Expression of inflammatory cytokines, Bcl2 and cathepsin D are altered in lymphoblasts of autistic subjects.

UNLABELLED: To determine whether inflammation and apoptosis are involved in the pathogenesis of autism, we examined cytokines, Bcl2 expression and cathepsin D protease activity in the lymphoblasts of autistic subjects and age-matched controls. We found increased expression levels of pro-inflammatory cytokines TNF-α and IL-6, but decreased Bcl2 expression in lymphoblasts of autistic subjects. We also found that cathepsin D mRNA and protein expression were significantly increased in autistic lymphoblasts. CONCLUSION: Our findings suggest that inflammation and apoptosis may play a significant role in the pathogenesis of autism, and cathepsin D may participate in the regulation of cytokine-induced inflammation and apoptosis in autistic lymphoblasts.

Fibrillar amyloid beta-protein inhibits the activity of high molecular weight brain protease and trypsin.

The effect of soluble amyloid beta-protein (sAbeta) and fibrillar amyloid beta-protein (fAbeta) on the casein-digesting activity of high molecular weight bovine brain protease (HMW protease) and trypsin was studied. While sAbeta stimulated the casein-digesting activity of HMW protease in a concentration-dependent manner, it did not affect trypsin activity. Structure-activity relationship was studied by testing different soluble and fibrillar Abeta peptides. Various Abeta peptides affected casein-digesting activity of HMW protease differently: sAbeta 1-40 > sAbeta 22-35 = sAbeta 1-11 = sAbeta1-16 > sAbeta 1-28 = sAbeta 31-35, while sAbeta 12-28 and sAbeta 25-35 had no effect. On the other hand, among the fibrillar beta peptides, only fAbeta 1-40 significantly inhibited the casein-digesting activity of HMW protease. Tricine gel electrophoresis showed that sAbeta was digested by trypsin while it remained un-cleaved in the presence of HMW protease. However, fAbeta, a major component of amyloid plaques in Alzheimer's disease, inhibited the casein-digesting activity of both HMW protease and trypsin. fAbetawas found to be resistant to proteolysis by HMW protease and trypsin. The trypsin resistance starts in the early stage of fibrillization of Abeta, i.e., aggregated Abeta. Taken together, these results suggest that fibrillization of Abeta may affect the clearance of Abeta by inhibiting the brain proteases, thereby increasing the concentration of circulating Abeta, that may further increase the Abeta fibrillization.

The 26S proteasome in garlic (Allium sativum): purification and partial characterization.

The 26S proteasome (multicatalytic protease complex, MPC) was purified from fresh garlic cloves (Allium sativum) to near homogeneity by ion exchange chromatography on DEAE-sephacel, gel filtration on Sepharose-4B, and glycerol density gradient centrifugation. Two alpha-type (20S proteasome "catalytic core") subunits were identified by the direct sequencing of peptide fragments (mass fingerprint analysis, Mass Spectrometry Lab, Stanford University) or the sequencing of a cloned cDNA generated using a garlic cDNA library as the template; these subunits were found to have a high homology to those from other plants. Polyacrylamide gel electrophoresis under denaturing conditions separated the garlic MPC into multiple polypeptides having molecular masses in the range of 21-35 (components of the 20S catalytic core) and 55-100 kDa (components of the 19S regulatory units). The banding pattern of the garlic MCP is similar to that of spinach and rat liver with minor differences in some components; however, polyclonal antibodies against mammalian proteasomes failed to significantly stain the enzyme from garlic. This is the first work to identify the garlic proteasome.

Elevated levels of serum alpha(2) macroglobulin in wild black bears during hibernation.

Bear serum alpha(2) macroglobulin (alpha(2)M) was purified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and partially characterized by tryptic digestion of alpha(2)M and analysis of the peptides by peptide mass fingerprinting. The molecular weight of bear serum alpha(2)M was 181 kDa, same as for human serum alpha(2)M, on SDS-PAGE. However, the MALDI mass spectrum of the tryptic digested bear serum alpha(2)M showed that it is different from human alpha(2)M or other data bank proteins. Liquid chromatography (LC)/mass spectrometry (MS)/MS of the proteolytic products of bear serum alpha(2)M showed eight peptides that had similarities to human alpha(2)M suggesting that the protein of interest was indeed alpha(2)M of bear. The polyclonal antibody against bear serum alpha(2)M recognized only one protein from the western blot of bear serum proteins. It also recognized human alpha(2)M. The levels of serum alpha(2)M were significantly increased during hibernating state as compared to active state of bears indicating its protective role from the consequences of the metabolic depression during hibernation.