amino-acid polypeptide which was identical to that of the human leukocyte PAF receptor. However, a portion of the 5' untranslated region of HV3 (or 4) was different from that of the leukocyte receptor cDNA. Northern blotting of human ventricles and atria using the HV3 insert showed a single band of approximately 4 kb. These results suggest a tissue-specific translational mechanism responsible for regulation of the expression of the PAF receptor mRNA in these tissues.     

The Effect of the B Subunit of Cholera Toxin on the Action of Nerve Growth Factor on PC12 Cells

Abstract: Exogenous gangliosides, especially ganglioside GM1 (GM1), seem to potentiate the action of nerve growth factor (NGF). We have examined the possible regulation of the NGF signaling pathway in PC12 cells by the B subunit of cholera toxin (CTB), which binds to endogenous GM1 specifically and with a high affinity. CTB treatment (1 μg/ml) enhanced NGF-induced neurite outgrowth from PC12 cells, NGF-induced activation of ribosomal protein S6 kinase, and NGF-induced stimulation of trk phosphorylation. CTB plus NGF also caused a greater inhibition of [³H]-thymidine incorporation into DNA than did NGF alone. These enhancing effects of CTB were blocked by the presence of cytochalasin B in the culture medium but were not affected by the presence of colchicine or by the depletion of Ca²⁺ in the medium. ¹²⁵I-NGF binding experiments revealed that CTB treatment did not affect the specific binding of NGF to the cells. These results strongly suggest that the binding of cell surface GM1 by CTB modulates the pathway of intracellular signaling initiated by NGF and that the association of CTB with a cytoskeletal component is essential for these effects.     

Role of lipopolysaccharide and outer membrane protein of Escherichia coli K-12 in the receptor activity for bacteriophage T4.

Lipopolysaccharide isolated from Escherichia coli K-12 did not inactivate phage T4, although the cell envelopes with 1% sodium deoxycholate resulted in the release of cytoplasmic membrane proteins, 70% of the lipopolysaccharide, and almost all of the phospholipid. The reconstitution of phage receptor activity was achieved from deoxycholate-soluble and -insoluble fractions by dialysis against a solution of magnesium chloride. Lipopolysaccharide was the only essential component in the deoxycholate-soluble fraction. PhageT4-resistant mutants YA21-6 and YA21-82, having defects in the deoxycholate-soluble and -insoluble fractions, respectively, were isolated. The deoxycholate-soluble fraction of YA21-6 possessed heptoseless lipopolysaccharide, and this defect was responsible for the phage resistance. The deoxycholate-insoluble fraction of YA21-82 lacked outer membrane protein O-8. The addition of O-8 to this fraction together with the wild-type lipopolysaccharide resulted in the appearance of the receptor activity. Furthermore, the reconstitution was successfully achieved with only O-8 and the wild-type lipopolysaccharide, indicating that O-8 was an essential component in the deoxycholate-insoluble fraction.     

Differential Responses of the Phosphorylation of Ribosomal Protein S6 to Nerve Growth Factor and Epidermal Growth Factor in PC 12 Cells

Previous studies from this laboratory have shown that the phosphorylation of the S6 protein of the ribosomes is catalyzed by at least two different and separable kinase activities in PC12 cells. One of these activities is increased by treatment of the cells with nerve growth factor, the other by treatment of the cells with epidermal growth factor. The present work shows that these two factors stimulate the phosphorylation of S6 with quite different kinetics, and that both the number of phosphates incorporated into S6 and the phosphopeptide pattern of S6 are different in cells treated with nerve growth factor than in cells treated with epidermal growth factor. The characteristics of the nerve growth factor-sensitive S6 kinase and of the epidermal growth factor-sensitive kinase were also clearly different. Substrate specificity and inhibitor studies indicated that neither was identical to cyclic AMP-dependent kinase, kinase C, or the calcium/calmodulin-dependent kinases. However, two major phosphopeptides produced by S6 phosphorylation in nerve growth factor-treated cells were also seen on phosphorylation of S6 by cyclic AMP-dependent kinase in vitro. In addition, when rat liver 40S ribosomal subunits were pretreated with cyclic AMP-dependent kinase in vitro, the action of the nerve growth factor-sensitive S6 kinase was increased about twofold.     

