Probing the structure of the Escherichia coli 10Sa RNA (tmRNA).

The conformation of the Escherichia coli 10Sa RNA (tmRNA) in solution was investigated using chemical and enzymatic probes. Single- and double-stranded domains were identified by hydrolysis of tmRNA in imidazole buffer and by lead(II)-induced cleavages. Ribonucleases T1 and S1 were used to map unpaired nucleotides and ribonuclease V1 was used to identify paired bases or stacked nucleotides. Specific atomic positions of bases were probed with dimethylsulfate, a carbodiimide, and diethylpyrocarbonate. Covariations, identified by sequence alignment with nine other tmRNA sequences, suggest the presence of several tertiary interactions, including pseudoknots. Temperature-gradient gel electrophoresis experiments showed structural transitions of tmRNA starting around 40 degrees C, and enzymatic probing performed at selected temperatures revealed the progressive melting of several predicted interactions. Based on these data, a secondary structure is proposed, containing two stems, four stem-loops, four pseudoknots, and an unstable structural domain, some connected by single-stranded A-rich sequence stretches. A tRNA-like domain, including an already reported acceptor branch, is supported by the probing data. A second structural domain encompasses the coding sequence, which extends from the top of one stem-loop to the top of another, with a 7-nt single-stranded stretch between. A third structural module containing pseudoknots connects and probably orients the tRNA-like domain and the coding sequence. Several discrepancies between the probing data and the phylogeny suggest that E. coli tmRNA undergoes a conformational change.     

Malaria parasites require TLR9 signaling for immune evasion by activating regulatory T cells

Malaria is still a life-threatening infectious disease that continues to produce 2 million deaths annually. Malaria parasites have acquired immune escape mechanisms and prevent the development of sterile immunity. Regulatory T cells (Tregs) have been reported to contribute to immune evasion during malaria in mice and humans, suggesting that activating Tregs is one of the mechanisms by which malaria parasites subvert host immune systems. However, little is known about how these parasites activate Tregs. We herein show that TLR9 signaling to dendritic cells (DCs) is crucial for activation of Tregs. Infection of mice with the rodent malaria parasite Plasmodium yoelii activates Tregs, leading to enhancement of their suppressive function. In vitro activation of Tregs requires the interaction of DCs with parasites in a TLR9-dependent manner. Furthermore, TLR9(-/-) mice are partially resistant to lethal infection, and this is associated with impaired activation of Tregs and subsequent development of effector T cells. Thus, malaria parasites require TLR9 to activate Tregs for immune escape.     

Identification of proteins that bind to the neuroprotective agent neoechinulin A

Neoechinulin A is an indole alkaloid with several biological activities. We previously reported that this compound protects neuronal PC12 cells from cytotoxicity induced by the peroxynitrite generator 3-morpholinosydnonimine (SIN-1), but the target proteins and precise mechanism of action of neoechinulin A were unclear. Here, we employed a phage display screen to identify proteins that bind directly with neoechinulin A. Our findings identified two proteins, chromogranin B and glutaredoxin 3, as candidate target binding partners for the alkaloid. QCM analyses revealed that neoechinulin A displays high affinity for both chromogranin B and glutaredoxin 3. RNA interference-mediated depletion of chromogranin B decreased the sensitivity of PC12 cells against SIN-1. Our results suggested chromogranin B is a plausible target of neoechinulin A.     

Polypeptide composition of flacherie viruses of the silkworm

The purified flacherie viruses of the silkworm, Bombyx mori, (FVS I, FVS II, FVS III, and FVS IV) were iodinated by using chloramine-T. The iodinated FVSes were purified by sucrose density gradient centrifugation or 2.4% polyacrylamide gel electrophoresis. FVS IV was found in the sedimentation analysis of FVS I, FVS II, and FVS IV. Electrophoretic patterns of FVS IV showed that it was a mixture of components having identical mobilities with FVS I, EVS IIa, and FVS IIb. FVS IV was a decomposed particle of FVS I, FVS II, and/or FVS III. All of these particles contained three polypeptides with molecular weights of about 51,000, 31,000, and 12,000 daltons. FVS I composed of six polypeptides with molecular weights of 67,000, 51,000, 39,000, 31,000, 14,000, and 12,000 daltons. The maturation process of FVS I was discussed and was suggested as the following process, FVS IIb→FVS IIa→FVS I. It is not clear whether FVS III is an intermediate for FVS IIa to convert into FVS I, or FVS III is a decomposed particle of FVS I.     

