Apolipoprotein E in Synaptic Plasticity and Alzheimer’s Disease: Potential Cellular and Molecular Mechanisms

Alzheimer’s disease (AD) is clinically characterized with progressive memory loss and cognitive decline. Synaptic dysfunction is an early pathological feature that occurs prior to neurodegeneration and memory dysfunction. Mounting evidence suggests that aggregation of amyloid-β (Aβ) and hyperphosphorylated tau leads to synaptic deficits and neurodegeneration, thereby to memory loss. Among the established genetic risk factors for AD, the ɛ4 allele of apolipoprotein E (APOE) is the strongest genetic risk factor. We and others previously demonstrated that apoE regulates Aβ aggregation and clearance in an isoform-dependent manner. While the effect of apoE on Aβ may explain how apoE isoforms differentially affect AD pathogenesis, there are also other underexplored pathogenic mechanisms. They include differential effects of apoE on cerebral energy metabolism, neuroinflammation, neurovascular function, neurogenesis, and synaptic plasticity. ApoE is a major carrier of cholesterols that are required for neuronal activity and injury repair in the brain. Although there are a few conflicting findings and the underlying mechanism is still unclear, several lines of studies demonstrated that apoE4 leads to synaptic deficits and impairment in long-term potentiation, memory and cognition. In this review, we summarize current understanding of apoE function in the brain, with a particular emphasis on its role in synaptic plasticity and the underlying cellular and molecular mechanisms, involving low-density lipoprotein receptor-related protein 1 (LRP1), syndecan, and LRP8/ApoER2.     

A FTIR Imaging Characterization of Fibroblasts Stimulated by Various Breast Cancer Cell Lines

It is well known that the microenvironment plays a major role in breast cancer progression. Yet, the mechanism explaining the transition from normal fibroblasts to cancer-stimulated fibroblasts remains to be elucidated. Here we report a FTIR imaging study of the effects of three different breast cancer cell lines on normal fibroblasts in culture. Fibroblast activation process was monitored by FTIR imaging and spectra compared by multivariate statistical analyses. Principal component analysis evidenced that the fibroblasts stimulated by these cancer cell lines grouped together and remained distinctly separated from normal fibroblasts indicating a modified different chemical composition in the cancer-stimulated fibroblasts. Similar changes in fibroblasts were induced by the various breast cancer cell lines belonging to different sub-types. Most significant changes were observed in the region of 2950 and 1230 cm⁻¹, possibly related to changes in lipids and in the 1230 cm⁻¹ area assigned to phosphate vibrations (nucleotides). Interestingly, the cancer-cell induced changes in the fibroblasts also occurred when there was no possible direct contact between the two cell lines in the co-culture. When contact was possible, the spectral changes were similar, suggesting that soluble factors but not direct cell-cell interactions were responsible for fibroblast activation. Overall, the results indicate that IR imaging could be used in the future for analyzing the microenvironment of breast tumors.     

Application of the multiple antigenic peptides (MAP) strategy to the production of prophormone convertases antibodies: Synthesis,characterization and use of 8-branched immunogenic peptides

Antiserum against an N-terminal sequence of murine prohormone convertase-1 (mPC1) incorporating the sequence immediatley following the junction between the putative pro-region and the active enzyme was obtained. This was accomplished using the multiple antigenic peptide (MAP) approach whereupon an 8-branched polylysine core to which are grafted multiple copies of a 16 amino acid peptide representing the N-terminal sequence of mPC1 (positions 84–99) was synthesized by solid-phase Fmoc chemistry. The ensuing peptide was purified and fully characterized by RP-HPLC, ¹H-NMR, amino acid composition, peptide sequencing and ion-spray mass spectrometry. The immunological properties of the resulting antibodies in detecting recombinant PC1 in both crude and purified preparations were compared with antibodies raised against a similar N-terminal segment of PC1 but using the conventioanl method of peptide–carrier protein conjugation and also developed against a C-terminal fusion protein of PC1. Our data indicate that the MAP antibody was as efficient as both the amino and carboxy-terminal antibodies in qualitative as well as quantitative analysis of PC1 encoded protein by radioimmunoassay. Following an identical approach, antibodies against other prohormone convertases like furin, PC5/6 and PACE4 were also developed and subsequently applied to a number of biochemical and immunological studies. In each case, the ease of preparation and high immunogenicity of the MAP approach were confirmed and reside in the simplicity and rapidity with which a potent and useful antiserum is obtained.     

