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Although amyloid ββ (Aββ) deposition has been a hallmark of Alzheimer's disease (AD), the absence of a phenotype in the ββ amyloid precursor protein (APP) knockout mouse, tends to detract our attention away from the physiological functions of APP. Although much attention has been focused on the neurotoxicity of Aββ, many studies suggest the involvement of APP in neuroplasticity. We found that secreted amyloid precursor protein (sAPP) increased the differentiation of human neural stem cells (hNSCs) in vitro, while an antibody-recognizing APP dose-dependently inhibited these activities. With a high dose of sAPP treatment or wild-type APP gene transfection, hNSCs were differentiated into astrocytes rather than neurons. In vivo, hNSCs transplanted into APP-transgenic mouse brain exhibited glial differentiation rather than neural differentiation. Our results suggest that APP regulates neural stem cell biology in the adult brain, and that altered APP metabolism in Down syndrome or AD may have implications for the pathophysiology of these diseases.

Abstract. The beta amyloid peptide is the major component of the neuritic plaques, the characteristic lesions in Alzheimer's disease. Mutations in three genes (APP, PS-1, and PS-2) cause familial Alzheimer's disease by alteration of the rate of generation of amyloid peptide or the length of this peptide. However, in the 90% non-familial cases, other factors play a major pathogenetic role. These include the apolipoprotein E genotype, the "plaque-associated" proteins promoting the formation of toxic fibrillar aggregates or the chronic inflammatory responses. The aim of this review is to explain the steps in the complex cascade leading to Alzheimer's disease and, based on this, to report the current efforts to intervene in these different pathophysiological events in order to prevent progression of Alzheimer's disease. Whereas acetylcholine substitution is currently used in clinical practice, future therapeutical strategies to combat Alzheimer's disease may include anti-inflammatory treatments, vaccination against beta amyloid peptide, or treatment with cholesterol-lowering drugs.

Glial activation contiguous to deposits of amyloid peptide (Aβ) is a characteristic feature in Alzheimer's disease. We performed complementary in vitroand in vivoexperiments to study the extent, kinetics, and mechanisms of microglial generation of nitric oxide (NO) induced by challenge with Aβ. We showed that Aβ fibrils dose‐dependently induced a marked release of stable metabolites of NO in vivothat was strikingly similar regarding extent and temporal profile to the one in the parallel designed microglial cell culture experiments. However, costimulation with interferon γ, which was a prerequisite for Aβ‐induced NO generation in vitro, was not required in vivo, demonstrating that factors are present in the living brain that activate glial cells synergistically with Aβ. Therefore, in Alzheimer's disease, deposits of Aβ fibrils alone may be sufficient to induce a chronic release of neurotoxic microglial products, explaining the progressive neurodegeneration associated with this disease. Our observation that systemic administration of selective iNOS inhibitors abolishes Aβ‐induced NO generation in vivomay have implications for therapy of Alzheimer's disease.–Ishii, K., Muelhauser, F., Liebl, U., Picard, M., Kühl, S., Penke, B., Bayer, T., Wiessler, M., Hennerici, M., Beyreuther, K., Hartmann, T., Fassbender, K. Subacute NO generation induced by Alzheimer's β‐amyloid in the living brain: reversal by inhibition of the inducible NO synthase. FASEB J.14, 1485–1489 (2000)

Alternative splicing of exon 15 of the amyloid precursor protein (APP) pre-mRNA generates two APP isoform groups APP(ex15) (containing exon 15) and L-APP (without exon 15), which show a cell-specific distribution in non-neuronal cells and neurons of rat. Both APP isoforms differ in regard to functional properties like post-translational modification, APP secretion, and proteolytic production of Abeta peptide from APP molecules. Since Abeta generation is an important factor in the development of Alzheimer's disease, one could anticipate that these major APP isoforms might contribute differentially to the mechanisms underlying neurodegeneration in Alzheimer's disease. In this study, we established an APP minigene system in a murine cell system to identify cis-acting elements controlling exon 15 recognition. A 12. 5-kilobase pair genomic fragment of the murine APP gene contained all cis-regulatory elements to reproduce the splicing pattern of the endogenous APP transcripts. By using this approach, two intronic cis-elements flanking exon 15 were identified that block the inclusion of exon 15 in APP transcripts of non-neuronal cells. Point mutation analysis of these intronic regions indicated that pyrimidine-rich sequences are involved in the splice repressor function. Finally, grafting experiments demonstrated that these regulatory regions cell-specifically enhance the blockage of a chimeric exon in the non-neuronal splicing system.

