Is experimental autoimmune myasthenia gravis induced only by acetylcholine receptors?

EAMG has been induced in a wide variety of animals by using AcChR purified from electric organ and muscle sources. Electrophoresis of SDS polyacrylamide gels heavily loaded with purified AcChR often reveals the presence of minor contaminants. To test whether these contaminants or any other components present in Torpedo californica AcChR preparations could induce EAMG, solubilized Torpedo membrane fragments were depleted of AcChR by passage over an alpha-BuTx-conjugated resin and then injected into Lewis rats in an attempt to induce EAMG. The results demonstrated that some of the minor contaminants present in purified AcChR preparations were antigenic, but EAMG could not be induced with preparations enriched in these contaminants or containing other Torpedo non-AcChR components and lacking AcChR. The conclusion drawn from this study was that the acetylcholine receptor was the only component present in Triton X-100-solubilized Torpedo californica membrane fragments that could induce EAMG.     

A new method for determining sulfoxides in peptide molecules using x-ray photoelectron spectroscopy

The problem of the quantitative determination of sulfoxide groups in peptide molecules has been re-examined. The approaches currently available for the estimation of δ-sulfoxide amino acids are limited in number and characterized by serious shortcomings; in addition, the choice of methods for the estimation of γ-sulfoxide amino acids is even more restricted. A new, rapid, and nondestructive direct method for determining quantitatively all types of sulfoxides in peptide molecules by using x-ray photoelectron spectroscopy is described.     

Circular dichroism analysis of the ribosomal binding of initiation factor IF-3

The circular dichroism spectra of Escherichia coli 30 S ribosomal subunits have been determined between 200 and 320 nm in the presence and in the absence of initiation factor IF-3. The addition of IF-3 did not produce any major alteration of the circular dichroism spectrum of the 30 S subunits between 320 and 240 nm, but resulted in an increase of the negative ellipticity between 240 and 205 nm. The effect was maximal for an IF-3:30 S molar ratio of approximately one, and further addition of IF-3 did not lead to a further increase of ellipticity. A similar effect was not seen when the 30 S ribosomal subunits were previously heat-inactivated to destroy their IF-3 binding capacity. These data indicate that the ribosomal binding of IF-3 may be accompanied by an increase in the secondary structure of the ribosomal proteins, but does not involve any major net change in the secondary structure of the rRNA.     

Deletion of the gliP gene of Aspergillus fumigatus results in loss of gliotoxin production but has no effect on virulence of the fungus in a low-dose mouse infection model

Gliotoxin is a secondary metabolite produced by several fungi including the opportunistic human pathogen Aspergillus fumigatus. As gliotoxin exerts immunosuppressive effects in vitro and in vivo, a role as a virulence determinant in invasive aspergillosis has been discussed for a long time but evidence has not been provided until now. Here, by the use of different selection marker genes A. fumigatus knock-out strains were generated that are deficient for the non-ribosomal peptide synthetase GliP, the putative key enzyme of the gliotoxin biosynthesis. Deletion of the gliP gene resulted in loss of gliotoxin production, as analysed by high performance liquid chromatography and tandem mass spectrometry. No differences in morphology or growth kinetics between wild-type and gliP-deletion strains were observed. In vitro, the culture supernatant of the gliP-deficient strains showed a reduced cytotoxic effect on both macrophage-like cells and T cell lines. In a low-dose murine infection model of invasive aspergillosis, gliotoxin was detected in the lung and absent when mice were infected with the gliP deletion strain. However, gliP deletion strains showed no difference in virulence compared with the corresponding wild-type strains. Taken together, the non-ribosomal peptide synthetase GliP is essential for gliotoxin production in A. fumigatus. Gliotoxin is not required for pathogenicity of the fungus in immunocompromised mice, despite the fact that a reduced cytotoxicity of the culture supernatant of gliP deletion strains was demonstrated.     

The RB-related gene Rb2/p130 in neuroblastoma differentiation and in B-myb promoter down-regulation

The retinoblastoma family of nuclear factors is composed of RB, the prototype of the tumour suppressor genes and of the strictly related genes p107 and Rb2/p130. The three genes code for proteins, namely pRb, p107 and pRb2/p130, that share similar structures and functions. These proteins are expressed, often simultaneously, in many cell types and are involved in the regulation of proliferation and differentiation. We determined the expression and the phosphorylation of the RB family gene products during the DMSO-induced differentiation of the N1E-115 murine neuroblastoma cells. In this system, pRb2/p130 was strongly up-regulated during mid-late differentiation stages, while, on the contrary, pRb and p107 resulted markedly decreased at late stages. Differentiating N1E-115 cells also showed a progressive decrease in B-myb levels, a proliferation-related protein whose constitutive expression inhibits neuronal differentiation. Transfection of each of the RB family genes in these cells was able, at different degrees, to induce neuronal differentiation, to inhibit [3H]thymidine incorporation and to down-regulate the activity of the B-myb promoter.     

Blocking VLDL secretion causes hepatic steatosis but does not affect peripheral lipid stores or insulin sensitivity in mice

The liver secretes triglyceride-rich VLDLs, and the triglycerides in these particles are taken up by peripheral tissues, mainly heart, skeletal muscle, and adipose tissue. Blocking hepatic VLDL secretion interferes with the delivery of liver-derived triglycerides to peripheral tissues and results in an accumulation of triglycerides in the liver. However, it is unclear how interfering with hepatic triglyceride secretion affects adiposity, muscle triglyceride stores, and insulin sensitivity. To explore these issues, we examined mice that cannot secrete VLDL [due to the absence of microsomal triglyceride transfer protein (Mttp) in the liver]. These mice exhibit markedly reduced levels of apolipoprotein B-100 in the plasma, along with reduced levels of triglycerides in the plasma. Despite the low plasma triglyceride levels, triglyceride levels in skeletal muscle were unaffected. Adiposity and adipose tissue triglyceride synthesis rates were also normal, and body weight curves were unaffected. Even though the blockade of VLDL secretion caused hepatic steatosis accompanied by increased ceramides and diacylglycerols in the liver, the mice exhibited normal glucose tolerance and were sensitive to insulin at the whole-body level, as judged by hyperinsulinemic euglycemic clamp studies. Normal hepatic glucose production and insulin signaling were also maintained in the fatty liver induced by Mttp deletion. Thus, blocking VLDL secretion causes hepatic steatosis without insulin resistance, and there is little effect on muscle triglyceride stores or adiposity.     

Cell-free propagation of prion strains

Prions are the infectious agents responsible for prion diseases, which appear to be composed exclusively by the misfolded prion protein (PrP(Sc)). Disease is transmitted by the autocatalytic propagation of PrP(Sc) misfolding at the expense of the normal prion protein. The biggest challenge of the prion hypothesis has been to explain the molecular mechanism by which prions can exist as different strains, producing diseases with distinguishable characteristics. Here, we show that PrP(Sc) generated in vitro by protein misfolding cyclic amplification from five different mouse prion strains maintains the strain-specific properties. Inoculation of wild-type mice with in vitro-generated PrP(Sc) caused a disease with indistinguishable incubation times as well as neuropathological and biochemical characteristics as the parental strains. Biochemical features were also maintained upon replication of four human prion strains. These results provide additional support for the prion hypothesis and indicate that strain characteristics can be faithfully propagated in the absence of living cells, suggesting that strain variation is dependent on PrP(Sc) properties.