Cholesterol, a modulator of membrane-associated Abeta-fibrillogenesis.

One of the major pathological features of Alzheimer's disease is the presence of extracellular amyloid plaques that are predominantly composed of the amyloid-beta peptide (Abeta). Characterisation of plaques demonstrated the predominance of two peptides differing at the carboxyl terminus by 2 hydrophobic amino acids, Abeta40 and Abeta42. Diffuse plaques associated with AD are composed predominantly of Abeta42, whereas senile plaques contain both Abeta40 and Abeta42. Recently, it has been suggested that diffuse plaque formation is initiated as a plasma membrane bound Abeta species and that Abeta42 is the critical component. In order to investigate this hypothesis, we have examined Abeta40/42-lipid interactions using in situ atomic force microscopy, electron microscopy and fluorescence anisotropy. While the association of Abeta42 with planar bilayers resulted in peptide aggregation but no fibre formation, this was not the case for Abeta40 where we observed preferential fibre formation. Cholesterol, a key membrane component and modulating factor in AD, is inversely correlated with the extent of Abeta40/42-bilayer interaction. These results were confirmed using fluorescence anisotropy to evaluate the effect of Abeta on membrane fluidity and fluorimetry to confirm membrane integrity. Our results suggest that the enhanced amyloidogenic properties of Abeta42 are not correlated with fibril formation but aggregation on bilayer surfaces.

Cholesterol, a modulator of membrane-associated Abeta-fibrillogenesis.

One of the major pathological features of Alzheimer's disease (AD) is the presence of extracellular amyloid plaques that are predominantly composed of the amyloid-beta peptide (Abeta). Characterization of plaques demonstrated the predominance of two peptides differing at the carboxyl terminus by two hydrophobic amino acids, Abeta40 and Abeta42. Diffuse plaques associated with AD are composed predominantly of Abeta42, whereas senile plaques contain both Abeta40 and Abeta42. Recently, it has been suggested that diffuse plaque formation is initiated as a plasma membrane-bound Abeta species and that Abeta42 is the critical component. In order to investigate this hypothesis, we have examined Abeta40/42-lipid interactions using in situ atomic force microscopy, electron microscopy, and fluorescence anisotropy. While the association of Abeta42 with planar bilayers resulted in peptide aggregation, but no fiber formation, this was not the case for Abeta40, where we observed preferential fiber formation. Cholesterol, a key membrane component and modulating factor in AD, is inversely correlated with the extent of Abeta40/42-bilayer interaction. These results were confirmed using fluorescence anisotropy to evaluate the effect of Abeta on membrane fluidity and fluorimetry to confirm membrane integrity. Our results suggest that the enhanced amyloidogenic properties of Abeta42 are not correlated with fibril formation, but with aggregation on bilayer surfaces.

Cholesterol, a modulator of membrane-associated Abeta-fibrillogenesis and neurotoxicity.

Recent studies have suggested that cholesterol, an important determinant of the physical state of biological membranes, plays a significant role in the development of Alzheimer's disease. We have employed in situ scanning probe microscopy, fluorescence anisotropy, and electron microscopy to investigate how cholesterol levels within total brain lipid bilayers effect amyloid beta-peptide (Abeta)-assembly. Fluorescence anisotropy measurements revealed that the relative fluidity of the total brain lipid membranes was influenced by the level of cholesterol and the addition of Abeta40 resulted in a decrease in the overall vesicle fluidity. In situ scanning probe microscopy performed on supported planar bilayers of total brain lipid revealed a correlation between membrane fluidity, as influenced by cholesterol level, and the extent of Abeta-insertion and subsequent fibrillogenesis. These observations were consistent with fluorescence microscopy studies of PC-12 and SH-SY5Y cell lines exposed to exogenous Abeta, which revealed an inverse correlation between membrane cholesterol level, and Abeta-cell surface binding and subsequent cell death. These results collectively suggest that Abeta-cell surface interactions are mediated by cellular cholesterol levels, the distribution of cholesterol throughout the cell, and membrane fluidity.