beta-Catenin has previously been shown to interact with presenilin 1 (PS1) in transfected cells. Here we report that beta-catenin co-immunoprecipitates with the endogenous C-terminal fragment of presenilin 1 (PS1-CTF) but not with the endogenous CTF of presenilin 2 (PS2-CTF) in H4 human neuroglioma cells. During staurosporine (STS)-induced cell death, beta-catenin and PS1-CTF undergo a caspase-mediated cleavage. After 12 h of STS treatment, the beta-catenin.PS1-CTF interaction is abrogated. While PS1-CTF immunoprecipitated with all caspase-cleaved species of beta-catenin, beta-catenin holoprotein did not co-immunoprecipitate with the "alternative" caspase-derived PS1-CTF (PS1-aCTF). Thus, the abrogation of the beta-catenin.PS1-CTF complex was due to caspase cleavage of PS1-CTF. beta-Catenin co-immunoprecipitated with PS1-NTF, but only when PS1-NTF was associated with PS1-CTF. Even though PS1-NTF.CTF complex stability was not altered by caspase cleavage, its ability to bind beta-catenin was abolished. Thus, while the PS1-NTF.CTF complex is preserved after caspase cleavage, it may no longer be fully functional.

MUTATIONS in the presenilin 1 (PS1) gene are responsible for ∼50 of early onset autosomal-dominant Alzheimer's disease cases. A PCR based mutation detection method, chemical cleavage of mismatch, was used to detect a novel PS1 mutation in the coding sequence of the PS1 gene. Sequencing confirmed a T to C transition altering a leucine to proline at codon 219 of the PS1 gene. This is a novel mutation in exon 7 of the PS1 gene occurring outside the transmembrane regions of IV and V.

Zn(II) and Cu(II) precipitate Abeta in vitro into insoluble aggregates that are dissolved by metal chelators. We now report evidence that these biometals also mediate the deposition of Abeta amyloid in Alzheimer's disease, since the solubilization of Abeta from post-mortem brain tissue was significantly increased by the presence of chelators, EGTA, N,N,N',N'-tetrakis(2-pyridyl-methyl) ethylene diamine, and bathocuproine. Efficient extraction of Abeta also required Mg(II) and Ca(II). The chelators were more effective in extracting Abeta from Alzheimer's disease brain tissue than age-matched controls, suggesting that metal ions differentiate the chemical architecture of amyloid in Alzheimer's disease. Agents that specifically chelate copper and zinc ions but preserve Mg(II) and Ca(II) may be of therapeutic value in Alzheimer's disease.

A distinguishing feature of Alzheimer's disease (AD) is the deposition of amyloid plaques in brain parenchyma. These plaques arise by the abnormal accumulation of beta A4, a proteolytic fragment of amyloid precursor protein (APP). Despite the fact that neurons are dramatically affected in the course of the disease, little is known about the neuronal processing of APP. To address this question we have expressed in fully mature, synaptically active rat hippocampal neurons, the neuronal form of human APP (APP695), two mutant forms of human APP associated with AD, and the mouse form of APP (a species known not to develop amyloid plaques). Protein expression was achieved via the Semliki Forest Virus system. Expression of wild type human APP695 resulted in the secretion of beta A4‐amyloid peptide and the intracellular accumulation of potential amyloidogenic and non‐amyloidogenic fragments. The relative amount of amyloid‐containing fragments increased dramatically during expression of the clinical mutants, while it decreased strongly when the mouse form of APP was expressed. ‘Humanizing’ the rodent APP sequence by introducing three mutations in the beta A4‐region also led to increased production of amyloid peptide to levels similar to those obtained with human APP. The single Gly601 to Arg substitution alone was sufficient to triple the ratio of beta A4‐peptide to non‐amyloidogenic p3‐peptide. Due to the capacity of these cells to secrete and accumulate intracellular amyloid fragments, we hypothesize that in the pathogenesis of AD there is a positive feed‐back loop where neurons are both producers and victims of amyloid, leading to neuronal degeneration and dementia.(ABSTRACT TRUNCATED AT 250 WORDS)

Idiotope Ac38, a V region determinant of the lambda 1 chain‐bearing, germ line encoded antibody B1‐8, is expressed at high frequency (approximately 1/40) in lambda 1 chain‐bearing B cells. Here, we describe the isolation of lambda‐positive hybridomas from C57BL/6 mice which had been immunized with antibody Ac38, the antibody recognizing idiotope Ac38. In Northern blot analysis, mRNA isolated from 10 such hybridomas hybridizes with a cDNA probe from the VH gene expressed in the cell line B1‐8. Amino acid sequence analysis of the VH regions of four of the hybridoma proteins reveals that they are all derived from related, though distinct, germ line VH genes. In one case the sequence data suggest that extensive somatic mutation has taken place. Only one of the four sequences derives from the same VH gene that is expressed in the cell line B1‐8. Together with earlier evidence, the present data demonstrate that the Ac38 idiotope is a marker for at least five VH and three D region genes in the C57BL/6 germ line. This explains the high frequency at which this idiotope is expressed in the B cell population. In addition, our sequence determinations identify two VH genes in the C57BL/6 strain which are closely related (and possibly allelic) to two known BALB/c VH genes. One of these genes is the gene expressed in the BALB/c myeloma MOPC 104E.

