Bapineuzumab Alters Aβ Composition: Implications for the Amyloid Cascade Hypothesis and Anti-Amyloid Immunotherapy

The characteristic neuropathological changes associated with Alzheimer’s disease (AD) and other lines of evidence support the amyloid cascade hypothesis. Viewing amyloid deposits as the prime instigator of dementia has now led to clinical trials of multiple strategies to remove or prevent their formation. We performed neuropathological and biochemical assessments of 3 subjects treated with bapineuzumab infusions. Histological analyses were conducted to quantify amyloid plaque densities, Braak stages and the extent of cerebral amyloid angiopathy (CAA). Amyloid-β (Aβ) species in frontal and temporal lobe samples were quantified by ELISA. Western blots of amyloid-β precursor protein (AβPP) and its C-terminal (CT) fragments as well as tau species were performed. Bapineuzumab-treated (Bapi-AD) subjects were compared to non-immunized age-matched subjects with AD (NI-AD) and non-demented control (NDC) cases. Our study revealed that Bapi-AD subjects exhibited overall amyloid plaque densities similar to those of NI-AD cases. In addition, CAA was moderate to severe in NI-AD and Bapi-AD patients. Although histologically-demonstrable leptomeningeal, cerebrovascular and neuroparenchymal-amyloid densities all appeared unaffected by treatment, Aβ peptide profiles were significantly altered in Bapi-AD subjects. There was a trend for reduction in total Aβ₄₂ levels as well as an increase in Aβ₄₀ which led to a corresponding significant decrease in Aβ₄₂:Aβ₄₀ ratio in comparison to NI-AD subjects. There were no differences in the levels of AβPP, CT99 and CT83 or tau species between Bapi-AD and NI-AD subjects. The remarkable alteration in Aβ profiles reveals a dynamic amyloid production in which removal and depositional processes were apparently perturbed by bapineuzumab therapy. Despite the alteration in biochemical composition, all 3 immunized subjects exhibited continued cognitive decline.     

Apical transport and folding of prostate-specific membrane antigen occurs independent of glycan processing

Prostate-specific membrane antigen (PSMA) is an integral cell-surface membrane glycoprotein that is overexpressed in prostate carcinomas rendering it an appropriate target for antibody-based therapeutic strategies. The biosynthesis of PSMA in transfected COS-1 cells reveals a slow conversion of mannose-rich to complex glycosylated PSMA compatible with slow transport kinetics from the endoplasmic reticulum to the Golgi. Importantly, mannose-rich PSMA persists as a trypsin-sensitive protein throughout its entire life cycle, and only Golgi-located PSMA glycoforms acquire trypsin resistance. This resistance, used here as a tool to examine correct folding, does not depend on the type of glycosylation, because different PSMA glycoforms generated in the presence of inhibitors of carbohydrate processing in the Golgi are also trypsin resistant. The conformational transition of PSMA to a correctly folded molecule is likely to occur in the Golgi and does not implicate ER molecular chaperones, such as BiP. We show here that PSMA is not only heavily N-but also O-glycosylated. The question arising is whether glycans, which do not play a role in folding of PSMA, are implicated in its transport to the cell surface. Neither the cell-surface expression of PSMA nor its efficient apical sorting in polarized Madin-Darby canine kidney cells are influenced by modulators of N- and O-glycosylation. The acquisition of folding determinants in the Golgi, therefore, is an essential prerequisite for protein trafficking and sorting of PSMA and suggests that altered or aberrant glycosylation often occurring during tumorigenesis has no regulatory effect on the cell-surface expression of PSMA.     

De Novo Mutations in SIK1 Cause a Spectrum of Developmental Epilepsies

Developmental epilepsies are age-dependent seizure disorders for which genetic causes have been increasingly identified. Here we report six unrelated individuals with mutations in salt-inducible kinase 1 (SIK1) in a series of 101 persons with early myoclonic encephalopathy, Ohtahara syndrome, and infantile spasms. Individuals with SIK1 mutations had short survival in cases with neonatal epilepsy onset, and an autism plus developmental syndrome after infantile spasms in others. All six mutations occurred outside the kinase domain of SIK1 and each of the mutants displayed autophosphorylation and kinase activity toward HDAC5. Three mutations generated truncated forms of SIK1 that were resistant to degradation and also showed changes in sub-cellular localization compared to wild-type SIK1. We also report the human neuropathologic examination of SIK1-related developmental epilepsy, with normal neuronal morphology and lamination but abnormal SIK1 protein cellular localization. Therefore, these results expand the genetic etiologies of developmental epilepsies by demonstrating SIK1 mutations as a cause of severe developmental epilepsy.     