Polarographic and spectrophotometric studies on the equilibration of Mo(V) species in HCl solutions

The equilibration of Mo(V) species has been investigated in 0.5–5 M HCl (I = 5.0). The equilibria involve the dimer(D₁)-dimer(D₂) interconversion, followed by the decomposition of D₂ to a monomeric form at greater acidities:

The equilibrium constants have been determined as K₁ = 2 ± 0.3 and K₂ = (1.5 ± 0.4) X 10^?8 at 25 °C.     

Strain Differences in SA Gene Expression in Brain and Kidney of Normotensive and Hypertensive Rats

SUMMARY 1. In situ hybridization done using a ³⁵S-cRNA probe was carried out to obtain information on the expressions of the SA gene in brains and kidneys of the spontaneously hypertensive rat (SHR) strain obtained from the Izumo colony (/Izm) and from Charles River Laboratories (/Crj).2. In the brain, SA mRNA expression was most abundantly observed in epithelial cells of the choroid plexus. High to moderate levels was present on neurons of the CA1–CA4 pyramidal cell layer and the dentate gyrus of the hippocampus and the cerebellar Purkinje cell layer. The solitary tract nucleus and the dorsal motor nucleus of the vagus expressed the SA gene at very low levels. An increase in the expression was noted in the choroid plexus of WKY/Crj; there was no difference, however, in expression levels of other brain areas between WKY/Izm, SHR/Izm, and SHRSP/Izm, and between WKY/Crj and SHR/Crj.3. In the kidney, expression signals of SA mRNA were observed in renal medullary rays and focal cortex of WKY/Izm, SHR/Izm, SHRSP/Izm, and SHR/Crj, whereas mRNA expression in the WKY/Crj kidney was observed in medullary rays and outer strips of the outer medulla. Microscopically, hybridization signals were predominant in the proximal tubules.4. Expression densities decreased only in the kidney of WKY/Crj in 4-and 8-week-old rats, but not in the WKY/Izm kidney, compared with findings in SHR and SHRSP kidneys. These observations are in good agreement with data from Northern blot analysis.5. The SA gene expressions in the brain and the kidney seem not to relate to states of elevated blood pressure, but rather to strain differences. Abundant expressions in the brain and the kidney may mean that the SA gene plays a role in the water–electrolyte transport system. It is noteworthy that there are neuronal expressions of the SA gene in hippocampal pyramidal cells and cerebellar Purkinje cells.     

Cell filamentation in an Escherichia coli K-12 fic mutant caused by theophylline or an adenylate cyclase gene (cya)-containing plasmid

Abstract The bioconversion of 17α-ethynyl steroids was effected with 11α-hydroxylase of Rhizopus nigricans . 7β-Hydroxyethisterone was found after the bioconversion of ethisterone, and 10β- and 6β-hydroxy derivatives after the bioconversion of norethisterone. It seems that the ethynyl group prevents steroid-enzyme binding in the normal mode and thus inhibits the formation of an 11α-hydroxylated product.     

Endothelin receptor in microvessels isolated from human meningiomas: Quantification with radioluminography

Summary 1. We characterized specific¹²⁵I-endothelin-1 (¹²⁵I-ET-1) binding sites in microvessels isolated from human meningiomas, using anin vitro quantitative receptor autoradiographic technique coupled to a radioluminographic imaging plate system.2. This newly developed and highly sensitive method revealed high-affinity ET receptors present in pellet sections of the microvessels from all the meningiomas studied, regardless of histological subtypes (dissociation constant, 1.2 ± 0.3 nM; maximum binding capacity, 185 ± 56 fmol/mg; means ± SE for nine tumors).3. In five cases of meningiomas, ET-3 competed for¹²⁵I-ET-1 binding to microvessels from those tumors with a low affinity [50% inhibiting concentration (IC₅₀) of 1.6 ± 0.4 × 10^–6 M], and a selective ET_B receptor agonist, sarafotoxin S6c, up to 10^–6 M, did not displace ET binding from the sections.4. In the sections of microvessels from four other tumors, biphasic competition curves were obtained in the case of incubation in the presence of increasing concentrations of ET-3, with an IC₅₀ of 1.1 ± 0.2 × 10^–9 M for the high-affinity component and 1.6 ± 0.3 × 10^–6 M for the low-affinity component, respectively. In addition, S6c competed for ET binding to those sections (IC₅₀=2.3 ± 0.2 × 10^–10 M) and 10^–6 M S6c displaced 30% of the control, corresponding to the high-affinity component of competition curves obtained in the presence of ET-3.5. Our results suggest that (a) capillaries in human meningiomas express a large number of high-affinity ET_A (non-ET_B) receptors with a small proportion of ET_B receptors, and (b) ET may have a role in neovascularization, tumor blood flow, and/or function of the blood-tumor barrier in meningioma tissues by interacting with specific receptors present on the surface of the endothelium.