The protein component of Alzheimer's disease amyloid [neurofibrillary tangles (NFT), amyloid plaque core and congophilic angiopathy] is an aggregated polypeptide with a subunit mass of 4 kd (the A4 monomer). Based on the degree of N‐terminal heterogeneity, the amyloid is first deposited in the neuron, and later in the extracellular space. Using antisera raised against synthetic peptides, we show that the N terminus of A4 (residues 1‐11) contains an epitope for neurofibrillary tangles, and the inner region of the molecule (residues 11‐23) contains an epitope for plaque cores and vascular amyloid. The non‐protein component of the amyloid (aluminum silicate) may form the basis for the deposition or amplification (possible self‐replication) of the aggregated amyloid protein. The amyloid of Alzheimer's disease is similar in subunit size, composition but not sequence to the scrapie‐associated fibril and its constituent polypeptides. The sequence and composition of NFT are not homologous to those of any of the known components of normal neurofilaments.

The mouse hybridoma line B1‐8.delta 1 secretes a monoclonal IgD, lambda 1 anti‐(4‐hydroxy‐3‐nitrophenyl)acetyl (NP) antibody with defined idiotypic determinants. Two spontaneous V‐region variants (B1‐8.V1/V2) with altered idiotope pattern were selected and the structural variation was located to the variable region of the heavy chain. The amino acid sequences of the B1‐8. delta 1 and variant heavy chain V regions were determined. The variant VH regions are identical. Wild‐type and variant VH regions differ in 10 positions. Single amino acid exchanges are found in the first and second framework at positions 20 and 43. The majority of replacements (eight substitutions) is clustered in the second complementary‐determining region (CDR 2). There are no differences in CDR 1 and CRD 3 and the JH region. The variant, which at first glance appears to have undergone a series of point mutations, arose by recombination, possibly gene conversion, between the rearranged VDJ gene of the wild‐type (B1‐8.delta 1) and a neighbouring germ line VH gene encoding all of the substitutions.

We have analysed the axonal sorting signals of amyloid precursor protein (APP). Wild‐type and mutant versions of human APP were expressed in hippocampal neurons using the Semliki forest virus system. We show that wild‐type APP and mutations implicated in Alzheimer's disease and another brain beta‐amyloidosis are sorted to the axon. By analysis of deletion mutants we found that the membrane‐inserted APP ectodomain but not the cytoplasmic tail is required for axonal sorting. Systematic deletions of the APP ectodomain identified two regions required for axonal delivery: one encoded by exons 11–15 in the carbohydrate domain, the other encoded by exons 16–17 in the juxtamembraneous beta‐amyloid domain. Treatment of the cells with the N‐glycosylation inhibitor tunicamycin induced missorting of wild‐type APP, supporting the importance of glycosylation in axonal sorting of APP. The data revealed a hierarchy of sorting signals on APP: the beta‐amyloid‐dependent membrane proximal signal was the major contributor to axonal sorting, while N‐glycosylation had a weaker effect. Furthermore, recessive somatodendritic signals, most likely in the cytoplasmic tail, directed the protein to the dendrites when the ectodomain was deleted. Analysis of detergent solubility of APP and another axonally delivered protein, hemagglutinin, demonstrated that only hemagglutinin formed CHAPS‐insoluble complexes, suggesting distinct mechanisms of axonal sorting for these two proteins. This study is the first delineation of sorting requirements of an axonally targeted protein in polarized neurons and indicates that the beta‐amyloid domain plays a major role in axonal delivery of APP.

Alzheimer's disease, which is characterized by amyloid plaques and neurofibrillary tangles, may be attributed to the abnormal expression of gene(s) located on human chromosome 21. Genetic linkage studies have narrowed the region of candidate genes to 21q11.2–21q22 of the long arm of this chromosome. Several single copy sequences within this region, including the amyloid precursor protein (APP), have been mapped to mouse chromosome 16. Reliable strategies exist for breeding Trisomy 16 mice. However, the consequences of developmental overexpression of genes on chromosome 16 have not been previously investigated, because of the lethal effects of this aneuploidy during gestation. In the present report, we employ neural transplantation to study long‐term survival and pathogenesis in Trisomy 16 central nervous system tissues. Immunocytochemical staining with antiserum raised against the synthetic APP, beta‐A4 and alpha 1‐antichymotrypsin revealed numerous densely stained cells within hippocampal grafts of Trisomy 16 mice. Similarly, a population of grafted cells were positively stained following incubation with an antiserum raised against components of the pathological neurofibrillary tangle and with the monoclonal antibodies Tau 6.423 and ubiquitin.

