Expression of the Chondroitin Sulfate Proteoglycans of Amyloid Precursor (Appican) and Amyloid Precursor-Like Protein 2

Abstract: The Alzheimer amyloid precursor (APP) protein is a member of a family of glycoproteins that includes the amyloid precursor-like proteins (APLPs). Previously, we showed that in C6 glioma cell cultures, secreted APP nexin II occurs as the core protein of a chondroitin sulfate proteoglycan (CSPG). Here, we report that among seven untransfected cell lines, expression of secreted APP CSPG was restricted to two cell lines of neural origin, namely, C6 glioma and Neuro-2a neuroblastoma (N2a) cells. Addition of dibutyryl cyclic AMP in N2a cultures, a treatment that induces the neuronal phenotype in these cells, resulted in a significant reduction in the amount of the secreted APP CSPG, although secretion of APP was only marginally affected. Growth in the presence of serum increased the size of the secreted APP CSPG, suggesting that the number and/or length of the chondroitin sulfate (CS) chains attached to the core APP varies with growth conditions. Extensive mapping with epitope-specific anti-bodies suggested that a CS chain is attached within or proximal to the Aβ sequence of APP. In contrast to the restricted expression of the APP CSPG, expression of secreted APLP2 CSPGs was observed in all cell lines examined. After chondroitinase treatment, two core proteins of ∼100 and 110 kDa were obtained that reacted with an APLP2-specific antiserum, suggesting that non-transfected cell lines contain at least two endogenous APLP2 CSPGs, probably derived by alternative splicing of the APLP2 KPI domain. The fraction of the APLP2 proteins in the CSPG form was dependent on the particular cell line examined. The proteoglycan nature of APP and APLP2 suggests that addition of the CS glycosaminoglycan chains is important for the implementation of the biological function of these proteins. However, the differential expression of these two proteoglycans suggests that their physiological roles and their possible involvement in Alzheimer's disease may differ.     

Persistent Translocation and Inhibition of Ca2+/Calmodulin-Dependent Protein Kinase II in the Crude Synaptosomal Fraction of the Vulnerable Hippocampus Following Hypoglycemia

Abstract: Alterations in the levels and activity of Ca²⁺/calmodulin-dependent protein kinase II (CaM-kinase II) were studied in the rat hippocampus during and after insulin-induced hypoglycemic coma. A permanent loss of CaM-kinase II immunohistostaining in the neuronal layer begins at 10 min of isoelectricity in the tip of the dentate gyrus and at 30-min isoelectricity in the CA1 region. The reduction in immunohistostaining in the neurites is less pronounced. Immunoreactivity of CaM-kinase II on western blots increases in the crude synaptosomal fractions and decreases in cytosolic fraction, indicative of a translocation of CaM-kinase II. The translocation persists for at least 1 day of recovery after 30 min of isoelectricity in the vulnerable hippocampus (dorsomedial hippocampus) but not in the resistant hippocampus (dorsolateral hippocampus). Calmodulin binding to western blots shows changes similar to the immunoblots. Ca²⁺/calmodulin-dependent activity of CaM-kinase II in the crude synaptosomal fraction is elevated immediately before isoelectricity and is then inhibited during and after 30 min of isoelectricity, despite the increase of CaM-kinase II immunoreactivity. This was seen in the vulnerable hippocampus. The data indicate that stimulus of translocation and inhibition of CaM-kinase II persist during the recovery phase, preceding neuronal degeneration in the vulnerable hippocampus. This may be of significance for hypoglycemia-induced neuronal death.     

Molecular cloning, sequence analysis, and characterization of a new cell wall hydrolase, CwIL, of Bacillus licheniformis

We have cloned a DNA fragment containing the gene for a cell wall hydrolase from Bacillus licheniformis FD0120 into Escherichia coli. Sequencing of the fragment showed the presence of an open reading frame (ORF; designated as cwlL), which is different from the B. licheniformis cell wall hydrolase gene cwlM, and encodes a polypeptide of 360 amino acids with a molecular mass of 38 994. The enzyme purified from the E. coli clone is an N-acetylmuramoyl-l-alanine amidase, which has a M_r value of 41 kDa as determined by SDS-polyacrylamide gel electrophoresis, and is able to digest B. licheniformis, B. subtilis and Micrococcus luteus cell walls. The nucleotide and deduced amino acid sequences of cwlL are very similar to those of ORF3 in the putative operon xpaL1-xpaL2-ORF3 in B. licheniformis MC14. Moreover, the amino acid sequence homology of CwlL with the B. subtilis amidase CwlA indicates two evolutionarily distinguishable regions in CwlL. The sequence homology of CwlL with other cell wall hydrolases and the regulation of cwlL are discussed.     

Gibberellin-enhanced sugar accumulation in growing subhooks of etiolated Pisum sativum seedlings. Effects of gibberellic acid, indoleacetic acid and cycloheximide on invertase activity, sugar accumulation and growth

The possible involvement of invertase in the action of gibberellic acid (GA) on stimulating sugar accumulation in growing subhooks of Alaska pea ( Pisum sativum L. cv. Alaska) was studied. GA and indoleacetic acid (IAA) stimulated elongation growth to a similar extent. GA, in contrast to IAA, increased the amount of soluble sugars in the subhook. GA substantially increased invertase activity whereas IAA did not. These results suggest that the mode of action of GA and IAA differs, although both stimulate pea subhook growth.
Cycloheximide (CH) inhibited the effect of GA on invertase activity, accumulation of soluble sugars, and elongation growth. Good correlations were found between invertase activity, the amount of soluble sugars and growth. The results suggest that GA-induced enhancement of sugar accumulation in the subhook cells is dependent on increased invertase activity. The sugar accumulated in the subhook may be involved in growth promotion by GA.     