Identification of three functions of the adenovirus e4orf6 protein that mediate p53 degradation by the E4orf6-E1B55K complex.

Complexes containing adenovirus E4orf6 and E1B55K proteins play critical roles in productive infection. Both proteins interact directly with the cellular tumor suppressor p53, and in combination they promote its rapid degradation. To examine the mechanism of this process, degradation of exogenously expressed p53 was analyzed in p53-null human cells infected with adenovirus vectors encoding E4orf6 and/or E1B55K. Coexpression of E4orf6 and E1B55K greatly reduced both the level and the half-life of wild-type p53. No effect was observed with the p53-related p73 proteins, which did not appear to interact with E4orf6 or E1B55K. Mutant forms of p53 were not degraded if they could not efficiently bind E1B55K, suggesting that direct interaction between p53 and E1B55K may be required. Degradation of p53 was independent of both MDM2 and p19ARF, regulators of p53 stability in mammalian cells, but required an extended region of E4orf6 from residues 44 to 274, which appeared to possess three separate biological functions. First, residues 39 to 107 were necessary to interact with E1B55K. Second, an overlapping region from about residues 44 to 218 corresponded to the ability of E4orf6 to form complexes with cellular proteins of 19 and 14 kDa. Third, the nuclear retention signal/amphipathic arginine-rich alpha-helical region from residues 239 to 253 was required. Interestingly, neither the E4orf6 nuclear localization signal nor the nuclear export signal was essential. These results suggested that if nuclear-cytoplasmic shuttling is involved in this process, it must involve another export signal. Degradation was significantly blocked by the 26S proteasome inhibitor MG132, but unlike the HPV E6 protein, E4orf6 and E1B55K were unable to induce p53 degradation in vitro in reticulocyte lysates. Thus, this study implies that the E4orf6-E1B55K complex may direct p53 for degradation by a novel mechanism.

Expression, purification, and characterization of recombinant forms of membrane-bound cytochrome c-550nm from Bacillus subtilis.

Bacillus subtilis expresses a cytochrome c-550nm that participates in respiratory electron transfer and is an integral membrane protein. Analysis of the B. subtilis cytochrome c-550nm amino acid sequence predicts a single N-terminal transmembrane helix attached to a water-soluble heme binding domain [C. von Wachenfeldt and L. Hederstedt (1990) J. Biol. Chem. 265, 13939-13948]. We have purified cytochrome c-550nm from wild-type B. subtilis and B. subtilis transformed with the shuttle vector pHP13 containing the gene for B. subtilis cytochrome c-550nm (cccA). In B. subtilis transformed with pHP13/cccA there is better than eightfold more membrane-bound cytochrome c-550nm than in wild-type B. subtilis. The overexpressed cytochrome c-550nm can be purified by chromatography on hydroxylapatite and Q-Sepharose media. A six-histidine tag has been added to the C-terminus of cytochrome c-550nm from B. subtilis as a further aid for purification. This strain produces cytochrome c-550nm to a level fourfold greater than wild type and allows for one-step purification using metal affinity chromatography. UV-Vis spectroscopy detects no change in the heme C spectrum due to the addition of six histidines. Neither form of B. subtilis cytochrome c-550nm is stable in its reduced state in aerated buffer, unless EDTA is added. The two forms, wild-type and his-tagged, of cytochromes c have similar midpoint redox potentials of 195 and 185 mV, respectively, and are equally good substrates for B. subtilis cytochrome c oxidase. We conclude that the addition of the histidine tag eases the purification of cytochrome c-550nm from B. subtilis plasma membranes and that the additional metal binding site does not compromise the stability or functional properties of the protein.

Analysis of synthesis, stability, phosphorylation, and interacting polypeptides of the 34-kilodalton product of open reading frame 6 of the early region 4 protein of human adenovirus type 5.

