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Recent studies suggest that one of the major pathways to the pathogenesis of autism is reduced cell migration. Focal adhesion kinase (FAK) has an important role in neural migration, dendritic morphological characteristics, axonal branching, and synapse formation. The FAK-Src complex, activated by upstream reelin and integrin β1, can initiate a cascade of phosphorylation events to trigger multiple intracellular pathways, including mitogen-activated protein kinase–extracellular signal–regulated kinase and phosphatidylinositol 3-kinase–Akt signaling. In this study, by using B lymphoblasts as a model, we tested whether integrin β1 and FAK-Src signaling are abnormally regulated in autism and whether abnormal FAK-Src signaling leads to defects in B-lymphoblast adhesion, migration, proliferation, and IgG production. To our knowledge, for the first time, we show that protein expression levels of both integrin β1 and FAK are significantly decreased in autistic lymphoblasts and that Src protein expression and the phosphorylation of an active site (Y416) are also significantly decreased. We also found that lymphoblasts from autistic subjects exhibit significantly decreased migration, increased adhesion properties, and an impaired capacity for IgG production. The overexpression of FAK in autistic lymphoblasts countered the adhesion and migration defects. In addition, we demonstrate that FAK mediates its effect through the activation of Src, phosphatidylinositol 3-kinase–Akt, and mitogen-activated protein kinase signaling cascades and that paxillin is also likely involved in the regulation of adhesion and migration in autistic lymphoblasts.

This study determined immune activities in the brain of ASD patients and matched normal subjects by examining cytokines in the brain tissue. Our results showed that proinflammatory cytokines (TNF-α, IL-6 and GM-CSF), Th1 cytokine (IFN-γ) and chemokine (IL-8) were significantly increased in the brains of ASD patients compared with the controls. However the Th2 cytokines (IL-4, IL-5 and IL-10) showed no significant difference. The Th1/Th2 ratio was also significantly increased in ASD patients. Conclusion: ASD patients displayed an increased innate and adaptive immune response through the Th1 pathway, suggesting that localized brain inflammation and autoimmune disorder may be involved in the pathogenesis of ASD.

We have previously reported that levels of phosphatidylethanolamine (PE) in the erythrocyte membrane and of ceruloplasmin, a copper-transport antioxidant protein, in the serum are lower in children with autism than in control subjects. In the present study, we report that (a) copper oxidizes and reduces the levels of membrane PE and (b) copper-mediated oxidation of PE is higher in lymphoblasts from autistic subjects than from control subjects. The effect of copper was examined on the oxidation of liposomes composed of brain lipids from mice and also on the lymphoblasts from autism and control subjects. Among the various metal cations (copper, iron, calcium, cadmium and zinc), only copper was found to oxidize and decrease the levels of PE. The metal cations did not affect the levels of other phospholipids. The action of copper on PE oxidation was time-dependent and concentration-dependent. No difference was observed between copper-mediated oxidation of diacyl-PE and alkenyl-PE (plasmalogen), suggesting that plasmalogenic and non-plasmalogenic PE are equally oxidized by copper. Together, these studies suggest that ceruloplasmin and copper may contribute to oxidative stress and to reduced levels of membrane PE in autism.

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.

In the original paper, mistakes occurred in Figures ​Figures1,1, ​,2,2, and ​and33 should be replaced with the figures below. In addition, a new author Fujiang Cao has been added as the second author in this paper. Figure 1 IKKα protein expression in the cerebellum of autistic subjects. (a) Western blot studies on cerebellar homogenates using IKKα antibody (dilution 1 : 1000). Lanes 1–7 represent control subjects and lanes 8–14 ... Figure 2 IκBα inhibitory subunit expression in the cerebellum of autistic subjects. Western blot studies on cerebellar homogenates using IκBα antibody (dilution 1 : 1000). Lanes 1–7 represent control subjects ... Figure 3 NF-κB p65 protein expression in the cerebellum of autistic subjects. Western blot studies on cerebellar homogenates using IκBα antibody (dilution 1 : 500). Lanes 1–7 represent control subjects and lanes ...