Tissue-specific ceramide response in the chronically hypoxic rat model mimicking cyanotic heart disease

BACKGROUND AND OBJECTIVE: Acute hypoxia is associated with apoptosis and increase in ceramide levels in various organs. To assess the effect of chronic hypoxia on ceramide accumulation in the lungs and kidneys, we utilized an animal model mimicking cyanotic heart disease. METHODS: Rats were placed in a hypoxic environment at birth and oxygen levels were maintained at 10% in an air-tight Plexiglas chamber. Controls remained in room air. Animals were sacrificed and the lung and kidneys were harvested and weighed at 1 and 4 weeks, respectively. Ceramide levels were measured using a modified diacylglycerol kinase assay. RESULTS: Significant polycythemia developed in the hypoxic rats at 1 and 4 weeks. Indexed lung and kidney masses were significantly increased in the hypoxic animals as compared to controls at 1 and 4 weeks, respectively. The ceramide levels in the hypoxic lungs and kidneys were not significantly different from control groups at 1 and 4 weeks. [Ceramide/phosphate ratio in the kidneys was 1.28 +/- 0.17 (C) versus 1.18 +/- 0.12 (H) at 1 week; P = 0.39, and 1.46 +/- 0.08 (C) versus 1.33 +/- 0.15 (H) at 4 weeks (P = 0.44)] and [ceramide/phosphate ratio (pmol/nmol) in the lungs was 2.29 +/- 0.14 (C) versus 1.98 +/- 0.12 (H) at 1 week (P = 0.17), and 2.42 +/- 0.16 (C) versus 2.30 +/- 0.05 (H) at 4 weeks, P = 0.34]. CONCLUSION: The response of lungs and kidneys to chronic hypoxia includes increase in indexed mass and lack of ceramide accumulation. This is similar to the response previously reported in the chronically hypoxic brain and heart. Thus, various organs appear to have similar ceramide response pattern to chronic hypoxia.     

Comparative study of growth temperature impact on the susceptibility of biofilm-detached and planktonic Staphylococcus aureus cells to benzalkonium chloride

The present study investigated and compared the effect of growth temperature on the susceptibility of biofilm-detached and planktonic Staphylococcus aureus cells, to benzalkonium chloride (BAC). This study also highlights the impact of BAC on the bacterial physiology and the role of membrane fluidity regulation as a bacterial resistance mechanism. The minimum inhibitory concentration of BAC was characterized with micro-dilution growth inhibition assay. The BAC treatment was performed on S. aureus cultured at 20 °C and 37 °C, for 24 h. The morphology of S. aureus cells was examined using scanning electron microscopy. The loss of bacterial membrane integrity after BAC treatment was studied by monitoring the intracellular potassium ion leakage using the atomic absorption spectroscopy. The bacterial membrane total fatty acid composition, controlling the membrane fluidity, was analyzed by GC/MS. The results showed that the resistance of S. aureus cells to BAC increased with the increase of growth temperature. The planktonic cells were more susceptible to BAC than biofilm-detached ones. The rise of growth temperature resulted in an increase of S. aureus membrane rigidity. Furthermore, a higher membrane fluidity was observed in planktonic cells when compared to that in the biofilm-detached ones. The resistance of S. aureus seems to depend on the growth temperature. Compared to planktonic cells, biofilm-detached cells showed a greater resistance to BAC. The BAC targets and disturbs the bacterial membrane. Membrane fluidity modulation is likely a one of resistance mechanisms for S. aureus to BAC at the cellular scale. Therefore, disinfection procedures, in food sector, should be adapted for bacteria detached from biofilm.     

Regulation of Switching Frequency and Bias of the Bacterial Flagellar Motor by CheY and Fumarate

The effect of CheY and fumarate on switching frequency and rotational bias of the bacterial flagellar motor was analyzed by computer-aided tracking of tethered Escherichia coli. Plots of cells overexpressing CheY in a gutted background showed a bell-shaped correlation curve of switching frequency and bias centering at about 50% clockwise rotation. Gutted cells (i.e., with cheA to cheZ deleted) with a low CheY level but a high cytoplasmic fumarate concentration displayed the same correlation of switching frequency and bias as cells overexpressing CheY at the wild-type fumarate level. Hence, a high fumarate level can phenotypically mimic CheY overexpression by simultaneously changing the switching frequency and the bias. A linear correlation of cytoplasmic fumarate concentration and clockwise rotation bias was found and predicts exclusively counterclockwise rotation without switching when fumarate is absent. This suggests that (i) fumarate is essential for clockwise rotation in vivo and (ii) any metabolically induced fluctuation of its cytoplasmic concentration will result in a transient change in bias and switching probability. A high fumarate level resulted in a dose-response curve linking bias and cytoplasmic CheY concentration that was offset but with a slope similar to that for a low fumarate level. It is concluded that fumarate and CheY act additively presumably at different reaction steps in the conformational transition of the switch complex from counterclockwise to clockwise motor rotation.     