The 34-kDa early-region 4 open reading frame 6 (E4orf6) product of human adenovirus type 5 forms complexes with both the cellular tumor suppressor p53 and the viral E1B 55-kDa protein (E1B-55kDa). E4orf6 can inhibit p53 transactivation activity, as can E1B-55kDa, and in combination these viral proteins cause the rapid turnover of p53. In addition, E4orf6-55kDa complexes play a critical role at later times in the regulation of viral mRNA transport and shutoff of host cell protein synthesis. In the present study, we have further characterized some of the biological properties of E4orf6. Analysis of extracts from infected cells by Western blotting indicated that E4orf6, like E1A and E1B products, is present at high levels until very late times, suggesting that it is available to act throughout the infectious cycle. This pattern is similar to that of E4orf4 but differs markedly from that of another E4 product, E4orf6/7, which is present only transiently. Synthesis of E4orf6 is maximal at early stages but ceases completely with the onset of shutoff of host protein synthesis; however, it was found that unlike E4orf6/7, E4orf6 is very stable, thus allowing high levels to be maintained even at late times. E4orf6 was shown to be phosphorylated at low levels. Coimmunoprecipitation studies in cells lacking p53 indicated that E4orf6 interacts with a number of other proteins. Five of these were shown to be viral or virally induced proteins ranging in size from 102 to 27 kDa, including E1B-55kDa. One such species, of 72 kDa, was shown not to represent the E2 DNA-binding protein and thus remains to be identified. Another appeared to be the L4 100-kDa nonstructural adenovirus late product, but it appeared to be present nonspecifically and not as part of an E4orf6 complex. Apart from p53, three additional cellular proteins, of 84, 19, and 14 kDa were detected by using an adenovirus vector that expresses only E4orf6. The 19-kDa species and a 16-kDa cellular protein were also shown to interact with E4orf6/7. It is possible that complex formation with these viral and cellular proteins plays a role in one or more of the biological activities associated with E4orf6 and E4orf6/7.

Aerobiology of the Colorado Rockies: pollen count comparisons between Vail and Denver, Colorado.

Pollen patterns were compared between Vail, CO (8,200 feet elevation), Aspen, CO (7,900 feet) and Denver, CO (5,280 feet) from 1984 through 1988. Counts were obtained at all sites with a volumetric intermittent cycling rotating impaction sampler. Aspen and Denver were compared in 1984, and Vail and Denver from 1985 through 1988. While counts were generally lower in the mountain sites than Denver, certain pollens, especially trees, were quite high. Ragweed was essentially absent from Aspen and Vail, and chenopod-amaranth counts were very low. Cedar, pine, and aspen frequently pollinated despite active snowfall.

Champ Camp: the Colorado Children's Asthma Camp experience.

To encourage children with asthma to enjoy outdoor activities without physical or psychosocial impairment, children's asthma camps are established throughout the country sponsored by organizations including local and state allergy societies. We wish to describe our Colorado "Champ Camp" experience as a model and reference for future similar efforts and to encourage networking by medical leadership for information sharing and guidelines development nationally. Statistics from parents' satisfaction surveys over 8 years demonstrate a positive influence on attitudes toward asthma and confidence to enter activities and sports with children without asthma.

Attempts to immunoprecipitate the LHRH precursor synthesized in cell free systems.

In order to determine the molecular weight of the Luteinizing Hormone Releasing Hormone (LHRH) precursor, poly(A)-RNA from rat hypothalami and human placenta were translated in two mRNA dependent cell free translation systems. Total translation products were immunoprecipitated with two antisera that recognized LHRH high molecular weight forms. After SDS-polyacrylamide slab gel electrophoretic analysis of the immunoprecipitated material and fluorography, we detected in both tissues a protein of 50,000 daltons with the No. 1076 antiserum. This peptide was not immunoprecipitated by the No. 743 anti-LHRH antiserum or by non-immune rabbit serum. However, this protein was not displaced by excess LHRH added during the immunoprecipitation and seemed to be present in species where LHRH has not been reported. These data demonstrated that the LHRH mRNA is present in very low amounts in hypothalamus or placenta and that the sensitivity of the assay is not high enough to recognize it.

A rapid microprocedure for isolating RNA from multiple samples of human and rat brain.