The proteasome is a multicatalytic proteinase complex composed of nonidentical protein subunits. We have isolated a cDNA clone encoding the 35 kd proteasome subunit of Drosophila melanogaster and propose the designation PROS‐35 for the corresponding gene. The deduced amino acid sequence reveals a region of striking homology to a tyrosine phosphorylation site of viral and cellular proteins suggesting a potential regulatory function for the 35 kd subunit within the proteinase complex. Immunocytochemical experiments reveal a tissue‐dependent differential distribution of the proteasome between the nucleus and cytoplasm. In addition developmental analysis shows that the proteasome is highly expressed in the CNS of stage‐16 embryos and in cardia, ventriculus and ovaries of adult flies. These data suggest a tissue‐ and development‐dependent distribution of the proteasome in D. melanogaster.

The promoter of the gene for the human precursor of Alzheimer's disease A4 amyloid protein (PAD gene) resembles promoters of housekeeping genes. It lacks a typical TATA box and shows a high GC content of 72% in a DNA region that confers promoter activity to a reporter gene in an in vivo assay. Transcription initiates at multiple sites. Sequences homologous to the consensus binding sites of transcription factor AP‐1 and the heat shock control element binding protein were found upstream of the RNA start sites. Six copies of a 9‐bp‐long GC‐rich element are located between positions −200 and −100. A protein–DNA interaction could be mapped to this element. The 3.8 kb of the 5′ region of the PAD gene include two Alu‐type repetitive sequences. These findings suggest that four mechanisms may participate in the regulation of the PAD gene and could be of relevance for the progression of amyloid deposition in Alzheimer's disease.

Antibodies induced against mammalian single‐stranded DNA binding protein (ssDBP) UP I were shown to be cross‐reactive with most of the basic hnRNP core proteins, the main constituents of 40S hnRNP particles. This suggested a structural relationship between both groups of proteins. Using the anti‐ssDBP antibodies, a cDNA clone (pRP10) was isolated from a human liver cDNA library in plasmid expression vector pEX1. By DNA sequencing this clone was shown to encode in its 949 bp insert the last 72 carboxy terminal amino acids of the ssDBP UP I. Thereafter, an open reading frame continued for another 124 amino acids followed by a UAA (ochre) stop codon. Direct amino acid sequencing of a V8 protease peptide from hnRNP core protein A1 showed that this peptide contained at its amino terminus the last 11 amino acids of UP I followed by 19 amino acids which are encoded by the open reading frame of cDNA clone pRP10 immediately following the UP I sequence. This proves that ssDBP UP I arises by proteolysis from hnRNP core protein A1. This finding must lead to a re‐evaluation of the possible physiological role of UP I and related ssDBPs. The formerly assumed function in DNA replication, although not completely ruled out, should be reconsidered in the light of a possible alternative or complementary function in hnRNA processing where UP I could either be a simple degradation product of core protein A1 (as a consequence of controlling the levels of active A1) or may continue to function as an RNA binding protein which has lost the ability to interact with the other core proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Subunit II of cytochrome oxidase is encoded by the mitochondrial OXI1 gene in Saccharomyces cerevisiae. The temperature‐sensitive nuclear pet mutant ts2858 has an apparent higher mol. wt. subunit II when analyzed on lithium dodecylsulfate (LiDS) polyacrylamide gels. However, on LiDS‐6M urea gels the apparent mol. wt. of the wild‐type protein exceeds that of the mutant. Partial revertants of mutant ts2858 that produce both the wild‐type and mutant form of subunit II were isolated. The two forms of subunit II differ at the N‐terminal part of the molecule as shown by constructing and analyzing nuclear ts2858 and mitochondrial chain termination double mutants. The presence of the primary translation product in the mutant and of the processed form in the wild‐type lacking 15 amino‐terminal residues was demonstrated by radiolabel protein sequencing. Comparison of the known DNA sequence with the partial protein sequence obtained reveals that six of the 15 residues are hydrophilic and, unlike most signal sequences, this transient sequence does not contain extended hydrophobic parts. The nuclear mutation ts2858 preventing post‐translational processing of cytochrome oxidase subunit II lies either in the gene for a protease or an enzyme regulating a protease.

The cloned cDNA encoding the rat cognate of the human A4 amyloid precursor protein was isolated from a rat brain library. The predicted primary structure of the 695‐amino acid‐long protein displays 97% identity to its human homologue shown previously to resemble an integral membrane protein. The protein was detected in rodent brain and muscle by Western blot analysis. Using in situ hybridization and immunocytochemistry on rat brain sections, we discovered that rat amyloidogenic glycoprotein (rAG) and its mRNA are ubiquitously and abundantly expressed in neurons indicating a neuronal original for the amyloid deposits observed in humans with Alzheimer's disease (AD). The protein appears in patches on or near the plasma membranes of neurons suggesting a role for this protein in cell contact. Highest expression was seen in rat brain regions where amyloid is deposited in AD but also in areas which do not contain deposits in AD. Since amyloid deposits are rarely observed in rat brain, we conclude that high expression of AG is not the sole cause of amyloidosis.