Oviposition preference for spherical surfaces is shared among multiple Drosophila species except D. melanogaster (Diptera: Drosophilidae)

Oviposition preference for spherical substrates has been reported in some insects but not in Drosophila species until the recent finding that Drosophila suzukii preferentially lays eggs on spherical surfaces with a smaller radius, whereas D. melanogaster does not. This finding raised two questions: (i) Was this trait specifically acquired in D. suzukii or lost in D. melanogaster? (ii) In the latter case, is it due to the long-term laboratory culture using oviposition substrates with flat surfaces? To answer these questions, we examined the oviposition preference of three Drosophila species using the stocks recently established from wild individuals. As with D. suzukii, D. simulans and D. takahashii showed significant preference for spherical surfaces with a smaller radius, suggesting that this trait is shared by multiple Drosophila species. In contrast, D. melanogaster did not show any preference for either smaller or larger radii, showing that the preference already has been lost in the natural population of D. melanogaster. It may be possible that the loss of oviposition preference for spherical surfaces is involved in the evolutionary process of D. melanogaster becoming a human commensal.     

Molecular cloning,sequence analysis,and characterization of a new cell wall hydrolase,CwIL, of Bacillus licheniformis

We have cloned a DNA fragment containing the gene for a cell wall hydrolase from Bacillus licheniformis FD0120 into Escherichia coli. Sequencing of the fragment showed the presence of an open reading frame (ORF; designated as cwlL), which is different from the B. licheniformis cell wall hydrolase gene cwlM, and encodes a polypeptide of 360 amino acids with a molecular mass of 38 994. The enzyme purified from the E. coli clone is an N-acetylmuramoyl-l-alanine amidase, which has a M_r value of 41 kDa as determined by SDS-polyacrylamide gel electrophoresis, and is able to digest B. licheniformis, B. subtilis and Micrococcus luteus cell walls. The nucleotide and deduced amino acid sequences of cwlL are very similar to those of ORF3 in the putative operon xpaL1-xpaL2-ORF3 in B. licheniformis MC14. Moreover, the amino acid sequence homology of CwlL with the B. subtilis amidase CwlA indicates two evolutionarily distinguishable regions in CwlL. The sequence homology of CwlL with other cell wall hydrolases and the regulation of cwlL are discussed.     

Genetic structure, isolation and characterization of a Bacillus licheniformis cell wall hydrolase.

Summary A DNA fragment containing the gene for a cell wall hydrolase of Bacillus licheniformis was cloned into Escherichia coli. Sequencing of the fragment showed the presence of an open reading frame which encodes a polypeptide of 253 amino acids with a molecular mass of 27 513. The gene was designated as cwlM, for cell wall lysis. The deduced amino acid sequence indicated that there is a repeated sequence consisting of 33 amino acid residues in the C-terminal region. Deletion of the C-terminal region did not lead to any loss of cell wall lytic activity. The gene product purified from E. coli cells harboring a cwlM-bearing plasmid exhibited a M _r value of 29 kDa on SDS-polyacrylamide gels, and characterization of the specific substrate bond cleaved by CWLM indicated that the enzyme is an N-acetylmuramoyl-l-alanine amidase (EC 3.5.1.28). The enzyme hydrolyzed the cell wall of Micrococcus luteus more efficiently than those of B. licheniformis and B. subtilis, but the truncated CWLM (lacking the C-terminal region) had lost this preference. CWLM prepared from B. subtilis cells harboring a plasmid containing cwlM had a similar M _r value to that from E. coli. Amino acid sequence homologies between CWLM and other amidases, and their protein structures are discussed.     

Functional Structure of the Oxygen-Evolving Unit of Photosystem II as Determined by Radiation Inactivation

The size of the complex that is essential for the electron-transferactivity from the oxygen-evolving center to the secondary electronacceptor, Q_B, is about 250 kDa, as determined by target-sizeanalysis after the radiation inactivation of functions of photosystemII (PS II). Inter-Chl tranfer of excitation energy was insensitiveto the radiation inactivation indicating that the masses ofCP47, CP43, and light-harvesting Chi a/b proteins are not includedin the functional size of the oxygen-evolving PS II complex.The transfer of electrons from the secondary electron donor,Z, to Q_B was catalyzed by a unit of only 65 kDa. The sizes ofthe complexes involved in these light-induced functions of PSII were dependent on the intensity of actinic light. Under saturatingintensities of light, the functional size of the complex fortransfer of electrons from Z to Q_B was 38 kDa, with a correspondingdecrease in the size of the oxygen-evolving PS II from 250 kDato 125 kDa [Takahashi, Mano and Asada (1985) Plant Cell Physiol.26: 383]. The protein of about 30 kDa functions in the photoreductionof the pheophytin molecule, as well as in the electron transferfrom Z to Q_A. Under low-intensity light, complexes having thesame sizes as those of the basal functional complexes undersaturating-intensity light are further required, probably tostabilize separated charges in the PS II reaction center andthe oxygen-evolving center. (Received June 20, 1990; Accepted September 18, 1990)