In order to establish a routine procedure for isolating undegraded RNA from small amounts of rat and human brain tissue, several techniques were investigated. Initial studies demonstrated that undegraded RNA could not be reproducibly isolated from milligram amounts of brain tissue homogenized in an aqueous medium. Several isolation techniques utilizing tissue homogenization in the denaturing agent guanidinium chloride were compared. This method of homogenization, followed by sedimentation of RNA through cesium chloride, resulted in good yields of undegraded translationally active RNA. A maximum of 6 RNA samples could be processed simultaneously. In contrast, when homogenization in guanidinium chloride was followed by repeated guanidinium chloride-ethanol precipitations many samples could be processed simultaneously. The resulting RNA yields were low. The introduction of several modifications in the guanidinium chloride-ethanol precipitation technique resulted in a high yield of undegraded translationally active RNA. DNA was removed by two guanidinium-ethanol precipitations. Residual protein was digested with proteinase K. RNA was precipitated after extraction with phenol-chloroform-isoamyl alcohol. This refined procedure allows the recovery, in high yields, of translationally active undegraded RNA which is both DNA and protein free. Thirty-six samples can be processed in one day.

The differential distribution of beta tubulin mRNAs in individual mammalian brain cells.

It has been shown by in vitro translation of polyadenylated messenger RNAs (poly(A)+ mRNAs) that the mRNAs encoding both alpha and beta tubulin isotypes are present at much higher relative levels in the developing rat brain than they are in the adult, suggesting that the requirements for tubulin subunits vary with cell type and/or with the developmental stages of a particular cell type. The postnatally developing rat cerebellum, with its readily identifiable cell populations that perform the gamut of developmental tasks, is a suitable model for analyzing specific cellular mRNA distributions during development. In this report, by in situ hybridization techniques it is shown that, by comparison to total cellular poly(A)+ mRNA levels, there is relatively more of the total beta tubulin mRNAs in mitotically active external granule layer cells than in those in the internal granule layer. These results show that migration and differentiation of these granule cells is accompanied by a decrease in their beta tubulin mRNA levels relative to the levels in granule cells of the external granule cell layer. Furthermore, the relative levels of beta tubulin mRNA both in the prenatally formed Purkinje cells and the postnatally formed stellate cells are two to fourfold less than in the granule cells of the internal granule cell layer.

In situ hybridization--visualization and quantitation of genetic expression in mammalian brain.

In situ hybridization techniques have been developed that quantitate the relative levels of specific mRNAs in individual cell types of a heterogeneous tissue, the mammalian brain. Here, we discuss those special procedures and precautions necessary for hybridizing radiolabeled probes to developing and adult brain mRNAs. The probes discussed include double-stranded recombinant DNA, as well as single-stranded DNA, RNA, and polyuridylate. We detail the procedure for determining the relative numbers of hybrids formed and computing the ratio of specific mRNAs to total polyadenylated mRNA and discuss the importance of this ratio for comparison of relative levels of specific mRNAs within and among cell types in an individual brain or between brains.

Altered expression of different tubulin electrophoretic variants during human cortex development.

Two alpha and two beta tubulin subunits are synthesized in vitro by polyadenylated mRNAs isolated from fetal and adult human cortex. The relative levels of the mRNAs encoding the different subunits change dramatically during development. In the fetus, the mRNA for beta 1 tubulin is present at higher levels than that of the beta 2 electrophoretic variant. There are relatively high levels of the mRNAs encoding both alpha subunits. In the adult, the levels of the mRNAs encoding both the alpha subunits and the beta 1 subunit are decreased relative to those of the mRNAs encoding the beta 2 subunit. These results suggest that fetal and adult cortical cells have very different requirements for the different tubulin electrophoretic variants.

Specific messenger RNA changes in Joseph disease cerebella.