The precursor of the Alzheimer's disease‐specific amyloid A4 protein is an integral, glycosylated membrane protein which spans the bilayer once. The carboxy‐terminal domain of 47 residues was located at the cytoplasmic site of the membrane. The three domains following the transient signal sequence of 17 residues face the opposite side of the membrane. The C‐terminal 100 residues of the precursor comprising the amyloid A4 part and the cytoplasmic domain have a high tendency to aggregate, and proteinase K treatment results in peptides of the size of amyloid A4. This finding suggests that there is a precursor‐product relationship between precursor and amyloid A4 and we conclude that besides proteolytic cleavage other events such as post‐translational modification and membrane injury are primary events that precede the release of the small aggregating amyloid A4 subunit.

Scrapie‐associated fibrils (SAF) are unique structures characteristic of the group of unconventional slow infections which includes scrapie and Creutzfeldt‐Jakob disease. A major component of hamster fibrils has been described as a protease‐resistant glycoprotein with an apparent mol. wt of 27,000‐30,000 (PrP27‐30). However, we report here that if fibrils are prepared by procedures designed to minimise proteolysis the PrP proteins co‐purifying with hamster SAF have mol. wts of 33,000‐35,000 (PrP33‐35) and 26,000‐29,000 (PrP26‐29). We find a Lys‐Lys‐Arg‐Pro‐Lys sequence at the amino terminus of these SAF proteins, that is absent from PrP27‐30, and which has recently been predicted to be the N‐terminal sequence of the native PrP protein of uninfected brain. The major SAF protein (PrP33‐35) and its normal brain homologue are shown to have the same apparent mol. wt and ionic charge distribution by two‐dimensional gel analysis, silver staining and immunoblotting. These results support our view that PrP33‐35 and the normal brain PrP protein may have the same covalent structure, and that the PrP protein is recruited into these amyloid‐like SAF or into association with a non‐protein component of SAF by an irreversible event initiated directly or indirectly by scrapie infection.

In an attempt to assign secondary structure elements to protein primary structures with antibodies, we synthesized a model peptide (beta‐peptide: TVTVTDPGQTVTY) with a putative beta‐turn structure and analysed the anti‐peptide antibodies for their specificity towards the turn sequence. At least 50% of the peptide fraction adopts the intended conformation of a beta‐turn (DPGQ) inserted between the two segments of an antiparallel beta‐sheet structure. The specific anti‐beta‐peptide antibodies of the hyperimmune response bind the beta‐turn containing epitope of the immunogenic beta‐peptide with a three orders of magnitude higher affinity than the synthetic control peptide (Gly‐peptide: GGGGGDPGQGGGG). The affinity of the antibodies with specificity for the beta‐turn region increases from the primary to the hyperimmune response. Therefore, probing of secondary structure elements, i.e., of individual beta‐turn regions, by anti‐peptide antibodies now seems feasible for proteins of known sequence and may result in sequence assignments of secondary structures.

Scrapie‐associated fibril protein (SAF‐protein) extracted from infectious scrapie‐associated fibrils (SAF) isolated from scrapie hamster brains is not infectious. SAF‐protein is composed of various mol. wt. species of glycoproteins differing in carbohydrate content rather than amino acid composition. The N‐linked carbohydrate chains represent approximately 40‐60% of the mol. wt. of SAF‐protein. The deglycosylated SAF‐protein has a surprisingly low mol. wt. of approximately 7 kd, representing approximately 55 amino acid residues. This size and chemical analyses indicate that SAF‐protein is an amyloid‐type of protein. The simplest explanation for the available data is that SAF‐polypeptide is very likely not to be part of the scrapie agent but that it is, like other amyloid proteins, derived from host‐encoded proteins and not infectious. It is suggested that the infectivity of fractions rich in SAF is due to co‐purification of scrapie virus and SAF caused by the high carbohydrate content of SAF‐protein.