Purification and characterization of two β-glucosidases from a thermo-tolerant yeast Pichia etchellsii

The thermo-tolerant yeast Pichia etchellsii produced two cell-wall-bound inducible β-glucosidases, BGLI (molecular mass 186 kDa) and BGLII (molecular mass 340 kDa), which were purified by a simple, three-step method, comprising ammonium sulfate precipitation, ion-exchange and hydroxyapatite chromatography. The two enzymes exhibited a similar pH and temperature optima, inhibitory effect by glucose and gluconolactone, and stability in the pH range of 3.0–9.0. Placed in family 3 of glycosylhydrolase families, BGLI was more active on salicin, p-nitrophenyl β-d-glucopyranoside and alkyl β-d-glucosides whereas BGLII was most active on cellobiose. k_cat and K_M values were determined for a number of substrates and, for BGLI, it was established that the deglycosylation step was equally effective on aryl- and alkyl-glucosides while the glycosylation step varied depending on the substrate used. This information was used to synthesize alkyl-glucosides (up to a chain length of C₁₀) using dimethyl sulfoxide stabilized single-phase reaction microenvironment. About 12% molar yield of octyl-glucoside was calculated based on a simple spectrophotometric method developed for its estimation. Further, detailed comparison of properties of the enzymes indicated these to be different from the previously cloned β-glucosidases from this yeast.     

Occurrence of proteins immunoreactive with anti-coupling factor B in phosphorylating membrane preparations

Coupling factor B has been isolated from beef heart mitochondria, apparently in multiple forms which differ in molecular weight and specific activity. Since it has no known intrinsic catalytic activity, detection and quantitation have been based upon the factor B-dependent stimulation of ATP-linked activities in factor B-deficient submitochondrial particles. This communication reports the development of a reliable and more universally applicable enzyme-linked immunosorbent assay (ELISA) for detection and quantitation of factor B in soluble or membranous preparations. The assay requires nanoliter volumes of rabbit antiserum raised against purified factor B and will detect nanogram amounts of the coupling factor. Analysis of beef heart submitochondrial particles using a competitive binding ELISA indicated a factor B content of 0.27 nmol/mg protein, making factor B stoichiometric with F₁ (0.3–0.6 nmol/mg). Furthermore, application of the factor B ELISA has indicated the presence of material cross-reacting with the beef heart factor B-antiserum in phosphorylating membranes from chloroplasts, Escherichia coli, Paracoccus denitrificans and the thermophilic bacterium, PS3. Negative results were obtained with mitochondria and microsomes from rat liver, purple membranes from Halobium halobacterium and sarcoplasmic reticulum from rabbit skeletal muscle.     

Uses and limitations of monoclonal antibodies to Giardia lamblia-specific 66-kDa copro-antigen in copro-immunodiagnosis of giardiasis

Abstract Antigen-capture enzyme-linked immunosorbent assay for the detection of Giardia lamblia -specific antigen in stool eluates from clinical subjects employing monoclonal antibody directed at 66-kDa G. lamblia copro-antigen has been evaluated. The G. lamblia copro-antigen was detected in 67% (31 of the 46 cases) of stool eluates from clinical cases, while none of the stool eluates from subjects with other intestinal parasites or from apparently healthy individuals, had detectable levels of G. lamblia copro-antigen. Monoclonal antibodies secreted by clones B4C5 and D3F4 recognised the periodate-sensitive and -insensitive epitopes of 66-kDa G. lamblia specific copro-antigen, respectively. Eight (73%) of the 11 symptomatic cases of giardiasis had trypsin-/periodate-sensitive epitopes of 66-kDa copro-antigen while 9 (92%) of 11 of the symptomatic cases and asymptomatic G. lamblia cyst carriers had trypsin-sensitive periodate-insensitive G. lamblia specific copro-antigen. The data tend to suggest that detection of periodate-insensitive epitopes of G. lamblia copro-antigen would indicate the presence of the parasite while the detection of periodate sensitive epitopes of G. lamblia copro-antigen would suggest symptomatic active giardial infection.     

Inhibitory specificity and potency of proSAAS-derived peptides toward proprotein convertase 1.

Prohormone convertase 1 (PC1), mediating the proteolytic processing of neural and endocrine precursors, is thought to be regulated by the neuroendocrine protein proSAAS. The PC1 inhibitory sequence is mostly confined within a 10-12-amino acid segment near the C terminus of the conserved human proSAAS and contains the critical KR(244) dibasic motif. Our results show that the decapeptide proSAAS-(235-244)( 235)VLGALLRVKR(244) is the most potent reversible competitive PC1-inhibitor (K(i) approximately 9 nm). The C-terminally extended proSAAS-(235-246) exhibits a 5-6-fold higher K(i) ( approximately 51 nm). The additional LE sequence at P1'-P2', resulted in a competitive substrate cleaved by PC1 at KR(244) downward arrowLE(246). Systematic alanine scanning and in some cases lysine scanning tested the contribution of each residue within proSAAS-(235-246) toward the PC1-inhibition's specificity and potency. The amino acids P1 Arg, P2 Lys, and P4 Arg are all critical for inhibition. Moreover, the aliphatic P3 Val and P5, P6, and P1' Leu significantly affect the degree of enzyme inactivation and PC1 specificity. Interestingly, a much longer N- and C-terminally extended endogenous rat proSAAS-(221-254) called little PenLen, was found to be a 3-fold less potent PC1 inhibitor with reduced selectivity but a much better substrate than proSAAS-(235-246). Molecular modeling studies and circular dichroism analysis indicate an extended and poly-l-proline II type structural conformation for proSAAS-(235-244), the most potent PC1 inhibitor, a feature not present in poor PC1 inhibitors.