In Saccharomyces cerevisiae, the phosphate signal transduction pathway (PHO pathway) is known to regulate the expression of several phosphate-responsive genes, such as PHO5 and PHO84. However, the fundamental issue of whether cells sense intracellular or extracellular phosphate remains unresolved. To address this issue, we have directly measured intracellular phosphate concentrations by 31 P NMR spectroscopy. We find that PHO5 expression is strongly correlated with the levels of both intracellular orthophosphate and intracellular polyphosphate and that the signaling defect in the ⌬pho84 strain is likely to result from insufficient intracellular phosphate caused by a defect in phosphate uptake. Furthermore, the ⌬phm1⌬phm2, ⌬phm3, and ⌬phm4 strains, which lack intracellular polyphosphate, have higher intracellular orthophosphate levels and lower expression of PHO5 than the wild-type strain. By contrast, the ⌬phm5 strain, which has lower intracellular orthophosphate and higher polyphosphate levels than the wild-type strain, shows repressed expression of PHO5, similar to the wildtype strain. These observations suggest that PHO5 expression is under the regulation of intracellular orthophosphate, although orthophosphate is not the sole signaling molecule. Moreover, the disruption of PHM3, PHM4, or of both PHM1 and PHM2 in the ⌬pho84 strain suppresses, although not completely, the PHO5 constitutive phenotype by increasing intracellular orthophosphate, suggesting that Pho84p affects phosphate signaling largely by functioning as a transporter.Inorganic phosphate is an essential nutrient needed in large amounts for nucleic acid and phospholipid biosynthesis, as well as for energy metabolism. It is therefore essential that organisms have appropriate regulatory mechanisms to respond and adapt rapidly to changes in phosphate availability (1). In Saccharomyces cerevisiae, the phosphate signal transduction pathway (PHO pathway) regulates the expression of several phosphate-responsive genes that are involved in the scavenging and specific uptake of phosphate from extracellular sources, including PHO5, which encodes repressible acid phosphatase (rAPase), 1 and PHO84, which encodes a high affinity phosphate transporter that acts in response to phosphate levels (2-4). Under high phosphate conditions, the transcription factor Pho4p is phosphorylated by the cyclin-dependent kinase (CDK) complex Pho80p-Pho85p (5) and exported from the nucleus to the cytoplasm (6), thereby turning off expression of the PHO5 and PHO84 genes. When yeast cells are starved of phosphate, the CDK inhibitor Pho81p inactivates 8). Under these conditions, Pho4p is unphosphorylated and active (9), leading to the induction of PHO5 and PHO84 expression to scavenge phosphate from the environment (2-4, 10). Although many components and the molecular mechanism of the PHO pathway have been extensively studied, fundamental questions, such as whether cells sense intracellular or extracellular phosphate and what phosphate metabolites function as signals for regulating t...

Teixobactin represents the first member of a newly discovered class of antibiotics that act through inhibition of cell wall synthesis. Teixobactin binds multiple bactoprenol-coupled cell wall precursors, inhibiting both peptidoglycan and teichoic acid synthesis. Here, we show that the impressive bactericidal activity of teixobactin is due to the synergistic inhibition of both targets, resulting in cell wall damage, delocalization of autolysins, and subsequent cell lysis. We also find that teixobactin does not bind mature peptidoglycan, further increasing its activity at high cell densities and against vancomycin-intermediate Staphylococcus aureus (VISA) isolates with thickened peptidoglycan layers. These findings add to the attractiveness of teixobactin as a potential therapeutic agent for the treatment of infection caused by antibiotic-resistant Gram-positive pathogens.