We have recently reported that fibrillar amyloid beta-protein (Abeta) inhibits the proteolytic activity of trypsin and high molecular weight bovine brain protease. We report here that trypsin binds to fibrillar Abeta (fAbeta) and the resulting complex of trypsin/fAbeta is sodium dodecyl sulfate (SDS)-stable. Electron microscopic analysis confirmed the binding of trypsin on the fibrils of both Abeta 1-40 and Abeta 1-42. SDS-polyacrylamide gel electrophoresis (PAGE) of fAbeta sample incubated in the presence of trypsin showed that major amount of trypsin was associated with fAbeta that did not enter the gel. The presence of trypsin in this protein complex was confirmed by Western blotting after its elution from the gel. Kinetic studies showed that the binding of trypsin to fibrillar Abeta was dependent on the degree of Abeta fibrillization and on the concentration of fAbeta. However, the trypsin binding to Abeta oligomers did not affect the fibril growth. The maximum binding (B(max)) of trypsin to fAbeta 1-40 and fAbeta 1-42 was 36 pmol and 40 pmol, and dissociation constant (K(d)) was 18.31 microM and 20 microM respectively. Similar to fAbeta, trypsin could also bind to fibrillar amylin. This binding was dependent on the concentration of fibrillar amylin. Under similar conditions, bovine serum albumin did not bind to fibrillar Abeta. These results suggest that fAbeta and fibrillar amylin have strong affinities for trypsin, and chelation of proteases by abnormal aggregated proteins may be a general mechanism for inflicting pathological conditions in various diseases.

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.

During hibernation, animals undergo metabolic changes that result in reduced utilization of glucose and oxygen. Fat is known to be the preferential source of energy for hibernating animals. Malonyldialdehyde (MDA) is an end product of fatty acid oxidation, and is generally used as an index of lipid peroxidation. We report here that peroxidation of lipids is increased in the plasma and in the membranes of red blood cells in black bears during hibernation. The plasma MDA content was about four fold higher during hibernation as compared to that during the active, non-hibernating state (P0.0001). Similarly, MDA content of erythrocyte membranes was significantly increased during hibernation (P0.025). The activity of Ca(2+)/Mg(2+)-ATPase in the erythrocyte membrane was significantly decreased in the hibernating state as compared to the active state. Na(+)/K(+)-ATPase activity was also decreased, though not significant, during hibernation. These results suggest that during hibernation, the bears are under increased oxidative stress, and have reduced activities of membrane-bound enzymes such as Ca(2+)/Mg(2+)-ATPase and Na(+)/K(+)-ATPase. These changes can be considered part of the adaptive for survival process of metabolic depression.

Polyclonal antiserum against a high molecular weight glycosylated protease, purified from calf brain cytosol, was raised in rabbit and purified by immunoaffinity. The antibody specifically immunoreacted with the M r = 165,000 and 155,000 polypeptides of the protease. Immunocytochemical localization data revealed that the protease is localized in the pyramidal neurons, granular and glial cells of the hippocampus. Microscopic analysis of the pyramidal neurons indicates that the protease is present in the cytoplasm and extends to the dendrite and axon. The nuclei of these remain unstained.

A third form (CANP3) of calcium-activated neutral proteinase (CANP) has been purified, 3900-fold, to near homogeneity from calf brain cortex. The purification procedure is based on the one recently developed for the purification of CANP1 and CANP2. The molecular weight of CANP3, as judged on SDS-polyacrylamide gel electrophoresis was Mr 78,000. A protein with an apparent Mr 17,000 co-purified with the proteinase. At neutral pH (7.2), it was maximally active at 260 microM CaCl2. In the presence of CaCl2, CANP1 and CANP3 were autolyzed very rapidly, whereas the autolysis of CANP2 was slow and gradual. The autolyzed CANP1 and CANP3 responded differently to CaCl2; CANP1 lost activity completely, whereas CANP3 was fully active at 0.5 microM CaCl2. Despite the opposite behavior of these proteinases in the presence of Ca2+, no significant differences in the peptide maps of the three proteinases were observed. Neurofilaments, neurotubules and myelin basic protein (MBP) were degraded by each of the proteinases. Monoclonal antibodies raised against CANP2 reacted almost equally with CANP1 and CANP3. As with CANP1 and CANP2, leupeptin and sulfhydryl-modifying compounds, NEM and iodoacetic acid, inhibited the activity of CANP3.

A Ca 2+ activated protease(s) capable of hydrolyzing several polypeptides at neutral pH including cytoskeletal proteins, actin, myosin, tubulin and neurofilament triplet was identified in calf brain cortex. The enzyme activity precipitates at 75 mM KCl, pH 6.5 – 7.0 and is inhibited by the sulfhydryl inhibitors, N-ethylmaleimide and para-chloromercuribenzoate and the protease inhibitors, antipain, pepstatin and leupeptin, leupeptin being the most effective.