Human monoclonal antibodies (MoAbs) directed against the blood group Rh(D), (c), and (E) antigens produced by Epstein-Barr virus (EBV)- transformed lymphoblastoid cell lines have been used to characterize the Rh components of human red cell membranes and to determine whether the Rh(D), (c), and (E) epitopes are carried by distinct polypeptides. After immunoprecipitation with the anti-Rh(D) antibody and preparative gel electrophoresis, a homogenous preparation of the Rh(D) protein was obtained from two different erythrocyte samples (Blo and D--/D--), which have an increased density of Rh(D) antigen. Both preparations exhibited the same N-terminal sequence (S)-(S)-K-Y-P-R-S-V-R-R-(L)-L-P- L-X-A, indicating that different Rh(D)-positive red cells are carrying a similar Rh(D) protein. Comparative immunoprecipitation studies with the human monoclonal anti-Rh(D), (c), and (E) antibodies have also shown that Rh components from intact and papain-treated erythrocytes have similar apparent mol wt of 30 to 32 Kd and are buried in the lipid bilayer and are not readily available to the proteolytic enzyme. Further investigations by indirect affinity chromatography and one- dimensional peptide mapping of the Rh(D), (c), and (E) molecules immunopurified from a single red cell sample demonstrate that a common Rh haplotype encodes three distinct polypeptide chains carrying the Rh(D), (c), and (E) epitopes, respectively.

The histologic diagnosis of Alzheimer's disease (AD) might be aided if a more sensitive marker of aberrant A4 amyloid protein deposition were available. We screened a sample of aged brains, using immunocytochemical methods to detect the A4 protein deposition, and found that, in comparison with conventional histologic techniques (silver impregnation and Congo red), immunocytochemistry is more sensitive and allows an easier demarcation between “normal” and “abnormal.” If A4 protein deposition is accepted as a definitive marker for AD, then the age-related prevalence of AD increases dramatically. To what degree these prevalence rates are reflected in clinically detectable impairment of higher cortical function remains to be determined.

Genetic information from the bacterium Escherichia coli was transferred to human cells by means of the specialized transducing phage lambda plac carrying the bacterial z gene for the enzyme beta-galactosidase (geta-D-galactoside galactohydrolase, EC 3.2.1.23). As recipient cells, cultured skin fibroblasts from a patient with generalized gangliosidosis (GMI-gangliosidosis Type I) characterized by a severe deficiency of beta-galactosidase activity were used. The deficient human cells were incubated with the bacteriophage lambda plac or lambda plac DNA and beta-galactosidase activity was measured in order to detect gene transfer and acceptance of the prokaryotic information in the mammalian system for transcription and translation. The expression of the phage genome in the deficient fibroblasts could be demonstrated by detection of higher beta-galactosidase activity after incubation with phage lambda plac in three out of 19 experiments and in four out of 16 experiments after treatment with lambda plac DNA. Lambda plac DNA induced much higher enzyme activities than infective phage particles. Immunochemical and physicochemical assays could not distinguish the induced beta-galactosidase activity from that of the z-gene product of E. coli.

The interaction of the NH2-terminal DNA-binding domain of lac repressor with synthetic oligo[d(AT)] was studied by a photo-CIDNP technique (CIDNP is chemically induced dynamic nuclear polarization). Three of the four tyrosines of the NH2-terminal region were found to be accessible to the photosensitive dye. The corresponding ring proton resonances were enhanced in the photo-CIDNP 1H NMR spectrum, and the only histidine (histidine 29) was located at the surface of the domain, which is supposed to be linked to the core protein of lac repressor by a flexible hinge region. After complex formation of the NH2-terminal region with oligo[d(AT)], two of the three tyrosine residues were no longer accessible to solvent or to photosensitive dye, which is strong evidence that the two tyrosines are part of the contact region.

The first Caucasian (MD) shown to exhibit the low-frequency MNSs system antigen, Dantu was detected due to an increased tendency of erythrocytes to be aggregated by substances that promote red cell agglutination. The donor was found to exhibit a novel variety of the Dantu gene complex (DantuMD), as judged from biochemical, immunochemical, and serological studies. The glycophorin (GP) A level of MD's erythrocyte membranes were slightly decreased (about 17%) but GP B was not significantly different from normal. GP A and GP B of MD's cells were shown to carry M and N or S and s antigens, respectively, indicating that MD exhibits two genes encoding GP A and two genes encoding GP B. MD's cells contain a Dantu-, N- and s-specific GP B-GP A hybrid GP (molar ratio to GP A approx. 0.6 : 1.0). Partial amino-acid sequence analysis indicates that the structure of this molecule is rather similar to, or completely identical with, that of the hybrid GP in DantuNE erythrocytes. The residues 1–39 or 40–99 of the latter molecule correspond to the residues 1–39 of s-specific GP B and the residues 72–131 of GP A, respectively. Statistical evidence suggests that MD exhibits a single gene encoding the hybrid GP. Thus, MD appears to be heterozygous for a typical anti-Lepore type gene complex that seems to comprise genes for GP A, GP B, and the GP B-GP A hybrid. The diminished GP A level and a decreased galactose-oxidase labelling of the major membrane protein (anion channel protein, band 3) in MD's cells is in accordance with previous data suggesting that band 3 might form a complex with GP A and the Dantu-specific hybrid GP. This complex formation may be necessary for optimum incorporation of the latter molecules into the membrane.