Joseph disease is an autosomal-dominant, spinocerebellar degeneration characterized at the biochemical level by elevations in the steady-state levels of several abundant proteins (H, J, and L) in affected brain areas such as the cerebellar cortex. The increased levels of these proteins could either be a consequence of a relative increase in their de novo synthesis or result from altered rates of proteolysis in degenerating brain cells. These alternatives can be distinguished by comparing the in vitro protein-synthetic capacities of the messenger ribonucleic acid populations isolated from cerebellar cortex of control subjects and patients with Joseph disease. Protein H (glial fibrillary acidic protein) is synthesized at detectable levels by all messenger ribonucleic acid isolates, and the levels of its translatable messenger ribonucleic acid are reproducibly increased in ribonucleic acids isolated from cerebellar cortex of patients with Joseph disease as compared with those isolated from cerebellar cortex of control subjects. Thus, the increased level of protein H in Joseph disease is a consequence of an increase in its de novo synthesis and is correlated with the increased number of cerebellar glial cells. In contrast to these results, there is no detectable synthesis of proteins J and L by messenger ribonucleic acid populations isolated from cerebellar cortex of either Joseph disease patients or control subjects, suggesting that the increased levels of these proteins in affected cerebellar cortex are a consequence of posttranslational protein modifications.

The enzymes of acetyl-CoA metabolism in differentiating cholinergic (s-20) and noncholinergic (NIE-115) neuroblastoma cells.

Dibutyryl cyclic AMP and butyrate inhibited growth of S-20 (cholinergic) and NIE-115 (adrenergic) neuroblastoma clones. Both these drugs resulted in a parallel increase of choline acetyltransferase and ATP-citrate lyase activities in S-20 neuroblastoma cells. On the other hand, the increase in tyrosine hydroxylase activity in NIE-115 caused by these drugs was not accompanied by a significant change in ATP-citrate lyase activity. Both dibutyryl cyclic AMP and butyrate caused a decrease in fatty acid synthetase activity in both cell lines. The activities of pyruvate dehydrogenase, citrate synthase, choline acetyltransferase, and lactate dehydrogenase in both S-20 and NIE-115 cells were not significantly influenced by the drugs. ATP-citrate lyases from S-20 and NIE-115 had similar kinetic and immunological properties, and their subunits had the same molecular weight as the rat liver enzyme. These data indicate that the differential regulation of ATP-citrate lyase activity in cholinergic and adrenergic cells does not result from the existence of different molecular forms of the enzyme in these cell lines. They also provide further evidence to support the hypothesis that ATP-citrate lyase activity increases during maturation of normal cholinergic neurons and decreases in noncholinergic cells of the brain.

Brain protein and messenger RNA identification in the same cell.

We have devised techniques with which to detect specific proteins as well as mRNAs in individual cells of the nervous system. We have localized proteins to specific cells in sections of the neonatal rat cerebellum by immunofluorescence and in the same cells have localized mRNAs by in situ hybridization. The specific protein identified was glial fibrillary acidic protein. The [3H] complementary DNA probes used in the in situ hybridization experiments were synthesized using as templates polyadenylated mRNAs isolated from neonatal rat cerebellum. Such dual detection can be used to assess the cellular sites of synthesis of the many proteins that have been localized in brain and other tissues by immunohistochemistry.

Normalization of cerebellar RNA synthesis and mRNA levels after treatment of graft versus host disease.

We have previously shown that neonatal rats with graft versus host disease (GVHD) (1) synthesize significantly less cerebellar RNA, (2) have RNA that is less translationally active, and (3) have changes in the relative abundance of certain mRNAs, including the induction of one coding for protein r that is present neither in control cerebellum nor in other brain regions at any age. Here we report on the ability of the cerebellum to recover from GVHD-induced changes in the synthesis of total RNA and in the relative levels of specific mRNAs. In order to halt the disease, 11-day-old diseased Fischer animals were injected with hyperimmune alloantiserum daily for 3 days. Cytoplasmic RNAs were isolated from the cerebella of 14-day-old serum-treated animals, their diseased littermates that were not treated with serum, and littermate controls. Comparison, by two-dimensional gel analysis, of the in vitro synthesized mRNA translation products showed that most GVHD-induced alterations in the levels of specific mRNAs were not present in serum-treated animals. In particular, protein r was not synthesized by cerebellar RNAs isolated from serum-treated animals. These results show that the adverse effects of this disease are reversible at the molecular level.