Antibodies to Cholinergic Neurons in Alzheimer''s Disease

Alzheimer's disease (AD) is associated with degenerative changes in nuclei of the basal forebrain which provide most of the cholinergic input to the cortex and hippocampus and with a reduction in presynaptic cholinergic parameters in these areas. Although the etiology and pathogenesis of AD are not known, several reports indicate the involvement of immunological mechanisms. In the present work we examined the existence of antibodies in sera of AD patients that bind specifically to cholinergic neurons. As antigens we employed the purely cholinergic electromotor neurons of the electric fish Torpedo which are chemically homogeneous and cross-react antigenically with human and other mammalian cholinergic neurons. Our findings show that immunoglobulins from sera of AD patients bind to a specific antigen (molecular mass 200 kilodaltons) in the cell bodies and axons of Torpedo electromotor neurons and that the levels of such antibodies are significantly higher in AD patients than in controls. The possible role of these antibodies in the cholinergic dysfunction in AD and their diagnostic potential are discussed.     

Effects of Extracellular Potassium on Glycogen Stores of Astrocytes In Vitro

Astrocyte-enriched and meningeal cell cultures of the rat cerebral cortex were prepared, and their glycogen content was measured after 10-90 min under control (2.5 mM) concentrations of potassium after prefeeding with 20 mM glucose. No net change in glycogen level was noted in either culture over this period. Cell cultures were then exposed to increased concentrations of potassium (5, 10, and 15 mM), and their glycogen content was measured after 10-90 min. Both types of cell culture showed complex and variable changes in glycogen content. In general, increased potassium concentrations caused astrocyte glycogen stores to be reduced at physiological increases of potassium levels (from 2.5 to 5 mM and above), although a period of resynthesis was evident at all potassium concentrations. Meningeal cell glycogen levels were highly variable and only affected by high (10 and 15 mM) levels of potassium. These results are discussed with respect to the theory that changes in the external potassium concentration caused by neuronal activity might act as a signal controlling astrocyte glycogen stores.     

Novel hemoglobin components and their amino acid sequences from the crab-eating macaque (Macaca fascicularis)

Summary We found two types of hemoglobin, T and R, from the crab-eating macaque and compared those to A and Q previously reported. The 22 animals studied showed six different phenotypes, A, R, QA, QT, QAT, and QAR. Analysis of the complete amino acid sequences for the chains of hemoglobins Q, A, T, and R revealed that amino acids at four positions, 8, 55, 71, and 78 from the N-terminal, are variable. In the ^A chain, Thr, Val, Gly, and Gln occupy these positions, and in the ^Q chain the analogous amino acids are Thr, Val, Asp, and Gln, respectively. In the newly found ^T chain they are Thr, Val, Gly, and His; and in the ^R chain, they are Ser, Ile, Gly, and His, respectively. Two amino acids (8 Thr and 79 Gln) in ^A of the crab-eating macaque were found to be different from those in the chain of the Japanese macaque.     

On the appearance and role of a spacer group in the protein amino acids

Summary Of the 20 protein amino acids, 16 have a methylene group at the position, and a further three bear a methine group. No aromatic, carboxamido, carboxylic carbon, or hetero atoms are attached directly to the carbon, but they are separated by this methylene or occasionally by a longern-alkylene spacer group. Therefore, the structure of the protein amino acids should rather be formulated as H₂N–CH((CH₂)_n–R)–COOH instead of the generally accepted H₂N–CH(R)–COOH. The appearance of and the role played by the spacer group are discussed in an evolutionary context. It is suggested that the spacer group appeared as a result of prebiotic selection, based on the relative abundance, racemization rate, and suitability for thermal polymerization of the protein amino acids and their homologs with various spacer group lengths. At the biotic level of evolution the requirements for ribosomal polymerization, as well as the abilities of polypeptides to maintain a stable and flexible threedimensional structure and to bind ligands are considered and are proposed to have been responsible for the possible exclusion of longer spacer groups. It is concluded that the general role of the spacer group is to ensure the uniformity of the constant regions H₂N–CH(-)–COOH and the individuality of the R contact groups by spatially separating them.     

Organization and structure of Volvox beta-tubulin genes

Summary Genomic clones encoding two Volvox -tubulin genes have been isolated and shown to represent the only two -tubulin genes in the genome. Restriction fragment length polymorphism analysis was used to demonstrate that the two genes are genetically linked. One of these genes was sequenced and the mRNA start site(s) determined by primer extension. A comparison of its sequence to those of the two -tubulin genes of Chlamydomonas revealed: (1) a high degree of conservation of the coding region, with the predicted amino acid sequence differing only in the C-terminal residue; (2) extensive sequence conservation in the 5 untranslated leader region and a 16 bp (putative regulatory) sequence in the promoter region; (3) the same number and location of introns, with a short region of homology in intron 1, but little significant homology in introns 2 and 3.     