Performance bonuses and the quality of primary health care delivered by family health teams in Brazil: A difference-in-differences analysis

BackgroundPay-for-performance (P4P) programmes to incentivise health providers to improve quality of care have been widely implemented globally. Despite intuitive appeal, evidence on the effectiveness of P4P is mixed, potentially due to differences in how schemes are designed. We exploited municipality variation in the design features of Brazil’s National Programme for Improving Primary Care Access and Quality (PMAQ) to examine whether performance bonuses given to family health team workers were associated with changes in the quality of care and whether the size of bonus mattered.Methods and findingsFor this quasi-experimental study, we used a difference-in-differences approach combined with matching. We compared changes over time in the quality of care delivered by family health teams between (bonus) municipalities that chose to use some or all of the PMAQ money to provide performance-related bonuses to team workers with (nonbonus) municipalities that invested the funds using traditional input-based budgets. The primary outcome was the PMAQ score, a quality of care index on a scale of 0 to 100, based on several hundred indicators (ranging from 598 to 660) of health care delivery. We did one-to-one matching of bonus municipalities to nonbonus municipalities based on baseline demographic and economic characteristics. On the matched sample, we used ordinary least squares regression to estimate the association of any bonus and size of bonus with the prepost change over time (between November 2011 and October 2015) in the PMAQ score. We performed subgroup analyses with respect to the local area income of the family health team. The matched analytical sample comprised 2,346 municipalities (1,173 nonbonus municipalities; 1,173 bonus municipalities), containing 10,275 family health teams that participated in PMAQ from the outset. Bonus municipalities were associated with a 4.6 (95% CI: 2.7 to 6.4; p < 0.001) percentage point increase in the PMAQ score compared with nonbonus municipalities. The association with quality of care increased with the size of bonus: the largest bonus group saw an improvement of 8.2 percentage points (95% CI: 6.2 to 10.2; p < 0.001) compared with the control. The subgroup analysis showed that the observed improvement in performance was most pronounced in the poorest two-fifths of localities. The limitations of the study include the potential for bias from unmeasured time-varying confounding and the fact that the PMAQ score has not been validated as a measure of quality of care.ConclusionsPerformance bonuses to family health team workers compared with traditional input-based budgets were associated with an improvement in the quality of care.

Nasser Fardousi and colleagues investigate the association between performance bonuses and the quality of primary health care delivered by family health teams in Brazil.     

Cloning of the C-terminal cytoplasmic fragment of the tar protein and effects of the fragment on chemotaxis of Escherichia coli.

A gene encoding only the C-terminal portion of the receptor-transducer protein Tar of Escherichia coli was constructed. The gene product was detected and localized in the cytoplasmic fraction of the cell by immunoblotting with anti-Tar antibodies. The C-terminal fragments from wild-type and mutant tar genes were characterized in vivo. The C-terminal fragment generated from tar-526, a mutation that results in a dominant "tumble" phenotype, was found to be deamidated and methylated by the CheB and CheR proteins, respectively. The C-terminal fragment derived from a wild-type gene was poorly deamidated, and the C-terminal fragment derived from tar-529, a dominant mutant with a "smooth swimming" phenotype, was not apparently modified. Cells carrying the C-terminal fragment with the tar-526 mutation as the sole receptor-transducer protein showed a high frequency of tumbling and chemotaxis responses to changes in intracellular pH. These results suggest that the cytoplasmic C-terminal fragment of Tar retains some of the functions of the whole protein in vivo.     

Spatiotemporal characteristics and mechanisms of intracellular Ca(2+) increases at fertilization in eggs of jellyfish (Phylum Cnidaria, Class Hydrozoa)

We have clarified, for the first time, the spatiotemporal patterns of intracellular Ca(2+) increases at fertilization and the Ca(2+)-mobilizing mechanisms in eggs of hydrozoan jellyfish, which belong to the evolutionarily old diploblastic phylum, Cnidaria. An initial Ca(2+) increase just after fertilization took the form of a Ca(2+) wave starting from one cortical region of the egg and propagating to its antipode in all of four hydrozoan species tested: Cytaeis uchidae, Cladonema pacificum, Clytia sp., and Gonionema vertens. The initiation site of the Ca(2+) wave was restricted to the animal pole, which is known to be the only area of sperm-egg fusion in hydrozoan eggs, and the wave propagating velocity was estimated to be 4.2-5.9 mum/s. After a Ca(2+) peak had been attained by the initial Ca(2+) wave, the elevated Ca(2+) gradually declined and returned nearly to the resting value at 7-10 min following fertilization. Injection of inositol 1,4,5-trisphosphate (IP(3)), an agonist of IP(3) receptors (IP(3)R), was highly effective in inducing a Ca(2+) increase in unfertilized eggs; IP(3) at a final intracellular concentration of 12-60 nM produced a fully propagating Ca(2+) wave equivalent to that observed at fertilization. In contrast, a higher concentration of cyclic ADP-ribose (cADPR), an agonist of ryanodine receptors (RyR), only generated a localized Ca(2+) increase that did not propagate in the egg. In addition, caffeine, another stimulator of RyR, was completely without effect. Sperm-induced Ca(2+) increases in Gonionema eggs were severely affected by preinjection of heparin, an inhibitor of Ca(2+) release from IP(3)R. These results strongly suggest that there is a well-developed IP(3)R-, but not RyR-mediated Ca(2+) release mechanism in hydrozoan eggs and that the former system primarily functions at fertilization. Our present data also demonstrate that the spatial characteristics and mechanisms of Ca(2+) increases at fertilization in hydrozoan eggs resemble those reported in higher triploblastic animals.