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We have recently developed a micropuncture technique to assess repeatedly function of the same nephrons in chronic renal disease and subsequently examine the morphology of their glomeruli by serial thin-section histological analysis. Using this approach, a potential causal linkage between early functional patterns and late structural abnormalities was examined in glomeruli of two established rat models of glomerular sclerosis. The models are (a) puromycin aminonucleoside (PAN) administration in unilaterally nephrectomized Munich-Wistar rats and (b) adriamycin (ADM) treatment in nonnephrectomized Munich-Wistar rats. Single nephron GFR (SNGFR) and glomerular capillary hydraulic pressure (PGc) were measured repeatedly for 8 (PAN rats) or 31 wk (ADM rats). In all animals studied, values for PGC remained at, or slightly below, levels measured before PAN or ADM administration. SNGFR values declined progressively in all glomeruli in PAN rats. Although some glomeruli in ADM rats had an increase in SNGFR above levels observed in nonnephrectomized control rats, these hyperfiltering glomeruli did not have abnormally high PGC nor did they exhibit glomerular sclerosis at the completion of the study. Histological analysis revealed the existence of a significant inverse correlation between the degree of sclerosis and SNGFR assessed at the time of sacrifice in both PAN and ADM groups. Chronic administration of captopril, an angiotensin I converting enzyme inhibitor, in PAN rats substantially attenuated development of glomerular sclerosis without affecting PGC in earlier stages. The observations in these models indicate that glomerular hyperfiltration and hypertension are not required for the development of glomerular sclerosis in renal diseases, and angiotensin I converting enzyme inhibitor can exert its protective effect independently of its effect on glomerular capillary pressure.

Cyclodextrins are commonly used as a safe excipient to enhance the solubility and bioavailability of hydrophobic pharmaceutical agents. Their efficacies and mechanisms as drug-delivery systems have been investigated for decades, but their immunological properties have not been examined. In this study, we reprofiled hydroxypropyl-β-cyclodextrin (HP-β-CD) as a vaccine adjuvant and found that it acts as a potent and unique adjuvant. HP-β-CD triggered the innate immune response at the injection site, was trapped by MARCO+ macrophages, increased Ag uptake by dendritic cells, and facilitated the generation of T follicular helper cells in the draining lymph nodes. It significantly enhanced Ag-specific Th2 and IgG Ab responses as potently as did the conventional adjuvant, aluminum salt (alum), whereas its ability to induce Ag-specific IgE was less than that of alum. At the injection site, HP-β-CD induced the temporary release of host dsDNA, a damage-associated molecular pattern. DNase-treated mice, MyD88-deficient mice, and TBK1-deficient mice showed significantly reduced Ab responses after immunization with this adjuvant. Finally, we demonstrated that HP-β-CD–adjuvanted influenza hemagglutinin split vaccine protected against a lethal challenge with a clinically isolated pandemic H1N1 influenza virus, and the adjuvant effect of HP-β-CD was demonstrated in cynomolgus macaques. Our results suggest that HP-β-CD acts as a potent MyD88- and TBK1-dependent T follicular helper cell adjuvant and is readily applicable to various vaccines.

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The present study was designed to assess both preventive and therapeutic effects of (S)-1-(2-Hydroxyethyl)-4-methyl-N-[4-(methylsulfonyl) phenyl]-5-[2-(trifluoromethyl) phenyl]-1H-pyrrole-3-carboxamide (CS-3150), a novel nonsteroidal mineralocorticoid receptor antagonist, on renal injury in deoxycorticosterone acetate (DOCA)/salt-induced hypertensive rats (DOCA rats). From 7 weeks of age, DOCA was subcutaneously administered once a week for 4 weeks to uninephrectomized rats fed a high-salt diet. In experiment 1, CS-3150 (0.3-3 mg/kg) was orally administered once a day for 4 weeks coincident with DOCA administration. In experiment 2, after establishment of renal injury by 4 weeks of DOCA/salt loading, CS-3150 (3 mg/kg) was orally administered once a day for 4 weeks with or without continuous DOCA administration. In experiment 1, DOCA/salt loading significantly increased systolic blood pressure (SBP), which was prevented by CS-3150 in a dose-dependent manner. Development of renal injury (proteinuria, renal hypertrophy, and histopathological changes in glomeruli and tubule) was also suppressed by CS-3150 with inhibition of mRNA expression of fibrosis, inflammation, and oxidative stress markers. In experiment 2, under continuous DOCA treatment, CS-3150 clearly ameliorated existing renal injury without lowering SBP, indicating that CS-3150 regressed renal injury independent of its antihypertensive action. Moreover, CS-3150 treatment in combination with withdrawal of DOCA showed further therapeutic effect on renal injury accompanied by reduction in SBP. These results demonstrate that CS-3150 not only prevents but also ameliorates hypertension and renal injury in DOCA rats. Therefore, CS-3150 could be a promising agent for the treatment of hypertension and renal disorders, and may have potential to promote regression of renal injury.