Secretion and export of IGF-1 in Escherichia coli strain JM101

Summary The processing of LamB-IGF-1 fusion protein and the export of processed IGF-1 (insulin-like growth-factor-1) into the growth medium was examined in the Escherichia coli host strain, JM101. Several strain or plasmid modifications were tried to increase export of periplasmic (Processed) IGF-1 into the growth medium of JM101. These included: (1) use of a lon null mutant strain to increase accumulation levels of unprocessed LamB-IGF-1 fusion protein; (2) use of an alternative drug resistance marker on the expression plasmid rather than beta-lactamase, thereby reducing any competition for processing of LamB-IGF-1 by signal peptidase; (3) examination of whether phage M13 gene III protein expression caused more periplasmic IGF-1 to be exported into the growth medium due to increased outer membrane permeability; and (4) examination of the effect of E. coli or yeast optimized IGF-1 codons. None of these strain or plasmid modifications caused any significant increase in export of IGF-1 into the growth medium of JM101. Solubility studies of LamB-IGF-1 and processed IGF-1 showed that virtually all of the LamB-IGF-1 and IGF-1 remaining within the cell after a 2 h induction period was insoluble. This implied that only soluble LamB-IGF-1 was processed to IGF-1 and that only soluble IGF-1 was exported into the growth medium. Taken together, the results indicated that LamB-IGF-1 and IGF-1 solubility were the limiting factors in secretion of IGF-1 into the periplasm and export of IGF-1 into the growth medium.     

Correlation of physical maps and some genetic functions in the genomes of the kappa-theta phage family of Bacillus licheniformis

Summary Natural recombinant genomes between several, phenotypically distinct forms of phages and were isolated and characterized by DNA restriction fragment mapping and electron microscopic heteroduplex analysis. The phenotypes of the recombinants were correlated with the physical maps of the genomes, and several genetic functions were therfore defined and mapped. All genes necessary for the assembly of infectious virus particles map in a contiguous tract of DNA comprising about 20 kb, or nearly one third of the genome length. No DNA homology occurs within these domains of the two genomes, so that homologous recombination does not take place here and phenotypic mixing of the phages is eo ipso excluded. Other regions of heterology contain regulatory genes responsible for the lytic or temperate character of the phages, and for exclusion of phage by .     

Bacteriophage lambda DNA packaging: A mutant terminase that is independent of integration host factor

Summary ⁺ is able to grow in Escherichia coli cells lacking integration host factor (IHF), producing a burst of approximately 25% that produced in IHF⁺ cells. In vitro, however, we find that the DNA packaging enzyme terminase is strongly dependent on IHF in both cos cleavage reactions and DNA packaging reactions. The cos59 mutation renders dependent on IHF in vivo. The cos59 mutation is a deletion of 3 base pairs at the XmnI site in the cohesive end site (cos) of . Variants of cos59 that were able to grow in the absence of IHF were isolated and found to carry a mutation, called ms1, in the Nu1 gene, which codes for the small subunit of terminase. The Nu1ms1 mutation results in a change of the 40th amino acid of the Nu1 gene product from leucine to phenylalanine. The Nu1ms1 terminase was independent of IHF in packaging reactions in vitro. The results indicate that the mutation either renders terminase: (1) able to utilize some host protein other than IHF, or (2) totally independent of host factors.     

2-Aminopurine and 5-bromouracil induce alleviation of type I restriction in Escherichia coli: Mismatches function as inducing signals?

Summary The EcoK restriction of unmodified phage is 1000-fold alleviated in Escherichia coli grown in the presence of base analogs 2-aminopurine (2AP) and 5-bromouracil (5BU). 2AP treatment of bacteria affects specificially the type I restriction systems (EcoA, EcoB, EcoD and EcoK) and does not influence type II (EcoRI) and type III (EcoP1) restriction. 2AP-induced alleviation of restriction occurs in bacteria which are deficient in the SOS response (recA and lexA) and mismatch repair (mutH, mutL and mutS) and can be distinguished from the alleviation of restriction observed in dam ^- strains. We suggest that mismatches induced by 2AP and 5BU may function as an inducing signal for the alleviation of restriction observed in the presence of base analogs.     

The promoter of the beta-glucosidase gene from Kluyveromyces fragilis contains sequences that act as upstream repressing sequences in Saccharomyces cerevisiae

Summary The relationship between the promoter length of the Kluyveromyces fragilis -glucosidase gene and the level of its expression in Saccharomyces cerevisiae was studied by gene fusion between deleted promoter fragments of various lengths and the promoterless -galactosidase gene of Escherichia coli. The removal of a region from position-425 to-232 led to a tenfold increase in the expression of the gene. The same results were obtained for the reconstructed -glucosidase gene with the same promoter length. It is likely that the deletion of this part of the promoter removes negative regulatory elements which are functional in Saccharomyces cerevisiae. This increase in activity is the main event which may explain the high increase in gene expression (60-fold) previously observed for an upstream deletion obtained during subcloning experiments of the -glucosidase gene. It is also shown that the expression of the gene greatly depends upon the nature of the recipient strain, the growth phase of the cell and that of the vector carrying it.