Macrophage cholesterol efflux and the active domains of serum amyloid A 2.1

Serum amyloid A 2.1 (SAA2.1) suppresses ACAT and stimulates cholesteryl ester hydrolase (CEH) activities in cholesterol-laden macrophages, and in the presence of a cholesterol transporter and an extracellular acceptor, there is a marked increase in the rate of cholesterol export in culture and in vivo. The stimulation of CEH activity by SAA2.1 is not affected by chloroquine, suggesting that it operates on neutral CEH rather than the lysosomal form. With liposomes containing individual peptides of SAA2.1, residues 1-20 inhibit ACAT activity, residues 74-103 stimulate CEH activity, and each of residues 1-20 and 74-103 promotes macrophage cholesterol efflux to HDL in culture media. In combination, these peptides exhibit a profound effect, so that 55-70% of cholesterol is exported to media HDL in 24 h. The effect is also demonstrable in vivo. [3H]cholesterol-laden macrophages injected intravenously into mice were allowed to establish themselves for 24 h. Thereafter, the mice received a single intravenous injection of liposomes containing intact SAA1.1, SAA2.1, peptides composed of SAA2.1 residues 1-20, 21-50, 51-80, 74-103, or SAA1.1 residues 1-20. Only liposomes containing intact SAA2.1 or its residues 1-20 or 74-103 promoted the efflux of cholesterol in vivo. A single injection of each of the active peptides is effective in promoting cholesterol efflux in vivo for at least 4 days.     

Mutations of Arg440 and Gly455/Gly456 oppositely change pH sensing of Na+/H+ exchanger 1

To identify important amino acid residues involved in intracellular pH (pH(i)) sensing of Na(+)/H(+) exchanger 1, we produced single-residue substitution mutants in the region of the exchanger encompassing the putative 11th transmembrane segment (TM11) and its adjacent intracellular (intracellular loop (IL) 5) and extracellular loops (extracellular loop 6). Substitution of Arg(440) in IL5 with other residues except positively charged Lys caused a large shift in pH(i) dependence of (22)Na(+) uptake to an acidic side, whereas substitution of Gly(455) or Gly(456) within the highly conserved glycine-rich sequence of TM11 shifted pH(i) dependence to an alkaline side. The observed alkaline shift was larger with substitution of Gly(455) with residues with increasing sizes, suggesting the involvement of the steric effect. Interestingly, mutation of Arg(440) (R440D) abolished the ATP depletion-induced acidic shift in pH(i) dependence of (22)Na(+) uptake as well as the cytoplasmic alkalinization induced by various extracellular stimuli, whereas with that of Gly(455) (G455Q) these functions were preserved. These mutant exchangers did not alter apparent affinities for extracellular transport substrates Na(+) and H(+) and the inhibitor 5-(N-ethyl-N-isopropyl)amiloride. These results suggest that positive charge at Arg(440) is required for normal pH(i) sensing, whereas mutation-induced perturbation of the TM11 structure may be involved in the effects of Gly mutations. Thus, both Arg(440) in IL5 and Gly residues in the conserved segment of TM11 appear to constitute important elements for proper functioning of the putative "pH(i) sensor" of Na(+)/H(+) exchanger 1.     

Kinetic dissection of two distinct proton binding sites in Na+/H+ exchangers by measurement of reverse mode reaction