Mutations in the presenilin (PS) genes are linked to early onset familial Alzheimer's disease (FAD). PS-1 proteins are proteolytically processed by an unknown protease to two stable fragments of approximately 30 kDa (N-terminal fragment (NTF)) and approximately 20 kDa (C-terminal fragment (CTF)) (Thinakaran, G., Borchelt, D. R., Lee, M. K., Slunt, H. H., Spitzer, L., Kim, G., Ratovitsky, T., Davenport, F., Nordstedt, C., Seeger, M., Hardy, J., Levey, A. I., Gandy, S. E., Jenkins, N. A., Copeland, N. G., Price, D. L., and Sisodia, S. S. (1996) Neuron 17, 181-190). Here we show that the CTF and NTF of PS-1 bind to each other. Fractionating proteins from 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid-extracted membrane preparations by velocity sedimentation reveal a high molecular mass SDS and Triton X-100-sensitive complex of approximately 100-150 kDa. To prove if both proteolytic fragments of PS-1 are bound to the same complex, we performed co-immunoprecipitations using multiple antibodies specific to the CTF and NTF of PS-1. These experiments revealed that both fragments of PS-1 occur as a tightly bound non-covalent complex. Upon overexpression, unclipped wild type PS-1 sediments at a lower molecular weight in glycerol velocity gradients than the endogenous fragments. In contrast, the non-cleavable, FAD-associated PS-1 Deltaexon 9 sediments at a molecular weight similar to that observed for the endogenous proteolytic fragments. This result may indicate that the Deltaexon 9 mutation generates a mutant protein that exhibits biophysical properties similar to the naturally occurring PS-1 fragments. This could explain the surprising finding that the Deltaexon 9 mutation is functionally active, although it cannot be proteolytically processed (Baumeister, R., Leimer, U., Zweckbronner, I., Jakubek, C., Grünberg, J., and Haass, C. (1997) Genes & Function 1, 149-159; Levitan, D., Doyle, T., Brousseau, D., Lee, M., Thinakaran, G., Slunt, H., Sisodia, S., and Greenwald, I. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 14940-14944). Formation of a high molecular weight complex of PS-1 composed of both endogenous PS-1 fragments may also explain the recent finding that FAD-associated mutations within the N-terminal portion of PS-1 result in the hyperaccumulation not only of the NTF but also of the CTF (Lee, M. K., Borchelt, D. R., Kim, G., Thinakaran, G., Slunt, H. H., Ratovitski, T., Martin, L. J., Kittur, A., Gandy, S., Levey, A. I., Jenkins, N., Copeland, N., Price, D. L., and Sisodia, S. S. (1997) Nat. Med. 3, 756-760). Moreover, these results provide a model to understand the highly regulated expression and processing of PS proteins.

Familial Alzheimer's disease (FAD) is a dominantly inherited condition that may present with an early onset, and myoclonus occurs frequently in the course of the disease. We report clinical and neuropathologic data on 2 large Belgian families with FAD in which we obtained 17 autopsies of the CNS. In family A, each of 11 autopsies had the typical neuropathologic features of Alzheimer's disease (AD), and there were a few cerebellar plaques in the molecular layer. In family B, in addition to the typical characteristics of AD in 6 autopsies, there were numerous amyloid plaques in the cortical cerebellar layers. In both families, we immunostained the amyloid deposits for the A4 protein, and they were negative for prion-associated protein immunoreactivity.

We present the nucleotide sequences and derived amino acid sequences of cDNAs that encode the complete precursors of the extrinsic ‘23 kDa’ and ‘16 kDa’ polypeptides associated with the photosynthetic oxygen‐evolving complex from spinach. The luminal proteins consist of 267/186 (precursor/mature 23 kDa protein) and 232/149 (16 kDa polypeptide) amino acid residues corresponding to molecular masses of 28.5/20.2 and 24.9/16.5 kDa, respectively. Secondary structure predictions disclose epitopes that are potential candidates for two‐step processing of the precursors during import and intraorganelle routing as well as for calcium sequestering, chloride binding and subunit/subunit interaction.

In this immunocytochemical study, the brains of nine squirrel monkeys (Saimiri sciureus), ranging from 8 to 27 years of age, were examined for the presence and distribution of ß/A4 amyloid, a 4-kilodalton peptide. In aged squirrel monkeys, amyloid is associated primarily with intracerebral and meningeal capillaries and arterioles and occurs to a lesser degree as small and/or diffuse deposits in the neural parenchyma and in the dense cores of senile plaques. Cerebrovascular amyloid is found primarily in neocortex, amygdala, and septum verum and is rare or nonexistent in other subcortical gray structures, white matter, cerebellum, and spinal cord; this pattern of localization is comparable to that in humans with cerebral amyloid angiopathy. There is a significant correlation between cerebrovascular and parenchymal deposits of amyloid. However, cerebrovascular amyloid is always the most abundant form in squirrel monkeys, even in cases of severe cerebral amyloidosis. In contrast to squirrel monkeys, aged rhesus monkeys (Macaca mulatta) develop, mostly parenchymal deposits of amyloid and have relatively less vascular amyloid. This species difference in the histological distribution of amyloid suggests that separate mechanisms may influence the accumulation of amyloid in cerebral blood vessels and in the neural parenchyma. These data also indicate that the squirrel monkey can serve as a model for investigations of cerebrovascular amyloidosis.