The rotary mechanism of the ATP synthase

The F₀F₁ ATP synthase is a large complex of at least 22 subunits, more than half of which are in the membranous F₀ sector. This nearly ubiquitous transporter is responsible for the majority of ATP synthesis in oxidative and photo-phosphorylation, and its overall structure and mechanism have remained conserved throughout evolution. Most examples utilize the proton motive force to drive ATP synthesis except for a few bacteria, which use a sodium motive force. A remarkable feature of the complex is the rotary movement of an assembly of subunits that plays essential roles in both transport and catalytic mechanisms. This review addresses the role of rotation in catalysis of ATP synthesis/hydrolysis and the transport of protons or sodium.     

Connexins: a myriad of functions extending beyond assembly of gap junction channels

Connexins constitute a large family of trans-membrane proteins that allow intercellular communication and the transfer of ions and small signaling molecules between cells. Recent studies have revealed complex translational and post-translational mechanisms that regulate connexin synthesis, maturation, membrane transport and degradation that in turn modulate gap junction intercellular communication. With the growing myriad of connexin interacting proteins, including cytoskeletal elements, junctional proteins, and enzymes, gap junctions are now perceived, not only as channels between neighboring cells, but as signaling complexes that regulate cell function and transformation. Connexins have also been shown to form functional hemichannels and have roles altogether independent of channel functions, where they exert their effects on proliferation and other aspects of life and death of the cell through mostly-undefined mechanisms. This review provides an updated overview of current knowledge of connexins and their interacting proteins, and it describes connexin modulation in disease and tumorigenesis.     

A rotor-stator cross-link in the F1-ATPase blocks the rate-limiting step of rotational catalysis

The F(0)F(1)-ATP synthase couples the functions of H(+) transport and ATP synthesis/hydrolysis through the efficient transmission of energy mediated by rotation of the centrally located gamma, epsilon, and c subunits. To understand the gamma subunit role in the catalytic mechanism, we previously determined the partial rate constants and devised a minimal kinetic model for the rotational hydrolytic mode of the F(1)-ATPase enzyme that uniquely fits the pre-steady state and steady state data ( Baylis Scanlon, J. A., Al-Shawi, M. K., Le, N. P., and Nakamoto, R. K. (2007) Biochemistry 46, 8785-8797 ). Here we directly test the model using two single cysteine mutants, betaD380C and betaE381C, which can be used to reversibly inhibit rotation upon formation of a cross-link with the conserved gammaCys-87. In the pre-steady state, the gamma-beta cross-linked enzyme at high Mg.ATP conditions retained the burst of hydrolysis but was not able to release P(i). These data show that the rate-limiting rotation step, k(gamma), occurs after hydrolysis and before P(i) release. This analysis provides additional insights into how the enzyme achieves efficient coupling and implicates the betaGlu-381 residue for proper formation of the rate-limiting transition state involving gamma subunit rotation.     

Context dependent reversion of tumor phenotype by connexin-43 expression in MDA-MB231 cells and MCF-7 cells: Role of β-catenin/connexin43 association

Connexins (Cx), gap junction (GJ) proteins, are regarded as tumor suppressors, and Cx43 expression is often down regulated in breast tumors. We assessed the effect of Cx43 over-expression in 2D and 3D cultures of two breast adenocarcinoma cell lines: MCF-7 and MDA-MB-231. While Cx43 over-expression decreased proliferation of 2D and 3D cultures of MCF-7 by 56% and 80% respectively, MDA-MB-231 growth was not altered in 2D cultures, but exhibited 35% reduction in 3D cultures. C-terminus truncated Cx43 did not alter proliferation. Untransfected MCF-7 cells formed spherical aggregates in 3D cultures, and MDA-MB-231 cells formed stellar aggregates. However, MCF-7 cells over-expressing Cx43 formed smaller sized clusters and Cx43 expressing MDA-MB-231 cells lost their stellar morphology. Extravasation ability of both MCF-7 and MDA-MB-231 cells was reduced by 60% and 30% respectively. On the other hand, silencing Cx43 in MCF10A cells, nonneoplastic human mammary cell line, increased proliferation in both 2D and 3D cultures, and disrupted acinar morphology. Although Cx43 over-expression did not affect total levels of β-catenin, α-catenin and ZO-2, it decreased nuclear levels of β-catenin in 2D and 3D cultures of MCF-7 cells, and in 3D cultures of MDA-MB-231 cells. Cx43 associated at the membrane with α-catenin, β-catenin and ZO-2 in 2D and 3D cultures of MCF-7 cells, and only in 3D conditions in MDA-MB-231 cells. This study suggests that Cx43 exerts tumor suppressive effects in a context-dependent manner where GJ assembly with α-catenin, β-catenin and ZO-2 may be implicated in reducing growth rate, invasiveness, and, malignant phenotype of 2D and 3D cultures of MCF-7 cells, and 3D cultures of MDA-MB-231 cells, by sequestering β-catenin away from nucleus.