We examined the effect of intracellular acidification on the reverse mode of Na+/H+ exchange by measuring 22Na+ efflux from 22Na+-loaded PS120 cells expressing the Na+/H+ exchanger (NHE) isoforms NHE1, NHE2, and NHE3. The 5-(N-ethyl-N-isopropyl)amiloride (EIPA)- or amiloride-sensitive fraction of 22Na+ efflux was dramatically accelerated by cytosolic acidification as opposed to thermodynamic prediction, supporting the concept that these NHE isoforms are activated by protonation of an internal binding site(s) distinct from the H+ transport site. Intracellular pH (pHi) dependence of 22 Na+ efflux roughly exhibited a bell-shaped profile; mild acidification from pHi 7.5 to 7 dramatically accelerated 22Na+ efflux, whereas acidification from pHi 6.6 gradually decreased it. Alkalinization above pHi 7.5 completely suppressed EIPA-sensitive 22Na+ efflux. Cell ATP depletion and mutation of NHE1 at Arg440 (R440D) caused a large acidic shift of the pHi profile for 22Na+ efflux, whereas mutation at Gly455 (G455Q) caused a significant alkaline shift. Because these mutations and ATP depletion cause correspondingly similar effects on the forward mode of Na+/H+ exchange, it is most likely that they alter exchange activity by modulating affinity of the internal modifier site for protons. The data provide substantial evidence that a proton modifier site(s) distinct from the transport site controls activities of at least three NHE isoforms through cooperative interaction with multiple protons.     

Thin-layer cytology findings of small cell carcinoma of the lower female genital tract. Review of three cases with molecular analysis

OBJECTIVE: To describe the thin-layer cytology findings of small cell carcinoma of the low female genital tract, with histologic correlation and human papillomavirus (HPV) genotyping. STUDY DESIGN: The authors reviewed the clinical findings, thin-layer cytology and histologic features of small cell carcinoma of the lower female genital tract (cervix or vagina) occurring in three postmenopausal Chinese women at Pamela Youde Nethersole Eastern Hospital, Hong Kong, over a four-year period, from January 1998 to December 2001. Molecular techniques for HPV screening and genotyping using the polymerase chain reaction and restriction fragment length polymorphism were employed on the cytologic specimens. RESULTS: The thin-layer preparations were of moderate to high cellularity. There were loose aggregates of or isolated small round cells with a high nuclear/cytoplasmic ratio, thin but irregular nuclear membrane, hyperchromatic nuclei, inconspicuous nucleoli and scanty cytoplasm. Tumor cell cannibalism was commonly found. Small groups of tumor cells with nuclear molding were noted. There was also obvious tumor diathesis in the background. The necrotic debris was admixed with isolated small round cells, apoptotic bodies and nuclear dust. Associated koilocytosis or squamous intraepithelial lesions were not seen. Histologic examination of the tumor biopsies showed classic features of small cell carcinoma associated with squashing artifacts and vascularized stroma. Molecular analysis revealed the presence of HPV DNA (either type 18 or 16) in all the three liquid-based cytology samples. CONCLUSION: While the cytomorphologic features of small cell carcinoma of the cervix or vagina in thin-layer preparations are slightly different from those in conventional smears, due mainly to the absence of smearing effect, recognition of the subtle but characteristic appearance can enhance the accuracy of the cytologic diagnosis. The association between HPV and primary small cell carcinoma of the lower female genital tract was confirmed by this study.     

Successful molecular dynamics simulation of two zinc complexes bridged by a hydroxide in phosphotriesterase using the cationic dummy atom method

I report herein two 2.0 ns (1.0 fs time step) MD simulations of two zinc complexes bridged by a hydroxide in phosphotriesterase (PTE) employing the nonbonded method and the cationic dummy atom method that uses virtual atoms to impose orientational requirement for zinc ligands. The cationic dummy atom method was able to simulate the four-ligand coordination of the two zinc complexes in PTE. The distance (3.39 +/- 0.07A) between two nearby zinc ions in the time-average structure of PTE derived from the MD simulation using the cationic dummy atoms matched that in the X-ray structure (3.31 +/- 0.001A). Unequivocally, the time-average structure of PTE was able to fit into the experimentally determined difference electron density map of the corresponding X-ray structure. The results demonstrate the practicality of the cationic dummy atom method for MD simulations of zinc proteins bound with multiple zinc ions. In contrast, a 2.0 ns (1.0 fs time step) MD simulation using the nonbonded method revealed a striking difference in the active site between the X-ray structure and the time-average structure that was unable to fit into the density map of PTE. The results suggest that caution should be used in the MD simulations using the nonbonded method.     