In order to develop a system which allows infection by an epitope-specific phage-antibody via an F-pilus expressing that epitope, a study on the expression of foreign sequences on F-pilin was undertaken. Initially, a plasmid library was constructed with random sequences encoding one to five amino acid residues fused to the C terminus of F-pilin (traA) which was used to complement an F-plasmid with an amber mutation in traA. Functional F-pilin fusions were detected using the filamentous phage, fUSE2, which transduces tetracycline resistance, as well as immunoblots using a monoclonal antiserum specific for the acetylated N terminus of pilin. All the clones selected expressed the pilin-fusions and displayed full sensitivity towards fUSE2 infection, which was indistinguishable from the wild-type F-pilin. The sequences of fUSE2-sensitive clones when compared to randomly selected clones which were not fUSE2-sensitive, revealed no obvious pattern in the amino acid residues fused to the C terminus, except for a preference for a hydrophilic amino acid at position +1. Mutating the C-terminal Leu in wt (wild-type) pilin to Ser blocked pilus assembly and fUSE2 infection; the pilin was correctly processed but the level of acetylation at the N terminus appeared to decrease. Fusing a known epitope (myc) directly to the C terminus blocked processing of F-pilin leading to loss of F-pilus assembly and function. The introduction of random sequences betweentraA and this epitope yielded fully recombinant, functional F-pili but this appeared to be due to processing of the extension by an unidentified protease leading to loss of the epitope. Surface expression of another epitope (G2-10) was clearly demonstrated by immuno-electron microscopy of pili with a G2-10 monoclonal antibody. A different five amino acid residue spacer between the F-pilin C terminus and the G2-10 epitope produced a system that was transfer-proficient and fUSE2-sensitive, but the pili were barely detectable by immunoblots or by electron microscopy. While the underlying rules that govern successful epitope expression at the C terminus of F-pilin remain elusive, many types of foreign sequences can be displayed with varying degrees of success. Our results also suggest that pilin sequence determines a number of steps in the complex pathway for pilus assembly.

By screening lambda gt11 cDNA expression libraries prepared from poly(A+) RNA of illuminated (4 and 16 h) spinach seedlings (Spinacia oleracea var. Monatol), we have recovered phage containing inserts corresponding to the complete “10-kDa” precursor polypeptide associated with the water oxidizing photosystem II. The amino acid sequence deduced from the nucleotide sequence indicates a polypeptide of 140 amino acid residues (15.0 kDa). The mature protein is predicted to consist of 99 amino acid residues (10.8 kDa). The identity of the recombinant DNAs was established serologically and by determining an N-terminal sequence of 25 amino acid residues from the purified authentic protein. This sequence was found in the cDNA-derived amino acid sequence. In addition, a monospecific antiserum detected the appropriate polypeptides when copy-RNA was translated into protein which was imported into isolated unbroken chloroplasts. The transit sequence (41 amino acid residues; 4.2 kDa) is markedly dissimilar to those of the three extrinsic, luminal photosystem II proteins. Secondary structure prediction suggests a transmembrane arrangement of the 10-kDa polypeptide in the thylakoid membrane, with the N terminus in the thylakoid lumen and a 17-amino acid C-terminal sequence at the outside partitions. This is consistent with biochemical and functional data for the protein. A major transcript of 1.0 kilobases has been observed in polyadenylated RNA.

The gene encoding the alpha-subunit of the Na+ pump oxalacetate decarboxylase of Klebsiella pneumoniae was cloned and sequenced. The deduced primary structure of the protein was confirmed by protein sequencing of about 30% of the polypeptide chain. The gene has a GC content of 67% and codes for 596 amino acids. The N-terminal methionine is removed in the mature protein which has a calculated molecular mass of 63,600 daltons. The protein consists of two different domains that are connected by a stretch of amino acid residues susceptible to proteolytic cleavage. Limited proteolysis of the native enzyme with trypsin produced fragments of about 51 kDa and 10.2 kDa, the latter of which started with valine 491 and contained the biotin prosthetic group. Peptide sequencing indicated binding of the biotin prosthetic group to lysine 561, 35 residues from the C terminus. The decarboxylase contains an extended alanine- and proline-rich region (positions 502-532) on the N-terminal side of the 10.2-kDa biotin domain. This sequence includes a total of 16 alanine and 9 proline residues.