自发性高血压大鼠心肌和血管组织牛磺酸的转运障碍

在自发性高血压大鼠（SHR）的心肌和主动脉血管组织上观察牛磺酸（taurine)转运和牛磺酸转运体（taurine transporter,TAUT) mRNA 的改变,结果显示,与对照组WKY大鼠相比,SHR组血浆牛磺酸水平和牛磺酸释放量增加,而心肌和血管组织牛磺酸水平和TAUT mRNA含量均降低,牛磺酸最大转运速率（Vmax)分别低24％和35％（P＜0．05）,米氏常数（Km)值分别高16％和39％（P＜0．05）,这些结果提示,SHR的心肌和血管组织牛磺酸转运障碍可能与TAUT活性和亲和力降低及TAUT基因水平的下调有关。     

The effects of melatonin and Ginkgo biloba extract on memory loss and choline acetyltransferase activities in the brain of rats infused intracerebroventricularly with beta-amyloid 1-40

Intraventricular infusion of rats with beta-amyloid for 14 days resulted in memory deficit in the water maze as well as decreases in choline acetyltransferase activities and somatostatin levels in the cerebral cortex and hippocampus. These changes were not altered by daily intraperitoneal injection of 20 mg/Kg melatonin. Orally administered Ginkgo biloba extract, however, partially reversed the memory deficit and the decrease in choline actyltransferase activities in the hippocampus. The latter treatment failed to reverse the decrease in somatostatin levels. The results indicate that orally administered Ginkgo biloba extract can protect the brain against beta-amyloid from changes leading to memory deficit through its effect on the cholinergic system.     

Derivation and characterization of a dengue type 1 host range-restricted mutant virus that is attenuated and highly immunogenic in monkeys

We recently described the derivation of a dengue serotype 2 virus (DEN2mutF) that exhibited a host range-restricted phenotype; it was severely impaired for replication in cultured mosquito cells (C6/36 cells). DEN2mutF virus had selected mutations in genomic sequences predicted to form a 3' stem-loop structure (3'-SL) that is conserved among all flavivirus species. The 3'-SL constitutes the downstream terminal similar95 nucleotides of the 3' noncoding region in flavivirus RNA. Here we report the introduction of these same mutational changes into the analogous region of an infectious DNA derived from the genome of a human-virulent dengue serotype 1 virus (DEN1), strain Western Pacific (DEN1WP). The resulting DEN1 mutant (DEN1mutF) exhibited a host range-restricted phenotype similar to that of DEN2mutF virus. DEN1mutF virus was attenuated in a monkey model for dengue infection in which viremia is taken as a correlate of human virulence. In spite of the markedly reduced levels of viremia that it induced in monkeys compared to DEN1WP, DEN1mutF was highly immunogenic. In addition, DEN1mutF-immunized monkeys retained high levels of neutralizing antibodies in serum and were protected from challenge with high doses of the DEN1WP parent for as long as 17 months after the single immunizing dose. Phenotypic revertants of DEN1mutF and DEN2mutF were each detected after a total of 24 days in C6/36 cell cultures. Complete nucleotide sequence analysis of DEN1mutF RNA and that of a revertant virus, DEN1mutFRev, revealed that (i) the DEN1mutF genome contained no additional mutations upstream from the 3'-SL compared to the DEN1WP parent genome and (ii) the DEN1mutFRev genome contained de novo mutations, consistent with our previous hypothesis that the defect in DEN2mutF replication in C6/36 cells was at the level of RNA replication. A strategy for the development of a tetravalent dengue vaccine is discussed.     

Pyridazine based inhibitors of p38 MAPK

Trisubstituted pyridazines were synthesized and evaluated as in vitro inhibitors of p38MAPK. The most active isomers were those possessing an aryl group alpha and a heteroaryl group beta relative to the nitrogen atom in the 2-position of the central pyridazine. Additionally, substitution in the 6-position of the central pyridazine with a variety of dialkylamino substituents afforded a set of inhibitors having good (p38 IC50 1-20 nM) in vitro activity.